WorldWideScience

Sample records for excision repair gene

  1. Molecular cloning of the human excision repair gene ERCC-6.

    NARCIS (Netherlands)

    C. Troelstra (Christine); H. Odijk (Hanny); J. de Wit (Jan); A. Westerveld (Andries); L.H. Thompson; D. Bootsma (Dirk); J.H.J. Hoeijmakers (Jan)

    1990-01-01

    textabstractThe UV-sensitive, nucleotide excision repair-deficient Chinese hamster mutant cell line UV61 was used to identify and clone a correcting human gene, ERCC-6. UV61, belonging to rodent complementation group 6, is only moderately UV sensitive in comparison with mutant lines in groups 1 to

  2. Polymorphism of the DNA Base Excision Repair Genes in Keratoconus

    Science.gov (United States)

    Wojcik, Katarzyna A.; Synowiec, Ewelina; Sobierajczyk, Katarzyna; Izdebska, Justyna; Blasiak, Janusz; Szaflik, Jerzy; Szaflik, Jacek P.

    2014-01-01

    Keratoconus (KC) is a degenerative corneal disorder for which the exact pathogenesis is not yet known. Oxidative stress is reported to be associated with this disease. The stress may damage corneal biomolecules, including DNA, and such damage is primarily removed by base excision repair (BER). Variation in genes encoding BER components may influence the effectiveness of corneal cells to cope with oxidative stress. In the present work we genotyped 5 polymorphisms of 4 BER genes in 284 patients and 353 controls. The A/A genotype of the c.–1370T>A polymorphism of the DNA polymerase γ (POLG) gene was associated with increased occurrence of KC, while the A/T genotype was associated with decreased occurrence of KC. The A/G genotype and the A allele of the c.1196A>G polymorphism of the X-ray repair cross-complementing group 1 (XRCC1) were associated with increased, and the G/G genotype and the G allele, with decreased KC occurrence. Also, the C/T and T as well as C/C genotypes and alleles of the c.580C>T polymorphism of the same gene displayed relationship with KC occurrence. Neither the g.46438521G>C polymorphism of the Nei endonuclease VIII-like 1 (NEIL1) nor the c.2285T>C polymorphism of the poly(ADP-ribose) polymerase-1 (PARP-1) was associated with KC. In conclusion, the variability of the XRCC1 and POLG genes may play a role in KC pathogenesis and determine the risk of this disease. PMID:25356504

  3. Polymorphism of the DNA Base Excision Repair Genes in Keratoconus

    Directory of Open Access Journals (Sweden)

    Katarzyna A. Wojcik

    2014-10-01

    Full Text Available Keratoconus (KC is a degenerative corneal disorder for which the exact pathogenesis is not yet known. Oxidative stress is reported to be associated with this disease. The stress may damage corneal biomolecules, including DNA, and such damage is primarily removed by base excision repair (BER. Variation in genes encoding BER components may influence the effectiveness of corneal cells to cope with oxidative stress. In the present work we genotyped 5 polymorphisms of 4 BER genes in 284 patients and 353 controls. The A/A genotype of the c.–1370T>A polymorphism of the DNA polymerase γ (POLG gene was associated with increased occurrence of KC, while the A/T genotype was associated with decreased occurrence of KC. The A/G genotype and the A allele of the c.1196A>G polymorphism of the X-ray repair cross-complementing group 1 (XRCC1 were associated with increased, and the G/G genotype and the G allele, with decreased KC occurrence. Also, the C/T and T as well as C/C genotypes and alleles of the c.580C>T polymorphism of the same gene displayed relationship with KC occurrence. Neither the g.46438521G>C polymorphism of the Nei endonuclease VIII-like 1 (NEIL1 nor the c.2285T>C polymorphism of the poly(ADP-ribose polymerase-1 (PARP-1 was associated with KC. In conclusion, the variability of the XRCC1 and POLG genes may play a role in KC pathogenesis and determine the risk of this disease.

  4. Cloning and characterization of the human DNA-excision repair gene ERCC-1

    NARCIS (Netherlands)

    M. van Duin (Michel)

    1988-01-01

    textabstractIt is the aim of the work described in this thesis to isolate and characterize human genes involved DNA excision repair. This will facilitate the understanding of the mechanism of this repair process whereas it also provides an important step to better understand the relationship

  5. Localization of the nucleotide excision repair gene ERCC-6 to human chromosome 10q11-q21.

    NARCIS (Netherlands)

    C. Troelstra (Christine); R.M. Landsvater; J. Wiegant; M. van der Ploeg; G. Viel; C.H.C.M. Buys; J.H.J. Hoeijmakers (Jan)

    1992-01-01

    textabstractWe have cloned the human DNA excision repair gene ERCC6 by virtue of its ability to correct the uv sensitivity of Chinese hamster overy cell mutant UV61. This mutant is a member of complementation group 6 of the nucleotide excision repair-deficient rodent mutants. By means of in situ

  6. Structure and expression of the excision repair gene ERCC6, involved in the human disorder Cockayne's syndrome group B.

    NARCIS (Netherlands)

    C. Troelstra (Christine); W. Hesen; D. Bootsma (Dirk); J.H.J. Hoeijmakers (Jan)

    1993-01-01

    textabstractThe human repair gene ERCC6--a presumed DNA (or RNA) helicase--has recently been found to function specifically in preferential nucleotide excision repair (NER). This NER subpathway is primarily directed towards repair of (the transcribed strand of) active genes. Mutations in the ERCC6

  7. Transcriptional and post-transcriptional regulation of nucleotide excision repair genes in human cells

    Energy Technology Data Exchange (ETDEWEB)

    Lefkofsky, Hailey B. [Translational Oncology Program, University of Michigan Medical School, Ann Arbor, MI (United States); Veloso, Artur [Translational Oncology Program, University of Michigan Medical School, Ann Arbor, MI (United States); Department of Radiation Oncology, University of Michigan Medical School, Ann Arbor, MI (United States); Bioinformatics Program, Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, MI (United States); Ljungman, Mats, E-mail: ljungman@umich.edu [Translational Oncology Program, University of Michigan Medical School, Ann Arbor, MI (United States); Department of Radiation Oncology, University of Michigan Medical School, Ann Arbor, MI (United States); Department of Environmental Health Sciences, School of Public Health, University of Michigan, Ann Arbor, MI (United States)

    2015-06-15

    Nucleotide excision repair (NER) removes DNA helix-distorting lesions induced by UV light and various chemotherapeutic agents such as cisplatin. These lesions efficiently block the elongation of transcription and need to be rapidly removed by transcription-coupled NER (TC-NER) to avoid the induction of apoptosis. Twenty-nine genes have been classified to code for proteins participating in nucleotide excision repair (NER) in human cells. Here we explored the transcriptional and post-transcriptional regulation of these NER genes across 13 human cell lines using Bru-seq and BruChase-seq, respectively. Many NER genes are relatively large in size and therefore will be easily inactivated by UV-induced transcription-blocking lesions. Furthermore, many of these genes produce transcripts that are rather unstable. Thus, these genes are expected to rapidly lose expression leading to a diminished function of NER. One such gene is ERCC6 that codes for the CSB protein critical for TC-NER. Due to its large gene size and high RNA turnover rate, the ERCC6 gene may act as dosimeter of DNA damage so that at high levels of damage, ERCC6 RNA levels would be diminished leading to the loss of CSB expression, inhibition of TC-NER and the promotion of cell death.

  8. Molecular characterization of the human excision repair gene ERCC-1: cDNA cloning and aminoacid homology with the yeast DNA repair gene RAD10.

    NARCIS (Netherlands)

    M. van Duin (Mark); J. de Wit (Jan); H. Odijk (Hanny); A. Westerveld (Andries); A. Yasui (Akira); M.H.M. Koken (Marcel); J.H.J. Hoeijmakers (Jan); D. Bootsma (Dirk)

    1986-01-01

    textabstractThe human excision repair gene ERCC-7 was cloned after DNA mediated gene transfer to the CHO mutant 43-38, which is sensitive to ultraviolet light and mitomycin-C. We describe the cloning and sequence analysis of the ERCC-7 cDNA and partial characterization of the gene. ERCC.1 has a size

  9. Molecular cloning of the human nucleotide-excision-repair gene ERCC4

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, L.H.; Brookman, K.W.; Weber, C.A.; Salazar, E.P. [Lawrence Livermore National Lab., CA (United States); Reardon, J.T.; Sancar, A. [Univ. of North Carolina, Chapel Hill, NC (United States); Deng, Z.; Siciliano, M.J. [Univ. of Texas Cancer Center, Houston, TX (United States)

    1994-07-19

    ERCC4 was previously identified in somatic cell hybrids as a human gene that corrects the nucleotide-excision-repair deficiency in mutant hamster cells. The cloning strategy for ERCC4 involved transfection of the repair-deficient hamster cell line UV41 with a human sCos-1 cosmid library derived from chromosome 16. Enhanced UV resistance was seen with one cosmid-library transformant and two secondary transformants of UV41. Cosmid clones carrying a functional ERCC4 gene were isolated from a library of a second transformant by selecting in Escherichia coli for expression of a linked neomycin-resistance gene that was present in the sCos-1 vector. The cosmids mapped to 16p13.13-p13.2, the location assigned to ERCC4 by using somatic cell hybrids. Upon transfection into UV41, six cosmid clones gave partial correction ranging from 30% to 64%, although all appeared to contain the complete gene. The capacity for in vitro excision of thymine dimers from a plasmid by transformant cell extracts correlated qualitatively with enhanced UV resistance.

  10. Nucleotide Excision Repair in Cellular Chromatin: Studies with Yeast from Nucleotide to Gene to Genome

    Directory of Open Access Journals (Sweden)

    Simon Reed

    2012-09-01

    Full Text Available Here we review our development of, and results with, high resolution studies on global genome nucleotide excision repair (GGNER in Saccharomyces cerevisiae. We have focused on how GGNER relates to histone acetylation for its functioning and we have identified the histone acetyl tranferase Gcn5 and acetylation at lysines 9/14 of histone H3 as a major factor in enabling efficient repair. We consider results employing primarily MFA2 as a model gene, but also those with URA3 located at subtelomeric sequences. In the latter case we also see a role for acetylation at histone H4. We then go on to outline the development of a high resolution genome-wide approach that enables one to examine correlations between histone modifications and the nucleotide excision repair (NER of UV-induced cyclobutane pyrimidine dimers throughout entire genomes. This is an approach that will enable rapid advances in understanding the complexities of how compacted chromatin in chromosomes is processed to access DNA damage and then returned to its pre-damaged status to maintain epigenetic codes.

  11. Mutational analysis of the human nucleotide excision repair gene ERCC1.

    NARCIS (Netherlands)

    A.M. Sijbers (Anneke); P.J. van der Spek (Peter); H. Odijk (Hanny); J.H. van den Berg (Jan); M. van Duin (Mark); A. Westerveld (Andries); N.G.J. Jaspers (Nicolaas); D. Bootsma (Dirk); J.H.J. Hoeijmakers (Jan)

    1996-01-01

    textabstractThe human DNA repair protein ERCC1 resides in a complex together with the ERCC4, ERCC11 and XP-F correcting activities, thought to perform the 5' strand incision during nucleotide excision repair (NER). Its yeast counterpart, RAD1-RAD10, has an additional engagement in a mitotic

  12. EZH2 suppresses the nucleotide excision repair in nasopharyngeal carcinoma by silencing XPA gene.

    Science.gov (United States)

    Huang, Yuxiang; Wang, Xuanyi; Niu, Xiaoshuang; Wang, Xiaoshen; Jiang, Rui; Xu, Tingting; Liu, Yong; Liang, Liping; Ou, Xiaomin; Xing, Xing; Li, Weiwei; Hu, Chaosu

    2017-02-01

    The enhancer of zeste homolog 2 (EZH2) is involved in a number of fundamental pathological processes of cancer. However, its role in DNA repair pathway is still unclear. Here, we have identified XPA as a novel target gene of EZH2 via a DNA repair pathway PCR array. XPA plays a pivot role in nucleotide excision repair (NER). The expression of XPA was significantly increased by EZH2 specific inhibitor GSK126 or lentiviral shEZH2 in nasopharyngeal carcinoma (NPC) CNE and 8F cell lines. Chromatin immunoprecipitation assay demonstrated that EZH2 catalyzes H3K27 trimethylation at the XPA promoters. Furthermore, we validated the negative correlation of EZH2 and XPA in a NPC tissue microarray by immunohistochemistry staining. We also found that high expression of EZH2 was positively correlated with advanced T, N, and AJCC stage of NPC; and low expression of XPA was positively correlated with advanced T and N stage. In NPC cell lines, increased XPA expression by EZH2 inhibition resulted in a more rapid removal of UVC induced 6-4PP- and CPD-DNA adducts, as well as enhanced efficiency of DNA repair after UVC irradiation as detected by the Comet assay and immunofluorescence staining of γH2Ax. Consistently, increased cell clonogenic survival, decreased apoptosis, and necrosis after UVC irradiation, and increased resistance to DNA damaging agent cisplatin was also observed in EZH2 inhibited cells. These results illustrate that EZH2 may promote carcinogenesis and cancer development of NPC by transcriptional repression of XPA gene and inactivation of NER pathway. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  13. Cloning, comparative mapping, and RNA expression of the mouse homologues of the Saccharomyces cerevisiae nucleotide excision repair gene RAD23.

    NARCIS (Netherlands)

    P.J. van der Spek (Peter); C.E. Visser (Cécile); F. Hanaoka (Fumio); B. Smit (Bep); A. Hagemeijer (Anne); D. Bootsma (Dirk); J.H.J. Hoeijmakers (Jan)

    1996-01-01

    textabstractThe Saccharomyces cerevisiae RAD23 gene is involved in nucleotide excision repair (NER). Two human homologs of RAD23, HHR23A and HHR23B (HGMW-approved symbols RAD23A and RAD23B), were previously isolated. The HHR23B protein is complexed with the protein defective in the cancer-prone

  14. Polymorphisms in nucleotide excision repair genes, smoking and intake of fruit and vegetables in relation to lung cancer

    DEFF Research Database (Denmark)

    Raaschou-Nielsen, Ole; Sørensen, Mette; Overvad, Kim

    2007-01-01

    in the XPC, XPA and XPD genes involved in the nucleotide excision DNA repair pathway and analysed possible interactions with smoking and dietary intake of fruit and vegetables in relation to risk for lung cancer. We found that intake of fruit was associated with lower risk for lung cancer only among carriers...

  15. Nucleotide excision repair in yeast

    NARCIS (Netherlands)

    Eijk, Patrick van

    2012-01-01

    Nucleotide Excision Repair (NER) is a conserved DNA repair pathway capable of removing a broad spectrum of DNA damage. In human cells a defect in NER leads to the disorder Xeroderma pigmentosum (XP). The yeast Saccharomyces cerevisiae is an excellent model organism to study the mechanism of NER. The

  16. A presumed DNA helicase, encoded by the excision repair gene ERCC-3 is involved in the human repair disorders xeroderma pigmentosum and Cockayne's syndrome.

    NARCIS (Netherlands)

    G. Weeda (Geert); R.C.A. van Ham; W. Vermeulen (Wim); D. Bootsma (Dirk); A.J. van der Eb; J.H.J. Hoeijmakers (Jan)

    1990-01-01

    textabstractThe human gene ERCC-3 specifically corrects the defect in an early step of the DNA excision repair pathway of UV-sensitive rodent mutants of complementation group 3. The predicted 782 animo acid ERCC-3 protein harbors putative nucleotide, chromatin, and helix-turn-helix DNA binding

  17. Mitochondrial base excision repair assays

    DEFF Research Database (Denmark)

    Maynard, Scott; de Souza-Pinto, Nadja C; Scheibye-Knudsen, Morten

    2010-01-01

    The main source of mitochondrial DNA (mtDNA) damage is reactive oxygen species (ROS) generated during normal cellular metabolism. The main mtDNA lesions generated by ROS are base modifications, such as the ubiquitous 8-oxoguanine (8-oxoG) lesion; however, base loss and strand breaks may also occur....... Many human diseases are associated with mtDNA mutations and thus maintaining mtDNA integrity is critical. All of these lesions are repaired primarily by the base excision repair (BER) pathway. It is now known that mammalian mitochondria have BER, which, similarly to nuclear BER, is catalyzed by DNA...

  18. Molecular spectrum of excision repair cross-complementation group 8 gene defects in Chinese patients with Cockayne syndrome type A

    OpenAIRE

    Wang, Xiaozhu; Huang, Yu; Yan, Ming; Li, Jiuwei; Ding, Changhong; Jin, Hong; Fang, Fang; Yang, Yanling; Wu, Baiyan; Chen, Dafang

    2017-01-01

    There are two genetics complementary groups Cockayne syndrome type A and B (CS-A and CS-B OMIM 216400, 133540), which is a rare autosomal recessive segmental progeroid syndrome. Homozygous or compound heterozygous mutations in the excision repair cross-complementation group 8 gene (ERCC8) result in CS-A, and mutations in ERCC6 result in CS-B. Homozygous ERCC6/ERCC8 mutations also result in UV-sensitive syndrome. In this study, twenty-one Han Chinese patients with CS were investigated to ident...

  19. Incomplete complementation of the DNA repair defect in cockayne syndrome cells by the denV gene from bacteriophage T4 suggests a deficiency in base excision repair.

    Science.gov (United States)

    Francis, M A; Bagga, P S; Athwal, R S; Rainbow, A J

    1997-10-01

    Endonuclease V (denV) from bacteriophage T4 has been examined for its ability to complement the repair defect in Cockayne syndrome (CS) cells of complementation groups A and B. CS is an autosomal recessive disorder characterized by hypersensitivity to UV light and a defect in the preferential repair of UV-induced lesions in transcriptionally active DNA by the nucleotide excision repair (NER) pathway. The denV gene was introduced into non-transformed normal and CS fibroblasts transiently via a recombinant adenovirus (Ad) vector and into SV40-transformed normal and CS cells via a retroviral vector. Expression of denV in CS-A cells resulted in partial correction of the UV-sensitive phenotype in assays of gene-specific repair and cell viability, while correction of CS-B cells by expression of denV in the same assays was minimal or non-existent. In contrast, denV expression led to enhanced host cell reactivation (HCR) of viral DNA synthesis in both CS complementation groups to near normal levels. DenV is a glycosylase which is specific for cyclobutane-pyrimidine dimers (CPDs) but does not recognize other UV-induced lesions. Previous work has indicated that CS cells can efficiently repair all non-CPD UV-induced transcription blocking lesions (S.F. Barrett et al.. Mutation Res. 255 (1991) 281-291 [1]) and that denV incised lesions are believed to be processed via the base excision repair (BER) pathway. The inability of denV to complement the NER defect in CS cells to normal levels implies an impaired ability to process denV incised lesions by the BER pathway, and suggests a role for the CS genes, particularly the CS-B gene, in BER.

  20. Deficiency in nucleotide excision repair family gene activity, especially ERCC3, is associated with non-pigmented hair fiber growth.

    Directory of Open Access Journals (Sweden)

    Mei Yu

    Full Text Available We conducted a microarray study to discover gene expression patterns associated with a lack of melanogenesis in non-pigmented hair follicles (HF by microarray. Pigmented and non-pigmented HFs were collected and micro-dissected into the hair bulb (HB and the upper hair sheaths (HS including the bulge region. In comparison to pigmented HS and HBs, nucleotide excision repair (NER family genes ERCC1, ERCC2, ERCC3, ERCC4, ERCC5, ERCC6, XPA, NTPBP, HCNP, DDB2 and POLH exhibited statistically significantly lower expression in non- pigmented HS and HBs. Quantitative PCR verified microarray data and identified ERCC3 as highly differentially expressed. Immunohistochemistry confirmed ERCC3 expression in HF melanocytes. A reduction in ERCC3 by siRNA interference in human melanocytes in vitro reduced their tyrosinase production ability. Our results suggest that loss of NER gene function is associated with a loss of melanin production capacity. This may be due to reduced gene transcription and/or reduced DNA repair in melanocytes which may eventually lead to cell death. These results provide novel information with regard to melanogenesis and its regulation.

  1. Cloning, comparative mapping, and RNA expression of the mouse homologues of the Saccharomyces cerevisiae nucleotide excision repair gene RAD23

    Energy Technology Data Exchange (ETDEWEB)

    Spek, P.J. van der; Visser, C.E.; Bootsma, D. [Erasmus Univ., Rotterdam (Netherlands)] [and others

    1996-01-01

    The Saccharomyces cerevisiae RAD23 gene is involved in nucleotide excision repair (NER). Two human homologs of RAD23, HHR23A and HHR23B (HGMW-approved symbols RAD23A and RAD23B), were previously isolated. The HHR23B protein is complexed with the protein defective in the cancer-prone repair syndrome xeroderma pigmentosum, complementation group C, and is specifically involved in the global genome NER subpathway. The cloning of both mouse homologs (designated MHR23A and MHR23B) and detailed sequence comparison permitted the deduction of the following overall structure for all RAD23 homologs: an ubiquitin-like N-terminus followed by a strongly conserved 50-amino-acid domain that is repeated at the C-terminus. We also found this domain as a specific C-terminal extension of one of the ubiquitin-conjugating enzymes, providing a second link with the ubiquitin pathway. By means of in situ hybridization, MHR23A was assigned to mouse chromosome 8C3 and MHR23B to 4B3. Because of the close chromosomal proximity of human XPC and HHR23B, the mouse XPC chromosomal location was determined (6D). Physical disconnection of the genes in mouse argues against a functional significance of the colocalization of these genes in human. Northern blot analysis revealed constitutive expression of both MHR23 genes in all tissues examined. Elevated RNA expression of both MHR23 genes was observed in testis. Although the RAD23 equivalents are well conserved during evolution, the mammalian genes did not express the UV-inducible phenotype of their yeast counterpart. This may point to a fundamental difference between the UV responses of yeast and human. No stage-specific mRNA expression during the cell cycle was observed for the mammalian RAD23 homologs. 38 refs., 5 figs.

  2. Modulation of radiation-induced base excision repair pathway gene expression by melatonin

    Directory of Open Access Journals (Sweden)

    Saeed Rezapoor

    2017-01-01

    Full Text Available Objective: Approximately 70% of all cancer patients receive radiotherapy. Although radiotherapy is effective in killing cancer cells, it has adverse effects on normal cells as well. Melatonin (MLT as a potent antioxidant and anti-inflammatory agent has been proposed to stimulate DNA repair capacity. We investigated the capability of MLT in the modification of radiation-induced DNA damage in rat peripheral blood cells. Materials and Methods: In this experimental study, male rats (n = 162 were divided into 27 groups (n = 6 in each group including: irradiation only, vehicle only, vehicle with irradiation, 100 mg/kg MLT alone, 100 mg/kg MLT plus irradiation in 3 different time points, and control. Subsequently, they were irradiated with a single whole-body X-ray radiation dose of 2 and 8 Gy at a dose rate of 200 MU/min. Rats were given an intraperitoneal injection of MLT or the same volume of vehicle alone 1 h prior to irradiation. Blood samples were also taken 8, 24, and 48 h postirradiation, in order to measure the 8-oxoguanine glycosylase1 (Ogg1, Apex1, and Xrcc1 expression using quantitative real-time-polymerase chain reaction. Results: Exposing to the ionizing radiation resulted in downregulation of Ogg1, Apex1, and Xrcc1 gene expression. The most obvious suppression was observed in 8 h after exposure. Pretreatments with MLT were able to upregulate these genes when compared to the irradiation-only and vehicle plus irradiation groups (P < 0.05 in all time points. Conclusion: Our results suggested that MLT in mentioned dose may result in modulation of Ogg1, Apex1, and Xrcc1 gene expression in peripheral blood cells to reduce X-ray irradiation-induced DNA damage. Therefore, administration of MLT may increase the normal tissue tolerance to radiation through enhancing the cell DNA repair capacity. We believed that MLT could play a radiation toxicity reduction role in patients who have undergone radiation treatment as a part of cancer radiotherapy.

  3. DNA Base-Excision Repair Genes OGG1 and NTH1 in Brazilian Lung Cancer Patients.

    Science.gov (United States)

    Couto, Patricia G; Bastos-Rodrigues, Luciana; Carneiro, Juliana G; Guieiro, Fernanda; Bicalho, Maria Aparecida; Leidenz, Franciele B; Bicalho, Ana J; Friedman, Eitan; De Marco, Luiz

    2015-12-01

    Lung cancer is the leading global cause of cancer-related mortality and is associated with poor prognosis. To improve survival rates of lung cancer patients, better understanding of tumorigenic mechanisms is necessary, which may lead to development of new therapeutic strategies. The hOGG1 and NTH1 genes act in the DNA BER repair pathway and their involvement in lung cancer pathogenesis has been analyzed in several populations. We analyzed targeted regions of the hOGG1 and NTH1 genes in 96 Brazilian patients with non-small-cell lung cancer (NSCLC) and 89 cancer-free, ethnically matched controls. The NTH1 c.98G>T polymorphism rs2302172 (p = 0.02 and p = 0.02 for allele and genotype frequency between cases and controls, respectively) and the 140-17C> T variant (rs2233518) (p = 0.02 and p = 0.02 for allele and genotype frequency between cases and controls, respectively) were detected in four lung cancer cases (4 %) while the NTH1 Q131K (C391A) polymorphism was found in seven lung cancer cases (7 %) (p = 0.001 and p = 0.008, for allele and genotype frequency between cases and controls, respectively). None of these sequence variants were detected in controls. The Ser326Cys (C1245G, rs1052133) polymorphism in the OGG1 gene was detected in 42 % of analyzed NSCLC patients and in 34 % of the controls (p = 0.11 and p = 0.25 for allele and genotype frequency between cases and controls, respectively). Our study provides preliminary evidence that polymorphisms in OGG1 do not contribute to development of NSCLC in Brazilian patients and that NTH1 polymorphisms may be associated with NSCLC pathogenesis.

  4. Nucleotide excision repair and human syndromes

    NARCIS (Netherlands)

    J. de Boer (Jan); J.H.J. Hoeijmakers (Jan)

    2000-01-01

    textabstractDNA damage is implicated in cancer and aging, and several DNA repair mechanisms exist that safeguard the genome from these deleterious consequences. Nucleotide excision repair (NER) removes a wide diversity of lesions, the main of which include UV-induced lesions, bulky chemical adducts

  5. Cloning of a human homolog of the yeast nucleotide excision repair gene MMS19 and interaction with transcription repair factor TFIIH via the XPB and XPD helicases

    NARCIS (Netherlands)

    T. Seroz; G.S. Winkler (Sebastiaan); J. Auriol; R.A. Verhage; W. Vermeulen (Wim); B. Smit (Bep); J. Brouwer (Jaap); G. Weeda (Geert); J.H.J. Hoeijmakers (Jan); A.P.M. Eker (André); J-M. Egly (Jean-Marc)

    2000-01-01

    textabstractNucleotide excision repair (NER) removes UV-induced photoproducts and numerous other DNA lesions in a highly conserved 'cut-and-paste' reaction that involves approximately 25 core components. In addition, several other proteins have been identified which are dispensable for NER in vitro

  6. Mammalian Transcription-Coupled Excision Repair

    Science.gov (United States)

    Vermeulen, Wim; Fousteri, Maria

    2013-01-01

    Transcriptional arrest caused by DNA damage is detrimental for cells and organisms as it impinges on gene expression and thereby on cell growth and survival. To alleviate transcriptional arrest, cells trigger a transcription-dependent genome surveillance pathway, termed transcription-coupled nucleotide excision repair (TC-NER) that ensures rapid removal of such transcription-impeding DNA lesions and prevents persistent stalling of transcription. Defective TC-NER is causatively linked to Cockayne syndrome, a rare severe genetic disorder with multisystem abnormalities that results in patients’ death in early adulthood. Here we review recent data on how damage-arrested transcription is actively coupled to TC-NER in mammals and discuss new emerging models concerning the role of TC-NER-specific factors in this process. PMID:23906714

  7. Association of nucleotide excision repair pathway gene polymorphisms with gastric cancer and atrophic gastritis risks.

    Science.gov (United States)

    Liu, Jingwei; Sun, Liping; Xu, Qian; Tu, Huakang; He, Caiyun; Xing, Chengzhong; Yuan, Yuan

    2016-02-09

    Polymorphisms of NER genes could change NER ability, thereby altering individual susceptibility to GC. We systematically analyzed 39 SNPs of 8 key genes of NER pathway in 2686 subjects including 898 gastric cancer (GC), 851 atrophic gastritis (AG) and 937 controls (CON) in northern Chinese. SNP genotyping were performed using Sequenom MassARRAY platform. The results demonstrated that DDB2 rs830083 GG genotype was significantly associated with increased GC risk compared with wild-type CC (OR=2.32, P= 6.62 × 10-9); XPC rs2607775 CG genotype conferred a 1.73 increased odds of GC risk than non-cancer subjects compared with wild-type CC (OR=1.73, P= 3.04 × 10-4). The combined detection of these two polymorphisms demonstrated even higher GC risk (OR=3.05). Haplotype analysis suggested that DDB2 rs2029298-rs326222-rs3781619-rs830083 GTAG haplotype was significantly associated with disease risk in each step of CON→AG→GC development (AG vs. CON: OR=2.88, P= 7.51 × 10-7; GC vs. AG: OR=2.90, P=5.68 × 10-15; GC vs. CON: OR=8.42, P=2.22 × 10-15); DDB2 GTAC haplotype was associated with reduced risk of GC compared with CON (OR=0.63, P= 8.31 × 10-12). XPC rs1870134-rs2228000-rs2228001-rs2470352-rs2607775 GCAAG haplotype conferred increased risk of GC compared with AG (OR=1.88, P= 6.98 × 10-4). XPA rs2808668 and drinking, DDB2 rs326222, rs3781619, rs830083 and smoking demonstrated significant interactions in AG; XPC rs2607775 had significant interaction with smoking in GC. In conclusion, NER pathway polymorphisms especially in "damage incision" step were significantly associated with GC risk and had interactions with environment factors. The detection of NER pathway polymorphisms such as DDB2 and XPC might be applied in the prediction of GC risk and personalized prevention in the future. NER pathway polymorphisms especially in "damage incision" step were significantly associated with GC risk and had interactions with environment factors, which might be applied in the

  8. Molecular spectrum of excision repair cross-complementation group 8 gene defects in Chinese patients with Cockayne syndrome type A.

    Science.gov (United States)

    Wang, Xiaozhu; Huang, Yu; Yan, Ming; Li, Jiuwei; Ding, Changhong; Jin, Hong; Fang, Fang; Yang, Yanling; Wu, Baiyan; Chen, Dafang

    2017-10-20

    There are two genetics complementary groups Cockayne syndrome type A and B (CS-A and CS-B OMIM 216400, 133540), which is a rare autosomal recessive segmental progeroid syndrome. Homozygous or compound heterozygous mutations in the excision repair cross-complementation group 8 gene (ERCC8) result in CS-A, and mutations in ERCC6 result in CS-B. Homozygous ERCC6/ERCC8 mutations also result in UV-sensitive syndrome. In this study, twenty-one Han Chinese patients with CS were investigated to identify mutations in ERCC8/ERCC6, of which thirteen cases with CS-A were identified with the mutations of ERCC8. There are five types mutations of ERCC8 in our study, such as exon 4 rearrangement, c.394_398delTTACA, c.299insA, c.843 + 2 T > C, and c.2 T > A. An estimated frequency of exon 4 rearrangement accounts for 69.23% and c.394_398delTTACA accounts for 11.53% in our cohort. Haplotype analysis revealed that the exon 4 rearrangement and c.394_398delTTACA mutations originated from a common founder in the Chinese population respectively. With the identification of three novel ERCC8 mutations, this study expanded the molecular spectrum of known ERCC8 defects, and furthermore, suggests that the exon 4 rearrangement and c.394_398delTTACA mutations may be a common underlying cause of CS-A in the Chinese population, which is different from that in other populations.

  9. A polymorphism in the base excision repair gene PARP2 is associated with differential prognosis by chemotherapy among postmenopausal breast cancer patients

    NARCIS (Netherlands)

    P. Seibold (Petra); P. Schmezer (Peter); T.W. Behrens (Timothy); K. Michailidou (Kyriaki); M.K. Bolla (Manjeet); Q. Wang (Qing); D. Flesch-Janys (Dieter); H. Nevanlinna (Heli); R. Fagerholm (Rainer); K. Aittomäki (Kristiina); C. Blomqvist (Carl); S. Margolin (Sara); A. Mannermaa (Arto); V. Kataja (Vesa); V-M. Kosma (Veli-Matti); J.M. Hartikainen (J.); D. Lambrechts (Diether); H. Wildiers (Hans); V. Kristensen (Vessela); G.G. Alnæs (Grethe Grenaker); S. Nord (Silje); A.-L. Borresen-Dale (Anne-Lise); M.J. Hooning (Maartje); A. Hollestelle (Antoinette); A. Jager (Agnes); C.M. Seynaeve (Caroline); J. Li (Jingmei); J. Liu (Jianjun); M.K. Humphreys (Manjeet); A.M. Dunning (Alison); V. Rhenius (Valerie); M. Shah (Mitul); M. Kabisch (Maria); D. Torres (Diana); H.U. Ulmer (Hans); U. Hamann (Ute); J.M. Schildkraut (Joellen M.); K.S. Purrington (Kristen S.); F.J. Couch (Fergus); P. Hall (Per); P.D.P. Pharoah (Paul); D.F. Easton (Douglas); M.K. Schmidt (Marjanka); J. Chang-Claude (Jenny); O. Popanda (Odilia)

    2015-01-01

    textabstractBackground: Personalized therapy considering clinical and genetic patient characteristics will further improve breast cancer survival. Two widely used treatments, chemotherapy and radiotherapy, can induce oxidative DNA damage and, if not repaired, cell death. Since base excision repair

  10. Variation in Base Excision Repair Capacity

    OpenAIRE

    Wilson, David M.; Kim, Daemyung; Berquist, Brian R.; Sigurdson, Alice J.

    2010-01-01

    The major DNA repair pathway for coping with spontaneous forms of DNA damage, such as natural hydrolytic products or oxidative lesions, is base excision repair (BER). In particular, BER processes mutagenic and cytotoxic DNA lesions such as non-bulky base modifications, abasic sites, and a range of chemically distinct single-strand breaks. Defects in BER have been linked to cancer predisposition, neurodegenerative disorders, and immunodeficiency. Recent data indicate a large degree of sequence...

  11. Genetic polymorphisms in DNA base excision repair gene XRCC1 and the risk of squamous cell carcinoma of the head and neck

    Directory of Open Access Journals (Sweden)

    Pietruszewska Wioletta

    2009-03-01

    Full Text Available Abstract Background The genes of base excision repair (BER pathway have been extensively studied in the association with various human cancers. We performed a case-control study to test the association between two common single nucleotide polymorphisms (SNPs of XRCC1 gene with human head and neck squamous cell carcinoma (HNSCC. Methods The genotype analysis of Arg194Trp and Arg399Gln gene polymorphisms for 92 HNSCC patients and 124 controls of cancer free subjects, in Polish population were performed using the PCR-based restriction fragment length polymorphism (PCR-RFLP with endonuclease MspI. Results No altered risk has been found individually for these SNPs, however haplotypes analysis showed high association with head and neck cancer. The highest frequency, according to wild-type of Arg194Arg and Arg399Arg genotypes, was identified for Arg194Trp-Arg399Arg haplotype (OR, 2.96; 95% CI, 1.01–8.80. Conclusion Finally, we identified the combined Arg194Trp-Arg399Arg genotype of base excision repair gene XRCC1 that was associated with HNSCC and may have an impact on identification of a high-risk cancer population.

  12. The Mutyh base excision repair gene influences the inflammatory response in a mouse model of ulcerative colitis.

    Directory of Open Access Journals (Sweden)

    Ida Casorelli

    Full Text Available BACKGROUND: The Mutyh DNA glycosylase is involved in the repair of oxidized DNA bases. Mutations in the human MUTYH gene are responsible for colorectal cancer in familial adenomatous polyposis. Since defective DNA repair genes might contribute to the increased cancer risk associated with inflammatory bowel diseases, we compared the inflammatory response of wild-type and Mutyh(-/- mice to oxidative stress. METHODOLOGY/PRINCIPAL FINDINGS: The severity of colitis, changes in expression of genes involved in DNA repair and inflammation, DNA 8-oxoguanine levels and microsatellite instability were analysed in colon of mice treated with dextran sulfate sodium (DSS. The Mutyh(-/- phenotype was associated with a significant accumulation of 8-oxoguanine in colon DNA of treated mice. A single DSS cycle induced severe acute ulcerative colitis in wild-type mice, whereas lesions were modest in Mutyh(-/- mice, and this was associated with moderate variations in the expression of several cytokines. Eight DSS cycles caused chronic colitis in both wild-type and Mutyh(-/- mice. Lymphoid hyperplasia and a significant reduction in Foxp3(+ regulatory T cells were observed only in Mutyh(-/- mice. CONCLUSIONS: The findings indicate that, in this model of ulcerative colitis, Mutyh plays a major role in maintaining intestinal integrity by affecting the inflammatory response.

  13. B lymphocytes of xeroderma pigmentosum or Cockayne syndrome patients with inherited defects in nucleotide excision repair are fully capable of somatic hypermutation of immunoglobulin genes.

    Science.gov (United States)

    Kim, N; Kage, K; Matsuda, F; Lefranc, M P; Storb, U

    1997-08-04

    Recent experiments have strongly suggested that the process of somatic mutation is linked to transcription initiation. It was postulated that a mutator factor loads onto the RNA polymerase and, during elongation, causes transcriptional arrest that activates DNA repair, thus occasionally causing errors in the DNA sequence. We report the analysis of the role of one of the known DNA repair systems, nucleotide excision repair (NER), in somatic mutation. Epstein-Barrvirus-transformed B cells from patients with defects in NER (XP-B, XP-D, XP-V, and CS-A) were studied. Their heavy and light chain genes show a high frequency of point mutations in the variable (V), but not in the constant (C) regions. This suggests that these B cells can undergo somatic hypermutation despite significant defects in NER. Thus, it is doubtful that NER is an essential part of the mechanism of somatic hypermutation of Ig genes. As an aside, NER seems also not involved in Ig gene switch recombination.

  14. DNA excision repair and double-strand break repair gene polymorphisms and the level of chromosome aberration in children with long-term exposure to radon.

    Science.gov (United States)

    Larionov, Aleksey V; Sinitsky, Maxim Y; Druzhinin, Vladimir G; Volobaev, Valentin P; Minina, Varvara I; Asanov, Maxim A; Meyer, Alina V; Tolochko, Tatiana A; Kalyuzhnaya, Ekaterina E

    2016-08-01

    To study polymorphic variants of repair genes in people affected by long-term exposure to radon. The chromosome aberration frequency in peripheral blood lymphocytes was used as the biological marker of genotoxicity. Genotyping of 12 single nucleotide polymorphisms in DNA repair genes (APE, XRCC1, OGG1, ADPRT, XpC, XpD, XpG, Lig4 and NBS1) was performed in children with long-term resident exposure to radon. Quantification of the aberrations was performed using light microscopy. The total frequency of aberrations was increased in carriers of the G/G genotype for the XpD gene (rs13181) polymorphism in recessive model confirmed by the results of ROC-analysis ('satisfactory predictor', AUC = 0.609). Single chromosome fragments frequency was increased in carriers of the G/G genotype in comparison with the T/T genotype. In respect to the total frequency of aberrations, the G/G genotype for the XpG gene (rs17655) polymorphism was also identified as a 'satisfactory predictor' (AUC = 0.605). Carriers of the T/C genotype for the ADPRT gene (rs1136410) polymorphism were characterized by an increased level of single fragments relative to the T/T genotype. The relationships with several types of cytogenetic damage suggest these three SNP (rs13181, rs17655 and rs1136410) may be considered radiosensitivity markers.

  15. Nucleotide Excision Repair in Caenorhabditis elegans

    Directory of Open Access Journals (Sweden)

    Hannes Lans

    2011-01-01

    Full Text Available Nucleotide excision repair (NER plays an essential role in many organisms across life domains to preserve and faithfully transmit DNA to the next generation. In humans, NER is essential to prevent DNA damage-induced mutation accumulation and cell death leading to cancer and aging. NER is a versatile DNA repair pathway that repairs many types of DNA damage which distort the DNA helix, such as those induced by solar UV light. A detailed molecular model of the NER pathway has emerged from in vitro and live cell experiments, particularly using model systems such as bacteria, yeast, and mammalian cell cultures. In recent years, the versatility of the nematode C. elegans to study DNA damage response (DDR mechanisms including NER has become increasingly clear. In particular, C. elegans seems to be a convenient tool to study NER during the UV response in vivo, to analyze this process in the context of a developing and multicellular organism, and to perform genetic screening. Here, we will discuss current knowledge gained from the use of C. elegans to study NER and the response to UV-induced DNA damage.

  16. Polymorphisms in base excision repair genes as colorectal cancer risk factors and modifiers of the effect of diets high in red meat

    Science.gov (United States)

    Brevik, Asgeir; Joshi, Amit D.; Corral, Román; Onland-Moret, N. Charlotte; Siegmund, Kimberly D.; Le Marchand, Loïc; Baron, John A.; Martinez, Maria Elena; Haile, Robert W.; Ahnen, Dennis J.; Sandler, Robert S.; Lance, Peter; Stern, Mariana C.

    2010-01-01

    Background A diet high in red meat is an established colorectal cancer (CRC) risk factor. Carcinogens generated during meat cooking have been implicated as causal agents, and can induce oxidative DNA damage, which elicits repair by the base excision repair (BER) pathway. Methods Using a family-based study we investigated the role of polymorphisms in four BER genes (APEX1 Gln51His, Asp148Glu; OGG1 Ser236Cys; PARP Val742Ala; XRCC1 Arg194Trp, Arg280His, Arg399Gln) as potential CRC risk factors and modifiers of the association between high-red meat or poultry diets and CRC risk. We tested for gene-environment interactions using case-only analyses (N = 577) and compared statistically significant results to those obtained using case-unaffected sibling comparisons (N = 307 sibships). Results Carriers of the APEX1 codon 51 Gln/His genotype had a reduced CRC risk compared to carriers of the Gln/Gln genotype (OR 0.15, 95% CI 0.03-0.69, p = 0.015). The association between higher red meat intake (>3 servings/week) and CRC was modified by the PARP Val762Ala SNP (case-only interaction p = 0.026). This SNP also modified the association between higher intake of high-temperature cooked red meat (case-only interaction p = 0.0009). Conclusions We report evidence that the BER pathway PARP gene modifies the association of diets high in red meat cooked at high temperatures with risk of CRC. Impact Our findings suggest a contribution to colorectal carcinogenesis of free radical damage as one of the possible harmful effects of a high-red meat diet. PMID:21037106

  17. Variation within 3'-UTRs of base excision repair genes and response to therapy in colorectal cancer patients: A potential modulation of microRNAs binding.

    Science.gov (United States)

    Pardini, Barbara; Rosa, Fabio; Barone, Elisa; Di Gaetano, Cornelia; Slyskova, Jana; Novotny, Jan; Levy, Miroslav; Garritano, Sonia; Vodickova, Ludmila; Buchler, Tomas; Gemignani, Federica; Landi, Stefano; Vodicka, Pavel; Naccarati, Alessio

    2013-11-01

    Colorectal cancer is routinely treated with a 5-fluorouracil (5-FU)-based chemotherapy. 5-FU incorporates into DNA, and the base excision repair (BER) pathway specifically recognizes such damage. We investigated the association of single-nucleotide polymorphisms (SNP) in the 3'-untranslated regions (UTR) of BER genes, and potentially affecting the microRNA (miRNA) binding, on the risk of colorectal cancer, its progression, and prognosis. SNPs in miRNA-binding sites may modulate the posttranscriptional regulation of gene expression operated by miRNAs and explain interindividual variability in BER capacity and response to 5-FU. We tested 12 SNPs in the 3'-UTRs of five BER genes for colorectal cancer susceptibility in a case-control study (1,098 cases and 1,459 healthy controls). Subsequently, we analyzed the role of these SNPs on clinical outcomes of patients (866 in the Training set and 232 in the Replication set). SNPs in the SMUG1 and NEIL2 genes were associated with overall survival. In particular, SMUG1 rs2233921 TT carriers showed increased survival compared with those with GT/GG genotypes [HR, 0.54; 95% confidence interval (CI), 0.36-0.81; P = 0.003] in the Training set and after pooling results from the Replication set. The association was more significant following stratification for 5-FU-based chemotherapy (P = 5.6 × 10(-5)). A reduced expression of the reporter gene for the T allele of rs2233921 was observed when compared with the common G allele by in vitro assay. None of the genotyped BER polymorphisms were associated with colorectal cancer risk. We provide the first evidence that variations in miRNA-binding sites in BER genes 3'-UTR may modulate colorectal cancer prognosis and therapy response.

  18. Emerging roles for histone modifications in DNA excision repair.

    Science.gov (United States)

    Mao, Peng; Wyrick, John J

    2016-11-01

    DNA repair is critical to maintain genome stability. In eukaryotic cells, DNA repair is complicated by the packaging of the DNA 'substrate' into chromatin. DNA repair pathways utilize different mechanisms to overcome the barrier presented by chromatin to efficiently locate and remove DNA lesions in the genome. DNA excision repair pathways are responsible for repairing a majority of DNA lesions arising in the genome. Excision repair pathways include nucleotide excision repair (NER) and base excision repair (BER), which repair bulky and non-bulky DNA lesions, respectively. Numerous studies have suggested that chromatin inhibits both NER and BER in vitro and in vivo Growing evidence demonstrates that histone modifications have important roles in regulating the activity of NER and BER enzymes in chromatin. Here, we will discuss the roles of different histone modifications and the corresponding modifying enzymes in DNA excision repair, highlighting the role of yeast as a model organism for many of these studies. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  19. Base excision repair: a critical player in many games.

    Science.gov (United States)

    Wallace, Susan S

    2014-07-01

    This perspective reviews the many dimensions of base excision repair from a 10,000 foot vantage point and provides one person's view on where the field is headed. Enzyme function is considered under the lens of X-ray diffraction and single molecule studies. Base excision repair in chromatin and telomeres, regulation of expression and the role of posttranslational modifications are also discussed in the context of enzyme activities, cellular localization and interacting partners. The specialized roles that base excision repair play in transcriptional activation by active demethylation and targeted oxidation as well as how base excision repair functions in the immune processes of somatic hypermutation and class switch recombination and its possible involvement in retroviral infection are also discussed. Finally the complexities of oxidative damage and its repair and its link to neurodegenerative disorders, as well as the role of base excision repair as a tumor suppressor are examined in the context of damage, repair and aging. By outlining the many base excision repair-related mysteries that have yet to be unraveled, hopefully this perspective will stimulate further interest in the field. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. A polymorphism in the base excision repair gene PARP2 is associated with differential prognosis by chemotherapy among postmenopausal breast cancer patients.

    Science.gov (United States)

    Seibold, Petra; Schmezer, Peter; Behrens, Sabine; Michailidou, Kyriaki; Bolla, Manjeet K; Wang, Qin; Flesch-Janys, Dieter; Nevanlinna, Heli; Fagerholm, Rainer; Aittomäki, Kristiina; Blomqvist, Carl; Margolin, Sara; Mannermaa, Arto; Kataja, Vesa; Kosma, Veli-Matti; Hartikainen, Jaana M; Lambrechts, Diether; Wildiers, Hans; Kristensen, Vessela; Alnæs, Grethe Grenaker; Nord, Silje; Borresen-Dale, Anne-Lise; Hooning, Maartje J; Hollestelle, Antoinette; Jager, Agnes; Seynaeve, Caroline; Li, Jingmei; Liu, Jianjun; Humphreys, Keith; Dunning, Alison M; Rhenius, Valerie; Shah, Mitul; Kabisch, Maria; Torres, Diana; Ulmer, Hans-Ulrich; Hamann, Ute; Schildkraut, Joellen M; Purrington, Kristen S; Couch, Fergus J; Hall, Per; Pharoah, Paul; Easton, Doug F; Schmidt, Marjanka K; Chang-Claude, Jenny; Popanda, Odilia

    2015-12-16

    Personalized therapy considering clinical and genetic patient characteristics will further improve breast cancer survival. Two widely used treatments, chemotherapy and radiotherapy, can induce oxidative DNA damage and, if not repaired, cell death. Since base excision repair (BER) activity is specific for oxidative DNA damage, we hypothesized that germline genetic variation in this pathway will affect breast cancer-specific survival depending on treatment. We assessed in 1,408 postmenopausal breast cancer patients from the German MARIE study whether cancer specific survival after adjuvant chemotherapy, anthracycline chemotherapy, and radiotherapy is modulated by 127 Single Nucleotide Polymorphisms (SNPs) in 21 BER genes. For SNPs with interaction terms showing p<0.1 (likelihood ratio test) using multivariable Cox proportional hazard analyses, replication in 6,392 patients from nine studies of the Breast Cancer Association Consortium (BCAC) was performed. rs878156 in PARP2 showed a differential effect by chemotherapy (p=0.093) and was replicated in BCAC studies (p=0.009; combined analysis p=0.002). Compared to non-carriers, carriers of the variant G allele (minor allele frequency=0.07) showed better survival after chemotherapy (combined allelic hazard ratio (HR)=0.75, 95% 0.53-1.07) and poorer survival when not treated with chemotherapy (HR=1.42, 95% 1.08-1.85). A similar effect modification by rs878156 was observed for anthracycline-based chemotherapy in both MARIE and BCAC, with improved survival in carriers (combined allelic HR=0.73, 95% CI 0.40-1.32). None of the SNPs showed significant differential effects by radiotherapy. Our data suggest for the first time that a SNP in PARP2, rs878156, may together with other genetic variants modulate cancer specific survival in breast cancer patients depending on chemotherapy. These germline SNPs could contribute towards the design of predictive tests for breast cancer patients.

  1. Modulation of DNA base excision repair during neuronal differentiation

    DEFF Research Database (Denmark)

    Sykora, Peter; Yang, Jenq-Lin; Ferrarelli, Leslie K

    2013-01-01

    Neurons are terminally differentiated cells with a high rate of metabolism and multiple biological properties distinct from their undifferentiated precursors. Previous studies showed that nucleotide excision DNA repair is downregulated in postmitotic muscle cells and neurons. Here, we characterize...... DNA damage susceptibility and base excision DNA repair (BER) capacity in undifferentiated and differentiated human neural cells. The results show that undifferentiated human SH-SY5Y neuroblastoma cells are less sensitive to oxidative damage than their differentiated counterparts, in part because...

  2. True Lies: The Double Life of the Nucleotide Excision Repair Factors in Transcription and DNA Repair

    Directory of Open Access Journals (Sweden)

    Nicolas Le May

    2010-01-01

    Full Text Available Nucleotide excision repair (NER is a major DNA repair pathway in eukaryotic cells. NER removes structurally diverse lesions such as pyrimidine dimers, arising upon UV irradiation or bulky chemical adducts, arising upon exposure to carcinogens and some chemotherapeutic drugs. NER defects lead to three genetic disorders that result in predisposition to cancers, accelerated aging, neurological and developmental defects. During NER, more than 30 polypeptides cooperate to recognize, incise, and excise a damaged oligonucleotide from the genomic DNA. Recent papers reveal an additional and unexpected role for the NER factors. In the absence of a genotoxic attack, the promoters of RNA polymerases I- and II-dependent genes recruit XPA, XPC, XPG, and XPF to initiate gene expression. A model that includes the growth arrest and DNA damage 45α protein (Gadd45α and the NER factors, in order to maintain the promoter of active genes under a hypomethylated state, has been proposed but remains controversial. This paper focuses on the double life of the NER factors in DNA repair and transcription and describes the possible roles of these factors in the RNA synthesis process.

  3. Excision repair in mammalian cells. [uv radiation, N-acetoxy-2-acetylaminofluorene

    Energy Technology Data Exchange (ETDEWEB)

    Ahmed, F.E.; Setlow, R.B.

    1978-01-01

    Excision repair after combined treatments of uv and N-acetoxy-2-acetylaminofluorene (AAAF) was studied by three different techniques in cells proficient in uv excision repair and in cells deficient in uv repair. Two patterns of repair were observed: in repair proficient cells total repair was additive, and in repair deficient cells total repair was much less than additive--usually less than observed for separate treatments--and AAAF inhibited dimer excision. We conclude that in the 1st class of cells pathways for repair of uv and AAAF lesions are not identical, and in the 2nd class the residual excision enzymes are different from those in repair proficient cells.

  4. Regulation of nucleotide excision repair by nuclear lamin b1.

    Directory of Open Access Journals (Sweden)

    Veronika Butin-Israeli

    Full Text Available The nuclear lamins play important roles in the structural organization and function of the metazoan cell nucleus. Recent studies on B-type lamins identified a requirement for lamin B1 (LB1 in the regulation of cell proliferation in normal diploid cells. In order to further investigate the function of LB1 in proliferation, we disrupted its normal expression in U-2 OS human osteosarcoma and other tumor cell lines. Silencing LB1 expression induced G1 cell cycle arrest without significant apoptosis. The arrested cells are unable to mount a timely and effective response to DNA damage induced by UV irradiation. Several proteins involved in the detection and repair of UV damage by the nucleotide excision repair (NER pathway are down-regulated in LB1 silenced cells including DDB1, CSB and PCNA. We propose that LB1 regulates the DNA damage response to UV irradiation by modulating the expression of specific genes and activating persistent DNA damage signaling. Our findings are relevant to understanding the relationship between the loss of LB1 expression, DNA damage signaling, and replicative senescence.

  5. Base excision repair deficient mice lacking the Aag alkyladenine DNA glycosylase.

    NARCIS (Netherlands)

    B.P. Engelward (Bevin); G. Weeda (Geert); M.D. Wyatt; J.L.M. Broekhof (Jose'); J. de Wit (Jan); I. Donker (Ingrid); J.M. Allan (James); B. Gold (Bert); J.H.J. Hoeijmakers (Jan); L.D. Samson (Leona)

    1997-01-01

    textabstract3-methyladenine (3MeA) DNA glycosylases remove 3MeAs from alkylated DNA to initiate the base excision repair pathway. Here we report the generation of mice deficient in the 3MeA DNA glycosylase encoded by the Aag (Mpg) gene. Alkyladenine DNA glycosylase turns out to be the major DNA

  6. Histone displacement during nucleotide excision repair

    DEFF Research Database (Denmark)

    Dinant, C.; Bartek, J.; Bekker-Jensen, S.

    2012-01-01

    chromatin. The condensed nature of chromatin inhibits many DNA metabolizing activities, including NER. In order to promote efficient repair, detection of a lesion not only has to activate the NER pathway but also chromatin remodeling. In general, such remodeling is thought on the one hand to precede NER...... of histone variants and histone displacement (including nucleosome sliding). Here we review current knowledge, and speculate about current unknowns, regarding those chromatin remodeling activities that physically displace histones before, during and after NER....

  7. Conservation and Divergence in Nucleotide Excision Repair Lesion Recognition*

    Science.gov (United States)

    Wirth, Nicolas; Gross, Jonas; Roth, Heide M.; Buechner, Claudia N.; Kisker, Caroline; Tessmer, Ingrid

    2016-01-01

    Nucleotide excision repair is an important and highly conserved DNA repair mechanism with an exceptionally large range of chemically and structurally unrelated targets. Lesion verification is believed to be achieved by the helicases UvrB and XPD in the prokaryotic and eukaryotic processes, respectively. Using single molecule atomic force microscopy analyses, we demonstrate that UvrB and XPD are able to load onto DNA and pursue lesion verification in the absence of the initial lesion detection proteins. Interestingly, our studies show different lesion recognition strategies for the two functionally homologous helicases, as apparent from their distinct DNA strand preferences, which can be rationalized from the different structural features and interactions with other nucleotide excision repair protein factors of the two enzymes. PMID:27405761

  8. Chromatin Dynamics during Nucleotide Excision Repair: Histones on the Move

    Directory of Open Access Journals (Sweden)

    Sophie E. Polo

    2012-09-01

    Full Text Available It has been a long-standing question how DNA damage repair proceeds in a nuclear environment where DNA is packaged into chromatin. Several decades of analysis combining in vitro and in vivo studies in various model organisms ranging from yeast to human have markedly increased our understanding of the mechanisms underlying chromatin disorganization upon damage detection and re-assembly after repair. Here, we review the methods that have been developed over the years to delineate chromatin alterations in response to DNA damage by focusing on the well-characterized Nucleotide Excision Repair (NER pathway. We also highlight how these methods have provided key mechanistic insight into histone dynamics coupled to repair in mammals, raising new issues about the maintenance of chromatin integrity. In particular, we discuss how NER factors and central players in chromatin dynamics such as histone modifiers, nucleosome remodeling factors, and histone chaperones function to mobilize histones during repair.

  9. Chromatin Dynamics during Nucleotide Excision Repair: Histones on the Move

    Science.gov (United States)

    Adam, Salomé; Polo, Sophie E.

    2012-01-01

    It has been a long-standing question how DNA damage repair proceeds in a nuclear environment where DNA is packaged into chromatin. Several decades of analysis combining in vitro and in vivo studies in various model organisms ranging from yeast to human have markedly increased our understanding of the mechanisms underlying chromatin disorganization upon damage detection and re-assembly after repair. Here, we review the methods that have been developed over the years to delineate chromatin alterations in response to DNA damage by focusing on the well-characterized Nucleotide Excision Repair (NER) pathway. We also highlight how these methods have provided key mechanistic insight into histone dynamics coupled to repair in mammals, raising new issues about the maintenance of chromatin integrity. In particular, we discuss how NER factors and central players in chromatin dynamics such as histone modifiers, nucleosome remodeling factors, and histone chaperones function to mobilize histones during repair. PMID:23109890

  10. Nucleotide excision repair genes are expressed at low levels and are not detectably inducible in Caenorhabditis elegans somatic tissues, but their function is required for normal adult life after UVC exposure

    Energy Technology Data Exchange (ETDEWEB)

    Boyd, Windy A. [Biomolecular Screening Branch, National Toxicology Program, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC (United States); Crocker, Tracey L. [Nicholas School of the Environment, Duke University, Durham, NC 27708 (United States); Rodriguez, Ana M. [Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC (United States); Leung, Maxwell C.K. [Nicholas School of the Environment, Duke University, Durham, NC 27708 (United States); Wade Lehmann, D. [Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC (United States); Freedman, Jonathan H. [Laboratory of Molecular Toxicology, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC (United States); Van Houten, Ben [Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC (United States); Meyer, Joel N., E-mail: joel.meyer@duke.edu [Nicholas School of the Environment, Duke University, Durham, NC 27708 (United States)

    2010-01-05

    We performed experiments to characterize the inducibility of nucleotide excision repair (NER) in Caenorhabditis elegans, and to examine global gene expression in NER-deficient and -proficient strains as well as germline vs. somatic tissues, with and without genotoxic stress. We also carried out experiments to elucidate the importance of NER in the adult life of C. elegans under genotoxin-stressed and control conditions. Adult lifespan was not detectably different between wild-type and NER-deficient xpa-1 nematodes under control conditions. However, exposure to 6 J/m{sup 2}/day of ultraviolet C radiation (UVC) decreased lifespan in xpa-1 nematodes more than a dose of 100 J/m{sup 2}/day in wild-type. Similar differential sensitivities were observed for adult size and feeding. Remarkably, global gene expression was nearly identical in young adult wild-type and xpa-1 nematodes, both in control conditions and 3 h after exposure to 50 J/m{sup 2} UVC. Neither NER genes nor repair activity were detectably inducible in young adults that lacked germ cells and developing embryos (glp-1 strain). However, expression levels of dozens of NER and other DNA damage response genes were much (5-30-fold) lower in adults lacking germ cells and developing embryos, suggesting that somatic and post-mitotic cells have a much lower DNA repair ability. Finally, we describe a refinement of our DNA damage assay that allows damage measurement in single nematodes.

  11. Mammalian transcription-coupled excision repair

    NARCIS (Netherlands)

    W. Vermeulen (Wim); M.I. Fousteri (Maria)

    2013-01-01

    textabstractTranscriptional arrest caused by DNA damage is detrimental for cells and organisms as it impinges on gene expression and thereby on cell growth and survival. To alleviate transcrip-tional arrest, cells trigger a transcription-dependent genome surveillance pathway, termed

  12. Biomolecular Simulation of Base Excision Repair and Protein Signaling

    Energy Technology Data Exchange (ETDEWEB)

    Straatsma, TP; McCammon, J A; Miller, John H; Smith, Paul E; Vorpagel, Erich R; Wong, Chung F; Zacharias, Martin W

    2006-03-03

    The goal of the Biomolecular Simulation of Base Excision Repair and Protein Signaling project is to enhance our understanding of the mechanism of human polymerase-β, one of the key enzymes in base excision repair (BER) and the cell-signaling enzymes cyclic-AMP-dependent protein kinase. This work used molecular modeling and simulation studies to specifically focus on the • dynamics of DNA and damaged DNA • dynamics and energetics of base flipping in DNA • mechanism and fidelity of nucleotide insertion by BER enzyme human polymerase-β • mechanism and inhibitor design for cyclic-AMP-dependent protein kinase. Molecular dynamics simulations and electronic structure calculations have been performed using the computer resources at the Molecular Science Computing Facility at the Environmental Molecular Sciences Laboratory.

  13. Dynamic control of strand excision during human DNA mismatch repair.

    Science.gov (United States)

    Jeon, Yongmoon; Kim, Daehyung; Martín-López, Juana V; Lee, Ryanggeun; Oh, Jungsic; Hanne, Jeungphill; Fishel, Richard; Lee, Jong-Bong

    2016-03-22

    Mismatch repair (MMR) is activated by evolutionarily conserved MutS homologs (MSH) and MutL homologs (MLH/PMS). MSH recognizes mismatched nucleotides and form extremely stable sliding clamps that may be bound by MLH/PMS to ultimately authorize strand-specific excision starting at a distant 3'- or 5'-DNA scission. The mechanical processes associated with a complete MMR reaction remain enigmatic. The purified human (Homo sapien or Hs) 5'-MMR excision reaction requires the HsMSH2-HsMSH6 heterodimer, the 5' → 3' exonuclease HsEXOI, and the single-stranded binding heterotrimer HsRPA. The HsMLH1-HsPMS2 heterodimer substantially influences 5'-MMR excision in cell extracts but is not required in the purified system. Using real-time single-molecule imaging, we show that HsRPA or Escherichia coli EcSSB restricts HsEXOI excision activity on nicked or gapped DNA. HsMSH2-HsMSH6 activates HsEXOI by overcoming HsRPA/EcSSB inhibition and exploits multiple dynamic sliding clamps to increase tract length. Conversely, HsMLH1-HsPMS2 regulates tract length by controlling the number of excision complexes, providing a link to 5' MMR.

  14. Radiation induced base excision repair (BER): a mechanistic mathematical approach.

    Science.gov (United States)

    Rahmanian, Shirin; Taleei, Reza; Nikjoo, Hooshang

    2014-10-01

    This paper presents a mechanistic model of base excision repair (BER) pathway for the repair of single-stand breaks (SSBs) and oxidized base lesions produced by ionizing radiation (IR). The model is based on law of mass action kinetics to translate the biochemical processes involved, step-by-step, in the BER pathway to translate into mathematical equations. The BER is divided into two subpathways, short-patch repair (SPR) and long-patch repair (LPR). SPR involves in replacement of single nucleotide via Pol β and ligation of the ends via XRCC1 and Ligase III, while LPR involves in replacement of multiple nucleotides via PCNA, Pol δ/ɛ and FEN 1, and ligation via Ligase I. A hallmark of IR is the production of closely spaced lesions within a turn of DNA helix (named complex lesions), which have been attributed to a slower repair process. The model presented considers fast and slow component of BER kinetics by assigning SPR for simple lesions and LPR for complex lesions. In the absence of in vivo reaction rate constants for the BER proteins, we have deduced a set of rate constants based on different published experimental measurements including accumulation kinetics obtained from UVA irradiation, overall SSB repair kinetic experiments, and overall BER kinetics from live-cell imaging experiments. The model was further used to calculate the repair kinetics of complex base lesions via the LPR subpathway and compared to foci kinetic experiments for cells irradiated with γ rays, Si, and Fe ions. The model calculation show good agreement with experimental measurements for both overall repair and repair of complex lesions. Furthermore, using the model we explored different mechanisms responsible for inhibition of repair when higher LET and HZE particles are used and concluded that increasing the damage complexity can inhibit initiation of LPR after the AP site removal step in BER. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. The role of base excision repair genes OGG1, APN1 and APN2 in benzo[a]pyrene-7,8-dione induced p53 mutagenesis.

    Science.gov (United States)

    Abedin, Zahidur; Louis-Juste, Melissa; Stangl, Melissa; Field, Jeffrey

    2013-01-20

    Lung cancer is primarily caused by exposure to tobacco smoke. Tobacco smoke contains numerous carcinogens, including polycyclic aromatic hydrocarbons (PAH). The most common PAH studied is benzo[a]pyrene (B[a]P). B[a]P is metabolically activated through multiple routes, one of which is catalyzed by aldo-keto reductase (AKR) to B[a]P-7,8-dione (BPQ). BPQ undergoes a futile redox cycle in the presence of NADPH to generate reactive oxygen species (ROS). ROS, in turn, damages DNA. Studies with a yeast p53 mutagenesis system found that the generation of ROS by PAH o-quinones may contribute to lung carcinogenesis because of similarities between the patterns (types of mutations) and spectra (location of mutations) and those seen in lung cancer. The patterns were dominated by G to T transversions, and the spectra in the experimental system have mutations at lung cancer hotspots. To address repair mechanisms that are responsible for BPQ induced damage we observed the effect of mutating two DNA repair genes OGG1 and APE1 (APN1 in yeast) and tested them in a yeast reporter system for p53 mutagenesis. There was an increase in both the mutant frequency and the number of G:C/T:A transversions in p53 treated with BPQ in ogg1 yeast but not in apn1 yeast. Knocking out APN2 increased mutagenesis in the apn1 cells. In addition, we did not find a strand bias on p53 treated with BPQ in ogg1 yeast. These studies suggest that Ogg1 is involved in repairing the oxidative damage caused by BPQ, Apn1 and Apn2 have redundant functions and that the stand bias seen in lung cancer may not be due to impaired repair of oxidative lesions. Copyright © 2012 Elsevier B.V. All rights reserved.

  16. DNA base excision repair and nucleotide excision repair synergistically contribute to survival of stationary-phase cells of the fission yeast Schizosaccharomyces pombe.

    Science.gov (United States)

    Senoo, Takanori; Kawano, Shinji; Ikeda, Shogo

    2017-03-01

    Defects of genome maintenance may causally contribute to aging. In general, base excision repair (BER) is involved in the repair of subtle base lesions and AP sites, and bulky helix-distorting lesions are restored by nucleotide excision repair (NER). Here, we measured the chronological lifespan (CLS) of BER- and NER-deficient mutants of the fission yeast Schizosaccharomyces pombe, and observed the aging process of cells. The CLS of the nth1 (gene for DNA glycosylase/AP lyase) mutant and the rad16 (a homolog of human XPF) mutant were slightly shorter than that of the wild-type (WT) strain. However, survival of the nth1Δ rad16Δ double mutant was significantly reduced after entry into the stationary phase. Deletion of rad16 in an AP endonuclease mutant apn2Δ also accelerated chronological aging. These results indicate that BER and NER synergistically contribute to genome maintenance in non-dividing cells. Reactive oxygen species (ROS) accumulated in cells during the stationary phase, and nth1Δ rad16Δ cells produced more ROS than WT cells. High mutation frequencies and nuclear DNA fragmentation were observed in nth1Δ rad16Δ stationary-phase cells concurrent with apoptotic-like cell death. Calorie restriction significantly reduced the level of ROS in the stationary phase and extended the CLS of nth1Δ rad16Δ cells. Therefore, ROS production critically affects the survival of the DNA repair mutant during chronological aging. © 2017 International Federation for Cell Biology.

  17. Loss of Nucleotide Excision Repair as a Source of Genomic Instability in Breast Cancer

    Science.gov (United States)

    2005-06-01

    sequence-specific mechanism of nucleotide excision repair. Genes Dev., 13, 768-785. DNA binding by short single strands of DNA requires the p53 C... Arabidopsis thaliana . The advantage signal (27). CPD-3 cells were further subcloned by single cell dilution, of using XP-A cells completely deficient...developed and optimized a novel technique for the detection of localized DNA damage and damage binding proteins in individual cells, using targeted

  18. Laparoscopic Excision of a Scar Pregnancy and Isthmocele Repair.

    Science.gov (United States)

    Kiyak, Huseyin; Wetherilt, Lale Susan; Seckin, Kerem Doga; Polat, Ibrahim; Kadirogullari, Pınar; Karacan, Tolga

    2017-10-13

    Laparoscopic excision of a scar pregnancy and isthmocele repair with a barbed suture. A step-by-step explanation of the laparoscopic excision technique of a scar pregnancy and isthmocele repair. Cesarean scar pregnancy occurs as a result of attachment of the products of conception to the uterine scar [1-3]. In the present case, a 34-year-old, gravida 4, para 1 patient with a history of 1 miscarriage and 1 ectopic pregnancy was diagnosed with type 2 cesarean scar pregnancy at 7 weeks of gestation. Dilation and curretage was performed at the 8th week of gestation to terminate the pregnancy. On ultrasonography performed 1 month later, placental material underlying the isthmocele was observed. Her beta human chorionic gonadotropin level was 13 836 mIU/mL. She was followed up for 1.5 months until the beta human chorionic gonadotropin levels were negative. However, the mass underneath the scar had grown larger, measuring up to 5 × 6 cm. Laparoscopy was performed because the patient reported vaginal spotting and pelvic pain. The incision was sutured with a synthetic absorbable unidirectional barbed suture (Stratafix Knotless Tissue Control Device; Ethicon Inc., Somerville, NJ). No residual scar defect was visible on follow-up ultrasonography 1 week and 1 month after surgery. Barbed sutures ease the repair of uterine scar defects and can provide ideal reapproximation of thick myometrial tissue. Laparoscopic treatment of a scar pregnancy and isthmocele repair are effective and safe modes of treatment. Copyright © 2017 American Association of Gynecologic Laparoscopists. Published by Elsevier Inc. All rights reserved.

  19. Role of endonucleases XPF and XPG in nucleotide excision repair of platinated DNA and cisplatin/oxaliplatin cytotoxicity

    OpenAIRE

    Graf, Nora; Ang, Wee Han; Zhu, Guangyu; Myint, MyatNoeZin; Lippard, Stephen J.

    2011-01-01

    Resistance of tumor cells to platinum anticancer agents poses a major problem in cancer chemotherapy. One of the mechanisms associated with platinum-based drug resistance is the enhanced capacity of the cell to carry out nucleotide excision repair (NER) on platinum-damaged DNA. Endonucleases XPF and XPG are critical components of NER, responsible for excising the damaged DNA strand to remove the DNA lesion. Here, we investigated possible consequences of down-regulation of XPF and XPG gene exp...

  20. Irofulven cytotoxicity depends on transcription-coupled nucleotide excision repair and is correlated with XPG expression in solid tumor cells.

    Science.gov (United States)

    Koeppel, Florence; Poindessous, Virginie; Lazar, Vladimir; Raymond, Eric; Sarasin, Alain; Larsen, Annette K

    2004-08-15

    Irofulven is a novel alkylating agent with promising clinical activity, particularly toward ovarian and hormone-refractory prostate cancers. To facilitate additional clinical development, we have aimed to identify biological markers associated with sensitivity to the compound. Fibroblasts derived from patients with xeroderma pigmentosum or Cockayne's syndrome along with a panel of 20 human cancer cell lines (eight different tumor types) were examined to establish the importance of nucleotide excision repair proteins in the sensitivity to irofulven. Human cells deficient in nucleotide excision repair are up to 30-fold more sensitive to the cytotoxic effects of irofulven compared with repair-proficient controls, clearly indicating that nucleotide excision repair plays a crucial role in the sensitivity to the drug. Interestingly, our results show that irofulven-induced lesions are recognized by transcription-coupled repair but not by global genome repair. Another unique feature is the pronounced sensitivity of XPD and XPB helicase-deficient cells to the drug. Comparison of the IC50 values for irofulven, cisplatin, and ecteinascidin 743 with the expression levels of ERCC1, XPD, and XPG genes in different solid tumor cell lines shows no correlation between the expression levels of any of the three nucleotide excision repair proteins and the sensitivity to ecteinascidin 743. In contrast, expression of the XPG endonuclease was correlated with the cytotoxicity for irofulven and, to a lesser degree, for cisplatin. Importantly, XPG expression was also correlated with cellular nucleotide excision repair activity. Increasing evidence indicates that compromised nucleotide excision repair activity is frequent in several solid tumor types. The results presented here suggest that XPG expression in such tumors may be a useful marker to predict their sensitivity to irofulven.

  1. Ku80-deleted cells are defective at base excision repair

    Energy Technology Data Exchange (ETDEWEB)

    Li, Han [The University of Texas Health Science Center at San Antonio, The Institute of Biotechnology, The Department of Molecular Medicine, 15355 Lambda Drive, San Antonio, TX 78245-3207 (United States); Tumor Suppression Group, Spanish National Cancer Research Centre (CNIO), Madrid 28029 (Spain); Marple, Teresa [The University of Texas Health Science Center at San Antonio, The Institute of Biotechnology, The Department of Molecular Medicine, 15355 Lambda Drive, San Antonio, TX 78245-3207 (United States); Hasty, Paul, E-mail: hastye@uthscsa.edu [The University of Texas Health Science Center at San Antonio, The Institute of Biotechnology, The Department of Molecular Medicine, 15355 Lambda Drive, San Antonio, TX 78245-3207 (United States); Tumor Suppression Group, Spanish National Cancer Research Centre (CNIO), Madrid 28029 (Spain)

    2013-05-15

    Graphical abstract: - Highlights: • Ku80-deleted cells are hypersensitive to ROS and alkylating agents. • Cells deleted for Ku80, but not Ku70 or Lig4, have reduced BER capacity. • OGG1 rescues hypersensitivity to H{sub 2}O{sub 2} and paraquat in Ku80-mutant cells. • Cells deleted for Ku80, but not Lig4, are defective at repairing AP sites. • Cells deleted for Ku80, but not Lig4 or Brca2 exon 27, exhibit increased PAR. - Abstract: Ku80 forms a heterodimer with Ku70, called Ku, that repairs DNA double-strand breaks (DSBs) via the nonhomologous end joining (NHEJ) pathway. As a consequence of deleting NHEJ, Ku80-mutant cells are hypersensitive to agents that cause DNA DSBs like ionizing radiation. Here we show that Ku80 deletion also decreased resistance to ROS and alkylating agents that typically cause base lesions and single-strand breaks (SSBs). This is unusual since base excision repair (BER), not NHEJ, typically repairs these types of lesions. However, we show that deletion of another NHEJ protein, DNA ligase IV (Lig4), did not cause hypersensitivity to these agents. In addition, the ROS and alkylating agents did not induce γ-H2AX foci that are diagnostic of DSBs. Furthermore, deletion of Ku80, but not Lig4 or Ku70, reduced BER capacity. Ku80 deletion also impaired BER at the initial lesion recognition/strand scission step; thus, involvement of a DSB is unlikely. Therefore, our data suggests that Ku80 deletion impairs BER via a mechanism that does not repair DSBs.

  2. SPT4 increases UV-induced mutagenesis in yeast through impaired nucleotide excision repair

    National Research Council Canada - National Science Library

    Kang, Mi-Sun; Yu, Sung-Lim; Kim, Ho-Yeol; Lim, Hyun-Sook; Lee, Sung-Keun

    2013-01-01

    .... As unrepaired DNA lesions inhibit transcription, UV-induced damage to transcribed DNA is repaired preferentially versus non-transcribed DNA through transcription-coupled nucleotide excision repair (TCR...

  3. Base Sequence Context Effects on Nucleotide Excision Repair

    Science.gov (United States)

    Cai, Yuqin; Patel, Dinshaw J.; Broyde, Suse; Geacintov, Nicholas E.

    2010-01-01

    Nucleotide excision repair (NER) plays a critical role in maintaining the integrity of the genome when damaged by bulky DNA lesions, since inefficient repair can cause mutations and human diseases notably cancer. The structural properties of DNA lesions that determine their relative susceptibilities to NER are therefore of great interest. As a model system, we have investigated the major mutagenic lesion derived from the environmental carcinogen benzo[a]pyrene (B[a]P), 10S (+)-trans-anti-B[a]P-N2-dG in six different sequence contexts that differ in how the lesion is positioned in relation to nearby guanine amino groups. We have obtained molecular structural data by NMR and MD simulations, bending properties from gel electrophoresis studies, and NER data obtained from human HeLa cell extracts for our six investigated sequence contexts. This model system suggests that disturbed Watson-Crick base pairing is a better recognition signal than a flexible bend, and that these can act in concert to provide an enhanced signal. Steric hinderance between the minor groove-aligned lesion and nearby guanine amino groups determines the exact nature of the disturbances. Both nearest neighbor and more distant neighbor sequence contexts have an impact. Regardless of the exact distortions, we hypothesize that they provide a local thermodynamic destabilization signal for repair. PMID:20871811

  4. Implication of Posttranslational Histone Modifications in Nucleotide Excision Repair

    Directory of Open Access Journals (Sweden)

    Shisheng Li

    2012-09-01

    Full Text Available Histones are highly alkaline proteins that package and order the DNA into chromatin in eukaryotic cells. Nucleotide excision repair (NER is a conserved multistep reaction that removes a wide range of generally bulky and/or helix-distorting DNA lesions. Although the core biochemical mechanism of NER is relatively well known, how cells detect and repair lesions in diverse chromatin environments is still under intensive research. As with all DNA-related processes, the NER machinery must deal with the presence of organized chromatin and the physical obstacles it presents. A huge catalogue of posttranslational histone modifications has been documented. Although a comprehensive understanding of most of these modifications is still lacking, they are believed to be important regulatory elements for many biological processes, including DNA replication and repair, transcription and cell cycle control. Some of these modifications, including acetylation, methylation, phosphorylation and ubiquitination on the four core histones (H2A, H2B, H3 and H4 or the histone H2A variant H2AX, have been found to be implicated in different stages of the NER process. This review will summarize our recent understanding in this area.

  5. Nucleotide excision repair- and p53-deficient mouse models in cancer research

    Energy Technology Data Exchange (ETDEWEB)

    Hoogervorst, Esther M. [Laboratory of Toxicology, Pathology and Genetics, National Institute of Public Health and the Environment, P.O. Box 1, 3720 BA Bilthoven (Netherlands); Utrecht University, Department of Pathobiology, Utrecht (Netherlands); Steeg, Harry van [Laboratory of Toxicology, Pathology and Genetics, National Institute of Public Health and the Environment, P.O. Box 1, 3720 BA Bilthoven (Netherlands); Vries, Annemieke de [Laboratory of Toxicology, Pathology and Genetics, National Institute of Public Health and the Environment, P.O. Box 1, 3720 BA Bilthoven (Netherlands)]. E-mail: Annemieke.de.Vries@rivm.nl

    2005-07-01

    Cancer is caused by the loss of controlled cell growth due to mutational (in)activation of critical genes known to be involved in cell cycle regulation. Three main mechanisms are known to be involved in the prevention of cells from becoming cancerous; DNA repair and cell cycle control, important to remove DNA damage before it will be fixed into mutations and apoptosis, resulting in the elimination of cells containing severe DNA damage. Several human syndromes are known to have (partially) deficiencies in these pathways, and are therefore highly cancer prone. Examples are xeroderma pigmentosum (XP) caused by an inborn defect in the nucleotide excision repair (NER) pathway and the Li-Fraumeni syndrome, which is the result of a germ line mutation in the p53 gene. XP patients develop skin cancer on sun exposed areas at a relatively early age, whereas Li-Fraumeni patients spontaneously develop a wide variety of early onset tumors, including sarcomas, leukemia's and mammary gland carcinomas. Several mouse models have been generated to mimic these human syndromes, providing us information about the role of these particular gene defects in the tumorigenesis process. In this review, spontaneous phenotypes of mice deficient for nucleotide excision repair and/or the p53 gene will be described, together with their responses upon exposure to either chemical carcinogens or radiation. Furthermore, possible applications of these and newly generated mouse models for cancer will be given.

  6. DREMECELS: A Curated Database for Base Excision and Mismatch Repair Mechanisms Associated Human Malignancies.

    Directory of Open Access Journals (Sweden)

    Ankita Shukla

    Full Text Available DNA repair mechanisms act as a warrior combating various damaging processes that ensue critical malignancies. DREMECELS was designed considering the malignancies with frequent alterations in DNA repair pathways, that is, colorectal and endometrial cancers, associated with Lynch syndrome (also known as HNPCC. Since lynch syndrome carries high risk (~40-60% for both cancers, therefore we decided to cover all three diseases in this portal. Although a large population is presently affected by these malignancies, many resources are available for various cancer types but no database archives information on the genes specifically for only these cancers and disorders. The database contains 156 genes and two repair mechanisms, base excision repair (BER and mismatch repair (MMR. Other parameters include some of the regulatory processes that have roles in these disease progressions due to incompetent repair mechanisms, specifically BER and MMR. However, our unique database mainly provides qualitative and quantitative information on these cancer types along with methylation, drug sensitivity, miRNAs, copy number variation (CNV and somatic mutations data. This database would serve the scientific community by providing integrated information on these disease types, thus sustaining diagnostic and therapeutic processes. This repository would serve as an excellent accompaniment for researchers and biomedical professionals and facilitate in understanding such critical diseases. DREMECELS is publicly available at http://www.bioinfoindia.org/dremecels.

  7. A Ubiquitin-Binding Domain in Cockayne Syndrome B Required for Transcription-Coupled Nucleotide Excision Repair

    NARCIS (Netherlands)

    R. Anindya (Roy); P.O. Mari (Pierre-Olivier); U. Kristensen (Ulrik); H.J.M. Kool (Hanneke); G. Giglia-Mari (Giuseppina); L.H.F. Mullenders (Leon); M.I. Fousteri (Maria); W. Vermeulen (Wim); J-M. Egly (Jean-Marc); J.Q. Svejstrup (Jesper)

    2010-01-01

    textabstractTranscription-coupled nucleotide excision repair (TC-NER) allows RNA polymerase II (RNAPII)-blocking lesions to be rapidly removed from the transcribed strand of active genes. Defective TCR in humans is associated with Cockayne syndrome (CS), typically caused by defects in either CSA or

  8. Repair of Oxidative DNA Damage and Cancer: Recent Progress in DNA Base Excision Repair

    OpenAIRE

    Scott, Timothy L.; Rangaswamy, Suganya; Wicker, Christina A.; Izumi, Tadahide

    2014-01-01

    Significance: Reactive oxygen species (ROS) are generated by exogenous and environmental genotoxins, but also arise from mitochondria as byproducts of respiration in the body. ROS generate DNA damage of which pathological consequence, including cancer is well established. Research efforts are intense to understand the mechanism of DNA base excision repair, the primary mechanism to protect cells from genotoxicity caused by ROS. Recent Advances: In addition to the notion that oxidative DNA dama...

  9. Base excision repair imbalance in colorectal cancer has prognostic value and modulates response to chemotherapy

    Science.gov (United States)

    Leguisamo, Natalia M.; Gloria, Helena C.; Kalil, Antonio N.; Martins, Talita V.; Azambuja, Daniel B.

    2017-01-01

    Colorectal cancer (CRC) is prevalent worldwide, and treatment often involves surgery and genotoxic chemotherapy. DNA repair mechanisms, such as base excision repair (BER) and mismatch repair (MMR), may not only influence tumour characteristics and prognosis but also dictate chemotherapy response. Defective MMR contributes to chemoresistance in colorectal cancer. Moreover, BER affects cellular survival by repairing genotoxic base damage in a process that itself can disrupt metabolism. In this study, we characterized BER and MMR gene expression in colorectal tumours and the association between this repair profile with patients’ clinical and pathological features. In addition, we exploited the possible mechanisms underlying the association between altered DNA repair, metabolism and response to chemotherapy. Seventy pairs of sporadic colorectal tumour samples and adjacent non-tumour mucosal specimens were assessed for BER and MMR gene and protein expression and their association with pathological and clinical features. MMR-deficient colon cancer cells (HCT116) transiently overexpressing MPG or XRCC1 were treated with 5-FU or TMZ and evaluated for viability and metabolic intermediate levels. Increase in BER gene and protein expression is associated with more aggressive tumour features and poor pathological outcomes in CRC. However, tumours with reduced MMR gene expression also displayed low MPG, OGG1 and PARP1 expression. Imbalancing BER by overexpression of MPG, but not XRCC1, sensitises MMR-deficient colon cancer cells to 5-FU and TMZ and leads to ATP depletion and lactate accumulation. MPG overexpression alters DNA repair and metabolism and is a potential strategy to overcome 5-FU chemotherapeutic resistance in MMR-deficient CRC. PMID:28903334

  10. Use of in vivo and in vitro assays for the characterization of mammalian excision repair and isolation of repair proteins.

    NARCIS (Netherlands)

    J.H.J. Hoeijmakers (Jan); A.P.M. Eker (André); R.D. Wood (Richard); P. Robins

    1990-01-01

    textabstractElucidation of the molecular mechanism of mammalian nucleotide excision repair requires the availability of purified proteins, DNA substrates with defined lesions and suitable repair assays. Repair assays introduced in recent years vary from testing individual steps and successions of

  11. Polymorphisms within base and nucleotide excision repair pathways and risk of differentiated thyroid carcinoma.

    Science.gov (United States)

    Cipollini, Monica; Figlioli, Gisella; Maccari, Giuseppe; Garritano, Sonia; De Santi, Chiara; Melaiu, Ombretta; Barone, Elisa; Bambi, Franco; Ermini, Stefano; Pellegrini, Giovanni; Cristaudo, Alfonso; Foddis, Rudy; Bonotti, Alessandra; Romei, Cristina; Vivaldi, Agnese; Agate, Laura; Molinari, Eleonora; Barale, Roberto; Forsti, Asta; Hemminki, Kari; Elisei, Rossella; Gemignani, Federica; Landi, Stefano

    2016-05-01

    The thyrocytes are exposed to high levels of oxidative stress which could induce DNA damages. Base excision repair (BER) is one of the principal mechanisms of defense against oxidative DNA damage, however recent evidences suggest that also nucleotide excision repair (NER) could be involved. The aim of present work was to identify novel differentiated thyroid cancer (DTC) risk variants in BER and NER genes. For this purpose, the most strongly associated SNPs within NER and BER genes found in our previous GWAS on DTC were selected and replicated in an independent series of samples for a new case-control study. Although a positive signal was detected at the nominal level of 0.05 for rs7689099 (encoding for an aminoacid change proline to arginine at codon 117 within NEIL3), none of the considered SNPs (i.e. rs7990340 and rs690860 within RFC3, rs3744767 and rs1131636 within RPA1, rs16962916 and rs3136166 in ERCC4, and rs17739370 and rs7689099 in NEIL3) was associated with the risk of DTC when the correction of multiple testing was applied. In conclusion, a role of NER and BER pathways was evoked in the susceptibility to DTC. However, this seemed to be limited to few polymorphic genes and the overall effect size appeared weak. Copyright © 2016. Published by Elsevier B.V.

  12. E2F1 and p53 Transcription Factors as Accessory Factors for Nucleotide Excision Repair

    Directory of Open Access Journals (Sweden)

    David G. Johnson

    2012-10-01

    Full Text Available Many of the biochemical details of nucleotide excision repair (NER have been established using purified proteins and DNA substrates. In cells however, DNA is tightly packaged around histones and other chromatin-associated proteins, which can be an obstacle to efficient repair. Several cooperating mechanisms enhance the efficiency of NER by altering chromatin structure. Interestingly, many of the players involved in modifying chromatin at sites of DNA damage were originally identified as regulators of transcription. These include ATP-dependent chromatin remodelers, histone modifying enzymes and several transcription factors. The p53 and E2F1 transcription factors are well known for their abilities to regulate gene expression in response to DNA damage. This review will highlight the underappreciated, transcription-independent functions of p53 and E2F1 in modifying chromatin structure in response to DNA damage to promote global NER.

  13. Exposure to low dose of gamma radiation enhances the excision repair in Saccharomyces cerevisiae

    Energy Technology Data Exchange (ETDEWEB)

    Dutta, K.; Verma, N.C. [Bhabha Atomic Research Centre, Mumbai (India)

    1998-08-01

    The effect of low doses of ionizing and nonionizing radiation on the radiation response of yeast Saccharomyces cerevisiae toward ionizing and nonionizing radiation was studied. The wild-type strain D273-10B on exposure to 54 Gy gamma radiation (resulting in about 10% cell killing) showed enhanced resistance to subsequent exposure to UV radiation. This induced UV resistance increased with the incubation time between the initial gamma radiation stress and the UV irradiation. Exposure to low doses of UV light on the other hand showed no change in gamma or UV radiation response of this strain. The strains carrying a mutation at rad52 behaved in a way similar to the wild type, but with slightly reduced induced response. In contrast to this, the rad3 mutants, defective in excision repair, showed no induced UV resistance. Removal of UV-induced pyrimidine dimers in wild-type yeast DNA after UV irradiation was examined by analyzing the sites recognized by UV endonuclease from Micrococcus luteus. The samples that were exposed to low doses of gamma radiation before UV irradiation were able to repair the pyrimidine dimers more efficiently than the samples in which low gamma irradiation was omitted. The nature of enhanced repair was studied by scoring the frequency of induced gene conversion and reverse mutation at trp and ilv loci respectively in strain D7, which showed similar enhanced UV resistance induced by low-dose gamma irradiation. The induced repair was found to be essentially error-free. These results suggest that irradiation of strain D273-10B with low doses of gamma radiation enhances its capability for excision repair of UV-induced pyrimidine dimers. (author)

  14. Interactions involving the human RNA polymerase II transcription/nucleotide excision repair complex TFIIH, the nucleotide excision repair protein XPG, and Cockayne syndrome group B (CSB) protein.

    Science.gov (United States)

    Iyer, N; Reagan, M S; Wu, K J; Canagarajah, B; Friedberg, E C

    1996-02-20

    The human basal transcription factor TFIIH plays a central role in two distinct processes. TFIIH is an obligatory component of the RNA polymerase II (RNAP II) transcription initiation complex. Additionally, it is believed to be the core structure around which some if not all the components of the nucleotide excision repair (NER) machinery assemble to constitute a nucleotide excision repairosome. At least two of the subunits of TFIIH (XPB and XPD proteins) are implicated in the disease xeroderma pigmentosum (XP). We have exploited the availability of the cloned XPB, XPD, p62, p44, and p34 genes (all of which encode polypeptide subunits of TFIIH) to examine interactions between in vitro-translated polypeptides by co-immunoprecipitation. Additionally we have examined interactions between TFIIH components, the human NER protein XPG, and the CSB protein which is implicated in Cockayne syndrome (CS). Our analyses demonstrate that the XPB, XPD, p44, and p62 proteins interact with each other. XPG protein interacts with multiple subunits of TFIIH and with CSB protein.

  15. Mismatch repair and nucleotide excision repair proteins cooperate in the recognition of DNA interstrand crosslinks

    Science.gov (United States)

    Zhao, Junhua; Jain, Aklank; Iyer, Ravi R.; Modrich, Paul L.; Vasquez, Karen M.

    2009-01-01

    DNA interstrand crosslinks (ICLs) are among the most cytotoxic types of DNA damage, thus ICL-inducing agents such as psoralen, are clinically useful chemotherapeutics. Psoralen-modified triplex-forming oligonucleotides (TFOs) have been used to target ICLs to specific genomic sites to increase the selectivity of these agents. However, how TFO-directed psoralen ICLs (Tdp-ICLs) are recognized and processed in human cells is unclear. Previously, we reported that two essential nucleotide excision repair (NER) protein complexes, XPA–RPA and XPC–RAD23B, recognized ICLs in vitro, and that cells deficient in the DNA mismatch repair (MMR) complex MutSβ were sensitive to psoralen ICLs. To further investigate the role of MutSβ in ICL repair and the potential interaction between proteins from the MMR and NER pathways on these lesions, we performed electrophoretic mobility-shift assays and chromatin immunoprecipitation analysis of MutSβ and NER proteins with Tdp-ICLs. We found that MutSβ bound to Tdp-ICLs with high affinity and specificity in vitro and in vivo, and that MutSβ interacted with XPA–RPA or XPC–RAD23B in recognizing Tdp-ICLs. These data suggest that proteins from the MMR and NER pathways interact in the recognition of ICLs, and provide a mechanistic link by which proteins from multiple repair pathways contribute to ICL repair. PMID:19468048

  16. Repair of oxidative DNA damage and cancer: recent progress in DNA base excision repair.

    Science.gov (United States)

    Scott, Timothy L; Rangaswamy, Suganya; Wicker, Christina A; Izumi, Tadahide

    2014-02-01

    Reactive oxygen species (ROS) are generated by exogenous and environmental genotoxins, but also arise from mitochondria as byproducts of respiration in the body. ROS generate DNA damage of which pathological consequence, including cancer is well established. Research efforts are intense to understand the mechanism of DNA base excision repair, the primary mechanism to protect cells from genotoxicity caused by ROS. In addition to the notion that oxidative DNA damage causes transformation of cells, recent studies have revealed how the mitochondrial deficiencies and ROS generation alter cell growth during the cancer transformation. The emphasis of this review is to highlight the importance of the cellular response to oxidative DNA damage during carcinogenesis. Oxidative DNA damage, including 7,8-dihydro-8-oxoguanine, play an important role during the cellular transformation. It is also becoming apparent that the unusual activity and subcellular distribution of apurinic/apyrimidinic endonuclease 1, an essential DNA repair factor/redox sensor, affect cancer malignancy by increasing cellular resistance to oxidative stress and by positively influencing cell proliferation. Technological advancement in cancer cell biology and genetics has enabled us to monitor the detailed DNA repair activities in the microenvironment. Precise understanding of the intracellular activities of DNA repair proteins for oxidative DNA damage should provide help in understanding how mitochondria, ROS, DNA damage, and repair influence cancer transformation.

  17. The role of Cockayne syndrome group A (CSA) protein in transcription-coupled nucleotide excision repair.

    Science.gov (United States)

    Saijo, Masafumi

    2013-01-01

    Nucleotide excision repair (NER) removes a variety of DNA lesions, including ultraviolet-induced cyclobutane pyrimidine dimers. NER comprises two subpathways: transcription-coupled NER (TC-NER) and global genome NER. TC-NER efficiently removes lesions from the transcribed strands of active genes. Mutations in Cockayne syndrome groups A and B genes (CSA and CSB) result in defective TC-NER. In mammalian cells, TC-NER is presumably initiated by the arrest of RNA polymerase II at a lesion on the transcribed strand of an active gene, but the molecular mechanism underlying TC-NER remains unclear. The CSA protein has seven WD40 repeat motifs and beta-propeller architecture. A protein complex consisting of CSA, DDB1, cullin 4A, and Roc1 exhibits ubiquitin ligase activity. The role of CSA protein in TC-NER is described in this review. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  18. Excision repair of bulky lesions in the DNA of mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Setlow, R B; Grist, E

    1980-01-01

    The report examines the process of excision repair of pyrimidine dimers from uv-irradiated and chemically challenged human cells. It is shown by means of a sensitive endonuclease assay that the amount of excision observed depends upon the isotope used to label cells, and that XP heterozygotes are between normals and XPs. (ACR)

  19. Nucleotide excision repair is not induced in human embryonic lung fibroblasts treated with environmental pollutants.

    Directory of Open Access Journals (Sweden)

    Pavel Rossner

    Full Text Available The cellular response to genotoxic treatment depends on the cell line used. Although tumor cell lines are widely used for genotoxicity tests, the interpretation of the results may be potentially hampered by changes in cellular processes caused by malignant transformation. In our study we used normal human embryonic lung fibroblasts (HEL12469 cells and tested their response to treatment with benzo[a]pyrene (B[a]P and extractable organic matter (EOM from ambient air particles <2.5 µm (PM2.5 collected in two Czech cities differing in levels and sources of air pollution. We analyzed multiple endpoints associated with exposure to polycyclic aromatic hydrocarbons (PAHs including the levels of bulky DNA adducts and the nucleotide excision repair (NER response [expression of XPE, XPC and XPA genes on the level of mRNA and proteins, unscheduled DNA synthesis (UDS]. EOMs were collected in the winter and summer of 2011 in two Czech cities with different levels and sources of air pollution. The effects of the studied compounds were analyzed in the presence (+S9 and absence (-S9 of the rat liver microsomal S9 fraction. The levels of bulky DNA adducts were highest after treatment with B[a]P, followed by winter EOMs; their induction by summer EOMs was weak. The induction of both mRNA and protein expression was observed, with the most pronounced effects after treatment with B[a]P (-S9; the response induced by EOMs from both cities and seasons was substantially weaker. The expression of DNA repair genes was not accompanied by the induction of UDS activity. In summary, our results indicate that the tested compounds induced low levels of DNA damage and affected the expression of NER genes; however, nucleotide excision repair was not induced.

  20. Age-related neuronal degeneration: complementary roles of nucleotide excision repair and transcription-coupled repair in preventing neuropathology.

    Directory of Open Access Journals (Sweden)

    Dick Jaarsma

    2011-12-01

    Full Text Available Neuronal degeneration is a hallmark of many DNA repair syndromes. Yet, how DNA damage causes neuronal degeneration and whether defects in different repair systems affect the brain differently is largely unknown. Here, we performed a systematic detailed analysis of neurodegenerative changes in mouse models deficient in nucleotide excision repair (NER and transcription-coupled repair (TCR, two partially overlapping DNA repair systems that remove helix-distorting and transcription-blocking lesions, respectively, and that are associated with the UV-sensitive syndromes xeroderma pigmentosum (XP and Cockayne syndrome (CS. TCR-deficient Csa(-/- and Csb(-/- CS mice showed activated microglia cells surrounding oligodendrocytes in regions with myelinated axons throughout the nervous system. This white matter microglia activation was not observed in NER-deficient Xpa(-/- and Xpc(-/- XP mice, but also occurred in Xpd(XPCS mice carrying a point mutation (G602D in the Xpd gene that is associated with a combined XPCS disorder and causes a partial NER and TCR defect. The white matter abnormalities in TCR-deficient mice are compatible with focal dysmyelination in CS patients. Both TCR-deficient and NER-deficient mice showed no evidence for neuronal degeneration apart from p53 activation in sporadic (Csa(-/-, Csb(-/- or highly sporadic (Xpa(-/-, Xpc(-/- neurons and astrocytes. To examine to what extent overlap occurs between both repair systems, we generated TCR-deficient mice with selective inactivation of NER in postnatal neurons. These mice develop dramatic age-related cumulative neuronal loss indicating DNA damage substrate overlap and synergism between TCR and NER pathways in neurons, and they uncover the occurrence of spontaneous DNA injury that may trigger neuronal degeneration. We propose that, while Csa(-/- and Csb(-/- TCR-deficient mice represent powerful animal models to study the mechanisms underlying myelin abnormalities in CS, neuron

  1. Biochemical characterization and DNA repair pathway interactions of Mag1-mediated base excision repair in Schizosaccharomyces pombe.

    Science.gov (United States)

    Alseth, Ingrun; Osman, Fikret; Korvald, Hanne; Tsaneva, Irina; Whitby, Matthew C; Seeberg, Erling; Bjørås, Magnar

    2005-01-01

    The Schizosaccharomyces pombe mag1 gene encodes a DNA repair enzyme with sequence similarity to the AlkA family of DNA glycosylases, which are essential for the removal of cytotoxic alkylation products, the premutagenic deamination product hypoxanthine and certain cyclic ethenoadducts such as ethenoadenine. In this paper, we have purified the Mag1 protein and characterized its substrate specificity. It appears that the substrate range of Mag1 is limited to the major alkylation products, such as 3-mA, 3-mG and 7-mG, whereas no significant activity was found towards deamination products, ethenoadducts or oxidation products. The efficiency of 3-mA and 3-mG removal was 5-10 times slower for Mag1 than for Escherichia coli AlkA whereas the rate of 7-mG removal was similar to the two enzymes. The relatively low efficiency for the removal of cytotoxic 3-methylpurines is consistent with the moderate sensitivity of the mag1 mutant to methylating agents. Furthermore, we studied the initial steps of Mag1-dependent base excision repair (BER) and genetic interactions with other repair pathways by mutant analysis. The double mutants mag1 nth1, mag1 apn2 and mag1 rad2 displayed increased resistance to methyl methanesulfonate (MMS) compared with the single mutants nth1, apn2 and rad2, respectively, indicating that Mag1 initiates both short-patch (Nth1-dependent) and long-patch (Rad2-dependent) BER of MMS-induced damage. Spontaneous intrachromosomal recombination frequencies increased 3-fold in the mag1 mutant suggesting that Mag1 and recombinational repair (RR) are both involved in repair of alkylated bases. Finally, we show that the deletion of mag1 in the background of rad16, nth1 and rad2 single mutants reduced the total recombination frequencies of all three double mutants, indicating that abasic sites formed as a result of Mag1 removal of spontaneous base lesions are substrates for nucleotide excision repair, long- and short-patch BER and RR.

  2. Cloning and characterization of excision repair genes

    NARCIS (Netherlands)

    P.J. van der Spek (Peter)

    1996-01-01

    textabstractFor all living organisms, it is of vital importance to maintain intact the genetic information stored in the nucleotide sequence of DNA. Numerous environmental and genotoxic agents can affect the DNA and lead to, for example, mutagenesis or carcinogenesis. Study of the mechanism of

  3. Gastroesophageal junction adenocarcinoma displays abnormalities in homologous recombination and nucleotide excision repair

    Directory of Open Access Journals (Sweden)

    Dewalt RI

    2014-02-01

    Full Text Available Robin I Dewalt,1 Kenneth A Kesler,2 Zane T Hammoud,3 LeeAnn Baldridge,4 Eyas M Hattab,4 Shadia I Jalal1,5 1Division of Hematology/Oncology, Department of Medicine, 2Cardiothoracic Division, Department of Surgery, Indiana University School of Medicine, Indianapolis, IN, USA; 3Henry Ford Hospital, Detroit, MI, USA; 4Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, IN, USA; 5Indiana University Melvin and Bren Simon Cancer Center, Indianapolis, IN, USA Objective: Esophageal adenocarcinoma (EAC continues to be a disease associated with high mortality. Among the factors leading to poor outcomes are innate resistance to currently available therapies, advanced stage at diagnosis, and complex biology. Platinum and ionizing radiation form the backbone of treatment for the majority of patients with EAC. Of the multiple processes involved in response to platinum chemotherapy or ionizing radiation, deoxyribonucleic acid (DNA repair has been a major player in cancer sensitivity to these agents. DNA repair defects have been described in various malignancies. The purpose of this study was to determine whether alterations in DNA repair are present in EAC compared with normal gastroesophageal tissues. Methods: We analyzed the expression of genes involved in homologous recombination (HR, nonhomologous end-joining, and nucleotide excision repair (NER pathways in 12 EAC tumor samples with their matched normal counterparts. These pathways were chosen because they are the main pathways involved in the repair of platinum- or ionizing-radiation-induced damage. In addition, abnormalities in these pathways have not been well characterized in EAC. Results: We identified increased expression of at least one HR gene in eight of the EAC tumor samples. Alterations in the expression of EME1, a structure-specific endonuclease involved in HR, were the most prevalent, with messenger (mRNA overexpression in six of the EAC samples

  4. Base excision DNA repair in the embryonic development of the sea urchin, Strongylocentrotus intermedius.

    Science.gov (United States)

    Torgasheva, Natalya A; Menzorova, Natalya I; Sibirtsev, Yurii T; Rasskazov, Valery A; Zharkov, Dmitry O; Nevinsky, Georgy A

    2016-06-21

    In actively proliferating cells, such as the cells of the developing embryo, DNA repair is crucial for preventing the accumulation of mutations and synchronizing cell division. Sea urchin embryo growth was analyzed and extracts were prepared. The relative activity of DNA polymerase, apurinic/apyrimidinic (AP) endonuclease, uracil-DNA glycosylase, 8-oxoguanine-DNA glycosylase, and other glycosylases was analyzed using specific oligonucleotide substrates of these enzymes; the reaction products were resolved by denaturing 20% polyacrylamide gel electrophoresis. We have characterized the profile of several key base excision repair activities in the developing embryos (2 blastomers to mid-pluteus) of the grey sea urchin, Strongylocentrotus intermedius. The uracil-DNA glycosylase specific activity sharply increased after blastula hatching, whereas the specific activity of 8-oxoguanine-DNA glycosylase steadily decreased over the course of the development. The AP-endonuclease activity gradually increased but dropped at the last sampled stage (mid-pluteus 2). The DNA polymerase activity was high at the first cleavage division and then quickly decreased, showing a transient peak at blastula hatching. It seems that the developing sea urchin embryo encounters different DNA-damaging factors early in development within the protective envelope and later as a free-floating larva, with hatching necessitating adaptation to the shift in genotoxic stress conditions. No correlation was observed between the dynamics of the enzyme activities and published gene expression data from developing congeneric species, S. purpuratus. The results suggest that base excision repair enzymes may be regulated in the sea urchin embryos at the level of covalent modification or protein stability.

  5. Enhanced base excision repair capacity in carotid atherosclerosis may protect nuclear DNA but not mitochondrial DNA

    DEFF Research Database (Denmark)

    Skarpengland, Tonje; B. Dahl, Tuva; Skjelland, Mona

    2016-01-01

    disease-free carotid specimens from patients with carotid plaques and 10 non-atherosclerotic control arteries. Genomic integrity, mitochondrial (mt) DNA copy number, oxidative DNA damage and BER proteins were evaluated in a subgroup of plaques and controls. Our major findings were: (i) The BER pathway...... genes in atherosclerosis may contribute to lesional nuclear DNA stability but appears insufficient to maintain mtDNA integrity, potentially influencing mitochondrial function in cells within the atherosclerotic lesion.......Lesional and systemic oxidative stress has been implicated in the pathogenesis of atherosclerosis, potentially leading to accumulation of DNA base lesions within atherosclerotic plaques. Although base excision repair (BER) is a major pathway counteracting oxidative DNA damage, our knowledge on BER...

  6. DNA glycosylases involved in base excision repair may be associated with cancer risk in BRCA1 and BRCA2 mutation carriers

    DEFF Research Database (Denmark)

    Osorio, Ana; Milne, Roger L; Kuchenbaecker, Karoline

    2014-01-01

    Single Nucleotide Polymorphisms (SNPs) in genes involved in the DNA Base Excision Repair (BER) pathway could be associated with cancer risk in carriers of mutations in the high-penetrance susceptibility genes BRCA1 and BRCA2, given the relation of synthetic lethality that exists between one of th...

  7. Nuclear translocation contributes to regulation of DNA excision repair activities

    DEFF Research Database (Denmark)

    Knudsen, Nina Østergaard; Andersen, Sofie Dabros; Lützen, Anne

    2009-01-01

    .T. Tomicic, W.P. Roos, B. Kaina, Mechanisms of human DNA repair: an update, Toxicology 193 (2003) 3-34; N.B. Larsen, M. Rasmussen, L.J. Rasmussen, Nuclear and mitochondrial DNA repair: similar pathways? Mitochondrion 5 (2005) 89-108]. Protein interactions are not only important for function, but also...

  8. Nrf1 CNC-bZIP protein promotes cell survival and nucleotide excision repair through maintaining glutathione homeostasis.

    Science.gov (United States)

    Han, Weinong; Ming, Mei; Zhao, Rui; Pi, Jingbo; Wu, Chunli; He, Yu-Ying

    2012-05-25

    Skin cancer is the most common cancer in the United States. Its major environmental risk factor is UVB radiation in sunlight. In response to UVB damage, epidermal keratinocytes activate a specific repair pathway, i.e. nucleotide excision repair, to remove UVB-induced DNA lesions. However, the regulation of UVB response is not fully understood. Here we show that the long isoform of the nuclear factor erythroid 2-related factor 1 (Nrf1, also called NFE2L1), a cytoprotective transcription factor critical for the expression of multiple antioxidant response element-dependent genes, plays an important role in the response of keratinocytes to UVB. Nrf1 loss sensitized keratinocytes to UVB-induced apoptosis by up-regulating the expression of the proapoptotic Bcl-2 family member Bik through reducing glutathione levels. Knocking down Bik reduced UVB-induced apoptosis in Nrf1-inhibited cells. In UVB-irradiated surviving cells, however, disruption of Nrf1 impaired nucleotide excision repair through suppressing the transcription of xeroderma pigmentosum C (XPC), a factor essential for initiating the global genome nucleotide excision repair by recognizing the DNA lesion and recruiting downstream factors. Nrf1 enhanced XPC expression by increasing glutathione availability but was independent of the transcription repressor of XPC. Adding XPC or glutathione restored the DNA repair capacity in Nrf1-inhibited cells. Finally, we demonstrate that Nrf1 levels are significantly reduced by UVB radiation in mouse skin and are lower in human skin tumors than in normal skin. These results indicate a novel role of Nrf1 in UVB-induced DNA damage repair and suggest Nrf1 as a tumor suppressor in the skin.

  9. Uncommon nucleotide excision repair phenotypes revealed by targeted high-throughput sequencing.

    Science.gov (United States)

    Calmels, Nadège; Greff, Géraldine; Obringer, Cathy; Kempf, Nadine; Gasnier, Claire; Tarabeux, Julien; Miguet, Marguerite; Baujat, Geneviève; Bessis, Didier; Bretones, Patricia; Cavau, Anne; Digeon, Béatrice; Doco-Fenzy, Martine; Doray, Bérénice; Feillet, François; Gardeazabal, Jesus; Gener, Blanca; Julia, Sophie; Llano-Rivas, Isabel; Mazur, Artur; Michot, Caroline; Renaldo-Robin, Florence; Rossi, Massimiliano; Sabouraud, Pascal; Keren, Boris; Depienne, Christel; Muller, Jean; Mandel, Jean-Louis; Laugel, Vincent

    2016-03-22

    Deficient nucleotide excision repair (NER) activity causes a variety of autosomal recessive diseases including xeroderma pigmentosum (XP) a disorder which pre-disposes to skin cancer, and the severe multisystem condition known as Cockayne syndrome (CS). In view of the clinical overlap between NER-related disorders, as well as the existence of multiple phenotypes and the numerous genes involved, we developed a new diagnostic approach based on the enrichment of 16 NER-related genes by multiplex amplification coupled with next-generation sequencing (NGS). Our test cohort consisted of 11 DNA samples, all with known mutations and/or non pathogenic SNPs in two of the tested genes. We then used the same technique to analyse samples from a prospective cohort of 40 patients. Multiplex amplification and sequencing were performed using AmpliSeq protocol on the Ion Torrent PGM (Life Technologies). We identified causative mutations in 17 out of the 40 patients (43%). Four patients showed biallelic mutations in the ERCC6(CSB) gene, five in the ERCC8(CSA) gene: most of them had classical CS features but some had very mild and incomplete phenotypes. A small cohort of 4 unrelated classic XP patients from the Basque country (Northern Spain) revealed a common splicing mutation in POLH (XP-variant), demonstrating a new founder effect in this population. Interestingly, our results also found ERCC2(XPD), ERCC3(XPB) or ERCC5(XPG) mutations in two cases of UV-sensitive syndrome and in two cases with mixed XP/CS phenotypes. Our study confirms that NGS is an efficient technique for the analysis of NER-related disorders on a molecular level. It is particularly useful for phenotypes with combined features or unusually mild symptoms. Targeted NGS used in conjunction with DNA repair functional tests and precise clinical evaluation permits rapid and cost-effective diagnosis in patients with NER-defects.

  10. Nucleotide Excision Repair and Vitamin D--Relevance for Skin Cancer Therapy.

    Science.gov (United States)

    Pawlowska, Elzbieta; Wysokinski, Daniel; Blasiak, Janusz

    2016-04-06

    Ultraviolet (UV) radiation is involved in almost all skin cancer cases, but on the other hand, it stimulates the production of pre-vitamin D3, whose active metabolite, 1,25-dihydroxyvitamin D3 (1,25VD3), plays important physiological functions on binding with its receptor (vitamin D receptor, VDR). UV-induced DNA damages in the form of cyclobutane pyrimidine dimers or (6-4)-pyrimidine-pyrimidone photoproducts are frequently found in skin cancer and its precursors. Therefore, removing these lesions is essential for the prevention of skin cancer. As UV-induced DNA damages are repaired by nucleotide excision repair (NER), the interaction of 1,25VD3 with NER components can be important for skin cancer transformation. Several studies show that 1,25VD3 protects DNA against damage induced by UV, but the exact mechanism of this protection is not completely clear. 1,25VD3 was also shown to affect cell cycle regulation and apoptosis in several signaling pathways, so it can be considered as a potential modulator of the cellular DNA damage response, which is crucial for mutagenesis and cancer transformation. 1,25VD3 was shown to affect DNA repair and potentially NER through decreasing nitrosylation of DNA repair enzymes by NO overproduction by UV, but other mechanisms of the interaction between 1,25VD3 and NER machinery also are suggested. Therefore, the array of NER gene functioning could be analyzed and an appropriate amount of 1.25VD3 could be recommended to decrease UV-induced DNA damage important for skin cancer transformation.

  11. Age-Related Neuronal Degeneration: Complementary Roles of Nucleotide Excision Repair and Transcription-Coupled Repair in Preventing Neuropathology

    Science.gov (United States)

    de Waard, Monique C.; Haasdijk, Elize D.; Brandt, Renata; Vermeij, Marcel; Rijksen, Yvonne; Maas, Alex; van Steeg, Harry; Hoeijmakers, Jan H. J.; van der Horst, Gijsbertus T. J.

    2011-01-01

    Neuronal degeneration is a hallmark of many DNA repair syndromes. Yet, how DNA damage causes neuronal degeneration and whether defects in different repair systems affect the brain differently is largely unknown. Here, we performed a systematic detailed analysis of neurodegenerative changes in mouse models deficient in nucleotide excision repair (NER) and transcription-coupled repair (TCR), two partially overlapping DNA repair systems that remove helix-distorting and transcription-blocking lesions, respectively, and that are associated with the UV-sensitive syndromes xeroderma pigmentosum (XP) and Cockayne syndrome (CS). TCR–deficient Csa−/− and Csb−/− CS mice showed activated microglia cells surrounding oligodendrocytes in regions with myelinated axons throughout the nervous system. This white matter microglia activation was not observed in NER–deficient Xpa−/− and Xpc−/− XP mice, but also occurred in XpdXPCS mice carrying a point mutation (G602D) in the Xpd gene that is associated with a combined XPCS disorder and causes a partial NER and TCR defect. The white matter abnormalities in TCR–deficient mice are compatible with focal dysmyelination in CS patients. Both TCR–deficient and NER–deficient mice showed no evidence for neuronal degeneration apart from p53 activation in sporadic (Csa−/−, Csb−/−) or highly sporadic (Xpa−/−, Xpc−/−) neurons and astrocytes. To examine to what extent overlap occurs between both repair systems, we generated TCR–deficient mice with selective inactivation of NER in postnatal neurons. These mice develop dramatic age-related cumulative neuronal loss indicating DNA damage substrate overlap and synergism between TCR and NER pathways in neurons, and they uncover the occurrence of spontaneous DNA injury that may trigger neuronal degeneration. We propose that, while Csa−/− and Csb−/− TCR–deficient mice represent powerful animal models to study the mechanisms underlying myelin abnormalities

  12. POLB: A new role of DNA polymerase beta in mitochondrial base excision repair.

    Science.gov (United States)

    Kaufman, Brett A; Van Houten, Bennett

    2017-12-01

    The mitochondrial genome is a matrilineally inherited DNA that encodes numerous essential subunits of the respiratory chain in all metazoans. As such mitochondrial DNA (mtDNA) sequence integrity is vital to organismal survival, but it has a limited cadre of DNA repair activities, primarily base excision repair (BER). We have known that the mtDNA is significantly oxidized by both endogenous and exogenous sources, but this does not lead to the expected preferential formation of transversion mutations, which suggest a robust base excision repair (BER) system. This year, two different groups reported compelling evidence that what was believed to be exclusively nuclear DNA repair polymerase, POLB, is located in the mitochondria and plays a significant role in mitochondrial BER, mtDNA integrity and mitochondrial function. In this commentary, we review the findings and highlight remaining questions for the field. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Polynucleotide kinase/phosphatase, Pnk1, is involved in base excision repair in Schizosaccharomyces pombe.

    Science.gov (United States)

    Kashkina, Ekaterina; Qi, Tao; Weinfeld, Michael; Young, Dallan

    2012-08-01

    We previously reported that Schizosaccharomyces pombe pnk1 cells are more sensitive than wild-type cells to γ-radiation and camptothecin, indicating that Pnk1 is required for DNA repair. Here, we report that pnk1pku70 and pnk1rhp51 double mutants are more sensitive to γ-radiation than single mutants, from which we infer that Pnk1's primary role is independent of either homologous recombination or non-homologous end joining mechanisms. We also report that pnk1 cells are more sensitive than wild-type cells to oxidizing and alkylating agents, suggesting that Pnk1 is involved in base excision repair. Mutational analysis of Pnk1 revealed that the DNA 3'-phosphatase activity is necessary for repair of DNA damage, whereas the 5'-kinase activity is dispensable. A role for Pnk1 in base excision repair is supported by genetic analyses which revealed that pnk1apn2 is synthetically lethal, suggesting that Pnk1 and Apn2 may function in parallel pathways essential for the repair of endogenous DNA damage. Furthermore, the nth1pnk1apn2 and tdp1pnk1apn2 triple mutants are viable, implying that single-strand breaks with 3'-blocked termini produced by Nth1 and Tdp1 contribute to synthetic lethality. We also examined the sensitivity to methyl methanesulfonate of all single and double mutant combinations of nth1, apn2, tdp1 and pnk1. Together, our results support a model where Tdp1 and Pnk1 act in concert in an Apn2-independent base excision repair pathway to repair 3'-blocked termini produced by Nth1; and they also provide evidence that Pnk1 has additional roles in base excision repair. Copyright © 2012 Elsevier B.V. All rights reserved.

  14. Nucleotide excision repair I: from E.coli to yeast.

    NARCIS (Netherlands)

    J.H.J. Hoeijmakers (Jan)

    1993-01-01

    textabstractGenetic information is constantly deteriorating, mainly as a consequence of the action of numerous genotoxic agents. In order to cope with this fundamental problem, all living organisms have acquired a complex network of DNA repair systems to safeguard their genetic integrity. Nucleotide

  15. Conservation of the nucleotide excision repair pathway: characterization of hydra Xeroderma Pigmentosum group F homolog.

    Directory of Open Access Journals (Sweden)

    Apurva Barve

    Full Text Available Hydra, one of the earliest metazoans with tissue grade organization and nervous system, is an animal with a remarkable regeneration capacity and shows no signs of organismal aging. We have for the first time identified genes of the nucleotide excision repair (NER pathway from hydra. Here we report cloning and characterization of hydra homolog of xeroderma pigmentosum group F (XPF gene that encodes a structure-specific 5' endonuclease which is a crucial component of NER. In silico analysis shows that hydra XPF amino acid sequence is very similar to its counterparts from other animals, especially vertebrates, and shows all features essential for its function. By in situ hybridization, we show that hydra XPF is expressed prominently in the multipotent stem cell niche in the central region of the body column. Ectoderm of the diploblastic hydra was shown to express higher levels of XPF as compared to the endoderm by semi-quantitative RT-PCR. Semi-quantitative RT-PCR analysis also demonstrated that interstitial cells, a multipotent and rapidly cycling stem cell lineage of hydra, express higher levels of XPF mRNA than other cell types. Our data show that XPF and by extension, the NER pathway is highly conserved during evolution. The prominent expression of an NER gene in interstitial cells may have implications for the lack of senescence in hydra.

  16. Conservation of the nucleotide excision repair pathway: characterization of hydra Xeroderma Pigmentosum group F homolog.

    Science.gov (United States)

    Barve, Apurva; Ghaskadbi, Saroj; Ghaskadbi, Surendra

    2013-01-01

    Hydra, one of the earliest metazoans with tissue grade organization and nervous system, is an animal with a remarkable regeneration capacity and shows no signs of organismal aging. We have for the first time identified genes of the nucleotide excision repair (NER) pathway from hydra. Here we report cloning and characterization of hydra homolog of xeroderma pigmentosum group F (XPF) gene that encodes a structure-specific 5' endonuclease which is a crucial component of NER. In silico analysis shows that hydra XPF amino acid sequence is very similar to its counterparts from other animals, especially vertebrates, and shows all features essential for its function. By in situ hybridization, we show that hydra XPF is expressed prominently in the multipotent stem cell niche in the central region of the body column. Ectoderm of the diploblastic hydra was shown to express higher levels of XPF as compared to the endoderm by semi-quantitative RT-PCR. Semi-quantitative RT-PCR analysis also demonstrated that interstitial cells, a multipotent and rapidly cycling stem cell lineage of hydra, express higher levels of XPF mRNA than other cell types. Our data show that XPF and by extension, the NER pathway is highly conserved during evolution. The prominent expression of an NER gene in interstitial cells may have implications for the lack of senescence in hydra.

  17. Small margin (2 mm) excision of peri-ocular basal cell carcinoma with delayed repair.

    Science.gov (United States)

    David, D B.; Gimblett, M L.; Potts, M J.; Harrad, R A.

    1999-03-01

    Successful surgical treatment of peri-ocular basal cell carcinomas requires complete excision. Mohs' micrographic surgery achieves this, but is not readily available in all hospitals. The standard 3-4 mm margin does not guarantee complete excision and histology is often not available until after a repair has been undertaken. The 3-4 mm margin has evolved to deal with all forms of BCC. In our opinion, this margin is unnecessarily large for nodular/ulcerative BCC. We report our interim results of excision of localised BCCs using a 2 mm margin in conjunction with a delayed repair following confirmation of histological clearance. Thirty-one patients were treated in this manner; there have been no recurrences after an average follow-up period of 36 months (range 24-57 months).

  18. Single-molecule fluorescence microscopy on nucleotide excision repair complexes using GFP fusion proteins

    NARCIS (Netherlands)

    Segers-Nolten, Gezina M.J.; Rademakers, Suzanne; Vermeulen, Wim; Lenferink, Aufrid T.M.; Otto, Cornelis; Hoeijmakers, Jan; Greve, Jan; Koenig, Karsten; Tanke, Hans J.; Schneckenburger, Herbert

    2000-01-01

    Scanning Confocal Fluorescence Microscopy is used for single molecule studies on DNA-protein complexes that occur in Nucleotide Excision Repair (NER). During DNA-damage elimination by the NER-pathway, complex protein structures assemble over DNA. It is our aim to resolve the architecture of these

  19. The mitochondrial transcription factor A functions in mitochondrial base excision repair

    DEFF Research Database (Denmark)

    Canugovi, Chandrika; Maynard, Scott; Bayne, Anne-Cécile V

    2010-01-01

    Mitochondrial transcription factor A (TFAM) is an essential component of mitochondrial nucleoids. TFAM plays an important role in mitochondrial transcription and replication. TFAM has been previously reported to inhibit nucleotide excision repair (NER) in vitro but NER has not yet been detected i...

  20. Excision and crosslink repair of DNA and sister chromatid exchanges in cultured human fibroblasts with different repair capacities

    Energy Technology Data Exchange (ETDEWEB)

    Fujiwara, Y.; Kano, Y.; Paul, P.; Goto, K.; Yamamoto, K. (Kobe Univ. (Japan). School of Medicine)

    1981-01-01

    Xeroderma pigmentosum (XP) groups A to G lacked the initial stage of ultraviolet (UV) excision repair in the order of A = G > C > D > E asymptotically equals F, while the XP variant was weakly defective in the later repair steps. Killing sensitivities were in the orders of A >= G > D > C > E asymptotically equals F asymptotically equals variant > normal to UV, A = G > D > F > C = E > variant > normal to 4-nitroquinoline-1-oxide (4NQO), and A > C > D = E = F = variant > G = normal to decarbamoyl mitomycin-C(DCMC). The induced sister chromatid exchange (SCE) frequency was unrelated to the extent of repair deficiency. The SCE induction rate was consistently 3 - 6 fold higher by these UV-like mutagens in XP group A cells than in normal cells. However, repair-proficient Cockayne's syndrome (CS) cells showed a higher SCE induction by UV, which was normalized by NAD/sup +/, suggesting that chromatin lesions as well as DNA damage contribute to SCE. Two-step crosslink repair involves a first rapid half-excision and a second slow nucleotide-excision repair. Fanconi's anemia (FA) cells had an impaired first half-excision and were supersensitive to MC, but not to UV and DCMC. The SCE frequency induced by MC (1 hr) was higher in FA cells than in normal cells despite their normal response to DCMC, and vice versa in XP cells. FA cells lacked the first rapid decline and showed higher remaining SCEs. Thus, part of the crosslink seems to lead to SCE formation. Caffeine synergistically elevated UV-induced SCEs, but not UV induced mutations in V79 cells, implying that SCE may not necessarily involve mutation.

  1. The Association of Low-Penetrance Variants in DNA Repair Genes with Colorectal Cancer: A Systematic Review and Meta-Analysis

    OpenAIRE

    Aggarwal, Nikhil; Donald, Neil D; Malik, Salim; Selvendran, Subothini S; McPhail, Mark JW.; Monahan, Kevin J

    2017-01-01

    Objectives: Approximately 35% of colorectal cancer (CRC) risk is attributable to heritable factors known hereditary syndromes, accounting for 6%. The remainder may be due to lower penetrance polymorphisms particularly of DNA repair genes. DNA repair pathways, including base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), direct reversal repair (DRR), and double-strand break repair are complex, evolutionarily conserved, and critical in carcinogenesis. Germline m...

  2. Nucleotide excision repair, mismatch repair, and R-loops modulate convergent transcription-induced cell death and repeat instability.

    Directory of Open Access Journals (Sweden)

    Yunfu Lin

    Full Text Available Expansion of CAG•CTG tracts located in specific genes is responsible for 13 human neurodegenerative disorders, the pathogenic mechanisms of which are not yet well defined. These disease genes are ubiquitously expressed in human tissues, and transcription has been identified as one of the major pathways destabilizing the repeats. Transcription-induced repeat instability depends on transcription-coupled nucleotide excision repair (TC-NER, the mismatch repair (MMR recognition component MSH2/MSH3, and RNA/DNA hybrids (R-loops. Recently, we reported that simultaneous sense and antisense transcription-convergent transcription-through a CAG repeat not only promotes repeat instability, but also induces a cell stress response, which arrests the cell cycle and eventually leads to massive cell death via apoptosis. Here, we use siRNA knockdowns to investigate whether NER, MMR, and R-loops also modulate convergent-transcription-induced cell death and repeat instability. We find that siRNA-mediated depletion of TC-NER components increases convergent transcription-induced cell death, as does the simultaneous depletion of RNase H1 and RNase H2A. In contrast, depletion of MSH2 decreases cell death. These results identify TC-NER, MMR recognition, and R-loops as modulators of convergent transcription-induced cell death and shed light on the molecular mechanism involved. We also find that the TC-NER pathway, MSH2, and R-loops modulate convergent transcription-induced repeat instability. These observations link the mechanisms of convergent transcription-induced repeat instability and convergent transcription-induced cell death, suggesting that a common structure may trigger both outcomes.

  3. Alternative Excision Repair of Ultraviolet B- and C-Induced DNA Damage in Dormant and Developing Spores of Bacillus subtilis

    Science.gov (United States)

    Ramírez-Guadiana, Fernando H.; Barraza-Salas, Marcelo; Ramírez-Ramírez, Norma; Ortiz-Cortés, Mayte; Setlow, Peter

    2012-01-01

    The nucleotide excision repair (NER) and spore photoproduct lyase DNA repair pathways are major determinants of Bacillus subtilis spore resistance to UV radiation. We report here that a putative ultraviolet (UV) damage endonuclease encoded by ywjD confers protection to developing and dormant spores of B. subtilis against UV DNA damage. In agreement with its predicted function, a His6-YwjD recombinant protein catalyzed the specific incision of UV-irradiated DNA in vitro. The maximum expression of a reporter gene fusion to the ywjD opening reading frame occurred late in sporulation, and this maximal expression was dependent on the forespore-specific RNA polymerase sigma factor, σG. Although the absence of YwjD and/or UvrA, an essential protein of the NER pathway, sensitized developing spores to UV-C, this effect was lower when these cells were treated with UV-B. In contrast, UV-B but not UV-C radiation dramatically decreased the survival of dormant spores deficient in both YwjD and UvrA. The distinct range of lesions generated by UV-C and UV-B and the different DNA photochemistry in developing and dormant spores may cause these differences. We postulate that in addition to the UvrABC repair system, developing and dormant spores of B. subtilis also rely on an alternative excision repair pathway involving YwjD to deal with the deleterious effects of various UV photoproducts. PMID:22961846

  4. DNA Repair in Drosophila: Mutagens, Models, and Missing Genes.

    Science.gov (United States)

    Sekelsky, Jeff

    2017-02-01

    The numerous processes that damage DNA are counterbalanced by a complex network of repair pathways that, collectively, can mend diverse types of damage. Insights into these pathways have come from studies in many different organisms, including Drosophila melanogaster Indeed, the first ideas about chromosome and gene repair grew out of Drosophila research on the properties of mutations produced by ionizing radiation and mustard gas. Numerous methods have been developed to take advantage of Drosophila genetic tools to elucidate repair processes in whole animals, organs, tissues, and cells. These studies have led to the discovery of key DNA repair pathways, including synthesis-dependent strand annealing, and DNA polymerase theta-mediated end joining. Drosophila appear to utilize other major repair pathways as well, such as base excision repair, nucleotide excision repair, mismatch repair, and interstrand crosslink repair. In a surprising number of cases, however, DNA repair genes whose products play important roles in these pathways in other organisms are missing from the Drosophila genome, raising interesting questions for continued investigations. Copyright © 2017 by the Genetics Society of America.

  5. Histone H3 Lys79 methylation is required for efficient nucleotide excision repair in a silenced locus of Saccharomyces cerevisiae

    Science.gov (United States)

    Chaudhuri, Shubho; Wyrick, John J.; Smerdon, Michael J.

    2009-01-01

    Methylation of specific histone lysine residues regulates gene expression and heterochromatin function, but little is known about its role in DNA repair. To examine how changes in conserved methylated residues of histone H3 affect nucleotide excision repair (NER), viable H3K4R and H3K79R mutants were generated in Saccharomyces cerevisiae. These mutants show decreased UV survival and impaired NER at the transcriptionally silent HML locus, while maintaining normal NER in the constitutively expressed RPB2 gene and transcriptionally repressed, nucleosome loaded GAL10 gene. Moreover, the HML chromatin in these mutants has reduced accessibility to Micrococcal nuclease (MNase). Importantly, chromatin immunoprecipitation analysis demonstrates there is enhanced recruitment of the Sir complex at the HML locus of these mutants, and deletion of the SIR2 or SIR3 genes restores the MNase accessibility and DNA repair efficiency at this locus. Furthermore, following UV irradiation expression of NER genes in these mutants remains at wild type levels, with the exception of RAD16 which decreases by more than 2-fold. These results indicate that impaired NER occurs in the silenced chromatin of H3K79R and H3K4,79R mutants as a result of increased binding of Sir complexes, which may reduce DNA lesion accessibility to repair enzymes. PMID:19155276

  6. Low-Dose Formaldehyde Delays DNA Damage Recognition and DNA Excision Repair in Human Cells

    Science.gov (United States)

    Luch, Andreas; Frey, Flurina C. Clement; Meier, Regula; Fei, Jia; Naegeli, Hanspeter

    2014-01-01

    Objective Formaldehyde is still widely employed as a universal crosslinking agent, preservative and disinfectant, despite its proven carcinogenicity in occupationally exposed workers. Therefore, it is of paramount importance to understand the possible impact of low-dose formaldehyde exposures in the general population. Due to the concomitant occurrence of multiple indoor and outdoor toxicants, we tested how formaldehyde, at micromolar concentrations, interferes with general DNA damage recognition and excision processes that remove some of the most frequently inflicted DNA lesions. Methodology/Principal Findings The overall mobility of the DNA damage sensors UV-DDB (ultraviolet-damaged DNA-binding) and XPC (xeroderma pigmentosum group C) was analyzed by assessing real-time protein dynamics in the nucleus of cultured human cells exposed to non-cytotoxic (formaldehyde concentrations. The DNA lesion-specific recruitment of these damage sensors was tested by monitoring their accumulation at local irradiation spots. DNA repair activity was determined in host-cell reactivation assays and, more directly, by measuring the excision of DNA lesions from chromosomes. Taken together, these assays demonstrated that formaldehyde obstructs the rapid nuclear trafficking of DNA damage sensors and, consequently, slows down their relocation to DNA damage sites thus delaying the excision repair of target lesions. A concentration-dependent effect relationship established a threshold concentration of as low as 25 micromolar for the inhibition of DNA excision repair. Conclusions/Significance A main implication of the retarded repair activity is that low-dose formaldehyde may exert an adjuvant role in carcinogenesis by impeding the excision of multiple mutagenic base lesions. In view of this generally disruptive effect on DNA repair, we propose that formaldehyde exposures in the general population should be further decreased to help reducing cancer risks. PMID:24722772

  7. The hepatitis B virus x protein inhibits thymine DNA glycosylase initiated base excision repair.

    Directory of Open Access Journals (Sweden)

    Maarten A A van de Klundert

    Full Text Available The hepatitis B virus (HBV genome encodes the X protein (HBx, a ubiquitous transactivator that is required for HBV replication. Expression of the HBx protein has been associated with the development of HBV infection-related hepatocellular carcinoma (HCC. Previously, we generated a 3D structure of HBx by combined homology and ab initio in silico modelling. This structure showed a striking similarity to the human thymine DNA glycosylase (TDG, a key enzyme in the base excision repair (BER pathway. To further explore this finding, we investigated whether both proteins interfere with or complement each other's functions. Here we show that TDG does not affect HBV replication, but that HBx strongly inhibits TDG-initiated base excision repair (BER, a major DNA repair pathway. Inhibition of the BER pathway may contribute substantially to the oncogenic effect of HBV infection.

  8. Induction of a mutant phenotype in human repair proficient cells after overexpression of a mutated human DNA repair gene.

    NARCIS (Netherlands)

    P.B.G.M. Belt; M.F. van Oostenrijk; H. Odijk (Hanny); J.H.J. Hoeijmakers (Jan); C.M.P. Backendorf (Claude)

    1991-01-01

    textabstractAntisense and mutated cDNA of the human excision repair gene ERCC-1 were overexpressed in repair efficient HeLa cells by means of an Epstein-Barr-virus derived CDNA expression vector. Whereas antisense RNA did not influence the survival of the transfected cells, a mutated cDNA generating

  9. DNA polymerase β: A missing link of the base excision repair machinery in mammalian mitochondria.

    Science.gov (United States)

    Prasad, Rajendra; Çağlayan, Melike; Dai, Da-Peng; Nadalutti, Cristina A; Zhao, Ming-Lang; Gassman, Natalie R; Janoshazi, Agnes K; Stefanick, Donna F; Horton, Julie K; Krasich, Rachel; Longley, Matthew J; Copeland, William C; Griffith, Jack D; Wilson, Samuel H

    2017-12-01

    Mitochondrial genome integrity is fundamental to mammalian cell viability. Since mitochondrial DNA is constantly under attack from oxygen radicals released during ATP production, DNA repair is vital in removing oxidatively generated lesions in mitochondrial DNA, but the presence of a strong base excision repair system has not been demonstrated. Here, we addressed the presence of such a system in mammalian mitochondria involving the primary base lesion repair enzyme DNA polymerase (pol) β. Pol β was localized to mammalian mitochondria by electron microscopic-immunogold staining, immunofluorescence co-localization and biochemical experiments. Extracts from purified mitochondria exhibited base excision repair activity that was dependent on pol β. Mitochondria from pol β-deficient mouse fibroblasts had compromised DNA repair and showed elevated levels of superoxide radicals after hydrogen peroxide treatment. Mitochondria in pol β-deficient fibroblasts displayed altered morphology by electron microscopy. These results indicate that mammalian mitochondria contain an efficient base lesion repair system mediated in part by pol β and thus pol β plays a role in preserving mitochondrial genome stability. Published by Elsevier B.V.

  10. Crystal structure of the FeS cluster-containing nucleotide excision repair helicase XPD.

    Directory of Open Access Journals (Sweden)

    Stefanie C Wolski

    2008-06-01

    Full Text Available DNA damage recognition by the nucleotide excision repair pathway requires an initial step identifying helical distortions in the DNA and a proofreading step verifying the presence of a lesion. This proofreading step is accomplished in eukaryotes by the TFIIH complex. The critical damage recognition component of TFIIH is the XPD protein, a DNA helicase that unwinds DNA and identifies the damage. Here, we describe the crystal structure of an archaeal XPD protein with high sequence identity to the human XPD protein that reveals how the structural helicase framework is combined with additional elements for strand separation and DNA scanning. Two RecA-like helicase domains are complemented by a 4Fe4S cluster domain, which has been implicated in damage recognition, and an alpha-helical domain. The first helicase domain together with the helical and 4Fe4S-cluster-containing domains form a central hole with a diameter sufficient in size to allow passage of a single stranded DNA. Based on our results, we suggest a model of how DNA is bound to the XPD protein, and can rationalize several of the mutations in the human XPD gene that lead to one of three severe diseases, xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy.

  11. UV-induced de novo protein synthesis enhances nucleotide excision repair efficiency in a transcription-dependent manner in S. cerevisiae.

    Science.gov (United States)

    Al-Moghrabi, Nisreen M; Al-Sharif, Ibtehaj S; Aboussekhra, Abdelilah

    2003-11-21

    DNA damage results in the up-regulation of several genes involved in different cellular physiological processes, such as the nucleotide excision repair (NER) mechanism that copes with a broad range of DNA alterations, including the carcinogenic ultraviolet (UV) light-induced pyrimidine dimers (PDs). There are two NER sub-pathways: transcription coupled repair (TCR) that is specific for the transcribed strands (TS) of active genes and global genomic repair (GGR) that repairs non-transcribed DNA sequences (NTD) and the non-transcribed strands (NTS) of expressed genes. To elucidate the role of UV-dependent de novo protein synthesis in nucleotide excision repair in the budding yeast, we investigated the effect of the protein synthesis inhibitor, cycloheximide, on the removal of PDs. Log phase as well as G(1)-synchronized cells were treated with the drug shortly before UV irradiation and immediately thereafter, and the repair of damaged DNA was assessed with the high resolution primer extension technique. The results show that in both cellular conditions, the inhibition of UV-dependent de novo protein synthesis by cycloheximide impairs the excision repair of the transcriptionally active GAL10 and URA3 genes, with a greater effect on the non-transcribed strands. This indicates that UV-mediated de novo protein synthesis is required for efficient nucleotide excision repair, but not for the preferential repair of the TSs. On the other hand, cycloheximide did not affect the repair of either strand of the repressed GAL10 gene or the non-transcribed promoter region of the URA3 gene, showing that UV-induced de novo protein synthesis is not required for PD removal from transcriptionally inactive DNA sequences. Together, these data show that despite the fact that NTD and NTSs are normally repaired by the GGR sub-pathway, their requirement for UV-dependent de novo protein synthesis is different, which may suggest a difference in the processing of UV lesions in these non

  12. The endoperoxide ascaridol shows strong differential cytotoxicity in nucleotide excision repair-deficient cells

    Energy Technology Data Exchange (ETDEWEB)

    Abbasi, Rashda [Division of Epigenomics and Cancer Risk Factors, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg (Germany); Efferth, Thomas [Institute of Pharmacy und Biochemistry, Johannes Gutenberg University, Staudinger Weg 5, 55128 Mainz (Germany); Kuhmann, Christine [Division of Epigenomics and Cancer Risk Factors, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg (Germany); Opatz, Till [Institute of Organic Chemistry, Johannes Gutenberg University, Duesbergweg 10-14, 55128 Mainz (Germany); Hao, Xiaojiang [Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204 (China); Popanda, Odilia, E-mail: o.popanda@dkfz.de [Division of Epigenomics and Cancer Risk Factors, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg (Germany); Schmezer, Peter [Division of Epigenomics and Cancer Risk Factors, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg (Germany)

    2012-03-15

    Targeting synthetic lethality in DNA repair pathways has become a promising anti-cancer strategy. However little is known about such interactions with regard to the nucleotide excision repair (NER) pathway. Therefore, cell lines with a defect in the NER genes ERCC6 or XPC and their normal counterparts were screened with 53 chemically defined phytochemicals isolated from plants used in traditional Chinese medicine for differential cytotoxic effects. The screening revealed 12 drugs that killed NER-deficient cells more efficiently than proficient cells. Five drugs were further analyzed for IC{sub 50} values, effects on cell cycle distribution, and induction of DNA damage. Ascaridol was the most effective compound with a difference of > 1000-fold in resistance between normal and NER-deficient cells (IC{sub 50} values for cells with deficiency in ERCC6: 0.15 μM, XPC: 0.18 μM, and normal cells: > 180 μM). NER-deficiency combined with ascaridol treatment led to G2/M-phase arrest, an increased percentage of subG1 cells, and a substantially higher DNA damage induction. These results were confirmed in a second set of NER-deficient and -proficient cell lines with isogenic background. Finally, ascaridol was characterized for its ability to generate oxidative DNA damage. The drug led to a dose-dependent increase in intracellular levels of reactive oxygen species at cytotoxic concentrations, but only NER-deficient cells showed a strongly induced amount of 8-oxodG sites. In summary, ascaridol is a cytotoxic and DNA-damaging compound which generates intracellular reactive oxidative intermediates and which selectively affects NER-deficient cells. This could provide a new therapeutic option to treat cancer cells with mutations in NER genes. -- Highlights: ► Thousand-fold higher Ascaridol activity in NER-deficient versus proficient cells. ► Impaired repair of Ascaridol-induced oxidative DNA damage in NER-deficient cells. ► Selective activity of Ascaridol opens new therapy

  13. Isolating human DNA repair genes using rodent-cell mutants

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, L.H.; Weber, C.A.; Brookman, K.W.; Salazar, E.P.; Stewart, S.A.; Mitchell, D.L.

    1987-03-23

    The DNA repair systems of rodent and human cells appear to be at least as complex genetically as those in lower eukaryotes and bacteria. The use of mutant lines of rodent cells as a means of identifying human repair genes by functional complementation offers a new approach toward studying the role of repair in mutagenesis and carcinogenesis. In each of six cases examined using hybrid cells, specific human chromosomes have been identified that correct CHO cell mutations affecting repair of damage from uv or ionizing radiations. This finding suggests that both the repair genes and proteins may be virtually interchangeable between rodent and human cells. Using cosmid vectors, human repair genes that map to chromosome 19 have cloned as functional sequences: ERCC2 and XRCC1. ERCC1 was found to have homology with the yeast excision repair gene RAD10. Transformants of repair-deficient cell lines carrying the corresponding human gene show efficient correction of repair capacity by all criteria examined. 39 refs., 1 fig., 1 tab.

  14. Defects in Base Excision Repair Sensitize Cells to Manganese in S. cerevisiae

    Directory of Open Access Journals (Sweden)

    Adrienne P. Stephenson

    2013-01-01

    Full Text Available Manganese (Mn is essential for normal physiologic functioning; therefore, deficiencies and excess intake of manganese can result in disease. In humans, prolonged exposure to manganese causes neurotoxicity characterized by Parkinson-like symptoms. Mn2+ has been shown to mediate DNA damage possibly through the generation of reactive oxygen species. In a recent publication, we showed that Mn induced oxidative DNA damage and caused lesions in thymines. This study further investigates the mechanisms by which cells process Mn2+-mediated DNA damage using the yeast S. cerevisiae. The strains most sensitive to Mn2+ were those defective in base excision repair, glutathione synthesis, and superoxide dismutase mutants. Mn2+ caused a dose-dependent increase in the accumulation of mutations using the CAN1 and lys2-10A mutator assays. The spectrum of CAN1 mutants indicates that exposure to Mn results in accumulation of base substitutions and frameshift mutations. The sensitivity of cells to Mn2+ as well as its mutagenic effect was reduced by N-acetylcysteine, glutathione, and Mg2+. These data suggest that Mn2+ causes oxidative DNA damage that requires base excision repair for processing and that Mn interferes with polymerase fidelity. The status of base excision repair may provide a biomarker for the sensitivity of individuals to manganese.

  15. The recombination protein RAD52 cooperates with the excision repair protein OGG1 for the repair of oxidative lesions in mammalian cells

    DEFF Research Database (Denmark)

    de Souza-Pinto, Nadja C; Maynard, Scott; Hashiguchi, Kazunari

    2009-01-01

    Oxidized bases are common types of DNA modifications. Their accumulation in the genome is linked to aging and degenerative diseases. These modifications are commonly repaired by the base excision repair (BER) pathway. Oxoguanine DNA glycosylase (OGG1) initiates BER of oxidized purine bases. A small...... activities and RAD52 stimulates OGG1 incision activity, likely increasing its turnover rate. RAD52 colocalizes with OGG1 after oxidative stress to cultured cells, but not after the direct induction of double-strand breaks by ionizing radiation. Human cells depleted of RAD52 via small interfering RNA...... knockdown, and mouse cells lacking the protein via gene knockout showed increased sensitivity to oxidative stress. Moreover, cells depleted of RAD52 show higher accumulation of oxidized bases in their genome than cells with normal levels of RAD52. Our results indicate that RAD52 cooperates with OGG1...

  16. Removal of misincorporated ribonucleotides from prokaryotic genomes: an unexpected role for nucleotide excision repair.

    Directory of Open Access Journals (Sweden)

    Alexandra Vaisman

    2013-11-01

    Full Text Available Stringent steric exclusion mechanisms limit the misincorporation of ribonucleotides by high-fidelity DNA polymerases into genomic DNA. In contrast, low-fidelity Escherichia coli DNA polymerase V (pol V has relatively poor sugar discrimination and frequently misincorporates ribonucleotides. Substitution of a steric gate tyrosine residue with alanine (umuC_Y11A reduces sugar selectivity further and allows pol V to readily misincorporate ribonucleotides as easily as deoxynucleotides, whilst leaving its poor base-substitution fidelity essentially unchanged. However, the mutability of cells expressing the steric gate pol V mutant is very low due to efficient repair mechanisms that are triggered by the misincorporated rNMPs. Comparison of the mutation frequency between strains expressing wild-type and mutant pol V therefore allows us to identify pathways specifically directed at ribonucleotide excision repair (RER. We previously demonstrated that rNMPs incorporated by umuC_Y11A are efficiently removed from DNA in a repair pathway initiated by RNase HII. Using the same approach, we show here that mismatch repair and base excision repair play minimal back-up roles in RER in vivo. In contrast, in the absence of functional RNase HII, umuC_Y11A-dependent mutagenesis increases significantly in ΔuvrA, uvrB5 and ΔuvrC strains, suggesting that rNMPs misincorporated into DNA are actively repaired by nucleotide excision repair (NER in vivo. Participation of NER in RER was confirmed by reconstituting ribonucleotide-dependent NER in vitro. We show that UvrABC nuclease-catalyzed incisions are readily made on DNA templates containing one, two, or five rNMPs and that the reactions are stimulated by the presence of mispaired bases. Similar to NER of DNA lesions, excision of rNMPs proceeds through dual incisions made at the 8(th phosphodiester bond 5' and 4(th-5(th phosphodiester bonds 3' of the ribonucleotide. Ribonucleotides misinserted into DNA can therefore be

  17. Triple Negative Breast Cancers Have a Reduced Expression of DNA Repair Genes

    Science.gov (United States)

    Andreis, Daniele; Bertoni, Ramona; Giardini, Roberto; Fox, Stephen B.; Broggini, Massimo; Bottini, Alberto; Zanoni, Vanessa; Bazzola, Letizia; Foroni, Chiara; Generali, Daniele; Damia, Giovanna

    2013-01-01

    DNA repair is a key determinant in the cellular response to therapy and tumor repair status could play an important role in tailoring patient therapy. Our goal was to evaluate the mRNA of 13 genes involved in different DNA repair pathways (base excision, nucleotide excision, homologous recombination, and Fanconi anemia) in paraffin embedded samples of triple negative breast cancer (TNBC) compared to luminal A breast cancer (LABC). Most of the genes involved in nucleotide excision repair and Fanconi Anemia pathways, and CHK1 gene were significantly less expressed in TNBC than in LABC. PARP1 levels were higher in TNBC than in LABC. In univariate analysis high level of FANCA correlated with an increased overall survival and event free survival in TNBC; however multivariate analyses using Cox regression did not confirm FANCA as independent prognostic factor. These data support the evidence that TNBCs compared to LABCs harbour DNA repair defects. PMID:23825533

  18. 'Batman excision' of ventral skin in hypospadias repair, clue to aesthetic repair (point of technique).

    Science.gov (United States)

    Hoebeke, P B; De Kuyper, P; Van Laecke, E

    2002-11-01

    In the hypospadiac penis the ventral skin is poorly developed, while dorsal skin is redundant. The classical Byars' flaps are a way to use the excess dorsal skin to cover the penile shaft. The appearance after Byars' flaps however is not natural. We use a more natural looking skin allocation with superior aesthetic results. The clue in this reconstruction is an inverted triangle shaped excision of ventral skin expanding over the edges of the hooded prepuce (which makes it look like Batman). After excision of the ventral skin it is possible to close the penile skin in the midline, thus mimicking the natural raphe. In case of preputial reconstruction the excised ventral skin makes the prepuce look more natural. The trend of further refining aesthetic appearance of the hypospadiac penis often neglects the penile skin reconstruction. A technique is presented by which the total penile appearances after surgery ameliorates due to better skin reconstruction.

  19. Processing closely spaced lesions during Nucleotide Excision Repair triggers mutagenesis in E. coli.

    Directory of Open Access Journals (Sweden)

    Régine Janel-Bintz

    2017-07-01

    Full Text Available It is generally assumed that most point mutations are fixed when damage containing template DNA undergoes replication, either right at the fork or behind the fork during gap filling. Here we provide genetic evidence for a pathway, dependent on Nucleotide Excision Repair, that induces mutations when processing closely spaced lesions. This pathway, referred to as Nucleotide Excision Repair-induced Mutagenesis (NERiM, exhibits several characteristics distinct from mutations that occur within the course of replication: i following UV irradiation, NER-induced mutations are fixed much more rapidly (t ½ ≈ 30 min than replication dependent mutations (t ½ ≈ 80-100 min ii NERiM specifically requires DNA Pol IV in addition to Pol V iii NERiM exhibits a two-hit dose-response curve that suggests processing of closely spaced lesions. A mathematical model let us define the geometry (infer the structure of the toxic intermediate as being formed when NER incises a lesion that resides in close proximity of another lesion in the complementary strand. This critical NER intermediate requires Pol IV / Pol II for repair, it is either lethal if left unrepaired or mutation-prone when repaired. Finally, NERiM is found to operate in stationary phase cells providing an intriguing possibility for ongoing evolution in the absence of replication.

  20. Evidence for the involvement of nucleotide excision repair in the removal of abasic sites in yeast.

    Science.gov (United States)

    Torres-Ramos, C A; Johnson, R E; Prakash, L; Prakash, S

    2000-05-01

    In eukaryotes, DNA damage induced by ultraviolet light and other agents which distort the helix is removed by nucleotide excision repair (NER) in a fragment approximately 25 to 30 nucleotides long. In humans, a deficiency in NER causes xeroderma pigmentosum (XP), characterized by extreme sensitivity to sunlight and a high incidence of skin cancers. Abasic (AP) sites are formed in DNA as a result of spontaneous base loss and from the action of DNA glycosylases involved in base excision repair. In Saccharomyces cerevisiae, AP sites are removed via the action of two class II AP endonucleases, Apn1 and Apn2. Here, we provide evidence for the involvement of NER in the removal of AP sites and show that NER competes with Apn1 and Apn2 in this repair process. Inactivation of NER in the apn1Delta or apn1Delta apn2Delta strain enhances sensitivity to the monofunctional alkylating agent methyl methanesulfonate and leads to further impairment in the cellular ability to remove AP sites. A deficiency in the repair of AP sites may contribute to the internal cancers and progressive neurodegeneration that occur in XP patients.

  1. Structural and Functional Studies on Nucleotide Excision Repair From Recognition to Incision.

    Energy Technology Data Exchange (ETDEWEB)

    Caroline Kisker

    2001-01-01

    Maintenance of the correct genetic information is crucial for all living organisms because mutations are the primary cause of hereditary diseases, as well as cancer and may also be involved in aging. The importance of genomic integrity is underscored by the fact that 80 to 90% of all human cancers are ultimately due to DNA damage. Among the different repair mechanisms that have evolved to protect the genome, nucleotide excision repair (NER) is a universal pathway found in all organisms. NER removes a wide variety of bulky DNA adducts including the carcinogenic cyclobutane pyrimidine dimers induced by UV radiation, benzo(a)pyrene-guanine adducts caused by smoking and the guanine-cisplatin adducts induced by chemotherapy. The importance of this repair mechanism is reflected by three severe inherited diseases in humans, which are due to defects in NER: xeroderma pigmentosum, Cockayne's syndrome and trichothiodystrophy.

  2. Fluorodeoxyuridine modulates cellular expression of the DNA base excision repair enzyme uracil-DNA glycosylase.

    Science.gov (United States)

    Fischer, Jennifer A; Muller-Weeks, Susan; Caradonna, Salvatore J

    2006-09-01

    The thymidylate synthase inhibitor 5-fluorouracil (5-FU) continues to play a pivotal role in the treatment of cancer. A downstream event of thymidylate synthase inhibition involves the induction of a self-defeating base excision repair process. With the depletion of TTP pools, there is also an increase in dUMP. Metabolism of dUMP to the triphosphate dUTP results in elevated pools of this atypical precursor for DNA synthesis. Under these conditions, there is a destructive cycle of dUMP incorporation into DNA, removal of uracil by the base excision repair enzyme uracil-DNA glycosylase (UDG), and reincorporation of dUMP during the synthesis phase of DNA repair. The end point is DNA strand breaks and loss of DNA integrity, which contributes to cell death. Evidence presented here indicates that both the nuclear and the mitochondrial isoforms of UDG are modulated by FdUrd (and 5-FU) treatment in certain cell lines but not in others. Modulation occurs at the transcriptional and post-translational levels. Under normal conditions, nUDG protein appears in G(1) and is degraded during the S to G(2) phase transition. The present study provides evidence that, in certain cell lines, FdUrd mediates an atypical turnover of nUDG. Additional data indicate that, for cell lines that do not down-regulate nUDG, small interfering RNA-mediated knockdown of nUDG significantly increases resistance to the cytotoxic effects of FdUrd. Results from these studies show that nUDG is an additional determinant in FdUrd-mediated cytotoxicity and bolster the notion that the self-defeating base excision repair pathway, instigated by elevated dUTP (FdUTP) pools, contributes to the cytotoxic consequences of 5-FU chemotherapy.

  3. Differential role of base excision repair proteins in mediating cisplatin cytotoxicity.

    Science.gov (United States)

    Sawant, Akshada; Floyd, Ashley M; Dangeti, Mohan; Lei, Wen; Sobol, Robert W; Patrick, Steve M

    2017-03-01

    Interstrand crosslinks (ICLs) are covalent lesions formed by cisplatin. The mechanism for the processing and removal of ICLs by DNA repair proteins involves nucleotide excision repair (NER), homologous recombination (HR) and fanconi anemia (FA) pathways. In this report, we monitored the processing of a flanking uracil adjacent to a cisplatin ICL by the proteins involved in the base excision repair (BER) pathway. Using a combination of extracts, purified proteins, inhibitors, functional assays and cell culture studies, we determined the specific BER proteins required for processing a DNA substrate with a uracil adjacent to a cisplatin ICL. Uracil DNA glycosylase (UNG) is the primary glycosylase responsible for the removal of uracils adjacent to cisplatin ICLs, whereas other uracil glycosylases can process uracils in the context of undamaged DNA. Repair of the uracil adjacent to cisplatin ICLs proceeds through the classical BER pathway, highlighting the importance of specific proteins in this redundant pathway. Removal of uracil is followed by the generation of an abasic site and subsequent cleavage by AP endonuclease 1 (APE1). Inhibition of either the repair or redox domain of APE1 gives rise to cisplatin resistance. Inhibition of the lyase domain of Polymerase β (Polβ) does not influence cisplatin cytotoxicity. In addition, lack of XRCC1 leads to increased DNA damage and results in increased cisplatin cytotoxicity. Our results indicate that BER activation at cisplatin ICLs influences crosslink repair and modulates cisplatin cytotoxicity via specific UNG, APE1 and Polβ polymerase functions. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Extracts of proliferating and non-proliferating human cells display different base excision pathways and repair fidelity

    DEFF Research Database (Denmark)

    Akbari, Mansour; Pena Diaz, Javier; Andersen, Sonja

    2009-01-01

    Base excision repair (BER) of damaged or inappropriate bases in DNA has been reported to take place by single nucleotide insertion or through incorporation of several nucleotides, termed short-patch and long-patch repair, respectively. We found that extracts from proliferating and non-proliferati...

  5. New design of nucleotide excision repair (NER) inhibitors for combination cancer therapy.

    Science.gov (United States)

    Gentile, Francesco; Tuszynski, Jack A; Barakat, Khaled H

    2016-04-01

    Many cancer chemotherapy agents act by targeting the DNA of cancer cells, causing substantial damage within their genome and causing them to undergo apoptosis. An effective DNA repair pathway in cancer cells can act in a reverse way by removing these drug-induced DNA lesions, allowing cancer cells to survive, grow and proliferate. In this context, DNA repair inhibitors opened a new avenue in cancer treatment, by blocking the DNA repair mechanisms from removing the chemotherapy-mediated DNA damage. In particular, the nucleotide excision repair (NER) involves more than thirty protein-protein interactions and removes DNA adducts caused by platinum-based chemotherapy. The excision repair cross-complementation group 1 (ERCC1)-xeroderma pigmentosum, complementation group A (XPA) protein (XPA-ERCC1) complex seems to be one of the most promising targets in this pathway. ERCC1 is over expressed in cancer cells and the only known cellular function so far for XPA is to recruit ERCC1 to the damaged point. Here, we build upon our recent advances in identifying inhibitors for this interaction and continue our efforts to rationally design more effective and potent regulators for the NER pathway. We employed in silico drug design techniques to: (1) identify compounds similar to the recently discovered inhibitors, but more effective at inhibiting the XPA-ERCC1 interactions, and (2) identify different scaffolds to develop novel lead compounds. Two known inhibitor structures have been used as starting points for two ligand/structure-hybrid virtual screening approaches. The findings described here form a milestone in discovering novel inhibitors for the NER pathway aiming at improving the efficacy of current platinum-based therapy, by modulating the XPA-ERCC1 interaction. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Nucleotide excision repair and the 26S proteasome function together to promote trinucleotide repeat expansions.

    Science.gov (United States)

    Concannon, Claire; Lahue, Robert S

    2014-01-01

    Trinucleotide repeat (TNR) expansion underpins a number of inheritable neurological human disorders. Multiple mechanisms are thought to contribute to the expansion process. The incorrect processing of the repeat tract by DNA repair proteins can drive this mutation process forward, as expansions are suppressed following ablation of certain repair factors in mouse models and cell models of disease. Nucleotide excision repair (NER) is one repair pathway implicated in TNR instability, although most previous work focussed on TNR contractions, not expansions. Here we investigated the role of NER in modulating expansions of threshold-length (CTG·CAG) repeats in yeast. We show that both the global genome and transcription-coupled repair subpathways promote expansions of threshold-length TNRs. Furthermore, NER works with the 26S proteasome to drive expansions, based on analysis of double mutants defective in both pathways, and of Rad23, a protein involved in both NER and the shuttling of ubiquitinated proteins to the proteasome. This work provides the first evidence that both subpathways of NER can promote threshold-length TNR expansions and that NER interacts with the proteasome to drive expansions. Copyright © 2013 Elsevier B.V. All rights reserved.

  7. Abnormal Base Excision Repair at Trinucleotide Repeats Associated with Diseases: A Tissue-Selective Mechanism

    Directory of Open Access Journals (Sweden)

    Agathi-Vasiliki Goula

    2013-07-01

    Full Text Available More than fifteen genetic diseases, including Huntington’s disease, myotonic dystrophy 1, fragile X syndrome and Friedreich ataxia, are caused by the aberrant expansion of a trinucleotide repeat. The mutation is unstable and further expands in specific cells or tissues with time, which can accelerate disease progression. DNA damage and base excision repair (BER are involved in repeat instability and might contribute to the tissue selectivity of the process. In this review, we will discuss the mechanisms of trinucleotide repeat instability, focusing more specifically on the role of BER.

  8. A novel role for transcription-coupled nucleotide excision repair for the in vivo repair of 3,N4-ethenocytosine.

    Science.gov (United States)

    Chaim, Isaac A; Gardner, Alycia; Wu, Jie; Iyama, Teruaki; Wilson, David M; Samson, Leona D

    2017-04-07

    Etheno (ε) DNA base adducts are highly mutagenic lesions produced endogenously via reactions with lipid peroxidation (LPO) products. Cancer-promoting conditions, such as inflammation, can induce persistent oxidative stress and increased LPO, resulting in the accumulation of ε-adducts in different tissues. Using a recently described fluorescence multiplexed host cell reactivation assay, we show that a plasmid reporter bearing a site-specific 3,N4-ethenocytosine (εC) causes transcriptional blockage. Notably, this blockage is exacerbated in Cockayne Syndrome and xeroderma pigmentosum patient-derived lymphoblastoid and fibroblast cells. Parallel RNA-Seq expression analysis of the plasmid reporter identifies novel transcriptional mutagenesis properties of εC. Our studies reveal that beyond the known pathways, such as base excision repair, the process of transcription-coupled nucleotide excision repair plays a role in the removal of εC from the genome, and thus in the protection of cells and tissues from collateral damage induced by inflammatory responses. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  9. Cytosine deamination and base excision repair cause R-loop-induced CAG repeat fragility and instability in Saccharomyces cerevisiae.

    Science.gov (United States)

    Su, Xiaofeng A; Freudenreich, Catherine H

    2017-10-03

    CAG/CTG repeats are structure-forming repetitive DNA sequences, and expansion beyond a threshold of ∼35 CAG repeats is the cause of several human diseases. Expanded CAG repeats are prone to breakage, and repair of the breaks can cause repeat contractions and expansions. In this study, we found that cotranscriptional R-loops formed at a CAG-70 repeat inserted into a yeast chromosome. R-loops were further elevated upon deletion of yeast RNaseH genes and caused repeat fragility. A significant increase in CAG repeat contractions was also observed, consistent with previous human cell studies. Deletion of yeast cytosine deaminase Fcy1 significantly decreased the rate of CAG repeat fragility and contractions in the rnh1Δrnh201Δ background, indicating that Fcy1-mediated deamination is one cause of breakage and contractions in the presence of R-loops. Furthermore, base excision repair (BER) is responsible for causing CAG repeat contractions downstream of Fcy1, but not fragility. The Rad1/XPF and Rad2/XPG nucleases were also important in protecting against contractions, but through BER rather than nucleotide excision repair. Surprisingly, the MutLγ (Mlh1/Mlh3) endonuclease caused R-loop-dependent CAG fragility, defining an alternative function for this complex. These findings provide evidence that breakage at expanded CAG repeats occurs due to R-loop formation and reveal two mechanisms for CAG repeat instability: one mediated by cytosine deamination of DNA engaged in R-loops and the other by MutLγ cleavage. Since disease-causing CAG repeats occur in transcribed regions, our results suggest that R-loop-mediated fragility is a mechanism that could cause DNA damage and repeat-length changes in human cells.

  10. Oxidative and energy metabolism as potential clues for clinical heterogeneity in nucleotide excision repair disorders.

    Science.gov (United States)

    Hosseini, Mohsen; Ezzedine, Khaled; Taieb, Alain; Rezvani, Hamid R

    2015-02-01

    Nucleotide excision repair (NER) is an important DNA repair pathway involved in the removal of a wide array of DNA lesions. The absence or dysfunction of NER results in the following distinct disorders: xeroderma pigmentosum (XP), Cockayne syndrome (CS), cerebro-oculo-facio-skeletal (COFS) syndrome, UV-sensitive syndrome (UVSS), trichothiodystrophy (TTD), or combined syndromes including XP/CS, XP/TTD, CS/TTD, and COFS/TTD. In addition to their well-characterized role in the NER signaling pathway, NER factors also seem to be important in biological processes that are not directly associated with DNA damage responses, including mitochondrial function and redox homeostasis. The potential causative role of these factors in the large clinical spectrum seen in NER diseases is discussed in this review.

  11. The role of DNA base excision repair in brain homeostasis and disease

    DEFF Research Database (Denmark)

    Akbari, Mansour; Morevati, Marya; Croteau, Deborah

    2015-01-01

    of proteins required for BER or proteins that regulate BER have been consistently associated with neurological dysfunction and disease in humans. Recent studies suggest that DNA lesions in the nuclear and mitochondrial compartments and the cellular response to those lesions have a profound effect on cellular......Chemical modification and spontaneous loss of nucleotide bases from DNA are estimated to occur at the rate of thousands per human cell per day. DNA base excision repair (BER) is a critical mechanism for repairing such lesions in nuclear and mitochondrial DNA. Defective expression or function...... energy homeostasis, mitochondrial function and cellular bioenergetics, with especially strong influence on neurological function. Further studies in this area could lead to novel approaches to prevent and treat human neurodegenerative disease....

  12. Membrane association of mitochondrial DNA facilitates base excision repair in mammalian mitochondria.

    Science.gov (United States)

    Boesch, Pierre; Ibrahim, Noha; Dietrich, André; Lightowlers, Robert N

    2010-03-01

    Mitochondrial DNA encodes a set of 13 polypeptides and is subjected to constant oxidative stress due to ROS production within the organelle. It has been shown that DNA repair in the mitochondrion proceeds through both short- and long-patch base excision repair (BER). In the present article, we have used the natural competence of mammalian mitochondria to import DNA and study the sub-mitochondrial localization of the repair system in organello. Results demonstrate that sequences corresponding to the mtDNA non-coding region interact with the inner membrane in a rapid and saturable fashion. We show that uracil containing import substrates are taken into the mitochondrion and are used as templates for damage driven DNA synthesis. After further sub-fractionation, we show that the length of the repair synthesis patch differs in the soluble and the particulate fraction. Bona fide long patch BER synthesis occurs on the DNA associated with the particulate fraction, whereas a nick driven DNA synthesis occurs when the uracil containing DNA accesses the soluble fraction. Our results suggest that coordinate interactions of the different partners needed for BER is only found at sites where the DNA is associated with the membrane.

  13. Base excision repair of oxidative DNA damage: from mechanism to disease

    Science.gov (United States)

    Whitaker, Amy M.; Schaich, Matthew A.; Smith, Mallory S.; Flynn, Tony S.; Freudenthal, Bret. D.

    2017-01-01

    Reactive oxygen species continuously assault the structure of DNA resulting in oxidation and fragmentation of the nucleobases. Both oxidative DNA damage itself and its repair mediate the progression of many prevalent human maladies. The major pathway tasked with removal of oxidative DNA damage, and hence maintaining genomic integrity, is base excision repair (BER). The aphorism that structure often dictates function has proven true, as numerous recent structural biology studies have aided in clarifying the molecular mechanisms used by key BER enzymes during the repair of damaged DNA. This review focuses on the mechanistic details of the individual BER enzymes and the association of these enzymes during the development and progression of human diseases, including cancer and neurological diseases. Expanding on these structural and biochemical studies to further clarify still elusive BER mechanisms, and focusing our efforts toward gaining an improved appreciation of how these enzymes form co-complexes to facilitate DNA repair is a crucial next step toward understanding how BER contributes to human maladies and how it can be manipulated to alter patient outcomes. PMID:28199214

  14. Transcription factor TFIIH and DNA endonuclease Rad2 constitute yeast nucleotide excision repair factor 3: implications for nucleotide excision repair and Cockayne syndrome.

    Science.gov (United States)

    Habraken, Y; Sung, P; Prakash, S; Prakash, L

    1996-10-01

    Nucleotide excision repair (NER) of ultraviolet light-damaged DNA in eukaryotes requires a large number of highly conserved protein factors. Recent studies in yeast have suggested that NER involves the action of distinct protein subassemblies at the damage site rather than the placement there of a "preformed repairosome" containing all the essential NER factors. Neither of the two endonucleases, Rad1-Rad10 and Rad2, required for dual incision, shows any affinity for ultraviolet-damaged DNA. Rad1-Rad10 forms a ternary complex with the DNA damage recognition protein Rad14, providing a means for targeting this nuclease to the damage site. It has remained unclear how the Rad2 nuclease is targeted to the DNA damage site and why mutations in the human RAD2 counterpart, XPG, result in Cockayne syndrome. Here we examine whether Rad2 is part of a higher order subassembly. Interestingly, we find copurification of Rad2 protein with TFIIH, such that TFIIH purified from a strain that overexpresses Rad2 contains a stoichiometric amount of Rad2. By several independent criteria, we establish that Rad2 is tightly associated with TFIIH, exhibiting an apparent dissociation constant Cockayne syndrome.

  15. Modulation of base excision repair of 8-oxoguanine by the nucleotide sequence.

    Science.gov (United States)

    Allgayer, Julia; Kitsera, Nataliya; von der Lippen, Carina; Epe, Bernd; Khobta, Andriy

    2013-10-01

    8-Oxoguanine (8-oxoG) is a major product of oxidative DNA damage, which induces replication errors and interferes with transcription. By varying the position of single 8-oxoG in a functional gene and manipulating the nucleotide sequence surrounding the lesion, we found that the degree of transcriptional inhibition is independent of the distance from the transcription start or the localization within the transcribed or the non-transcribed DNA strand. However, it is strongly dependent on the sequence context and also proportional to cellular expression of 8-oxoguanine DNA glycosylase (OGG1)-demonstrating that transcriptional arrest does not take place at unrepaired 8-oxoG and proving a causal connection between 8-oxoG excision and the inhibition of transcription. We identified the 5'-CAGGGC[8-oxoG]GACTG-3' motif as having only minimal transcription-inhibitory potential in cells, based on which we predicted that 8-oxoG excision is particularly inefficient in this sequence context. This anticipation was fully confirmed by direct biochemical assays. Furthermore, in DNA containing a bistranded Cp[8-oxoG]/Cp[8-oxoG] clustered lesion, the excision rates differed between the two strands at least by a factor of 9, clearly demonstrating that the excision preference is defined by the DNA strand asymmetry rather than the overall geometry of the double helix or local duplex stability.

  16. MISMATCH REPAIR-DEPENDENT ITERATIVE EXCISION AT IRREPARABLE O6-METHYLGUANINE LESIONS IN HUMAN NUCLEAR EXTRACTS*

    Science.gov (United States)

    York, Sally J.; Modrich, Paul

    2008-01-01

    The response of mammalian cells to SN1 DNA methylators depends on functional MutSα and MutLα. Cells deficient in either of these activities are resistant to the cytotoxic effects of this class of chemotherapeutic drug. Because killing by SN1 methylators has been attributed to O6-methylguanine (MeG), we have constructed nicked circular heteroduplexes that contain a single MeG-T mispair and have examined processing of these molecules by mismatch repair in nuclear extracts of human cells. Excision provoked by MeG-T is restricted to the incised heteroduplex strand, leading to removal of the MeG when it resides on this strand. However, when the MeG is located on the continuous strand, the heteroduplex is irreparable. MeG-T-dependent repair DNA synthesis is observed on both reparable and irreparable, 3’ and 5’ heteroduplexes as judged by [32P]dAMP incorporation. Labeling with [α-32P]dATP followed by a cold dATP chase has demonstrated that newly synthesized DNA on irreparable molecules is subject to re-excision in a reaction that is MutLα-dependent, an effect attributable to presence of MeG on the template strand. Processing of the irreparable 3’ heteroduplex is also associated with incision of the discontinuous strand of a few percent of molecules near the thymidylate of the MeG-T base pair. These results provide the first direct evidence for mismatch repair-mediated iterative processing of DNA methylator damage, an effect that may be relevant to damage signaling events triggered by this class of chemotherapeutic agent. PMID:16772289

  17. Gene Therapy for Fracture Repair

    Science.gov (United States)

    2007-05-01

    forma - tion and finally remodeling [8]. Fracture callus formation eventually results in the bridging of the fracture and the restoration of skeletal...analysis was performed using ImaGene software (BioDiscovery, El Segundo, CA), that used an internal statistical analysis of the signal intensity of...expression during the normal repair of a simple femur fracture with the elimination of scar tissue from the healing bone. This model does not address

  18. Selective base excision repair of DNA damage by the non-base-flipping DNA glycosylase AlkC

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Rongxin; Mullins, Elwood A.; Shen, Xing; #8208; Xing; Lay, Kori T.; Yuen, Philip K.; David, Sheila S.; Rokas, Antonis; Eichman, Brandt F. (UCD); (Vanderbilt)

    2017-10-20

    DNA glycosylases preserve genome integrity and define the specificity of the base excision repair pathway for discreet, detrimental modifications, and thus, the mechanisms by which glycosylases locate DNA damage are of particular interest. Bacterial AlkC and AlkD are specific for cationic alkylated nucleobases and have a distinctive HEAT-like repeat (HLR) fold. AlkD uses a unique non-base-flipping mechanism that enables excision of bulky lesions more commonly associated with nucleotide excision repair. In contrast, AlkC has a much narrower specificity for small lesions, principally N3-methyladenine (3mA). Here, we describe how AlkC selects for and excises 3mA using a non-base-flipping strategy distinct from that of AlkD. A crystal structure resembling a catalytic intermediate complex shows how AlkC uses unique HLR and immunoglobulin-like domains to induce a sharp kink in the DNA, exposing the damaged nucleobase to active site residues that project into the DNA. This active site can accommodate and excise N3-methylcytosine (3mC) and N1-methyladenine (1mA), which are also repaired by AlkB-catalyzed oxidative demethylation, providing a potential alternative mechanism for repair of these lesions in bacteria.

  19. Up-regulation of nucleotide excision repair in mouse lung and liver following chronic exposure to aflatoxin B{sub 1} and its dependence on p53 genotype

    Energy Technology Data Exchange (ETDEWEB)

    Mulder, Jeanne E. [Pharmacology and Toxicology Graduate Program, Department of Biomedical and Molecular Sciences, Queen' s University Kingston, Ontario K7L 3N6 (Canada); Bondy, Genevieve S.; Mehta, Rekha [Toxicology Research Division, 2202D, Bureau of Chemical Safety, Food Directorate, Health Products and Food Branch, Health Canada, Ottawa, Ontario K1A 0K9 (Canada); Massey, Thomas E., E-mail: masseyt@queensu.ca [Pharmacology and Toxicology Graduate Program, Department of Biomedical and Molecular Sciences, Queen' s University Kingston, Ontario K7L 3N6 (Canada)

    2014-03-01

    Aflatoxin B{sub 1} (AFB{sub 1}) is biotransformed in vivo into an epoxide metabolite that forms DNA adducts that may induce cancer if not repaired. p53 is a tumor suppressor gene implicated in the regulation of global nucleotide excision repair (NER). Male heterozygous p53 knockout (B6.129-Trp53{sup tm1Brd}N5, Taconic) and wild-type mice were exposed to 0, 0.2 or 1.0 ppm AFB{sub 1} for 26 weeks. NER activity was assessed with an in vitro assay, using AFB{sub 1}-epoxide adducted plasmid DNA as a substrate. For wild-type mice, repair of AFB{sub 1}–N7-Gua adducts was 124% and 96% greater in lung extracts from mice exposed to 0.2 ppm and 1.0 ppm AFB{sub 1} respectively, and 224% greater in liver extracts from mice exposed to 0.2 ppm AFB{sub 1} (p < 0.05). In heterozygous p53 knockout mice, repair of AFB{sub 1}–N7-Gua was only 45% greater in lung extracts from mice exposed to 0.2 ppm AFB{sub 1} (p < 0.05), and no effect was observed in lung extracts from mice treated with 1.0 ppm AFB{sub 1} or in liver extracts from mice treated with either AFB{sub 1} concentration. p53 genotype did not affect basal levels of repair. AFB{sub 1} exposure did not alter repair of AFB{sub 1}-derived formamidopyrimidine adducts in lung or liver extracts of either mouse genotype nor did it affect XPA or XPB protein levels. In summary, chronic exposure to AFB{sub 1} increased NER activity in wild-type mice, and this response was diminished in heterozygous p53 knockout mice, indicating that loss of one allele of p53 limits the ability of NER to be up-regulated in response to DNA damage. - Highlights: • Mice are chronically exposed to low doses of the mycotoxin aflatoxin B{sub 1} (AFB{sub 1}). • The effects of AFB{sub 1} and p53 status on nucleotide excision repair are investigated. • AFB{sub 1} increases nucleotide excision repair in wild type mouse lung and liver. • This increase is attenuated in p53 heterozygous mouse lung and liver. • Results portray the role of p53 in

  20. Silymarin protects epidermal keratinocytes from ultraviolet radiation-induced apoptosis and DNA damage by nucleotide excision repair mechanism.

    Directory of Open Access Journals (Sweden)

    Santosh K Katiyar

    Full Text Available Solar ultraviolet (UV radiation is a well recognized epidemiologic risk factor for melanoma and non-melanoma skin cancers. This observation has been linked to the accumulation of UVB radiation-induced DNA lesions in cells, and that finally lead to the development of skin cancers. Earlier, we have shown that topical treatment of skin with silymarin, a plant flavanoid from milk thistle (Silybum marianum, inhibits photocarcinogenesis in mice; however it is less understood whether chemopreventive effect of silymarin is mediated through the repair of DNA lesions in skin cells and that protect the cells from apoptosis. Here, we show that treatment of normal human epidermal keratinocytes (NHEK with silymarin blocks UVB-induced apoptosis of NHEK in vitro. Silymarin reduces the amount of UVB radiation-induced DNA damage as demonstrated by reduced amounts of cyclobutane pyrimidine dimers (CPDs and as measured by comet assay, and that ultimately may lead to reduced apoptosis of NHEK. The reduction of UV radiation-induced DNA damage by silymarin appears to be related with induction of nucleotide excision repair (NER genes, because UV radiation-induced apoptosis was not blocked by silymarin in NER-deficient human fibroblasts. Cytostaining and dot-blot analysis revealed that silymarin repaired UV-induced CPDs in NER-proficient fibroblasts from a healthy individual but did not repair UV-induced CPD-positive cells in NER-deficient fibroblasts from patients suffering from xeroderma pigmentosum complementation-A disease. Similarly, immunohistochemical analysis revealed that silymarin did not reduce the number of UVB-induced sunburn/apoptotic cells in the skin of NER-deficient mice, but reduced the number of sunburn cells in their wild-type counterparts. Together, these results suggest that silymarin exert the capacity to reduce UV radiation-induced DNA damage and, thus, prevent the harmful effects of UV radiation on the genomic stability of epidermal cells.

  1. Polysulfide compounds as inhibitors of the key base excision repair enzymes

    Directory of Open Access Journals (Sweden)

    Salakhutdinov N. F.

    2012-06-01

    Full Text Available Aim. To increase the capacity of antitumor therapy based on DNA damage it is important to minimize the repair of DNA lesions that can be achieved by inhibiting the activity of key DNA repair enzymes. To this end several benzopentathiepine and benzo[1,3]dithiol derivatives were synthesized and tested as inhibitors of the key base excision repair (BER enzymes, PARP1, DNA polymerase β, and APE1. Methods. The procedure of synthesis of several new compounds was developed. The inhibitory capacity of the compounds was estimated by comparison of the enzyme activities in specific tests in the presence of compounds versus their absence. Results. Benzopentathiepine derivative bearing trifluoromethyl group at the 1st position was shown to be a weak inhibitor of PARP1. Cyclic substituents at the 1st position attached through amide bond bring about moderate enhancement of pol β inhibition. Each studied substituent at the 1st position considerably increases the inhibition of APE1-catalyzed hydrolysis of AP sites as compared to parent compound. Conclusions. Several new inhibitors of BER enzymes were revealed. The directions for further modification of compounds to improve their inhibitory activity were found out.

  2. Oxidative DNA damage background estimated by a system model of base excision repair

    Energy Technology Data Exchange (ETDEWEB)

    Sokhansanj, B A; Wilson, III, D M

    2004-05-13

    Human DNA can be damaged by natural metabolism through free radical production. It has been suggested that the equilibrium between innate damage and cellular DNA repair results in an oxidative DNA damage background that potentially contributes to disease and aging. Efforts to quantitatively characterize the human oxidative DNA damage background level based on measuring 8-oxoguanine lesions as a biomarker have led to estimates varying over 3-4 orders of magnitude, depending on the method of measurement. We applied a previously developed and validated quantitative pathway model of human DNA base excision repair, integrating experimentally determined endogenous damage rates and model parameters from multiple sources. Our estimates of at most 100 8-oxoguanine lesions per cell are consistent with the low end of data from biochemical and cell biology experiments, a result robust to model limitations and parameter variation. Our results show the power of quantitative system modeling to interpret composite experimental data and make biologically and physiologically relevant predictions for complex human DNA repair pathway mechanisms and capacity.

  3. Structure of UvrA nucleotide excision repair protein in complex with modified DNA

    Science.gov (United States)

    Jaciuk, Marcin; Nowak, Elżbieta; Skowronek, Krzysztof; Tańska, Anna; Nowotny, Marcin

    2012-01-01

    One of the primary pathways for removal of DNA damage is nucleotide excision repair (NER). In bacteria, the UvrA protein is the component of NER that locates the lesion. A notable feature of NER is its ability to act on many DNA modifications that vary in chemical structure. So far, the mechanism underlying this broad specificity has been unclear. Here, we report the first crystal structure of a UvrA protein in complex with a chemically modified oligonucleotide. The structure shows that the UvrA dimer does not contact the site of lesion directly, but rather binds the DNA regions on both sides of the modification. The DNA region harboring the modification is deformed, with the double helix bent and unwound. UvrA uses damage-induced deformations of the DNA and a less rigid structure of the modified double helix for indirect readout of the lesion. PMID:21240268

  4. Nucleotide excision repair pathway assessment in DNA exposed to low-intensity red and infrared lasers.

    Science.gov (United States)

    Fonseca, A S; Campos, V M A; Magalhães, L A G; Paoli, F

    2015-10-01

    Low-intensity lasers are used for prevention and management of oral mucositis induced by anticancer therapy, but the effectiveness of treatment depends on the genetic characteristics of affected cells. This study evaluated the survival and induction of filamentation of Escherichia coli cells deficient in the nucleotide excision repair pathway, and the action of T4endonuclease V on plasmid DNA exposed to low-intensity red and near-infrared laser light. Cultures of wild-type (strain AB1157) E. coli and strain AB1886 (deficient in uvrA protein) were exposed to red (660 nm) and infrared (808 nm) lasers at various fluences, powers and emission modes to study bacterial survival and filamentation. Also, plasmid DNA was exposed to laser light to study DNA lesions produced in vitro by T4endonuclease V. Low-intensity lasers:i) had no effect on survival of wild-type E. coli but decreased the survival of uvrA protein-deficient cells,ii) induced bacterial filamentation, iii) did not alter the electrophoretic profile of plasmids in agarose gels, andiv) did not alter the electrophoretic profile of plasmids incubated with T4 endonuclease V. These results increase our understanding of the effects of laser light on cells with various genetic characteristics, such as xeroderma pigmentosum cells deficient in nucleotide excision pathway activity in patients with mucositis treated by low-intensity lasers.

  5. Nucleotide excision repair pathway assessment in DNA exposed to low-intensity red and infrared lasers

    Energy Technology Data Exchange (ETDEWEB)

    Fonseca, A.S.; Campos, V.M.A.; Magalhaes, L.A.G., E-mail: adnfonseca@ig.com.br [Instituto de Biologia Roberto Alcantara Gomes, Rio de Janeiro, RJ (Brazil). Departamento de Biofisica e Biometria. Lab. de Ciencias Radiologicas; Paoli, F. [Universidade Federal de Juiz de Fora (UFJF), Juiz de Fora, MG (Brazil). Instituto de Ciencias Biologicas. Departamento de Morfologia

    2015-10-15

    Low-intensity lasers are used for prevention and management of oral mucositis induced by anticancer therapy, but the effectiveness of treatment depends on the genetic characteristics of affected cells. This study evaluated the survival and induction of filamentation of Escherichia coli cells deficient in the nucleotide excision repair pathway, and the action of T{sub 4} endonuclease V on plasmid DNA exposed to low-intensity red and near-infrared laser light. Cultures of wild-type (strain AB1157) E. coli and strain AB1886 (deficient in uvrA protein) were exposed to red (660 nm) and infrared (808 nm) lasers at various fluences, powers and emission modes to study bacterial survival and filamentation. Also, plasmid DNA was exposed to laser light to study DNA lesions produced in vitro by T{sub 4} endonuclease V. Low-intensity lasers: i) had no effect on survival of wild-type E. coli but decreased the survival of uvrA protein-deficient cells, ii) induced bacterial filamentation, iii) did not alter the electrophoretic profile of plasmids in agarose gels, and iv) did not alter the electrophoretic profile of plasmids incubated with T{sub 4} endonuclease V. These results increase our understanding of the effects of laser light on cells with various genetic characteristics, such as xeroderma pigmentosum cells deficient in nucleotide excision pathway activity in patients with mucositis treated by low-intensity lasers. (author)

  6. The Base Excision Repair system of Salmonella enterica serovar typhimurium counteracts DNA damage by host nitric oxide.

    Directory of Open Access Journals (Sweden)

    Anthony R Richardson

    2009-05-01

    Full Text Available Intracellular pathogens must withstand nitric oxide (NO. generated by host phagocytes. Salmonella enterica serovar Typhimurium interferes with intracellular trafficking of inducible nitric oxide synthase (iNOS and possesses multiple systems to detoxify NO.. Consequently, the level of NO. stress encountered by S. Typhimurium during infection in vivo has been unknown. The Base Excision Repair (BER system recognizes and repairs damaged DNA bases including cytosine and guanine residues modified by reactive nitrogen species. Apurinic/apyrimidinic (AP sites generated by BER glycosylases require subsequent processing by AP endonucleases. S. Typhimurium xth nfo mutants lacking AP endonuclease activity exhibit increased NO. sensitivity resulting from chromosomal fragmentation at unprocessed AP sites. BER mutant strains were thus used to probe the nature and extent of nitrosative damage sustained by intracellular bacteria during infection. Here we show that an xth nfo S. Typhimurium mutant is attenuated for virulence in C3H/HeN mice, and virulence can be completely restored by the iNOS inhibitor L-NIL. Inactivation of the ung or fpg glycosylase genes partially restores virulence to xth nfo mutant S. Typhimurium, demonstrating that NO. fluxes in vivo are sufficient to modify cytosine and guanine bases, respectively. Mutants lacking ung or fpg exhibit NO.-dependent hypermutability during infection, underscoring the importance of BER in protecting Salmonella from the genotoxic effects of host NO.. These observations demonstrate that host-derived NO. damages Salmonella DNA in vivo, and the BER system is required to maintain bacterial genomic integrity.

  7. Molecular Cloning and 3D Structure Modeling of APEX1, DNA Base Excision Repair Enzyme from the Camel, Camelus dromedarius

    OpenAIRE

    Dalia Fouad; Hesham Mahmoud Saeed; Farid Shokry Ataya; Ajamaluddin Malik

    2012-01-01

    The domesticated one-humped camel, Camelus dromedarius, is one of the most important animals in the Arabian Desert. It is exposed most of its life to both intrinsic and extrinsic genotoxic factors that are known to cause gross DNA alterations in many organisms. Ionic radiation and sunlight are known producers of Reactive Oxygen Species (ROS), one of the causes for DNA lesions. The damaged DNA is repaired by many enzymes, among of them Base Excision Repair enzymes, produci...

  8. Exposure of Human Lung Cells to Tobacco Smoke Condensate Inhibits the Nucleotide Excision Repair Pathway.

    Directory of Open Access Journals (Sweden)

    Nathaniel Holcomb

    Full Text Available Exposure to tobacco smoke is the number one risk factor for lung cancer. Although the DNA damaging properties of tobacco smoke have been well documented, relatively few studies have examined its effect on DNA repair pathways. This is especially true for the nucleotide excision repair (NER pathway which recognizes and removes many structurally diverse DNA lesions, including those introduced by chemical carcinogens present in tobacco smoke. The aim of the present study was to investigate the effect of tobacco smoke on NER in human lung cells. We studied the effect of cigarette smoke condensate (CSC, a surrogate for tobacco smoke, on the NER pathway in two different human lung cell lines; IMR-90 lung fibroblasts and BEAS-2B bronchial epithelial cells. To measure NER, we employed a slot-blot assay to quantify the introduction and removal of UV light-induced 6-4 photoproducts and cyclobutane pyrimidine dimers. We find a dose-dependent inhibition of 6-4 photoproduct repair in both cell lines treated with CSC. Additionally, the impact of CSC on the abundance of various NER proteins and their respective RNAs was investigated. The abundance of XPC protein, which is required for functional NER, is significantly reduced by treatment with CSC while the abundance of XPA protein, also required for NER, is unaffected. Both XPC and XPA RNA levels are modestly reduced by CSC treatment. Finally, treatment of cells with MG-132 abrogates the reduction in the abundance of XPC protein produced by treatment with CSC, suggesting that CSC enhances proteasome-dependent turnover of the protein that is mediated by ubiquitination. Together, these findings indicate that tobacco smoke can inhibit the same DNA repair pathway that is also essential for the removal of some of the carcinogenic DNA damage introduced by smoke itself, increasing the DNA damage burden of cells exposed to tobacco smoke.

  9. Recruitment of the nucleotide excision repair endonuclease XPG to sites of UV-induced DNA damage depends on functional TFIIH

    NARCIS (Netherlands)

    A. Zotter (Angelika); A.B. Houtsmuller (Adriaan); M.S. Luijsterburg (Martijn); D.O. Warmerdam (Daniël); S.M. Ibrahim (Shehu); A.L. Nigg (Alex); W.A. van Cappellen (Gert); J.H.J. Hoeijmakers (Jan); R. van Driel; W. Vermeulen (Wim)

    2006-01-01

    textabstractThe structure-specific endonuclease XPG is an indispensable core protein of the nucleotide excision repair (NER) machinery. XPG cleaves the DNA strand at the 3′ side of the DNA damage. XPG binding stabilizes the NER preincision complex and is essential for the 5′ incision by the

  10. Coupling of Human DNA Excision Repair and the DNA Damage Checkpoint in a Defined in Vitro System*

    Science.gov (United States)

    Lindsey-Boltz, Laura A.; Kemp, Michael G.; Reardon, Joyce T.; DeRocco, Vanessa; Iyer, Ravi R.; Modrich, Paul; Sancar, Aziz

    2014-01-01

    DNA repair and DNA damage checkpoints work in concert to help maintain genomic integrity. In vivo data suggest that these two global responses to DNA damage are coupled. It has been proposed that the canonical 30 nucleotide single-stranded DNA gap generated by nucleotide excision repair is the signal that activates the ATR-mediated DNA damage checkpoint response and that the signal is enhanced by gap enlargement by EXO1 (exonuclease 1) 5′ to 3′ exonuclease activity. Here we have used purified core nucleotide excision repair factors (RPA, XPA, XPC, TFIIH, XPG, and XPF-ERCC1), core DNA damage checkpoint proteins (ATR-ATRIP, TopBP1, RPA), and DNA damaged by a UV-mimetic agent to analyze the basic steps of DNA damage checkpoint response in a biochemically defined system. We find that checkpoint signaling as measured by phosphorylation of target proteins by the ATR kinase requires enlargement of the excision gap generated by the excision repair system by the 5′ to 3′ exonuclease activity of EXO1. We conclude that, in addition to damaged DNA, RPA, XPA, XPC, TFIIH, XPG, XPF-ERCC1, ATR-ATRIP, TopBP1, and EXO1 constitute the minimum essential set of factors for ATR-mediated DNA damage checkpoint response. PMID:24403078

  11. Nucleotide excision repair at the single-molecule level : analysis of the E. coli UvrA protein

    NARCIS (Netherlands)

    Wagner, Koen

    2011-01-01

    In this thesis, the characteristics of the Escherichia coli UvrA protein were analyzed with microscopy techniques that allow detection of protein complexes at the single-molecule level. Together with UvrB and UvrC, UvrA catalyzes the excision of damaged DNA from the bacterial genome. This DNA repair

  12. Base excision repair dysfunction in a subgroup of patients with myelodysplastic syndrome.

    Science.gov (United States)

    Jankowska, A M; Gondek, L P; Szpurka, H; Nearman, Z P; Tiu, R V; Maciejewski, J P

    2008-03-01

    In myelodysplastic syndromes (MDS) increased chromosomal breaks point toward defects in DNA repair machinery including base excision repair (BER) pathway involved in handling of oxidative DNA damage. We investigated whether defects in this pathway can be found in MDS. Elevated levels of 8-oxoguanine (8-OG) were found in a significant proportion of MDS patients, indicating increased oxidative DNA damage or defective handling of oxidative load. In a distinct subgroup of patients, increased 8-OG content was associated with increased hOGG1 mRNA expression and activity. In some patients, increased numbers of abasic sites (AP sites) correlated with low levels of POLbeta. To further investigate the nature of this defect, we examined genetic lesions potentially explaining accumulation of 8-OG and AP sites. We genotyped a large cohort of MDS patients and found a correlation between increased oxidative damage and the presence of the hOGG1-Cys326 allele suggesting inadequate compensatory feedback. Overall, this hOGG1 variant was more frequent in MDS, particularly in advanced forms, as compared to controls. In summary, we demonstrated that BER dysfunction in some MDS patients may be responsible for the increased 8-OG incorporation and explains one aspect of the propensity to chromosomal breaks in MDS but other mechanisms may also be involved.

  13. Effects of post mortem interval and gender in DNA base excision repair activities in rat brains

    Energy Technology Data Exchange (ETDEWEB)

    Soltys, Daniela Tathiana; Pereira, Carolina Parga Martins; Ishibe, Gabriela Naomi; Souza-Pinto, Nadja Cristhina de, E-mail: nadja@iq.usp.br

    2015-06-15

    Most human tissues used in research are of post mortem origin. This is the case for all brain samples, and due to the difficulty in obtaining a good number of samples, especially in the case of neurodegenerative diseases, male and female samples are often included in the same experimental group. However, the effects of post mortem interval (PMI) and gender differences in the endpoints being analyzed are not always fully understood, as is the case for DNA repair activities. To investigate these effects, in a controlled genetic background, base excision repair (BER) activities were measured in protein extracts obtained from Wistar rat brains from different genders and defined PMI up to 24 hours, using a novel fluorescent-based in vitro incision assay. Uracil and AP-site incision activity in nuclear and mitochondrial extracts were similar in all groups included in this study. Our results show that gender and PMI up to 24 hours have no influence in the activities of the BER proteins UDG and APE1 in rat brains. These findings demonstrate that these variables do not interfere on the BER activities included in these study, and provide a security window to work with UDG and APE1 proteins in samples of post mortem origin.

  14. PARP-1 enhances the mismatch-dependence of 5′-directed excision in human mismatch repair in vitro

    Science.gov (United States)

    Liu, Yiyong; Kadyrov, Farid A.; Modrich, Paul

    2011-01-01

    End-directed mismatch-provoked excision has been reconstituted in several purified systems. While 3′-directed excision displays a mismatch dependence similar to that observed in nuclear extracts (≈ 20-fold), the mismatch dependence of 5′-directed excision is only 3 to 4-fold, significantly less than that in extracts (8 to 10-fold). Utilizing a fractionation-based approach, we have isolated a single polypeptide that enhances mismatch dependence of reconstituted 5′-directed excision and have shown it to be identical to poly[ADP-ribose] polymerase 1 (PARP-1). Titration of reconstituted excision reactions or PARP-1-depleted HeLa nuclear extract with purified PARP-1 showed that the protein specifically enhances mismatch dependence of 5′-directed excision. Analysis of a set of PARP-1 mutants revealed that the DNA binding domain and BRCT fold contribute to the regulation of excision specificity. Involvement of the catalytic domain is restricted to its ability to poly(ADP-ribosyl)ate PARP-1 in the presence of NAD+, likely through interference with DNA binding. Analysis of protein-protein interactions demonstrated that PARP-1 interacts with mismatch repair proteins MutSα, exonuclease 1, replication protein A (RPA), and as previously shown by others, replication factor C (RFC) and proliferating cell nuclear antigen (PCNA) as well. The BRCT fold plays an important role in the interaction of PARP-1 with the former three proteins. PMID:21945626

  15. Decreased nucleotide excision repair in steatotic livers associates with myeloperoxidase-immunoreactivity

    Energy Technology Data Exchange (ETDEWEB)

    Schults, Marten A.; Nagle, Peter W. [Department of Toxicology, NUTRIM-School for Nutrition, Toxicology and Metabolism, Maastricht University Medical Centre, PO Box 616, 6200 MD Maastricht (Netherlands); Rensen, Sander S. [Department of Surgery, NUTRIM-School for Nutrition, Toxicology and Metabolism, Maastricht University Medical Centre, PO Box 616, 6200 MD Maastricht (Netherlands); Godschalk, Roger W. [Department of Toxicology, NUTRIM-School for Nutrition, Toxicology and Metabolism, Maastricht University Medical Centre, PO Box 616, 6200 MD Maastricht (Netherlands); Munnia, Armelle; Peluso, Marco [Cancer Risk Factor Branch, ISPO Cancer Prevention and Research Institute, Via Cosimo il Vecchio 2, 50139 Florence (Italy); Claessen, Sandra M. [Department of Toxicogenomics, GROW-School for Oncology and Developmental Biology, Maastricht University Medical Centre, PO Box 616, 6200 MD Maastricht (Netherlands); Greve, Jan W. [Department of Surgery, NUTRIM-School for Nutrition, Toxicology and Metabolism, Maastricht University Medical Centre, PO Box 616, 6200 MD Maastricht (Netherlands); Driessen, Ann [Department of Pathology, NUTRIM-School for Nutrition, Toxicology and Metabolism, Maastricht University Medical Centre, PO Box 616, 6200 MD Maastricht (Netherlands); Verdam, Froukje J.; Buurman, Wim A. [Department of Surgery, NUTRIM-School for Nutrition, Toxicology and Metabolism, Maastricht University Medical Centre, PO Box 616, 6200 MD Maastricht (Netherlands); Schooten, Frederik J. van [Department of Toxicology, NUTRIM-School for Nutrition, Toxicology and Metabolism, Maastricht University Medical Centre, PO Box 616, 6200 MD Maastricht (Netherlands); Chiu, Roland K., E-mail: r.k.chiu@med.umcg.nl [Department of Toxicology, NUTRIM-School for Nutrition, Toxicology and Metabolism, Maastricht University Medical Centre, PO Box 616, 6200 MD Maastricht (Netherlands)

    2012-08-01

    Chronic inflammation is characterized by the influx of neutrophils and is associated with an increased production of reactive oxygen species that can damage DNA. Oxidative DNA damage is generally thought to be involved in the increased risk of cancer in inflamed tissues. We previously demonstrated that activated neutrophil mediated oxidative stress results in a reduction in nucleotide excision repair (NER) capacity, which could further enhance mutagenesis. Inflammation and oxidative stress are critical factors in the progression of nonalcoholic fatty liver disease that is linked with enhanced liver cancer risk. In this report, we therefore evaluated the role of neutrophils and the associated oxidative stress in damage recognition and DNA repair in steatotic livers of 35 severely obese subjects with either nonalcoholic steatohepatitis (NASH) (n = 17) or steatosis alone (n = 18). The neutrophilic influx in liver was assessed by myeloperoxidase (MPO) staining and the amount of oxidative DNA damage by measuring M{sub 1}dG adducts. No differences in M{sub 1}dG adduct levels were observed between patients with or without NASH and also not between individuals with high or low MPO immunoreactivity. However, we found that high expression of MPO in the liver, irrespective of disease status, reduced the damage recognition capacity as determined by staining for histone 2AX phosphorylation ({gamma}H2AX). This reduction in {gamma}H2AX formation in individuals with high MPO immunoreactivity was paralleled by a significant decrease in NER capacity as assessed by a functional repair assay, and was not related to cell proliferation. Thus, the observed reduction in NER capacity upon hepatic inflammation is associated with and may be a consequence of reduced damage recognition. These findings suggest a novel mechanism of liver cancer development in patients with nonalcoholic fatty liver disease.

  16. The role of base excision repair in the development of primary open angle glaucoma in the Polish population

    Energy Technology Data Exchange (ETDEWEB)

    Cuchra, Magda; Markiewicz, Lukasz; Mucha, Bartosz [Department of Clinical Chemistry and Biochemistry, Medical University of Lodz (Poland); Pytel, Dariusz [The Abramson Family Cancer Research Institute, Department of Cancer Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104 (United States); Department of Biochemistry and Molecular Biology, Hollings Cancer Center, Medical University of South Carolina, Charleston, SC 29425 (United States); Szymanek, Katarzyna [Department of Ophthalmology, Medical University of Warsaw, SPKSO Hospital, Warsaw (Poland); Szemraj, Janusz [Department of Medical Biochemistry, Medical University of Lodz, Lodz (Poland); Szaflik, Jerzy; Szaflik, Jacek P. [Department of Ophthalmology, Medical University of Warsaw, SPKSO Hospital, Warsaw (Poland); Majsterek, Ireneusz, E-mail: ireneusz.majsterek@umed.lodz.pl [Department of Clinical Chemistry and Biochemistry, Medical University of Lodz (Poland)

    2015-08-15

    Highlights: • We suggested the association of XRCC1 gene with the increase risk of POAG development. • We indicated the association of clinical factor and XRCC1, MUTYH, ADPRT and APE1 genes with POAG progression. • We postulated the increase level of oxidative DNA damage in group of patients with POAG in relation to healthy controls. • We suggested the slightly decrease ability to repair of oxidative DNA damage. • This is the first data that showed the role of BER mechanism in POAG pathogenesis. - Abstract: Glaucoma is a leading cause of irreversible blindness in developing countries. Previous data have shown that progressive loss of human TM cells may be connected with chronic exposure to oxidative stress. This hypothesis may suggest a role of the base excision repair (BER) pathway of oxidative DNA damage in primary open angle glaucoma (POAG) patients. The aim of our study was to evaluate an association of BER gene polymorphism with a risk of POAG. Moreover, an association of clinical parameters was examined including cup disk ratio (c/d), rim area (RA) and retinal nerve fiber layer (RNFL) with glaucoma progression according to BER gene polymorphisms. Our research included 412 patients with POAG and 454 healthy controls. Gene polymorphisms were analyzed by PCR-RFLP. Heidelberg Retinal Tomography (HRT) clinical parameters were also analyzed. The 399Arg/Gln genotype of the XRCC1 gene (OR 1.38; 95% CI 1.02–1.89 p = 0.03) was associated with an increased risk of POAG occurrence. It was indicated that the 399Gln/Gln XRCC1 genotype might increase the risk of POAG progression according to the c/d ratio (OR 1.67; 95% CI 1.07–2.61 P = 0.02) clinical parameter. Moreover, the association of VF factor with 148Asp/Glu of APE1 genotype distribution and POAG progression (OR 2.25; 95% CI 1.30–3.89) was also found. Additionally, the analysis of the 324Gln/His MUTYH polymorphism gene distribution in the patient group according to RNFL factor showed that it might

  17. DNA excision repair in cell extracts from human cell lines exhibiting hypersensitivity to DNA-damaging agents

    Energy Technology Data Exchange (ETDEWEB)

    Hansson, J.; Keyse, S.M.; Lindahl, T.; Wood, R.D. (Imperial Cancer Research Fund, South Mimms, (United Kingdom))

    1991-07-01

    Whole cell extracts from human lymphoid cell lines can perform in vitro DNA repair synthesis in plasmids damaged by agents including UV or cis-diamminedichloroplatinum(II) (cis-DDP). Extracts from xeroderma pigmentosum (XP) cells are defective in repair synthesis. We have now studied in vitro DNA repair synthesis using extracts from lymphoblastoid cell lines representing four human hereditary syndromes with increased sensitivity to DNA-damaging agents. Extracts of cell lines from individuals with the sunlight-sensitive disorders dysplastic nevus syndrome or Cockayne's syndrome (complementation groups A and B) showed normal DNA repair synthesis in plasmids with UV photoproducts. This is consistent with in vivo measurements of the overall DNA repair capacity in such cell lines. A number of extracts were prepared from two cell lines representing the variant form of XP (XP-V). Half of the extracts prepared showed normal levels of in vitro DNA repair synthesis in plasmids containing UV lesions, but the remainder of the extracts from the same cell lines showed deficient repair synthesis, suggesting the possibility of an unusually labile excision repair protein in XP-V. Fanconi's anemia (FA) cells show cellular hypersensitivity to cross-linking agents including cis-DDP. Extracts from cell lines belonging to two different complementation groups of FA showed normal DNA repair synthesis in plasmids containing cis-DDP or UV adducts. Thus, there does not appear to be an overall excision repair defect in FA, but the data do not exclude a defect in the repair of interstrand DNA cross-links.

  18. Differential contributory roles of nucleotide excision and homologous recombination repair for enhancing cisplatin sensitivity in human ovarian cancer cells

    Directory of Open Access Journals (Sweden)

    Wani Gulzar

    2011-03-01

    Full Text Available Abstract Background While platinum-based chemotherapeutic agents are widely used to treat various solid tumors, the acquired platinum resistance is a major impediment in their successful treatment. Since enhanced DNA repair capacity is a major factor in conferring cisplatin resistance, targeting of DNA repair pathways is an effective stratagem for overcoming cisplatin resistance. This study was designed to delineate the role of nucleotide excision repair (NER, the principal mechanism for the removal of cisplatin-induced DNA intrastrand crosslinks, in cisplatin resistance and reveal the impact of DNA repair interference on cisplatin sensitivity in human ovarian cancer cells. Results We assessed the inherent NER efficiency of multiple matched pairs of cisplatin-sensitive and -resistant ovarian cancer cell lines and their expression of NER-related factors at mRNA and protein levels. Our results showed that only the cisplatin-resistant ovarian cancer cell line PEO4 possessed an increased NER capacity compared to its inherently NER-inefficient parental line PEO1. Several other cisplatin-resistant cell lines, including CP70, CDDP and 2008C13, exhibited a normal and parental cell-comparable NER capacity for removing cisplatin-induced DNA intrastrand cross-links (Pt-GG. Concomitant gene expression analysis revealed discordance in mRNA and protein levels of NER factors in various ovarian cancer cell lines and NER proteins level were unrelated to the cisplatin sensitivity of these cell lines. Although knockdown of NER factors was able to compromise the NER efficiency, it only caused a minimal effect on cisplatin sensitivity. On the contrary, downregulation of BRCA2, a critical protein for homologous recombination repair (HRR, significantly enhanced the efficacy of cisplatin in killing ovarian cancer cell line PEO4. Conclusion Our studies indicate that the level of NER factors in ovarian cancer cell lines is neither a determinant of their NER capacity nor

  19. Influence of Morinda citrifolia (Noni) on Expression of DNA Repair Genes in Cervical Cancer Cells.

    Science.gov (United States)

    Gupta, Rakesh Kumar; Bajpai, Deepti; Singh, Neeta

    2015-01-01

    Previous studies have suggested that Morinda citrifolia (Noni) has potential to reduce cancer risk. The purpose of this study was to investigate the effect of Noni, cisplatin, and their combination on DNA repair genes in the SiHa cervical cancer cell line. SiHa cells were cultured and treated with 10% Noni, 10 μg/dl cisplatin or their combination for 24 hours. Post culturing, the cells were pelleted, RNA extracted, and processed for investigating DNA repair genes by real time PCR. The expression of nucleotide excision repair genes ERCC1, ERCC2, and ERCC4 and base excision repair gene XRCC1 was increased 4 fold, 8.9 fold, 4 fold, and 5.5 fold, respectively, on treatment with Noni as compared to untreated controls (p<0.05). In contrast, expression was found to be decreased 22 fold, 13 fold, 16 fold, and 23 fold on treatment with cisplatin (p<0.05). However, the combination of Noni and cisplatin led to an increase of 2 fold, 1.6 fold, 3 fold, 1.2 fold, respectively (p<0.05). Noni enhanced the expression of DNA repair genes by itself and in combination with cisplatin. However, high expression of DNA repair genes at mRNA level only signifies efficient DNA transcription of the above mentioned genes; further investigations are needed to evaluate the DNA repair protein expression.

  20. Metal inhibition of human N-methylpurine-DNA glycosylase activity in base excision repair.

    Science.gov (United States)

    Wang, Ping; Guliaev, Anton B; Hang, Bo

    2006-10-25

    Cadmium (Cd2+), nickel (Ni2+) and cobalt (Co2+) are human and/or animal carcinogens. Zinc (Zn2+) is not categorized as a carcinogen, and rather an essential element to humans. Metals were recently shown to inhibit DNA repair proteins that use metals for their function and/or structure. Here we report that the divalent ions Cd2+, Ni2+, and Zn2+ can inhibit the activity of a recombinant human N-methylpurine-DNA glycosylase (MPG) toward a deoxyoligonucleotide with ethenoadenine (varepsilonA). MPG removes a variety of toxic/mutagenic alkylated bases and does not require metal for its catalytic activity or structural integrity. At concentrations starting from 50 to 1,000 microM, both Cd2+ and Zn2+ showed metal-dependent inhibition of the MPG catalytic activity. Ni2+ also inhibited MPG, but to a lesser extent. Such an effect can be reversed with EDTA addition. In contrast, Co2+ and Mg2+ did not inhibit the MPG activity in the same dose range. Experiments using HeLa cell-free extracts demonstrated similar patterns of inactivation of the varepsilonA excision activity by the same metals. Binding of MPG to the substrate was not significantly affected by Cd2+, Zn2+, and Ni2+ at concentrations that show strong inhibition of the catalytic function, suggesting that the reduced catalytic activity is not due to altered MPG binding affinity to the substrate. Molecular dynamics (MD) simulations with Zn2+ showed that the MPG active site has a potential binding site for Zn2+, formed by several catalytically important and conserved residues. Metal binding to such a site is expected to interfere with the catalytic mechanism of this protein. These data suggest that inhibition of MPG activity may contribute to metal genotoxicity and depressed repair of alkylation damage by metals in vivo.

  1. A human repair gene ERCC5 is involved in group G xeroderma pigmentosum

    Energy Technology Data Exchange (ETDEWEB)

    Shiomi, Tadahiro [National Inst. of Radiological Sciences, Chiba (Japan)

    1994-03-01

    In E. coli, ultraviolet-induced DNA damage is removed by the coordinated action of UVR A, B, C, and D proteins (1). In Saccharomyces cerevisiae, more than ten genes have been reported to be involved in excision repair (2). The nucleotide excision repair pathway has been extensively studied in these organisms. To facilitate studying nucleotide excision repair in mammalian cells. Ultraviolet-sensitive rodent cell mutants have been isolated and classified into 11 complementation groups (9,10). The human nucleotide excision repair genes which complement the defects of the mutants have been designated as the ERCC (excision repair cross-complementing) genes; a number is added to refer to the particular rodent complementation group that is corrected by the gene. Recently, several human DNA repair genes have been cloned using rodent cell lines sensitive to ultraviolet. These include ERCC2 (3), ERCC3 (4), and ERCC6 (5), which correspond to the defective genes in the ultraviolet-sensitive human disorders xeroderma pigmentosum (XP) group D (6) and group B (4), and Cockayne`s syndrome (CS) group B (7), respectively. The human excision repair gene ERCC5 was cloned after DNA-mediated gene transfer of human HeLa cell genomic DNA into the ultraviolet-sensitive mouse mutant XL216, a member of rodent complementation group 5 (11,12) and the gene was mapped on human chromosome 13q32.3-q33.1 by the replication R-banding fluorescence in situ hybridization method (13). The ERCC5 cDNA encodes a predicted 133 kDa nuclear protein that shares some homology with product of the yeast DNA repair gene RAD 2. Transfection with mouse ERCC5 cDNA restored normal levels of ultraviolet-resistance to XL216 cells. Microinjection of ERCC5 cDNA specifically restored the defect of XP group G cells (XP-G) as measured by unscheduled DNA synthesis (UDS), and XP-G cells stably transformed with ERCC5 cDNA showed nearly normal ultraviolet resistance. (J.P.N.).

  2. Chromosomal localization of three repair genes: the xeroderma pigmentosum group C gene and two human homologs of yeast RAD23.

    NARCIS (Netherlands)

    P.J. van der Spek (Peter); E.M.E. Smit (Elisabeth); H.B. Beverloo (Berna); K. Sugasawa (Kaoru); C. Matsutani; F. Hanaoka (Fumio); J.H.J. Hoeijmakers (Jan); A. Hagemeier

    1994-01-01

    textabstractThe nucleotide excision repair (NER) disorder xeroderma pigmentosum (XP) is characterized by sun (UV) sensitivity, predisposition to skin cancer, and extensive genetic heterogeneity. Recently, we reported the cloning and analysis of three human NER genes, XPC, HHR23A, and HHR23B. The

  3. APE1, the DNA base excision repair protein, regulates the removal of platinum adducts in sensory neuronal cultures by NER

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Hyun-Suk [Department of Biochemistry and Molecular Biology, Indianapolis, IN 46202 (United States); Guo, Chunlu; Thompson, Eric L. [Department of Pharmacology and Toxicology, Indianapolis, IN 46202 (United States); Jiang, Yanlin [Department of Pediatrics and Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202 (United States); Kelley, Mark R. [Department of Biochemistry and Molecular Biology, Indianapolis, IN 46202 (United States); Department of Pharmacology and Toxicology, Indianapolis, IN 46202 (United States); Department of Pediatrics and Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202 (United States); Vasko, Michael R. [Department of Pharmacology and Toxicology, Indianapolis, IN 46202 (United States); Lee, Suk-Hee, E-mail: slee@iu.edu [Department of Biochemistry and Molecular Biology, Indianapolis, IN 46202 (United States)

    2015-09-15

    Peripheral neuropathy is one of the major side effects of treatment with the anticancer drug, cisplatin. One proposed mechanism for this neurotoxicity is the formation of platinum adducts in sensory neurons that could contribute to DNA damage. Although this damage is largely repaired by nuclear excision repair (NER), our previous findings suggest that augmenting the base excision repair pathway (BER) by overexpressing the repair protein APE1 protects sensory neurons from cisplatin-induced neurotoxicity. The question remains whether APE1 contributes to the ability of the NER pathway to repair platinum-damage in neuronal cells. To examine this, we manipulated APE1 expression in sensory neuronal cultures and measured Pt-removal after exposure to cisplatin. When neuronal cultures were treated with increasing concentrations of cisplatin for two or three hours, there was a concentration-dependent increase in Pt-damage that peaked at four hours and returned to near baseline levels after 24 h. In cultures where APE1 expression was reduced by ∼80% using siRNA directed at APE1, there was a significant inhibition of Pt-removal over eight hours which was reversed by overexpressing APE1 using a lentiviral construct for human wtAPE1. Overexpressing a mutant APE1 (C65 APE1), which only has DNA repair activity, but not its other significant redox-signaling function, mimicked the effects of wtAPE1. Overexpressing DNA repair activity mutant APE1 (226 + 177APE1), with only redox activity was ineffective suggesting it is the DNA repair function of APE1 and not its redox-signaling, that restores the Pt-damage removal. Together, these data provide the first evidence that a critical BER enzyme, APE1, helps regulate the NER pathway in the repair of cisplatin damage in sensory neurons.

  4. Genome Instability in Development and Aging: Insights from Nucleotide Excision Repair in Humans, Mice, and Worms

    Directory of Open Access Journals (Sweden)

    Diletta Edifizi

    2015-08-01

    Full Text Available DNA damage causally contributes to aging and cancer. Congenital defects in nucleotide excision repair (NER lead to distinct cancer-prone and premature aging syndromes. The genetics of NER mutations have provided important insights into the distinct consequences of genome instability. Recent work in mice and C. elegans has shed new light on the mechanisms through which developing and aging animals respond to persistent DNA damage. The various NER mouse mutants have served as important disease models for Xeroderma pigmentosum (XP, Cockayne syndrome (CS, and trichothiodystrophy (TTD, while the traceable genetics of C. elegans have allowed the mechanistic delineation of the distinct outcomes of genome instability in metazoan development and aging. Intriguingly, highly conserved longevity assurance mechanisms respond to transcription-blocking DNA lesions in mammals as well as in worms and counteract the detrimental consequences of persistent DNA damage. The insulin-like growth factor signaling (IIS effector transcription factor DAF-16 could indeed overcome DNA damage-driven developmental growth delay and functional deterioration even when DNA damage persists. Longevity assurance mechanisms might thus delay DNA damage-driven aging by raising the threshold when accumulating DNA damage becomes detrimental for physiological tissue functioning.

  5. Genome Instability in Development and Aging: Insights from Nucleotide Excision Repair in Humans, Mice, and Worms.

    Science.gov (United States)

    Edifizi, Diletta; Schumacher, Björn

    2015-08-13

    DNA damage causally contributes to aging and cancer. Congenital defects in nucleotide excision repair (NER) lead to distinct cancer-prone and premature aging syndromes. The genetics of NER mutations have provided important insights into the distinct consequences of genome instability. Recent work in mice and C. elegans has shed new light on the mechanisms through which developing and aging animals respond to persistent DNA damage. The various NER mouse mutants have served as important disease models for Xeroderma pigmentosum (XP), Cockayne syndrome (CS), and trichothiodystrophy (TTD), while the traceable genetics of C. elegans have allowed the mechanistic delineation of the distinct outcomes of genome instability in metazoan development and aging. Intriguingly, highly conserved longevity assurance mechanisms respond to transcription-blocking DNA lesions in mammals as well as in worms and counteract the detrimental consequences of persistent DNA damage. The insulin-like growth factor signaling (IIS) effector transcription factor DAF-16 could indeed overcome DNA damage-driven developmental growth delay and functional deterioration even when DNA damage persists. Longevity assurance mechanisms might thus delay DNA damage-driven aging by raising the threshold when accumulating DNA damage becomes detrimental for physiological tissue functioning.

  6. Actual state of knowledge in the field of diseases related with defective nucleotide excision repair.

    Science.gov (United States)

    Bukowska, Barbara; Karwowski, Bolesław T

    2018-02-15

    Xeroderma pigmentosum (XP), trichothiodystrophy (TTD) and Cockayne syndrome (CS) are rare genetic diseases characterized by a large range of clinical symptoms. However, they are all associated with defects in nucleotide excision repair (NER), the system responsible for removing bulky DNA lesions such as those generated by UV light: cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidone photoproducts (6-4 PPs). Over the past years, detailed structural and biochemical information on NER-associated proteins has emerged. In the first part of the article we briefly present the main steps of the NER pathway with an emphasis on the precise role of certain proteins. Further, we focus on clinical manifestations of the disorders and describe the diagnostic procedures. Then we consider how current therapy and advanced technology could improve patients' quality of life. Although to date the discussed diseases remain incurable, effective sun protection, a well thought out diet, and holistic medical care provide longer life and better health. This review summarizes the current state of knowledge regarding the epidemiology of NER-associated diseases, their genetic background, clinical features, and treatment options. Copyright © 2018 Elsevier Inc. All rights reserved.

  7. The nucleotide excision repair (NER) system of Helicobacter pylori: role in mutation prevention and chromosomal import patterns after natural transformation.

    Science.gov (United States)

    Moccia, Claudia; Krebes, Juliane; Kulick, Stefan; Didelot, Xavier; Kraft, Christian; Bahlawane, Christelle; Suerbaum, Sebastian

    2012-05-06

    Extensive genetic diversity and rapid allelic diversification are characteristics of the human gastric pathogen Helicobacter pylori, and are believed to contribute to its ability to cause chronic infections. Both a high mutation rate and frequent imports of short fragments of exogenous DNA during mixed infections play important roles in generating this allelic diversity. In this study, we used a genetic approach to investigate the roles of nucleotide excision repair (NER) pathway components in H. pylori mutation and recombination. Inactivation of any of the four uvr genes strongly increased the susceptibility of H. pylori to DNA damage by ultraviolet light. Inactivation of uvrA and uvrB significantly decreased mutation frequencies whereas only the uvrA deficient mutant exhibited a significant decrease of the recombination frequency after natural transformation. A uvrC mutant did not show significant changes in mutation or recombination rates; however, inactivation of uvrC promoted the incorporation of significantly longer fragments of donor DNA (2.2-fold increase) into the recipient chromosome. A deletion of uvrD induced a hyper-recombinational phenotype. Our data suggest that the NER system has multiple functions in the genetic diversification of H. pylori, by contributing to its high mutation rate, and by controlling the incorporation of imported DNA fragments after natural transformation.

  8. Influence of some prostaglandins on DNA synthesis and DNA excision repair in mouse spleen cells in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Egg, D.; Altmann, H.; Guenther R.; Klein W.; Kocsis, F.

    1978-03-01

    In vitro experiments were performed on mouse spleen cells to establish possible influences of some naturally occurring prostaglandins on DNA synthesis and DNA excision repair. The prostaglandins A1, B1, E1, E2, and F2 alpha were tested in concentrations of lopg, 5 ng and 2.5 microgram per ml cell suspension. DNA synthesis was significantly increased by PgF2 alpha in all the three concentrations tested, while the other tested prostaglandins were essentially ineffective. DNA excision repair was significantly inhibited by PgE1 and PgE2 at 5 ng/ml and at 2.5 microgram/ml but increased by PgF2 alpha in the two lower concentrations. The rejoining of DNA-strand breaks after gamma-irradiation was slightly reduced by PgE1, PgE2 and PgF2 alpha at 2.5 microgram/ml.

  9. Role of Base Excision Repair (BER) in Transcription-associated Mutagenesis of Nutritionally Stressed Nongrowing Bacillus subtilis Cell Subpopulations.

    Science.gov (United States)

    Ambriz-Aviña, Verónica; Yasbin, Ronald E; Robleto, Eduardo A; Pedraza-Reyes, Mario

    2016-11-01

    Compelling evidence points to transcriptional processes as important factors contributing to stationary-phase associated mutagenesis. However, it has not been documented whether or not base excision repair mechanisms play a role in modulating mutagenesis under conditions of transcriptional derepression. Here, we report on a flow cytometry-based methodology that employs a fluorescent reporter system to measure at single-cell level, the occurrence of transcription-associated mutations in nutritionally stressed B. subtilis cultures. Using this approach, we demonstrate that (i) high levels of transcription correlates with augmented mutation frequency, and (ii) mutation frequency is enhanced in nongrowing population cells deficient for deaminated (Ung, YwqL) and oxidized guanine (GO) excision repair, strongly suggesting that accumulation of spontaneous DNA lesions enhance transcription-associated mutagenesis.

  10. Red meat and poultry intake, polymorphisms in the nucleotide excision repair and mismatch repair pathways and colorectal cancer risk

    Science.gov (United States)

    Joshi, Amit D.; Corral, Román; Siegmund, Kimberly D.; Haile, Robert W.; Le Marchand, Loïc; Martínez, Maria Elena; Ahnen, Dennis J.; Sandler, Robert S.; Lance, Peter; Stern, Mariana C.

    2009-01-01

    Diets high in red meat have been consistently associated with colorectal cancer (CRC) risk and may result in exposure to carcinogens that cause DNA damage [i.e polycyclic aromatic hydrocarbons, heterocyclic amines (HCAs) and N-nitroso compounds]. Using a family-based study, we investigated whether polymorphisms in the nucleotide excision repair (NER) (ERCC1 3′ untranslated region (UTR) G/T, XPD Asp312Asn and Lys751Gln, XPC intron 11 C/A, XPA 5′ UTR C/T, XPF Arg415Gln and XPG Asp1104His) and mismatch repair (MLH1 Ile219Val and MSH2 Gly322Asp) pathways modified the association with red meat and poultry intake. We tested for gene–environment interactions using case-only analyses (n = 577) and compared the results using case-unaffected sibling comparisons (n = 307 sibships). Increased risk of CRC was observed for intake of more than or equal to three servings per week of red meat [odds ratio (OR) = 1.8, 95% confidence interval (CI) = 1.3–2.5)] or high-temperature cooked red meat (OR = 1.6, 95% CI = 1.1–2.2). Intake of red meat heavily brown on the outside or inside increased CRC risk only among subjects who carried the XPD codon 751 Lys/Lys genotype (case-only interaction P = 0.006 and P = 0.001, respectively, for doneness outside or inside) or the XPD codon 312 Asp/Asp genotype (case-only interaction P = 0.090 and P < 0.001, respectively). These interactions were stronger for rectal cancer cases (heterogeneity test P = 0.002 for XPD Asp312Asn and P = 0.03 for XPD Lys751Gln) and remained statistically significant after accounting for multiple testing. Case-unaffected sibling analyses were generally supportive of the case-only results. These findings highlight the possible contribution of diets high in red meat to the formation of lesions that elicit the NER pathway, such as carcinogen-induced bulky adducts. PMID:19029193

  11. REPAIR OF LARGE SKULL BASE DEFECT FOLLOWING EXCISION OF BASALOID SQUAMOUS CELL CARCINOMA OF MAXILLO - ETHMOID REGION : A CASE REPORT

    Directory of Open Access Journals (Sweden)

    Monoj Mukherjee

    2015-02-01

    Full Text Available AIM: To present a case of basaloid squamous cell carcinoma of maxillo - ethmoid region with intracranial extradural extention and its surgical management including repair of the skull base defect. MATERIAL : A 30 year female presented with progressive bilateral nasal obstruction, facial deformity for 5 years duration. She developed blindness in last 6 months. Recent CT s can showed large heterogeneous enhancing soft tissue mass in right maxillary sinus, nasal cavity and right ethmoid sinus invading the skull base . INTERVENTION : She underwent excision of the mass by modified weber ferguson incision and repair of skull base defect with temporalis muscle flap. Skin defect over the face and nose was repaired by median forehead flap. RESULT : There was total tumor clearance and no CSF leakage following surgery. CONCLUSION : Sinonasal malignancy with intracranial extradural extenti on is not a contraindication for successful surgical management. Resultant skull base defect can be repaired by a temporalis muscle flap to prevent CSF leak and intracranial infection

  12. A ubiquitylation site in Cockayne syndrome B required for repair of oxidative DNA damage, but not for transcription-coupled nucleotide excision repair.

    Science.gov (United States)

    Ranes, Michael; Boeing, Stefan; Wang, Yuming; Wienholz, Franziska; Menoni, Hervé; Walker, Jane; Encheva, Vesela; Chakravarty, Probir; Mari, Pierre-Olivier; Stewart, Aengus; Giglia-Mari, Giuseppina; Snijders, Ambrosius P; Vermeulen, Wim; Svejstrup, Jesper Q

    2016-06-20

    Cockayne syndrome B (CSB), best known for its role in transcription-coupled nucleotide excision repair (TC-NER), contains a ubiquitin-binding domain (UBD), but the functional connection between protein ubiquitylation and this UBD remains unclear. Here, we show that CSB is regulated via site-specific ubiquitylation. Mass spectrometry analysis of CSB identified lysine (K) 991 as a ubiquitylation site. Intriguingly, mutation of this residue (K991R) does not affect CSB's catalytic activity or protein stability, but greatly affects genome stability, even in the absence of induced DNA damage. Moreover, cells expressing CSB K991R are sensitive to oxidative DNA damage, but proficient for TC-NER. K991 becomes ubiquitylated upon oxidative DNA damage, and while CSB K991R is recruited normally to such damage, it fails to dissociate in a timely manner, suggesting a requirement for K991 ubiquitylation in CSB activation. Interestingly, deletion of CSB's UBD gives rise to oxidative damage sensitivity as well, while CSB ΔUBD and CSB K991R affects expression of overlapping groups of genes, further indicating a functional connection. Together, these results shed new light on the regulation of CSB, with K991R representing an important separation-of-function-mutation in this multi-functional protein. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  13. Neil3-dependent base excision repair regulates lipid metabolism and prevents atherosclerosis in Apoe-deficient mice

    DEFF Research Database (Denmark)

    Skarpengland, Tonje; Holm, Sverre; Scheffler, Katja

    2016-01-01

    an atherogenic lipid profile, increased hepatic triglyceride levels and attenuated macrophage cholesterol efflux capacity. Apoe-/- Neil3-/- mice showed marked alterations in several pathways affecting hepatic lipid metabolism, but no genotypic alterations in genome integrity or genome-wide accumulation...... of oxidative DNA damage. These results suggest a novel role for the DNA glycosylase Neil3 in atherogenesis in balancing lipid metabolism and macrophage function, potentially independently of genome-wide canonical base excision repair of oxidative DNA damage....

  14. Metal inhibition of human alkylpurine-DNA-N-glycosylase activityin base excision repair

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Ping; Guliaev, Anton B.; Hang, Bo

    2006-02-28

    Cadmium (Cd{sup 2+}), nickel (Ni{sup 2+}) and cobalt (Co{sup 2+}) are human and/or animal carcinogens. Zinc (Zn{sup 2+}) is not categorized as a carcinogen, and rather an essential element to humans. Metals were recently shown to inhibit DNA repair proteins that use metals for their function and/or structure. Here we report that the divalent ions Cd{sup 2+}, Ni{sup 2+}, and Zn{sup 2+} can inhibit the activity of a recombinant human N-methylpurine-DNA glycosylase (MPG) toward a deoxyoligonucleotide with ethenoadenine (var epsilonA). MPG removes a variety of toxic/mutagenic alkylated bases and does not require metal for its catalytic activity or structural integrity. At concentrations starting from 50 to 1000 {micro}M, both Cd{sup 2+} and Zn{sup 2+} showed metal-dependent inhibition of the MPG catalytic activity. Ni{sup 2+} also inhibited MPG, but to a lesser extent. Such an effect can be reversed with EDTA addition. In contrast, Co{sup 2+} and Mg{sup 2+} did not inhibit the MPG activity in the same dose range. Experiments using HeLa cell-free extracts demonstrated similar patterns of inactivation of the var epsilonA excision activity by the same metals. Binding of MPG to the substrate was not significantly affected by Cd{sup 2+}, Zn{sup 2+}, and Ni{sup 2+} at concentrations that show strong inhibition of the catalytic function, suggesting that the reduced catalytic activity is not due to altered MPG binding affinity to the substrate. Molecular dynamics (MD) simulations with Zn{sup 2+} showed that the MPG active site has a potential binding site for Zn{sup 2+}, formed by several catalytically important and conserved residues. Metal binding to such a site is expected to interfere with the catalytic mechanism of this protein. These data suggest that inhibition of MPG activity may contribute to metal genotoxicity and depressed repair of alkylation damage by metals in vivo.

  15. DNA Repair Dysfunction and Neurodegeneration: Lessons From Rare Pediatric Disorders.

    Science.gov (United States)

    Shabbir, Syed H

    2016-03-01

    Nucleotide excision repair disorders display a wide range of clinical syndromes and presentations, all associated at the molecular level by dysfunction of genes participating in the nucleotide excision repair pathway. Genotype-phenotype relationships are remarkably complex and not well understood. This article outlines neurodegenerative symptoms seen in nucleotide excision repair disorders and explores the role that nucleotide excision repair dysfunction can play in the pathogenesis of chronic neurodegenerative diseases. © The Author(s) 2015.

  16. NDR1 modulates the UV-induced DNA-damage checkpoint and nucleotide excision repair

    Energy Technology Data Exchange (ETDEWEB)

    Park, Jeong-Min; Choi, Ji Ye [Department of Biological Science, Dong-A University, Busan (Korea, Republic of); Yi, Joo Mi [Research Center, Dongnam Institute of Radiological & Medical Sciences, Busan (Korea, Republic of); Chung, Jin Woong; Leem, Sun-Hee; Koh, Sang Seok [Department of Biological Science, Dong-A University, Busan (Korea, Republic of); Kang, Tae-Hong, E-mail: thkang@dau.ac.kr [Department of Biological Science, Dong-A University, Busan (Korea, Republic of)

    2015-06-05

    Nucleotide excision repair (NER) is the sole mechanism of UV-induced DNA lesion repair in mammals. A single round of NER requires multiple components including seven core NER factors, xeroderma pigmentosum A–G (XPA–XPG), and many auxiliary effector proteins including ATR serine/threonine kinase. The XPA protein helps to verify DNA damage and thus plays a rate-limiting role in NER. Hence, the regulation of XPA is important for the entire NER kinetic. We found that NDR1, a novel XPA-interacting protein, modulates NER by modulating the UV-induced DNA-damage checkpoint. In quiescent cells, NDR1 localized mainly in the cytoplasm. After UV irradiation, NDR1 accumulated in the nucleus. The siRNA knockdown of NDR1 delayed the repair of UV-induced cyclobutane pyrimidine dimers in both normal cells and cancer cells. It did not, however, alter the expression levels or the chromatin association levels of the core NER factors following UV irradiation. Instead, the NDR1-depleted cells displayed reduced activity of ATR for some set of its substrates including CHK1 and p53, suggesting that NDR1 modulates NER indirectly via the ATR pathway. - Highlights: • NDR1 is a novel XPA-interacting protein. • NDR1 accumulates in the nucleus in response to UV irradiation. • NDR1 modulates NER (nucleotide excision repair) by modulating the UV-induced DNA-damage checkpoint response.

  17. Haploinsufficiency for BRCA1 is associated with normal levels of DNA nucleotide excision repair in breast tissue and blood lymphocytes

    Directory of Open Access Journals (Sweden)

    Johnson Jennifer M

    2005-06-01

    Full Text Available Abstract Background Screening mammography has had a positive impact on breast cancer mortality but cannot detect all breast tumors. In a small study, we confirmed that low power magnetic resonance imaging (MRI could identify mammographically undetectable tumors by applying it to a high risk population. Tumors detected by this new technology could have unique etiologies and/or presentations, and may represent an increasing proportion of clinical practice as new screening methods are validated and applied. A very important aspect of this etiology is genomic instability, which is associated with the loss of activity of the breast cancer-predisposing genes BRCA1 and BRCA2. In sporadic breast cancer, however, there is evidence for the involvement of a different pathway of DNA repair, nucleotide excision repair (NER, which remediates lesions that cause a distortion of the DNA helix, including DNA cross-links. Case presentation We describe a breast cancer patient with a mammographically undetectable stage I tumor identified in our MRI screening study. She was originally considered to be at high risk due to the familial occurrence of breast and other types of cancer, and after diagnosis was confirmed as a carrier of a Q1200X mutation in the BRCA1 gene. In vitro analysis of her normal breast tissue showed no differences in growth rate or differentiation potential from disease-free controls. Analysis of cultured blood lymphocyte and breast epithelial cell samples with the unscheduled DNA synthesis (UDS assay revealed no deficiency in NER. Conclusion As new breast cancer screening methods become available and cost effective, patients such as this one will constitute an increasing proportion of the incident population, so it is important to determine whether they differ from current patients in any clinically important ways. Despite her status as a BRCA1 mutation carrier, and her mammographically dense breast tissue, we did not find increased cell

  18. Stabilization of Ultraviolet (UV)-stimulated Scaffold Protein A by Interaction with Ubiquitin-specific Peptidase 7 Is Essential for Transcription-coupled Nucleotide Excision Repair*

    Science.gov (United States)

    Higa, Mitsuru; Zhang, Xue; Tanaka, Kiyoji; Saijo, Masafumi

    2016-01-01

    UV-sensitive syndrome is an autosomal recessive disorder characterized by hypersensitivity to UV light and deficiency in transcription-coupled nucleotide excision repair (TC-NER), a subpathway of nucleotide excision repair that rapidly removes transcription-blocking DNA damage. UV-sensitive syndrome consists of three genetic complementation groups caused by mutations in the CSA, CSB, and UVSSA genes. UV-stimulated scaffold protein A (UVSSA), the product of UVSSA, which is required for stabilization of Cockayne syndrome group B (CSB) protein and reappearance of the hypophosphorylated form of RNA polymerase II after UV irradiation, forms a complex with ubiquitin-specific peptidase 7 (USP7). In this study, we demonstrated that the deubiquitination activity of USP7 is suppressed by its interaction with UVSSA. The interaction required the tumor necrosis factor receptor-associated factor domain of USP7 and the central region of UVSSA and was disrupted by an amino acid substitution in the tumor necrosis factor receptor-associated factor-binding motif of UVSSA. Cells expressing mutant UVSSA were highly sensitive to UV irradiation and defective in recovery of RNA synthesis after UV irradiation. These results indicate that the interaction between UVSSA and USP7 is important for TC-NER. Furthermore, the mutant UVSSA was rapidly degraded by the proteasome, and CSB was also degraded after UV irradiation as observed in UVSSA-deficient cells. Thus, stabilization of UVSSA by interaction with USP7 is essential for TC-NER. PMID:27129218

  19. High-Resolution Mapping of Modified DNA Nucleobases Using Excision Repair Enzymes.

    Science.gov (United States)

    Ransom, Monica; Bryan, D Suzi; Hesselberth, Jay R

    2018-01-01

    Modification of DNA nucleobases has a profound effect on genome function. We developed a method that maps the positions of the modified DNA nucleobases throughout genomic DNA. This method couples in vitro nucleobase excision with massively parallel DNA sequencing to determine the location of modified DNA nucleobases with single base precision. This protocol was used to map uracil incorporation and UV photodimers in DNA, and a modification of the protocol has been used to map sparse modification events in cells. The Excision-seq protocol is broadly applicable to a variety of base modifications for which an excision enzyme is available.

  20. Recognition and repair of the cyclobutane thymine dimer, a major cause of skin cancers, by the human excision nuclease.

    Science.gov (United States)

    Reardon, Joyce T; Sancar, Aziz

    2003-10-15

    The cyclobutane thymine dimer is the major DNA lesion induced in human skin by sunlight and is a primary cause of skin cancer, the most prevalent form of cancer in the Northern Hemisphere. In humans, the only known cellular repair mechanism for eliminating the dimer from DNA is nucleotide excision repair. Yet the mechanism by which the dimer is recognized and removed by this repair system is not known. Here we demonstrate that the six-factor human excision nuclease recognizes and removes the dimer at a rate consistent with the in vivo rate of removal of this lesion, even though none of the six factors alone is capable of efficiently discriminating the dimer from undamaged DNA. We propose a recognition mechanism by which the low-specificity recognition factors, RPA, XPA, and XPC, act in a cooperative manner to locate the lesion and, aided by the kinetic proofreading provided by TFIIH, form a high-specificity complex at the damage site that initiates removal of thymine dimers at a physiologically relevant rate and specificity.

  1. Site-specific analysis of UV-induced cyclobutane pyrimidine dimers in nucleotide excision repair-proficient and -deficient hamster cells: Lack of correlation with mutational spectra

    Energy Technology Data Exchange (ETDEWEB)

    Vreeswijk, Maaike P.G., E-mail: vreeswijk@lumc.nl [Department of Toxicogenetics, Leiden University Medical Center, Einthovenweg 20, P.O. Box 9600, Postzone S4-P, 2300 RC Leiden (Netherlands); Department of Human Genetics, Center for Human and Clinical Genetics, Leiden University Medical Center, Building 2, Postzone S-04, P.O. Box 9600, 2300 RC Leiden (Netherlands); Meijers, Caro M.; Giphart-Gassler, Micheline; Vrieling, Harry; Zeeland, Albert A. van; Mullenders, Leon H.F.; Loenen, Wil A.M. [Department of Toxicogenetics, Leiden University Medical Center, Einthovenweg 20, P.O. Box 9600, Postzone S4-P, 2300 RC Leiden (Netherlands)

    2009-04-26

    Irradiation of cells with UVC light induces two types of mutagenic DNA photoproducts, i.e. cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidone photoproducts (6-4PP). To investigate the relationship between the frequency of UV-induced photolesions at specific sites and their ability to induce mutations, we quantified CPD formation at the nucleotide level along exons 3 and 8 of the hprt gene using ligation-mediated PCR, and determined the mutational spectrum of 132 UV-induced hprt mutants in the AA8 hamster cell line and of 165 mutants in its nucleotide excision repair-defective derivative UV5. In AA8 cells, transversions predominated with a strong strand bias towards thymine-containing photolesions in the non-transcribed strand. As hamster AA8 cells are proficient in global genome repair of 6-4PP but selectively repair CPD from the transcribed strand of active genes, most mutations probably resulted from erroneous bypass of CPD in the non-transcribed strand. However, the relative incidence of CPD and the positions where mutations most frequently arose do not correlate. In fact some major damage sites hardly gave rise to the formation of mutations. In the repair-defective UV5 cells, mutations were almost exclusively C > T transitions caused by photoproducts at PyC sites in the transcribed strand. Even though CPD were formed at high frequencies at some TT sites in UV5, these photoproducts did not contribute to mutation induction at all. We conclude that, even in the absence of repair, large variations in the level of induction of CPD at different sites throughout the two exons do not correspond to frequencies of mutation induction.

  2. Yeast DNA-repair gene RAD14 encodes a zinc metalloprotein with affinity for ultraviolet-damaged DNA.

    OpenAIRE

    Guzder, S N; Sung, P; Prakash, L; Prakash, S

    1993-01-01

    Xeroderma pigmentosum (XP) patients suffer from a high incidence of skin cancers due to a defect in excision repair of UV light-damaged DNA. Of the seven XP complementation groups, A-G, group A represents a severe and frequent form of the disease. The Saccharomyces cerevisiae RAD14 gene is a homolog of the XP-A correcting (XPAC) gene. Like XP-A cells, rad14-null mutants are defective in the incision step of excision repair of UV-damaged DNA. We have purified RAD14 protein to homogeneity from ...

  3. International congress on DNA damage and repair: Book of abstracts

    Energy Technology Data Exchange (ETDEWEB)

    1987-01-01

    This document contains the abstracts of 105 papers presented at the Congress. Topics covered include the Escherichia coli nucleotide excision repair system, DNA repair in malignant transformations, defective DNA repair, and gene regulation. (TEM)

  4. Role of nucleotide excision repair and photoreactivation in the solar UVB radiation survival of Pseudomonas syringae pv. syringae B728a.

    Science.gov (United States)

    Gunasekera, T S; Sundin, G W

    2006-05-01

    To assess the role of DNA repair and photoreactivation in the solar radiation survival of the plant pathogen and leaf surface epiphyte Pseudomonas syringae pv. syringae (Pss). Mutants of Pss B728a, with insertional mutations within the nucleotide excision repair gene uvrA, photolyase gene phr, or uvrA phr double mutants, were constructed to examine the importance of individual repair mechanisms in solar UV radiation (UVR) survival. The survival of either the uvrA mutant or the phr mutant was reduced by approx. 10(2)-fold following exposure to a dose of 4.5 kJ m(-2) solar UVB (290-320 nm wavelengths) while the uvrA phr double mutant was reduced >10(6)-fold by the same dose. We constructed a transcriptional fusion between the Pss recA promoter and gfp to examine the induction of the SOS response in wild-type and mutant strains. Initiation of the recA mediated SOS response was more rapid and peaked at higher levels in mutant strains suggesting both increased DNA damage in mutant strains and also that photoreactivation and nucleotide excision repair remove DNA damage as it is incurred which is reflected in a delay of recA expression. Visualization of expression of B728a cells containing the recA::gfp reporter on UVB-irradiated bean leaves highlighted the movement of cells to intercellular spaces over time and that SOS induction was detectable when leaves were irradiated 48 h following leaf inoculation. This study indicated that solar UVB is detrimental to Pss B728a, DNA repair mechanisms play an important role in strain survival and expression of the SOS regulon on leaf surfaces contributes to survival of UVR-exposed cells during plant colonization. This work links previous laboratory-based UVR analyses with solar UVB dose-response analyses and highlights the role of photoreactivation in delaying induction of the SOS response following solar irradiation. Knowledge of population dynamics following direct solar irradiation will enhance our understanding of the biology of

  5. Association of DNA repair gene XRCC1 and lung cancer susceptibility among nonsmoking Chinese women

    DEFF Research Database (Denmark)

    Yin, J.; Vogel, Ulla Birgitte; Ma, Y.

    2009-01-01

    Nonsmokers who develop lung cancer provide a good model for investigating the effect of genetic polymorphisms. XRCC1 is one of the major DNA repair proteins involved in the base-excision repair pathway. XRCC1 gene variations may lead to lower DNA repair capacity and thus confer inherited predispo......Nonsmokers who develop lung cancer provide a good model for investigating the effect of genetic polymorphisms. XRCC1 is one of the major DNA repair proteins involved in the base-excision repair pathway. XRCC1 gene variations may lead to lower DNA repair capacity and thus confer inherited...... predisposition to cancer risk. To address this question in more detail, we conducted a hospital-based case-control study consisting of 55 lung cancer cases and 74 cancer-free controls matched on age and ethnicity among nonsmoking Chinese women. We analyzed five coding single-nucleotide polymorphisms in the XRCC1...... gene: Agr194Trp, Arg280His, Arg399Gln, Pro206Pro, and Gln632Gln. Polymerase chain reaction-restriction fragment length polymorphism was used for genotyping. Carriers of the variant T-allele of Arg194Trp had a lower lung cancer risk than carriers of CC genotypes [odds ratio (OR)=0.46, 95% confidence...

  6. Mitochondrial base excision repair in mouse synaptosomes during normal aging and in a model of Alzheimer's disease

    DEFF Research Database (Denmark)

    Diaz, Ricardo Gredilla; Weissman, Lior; Yang, Jenq-Lin

    2010-01-01

    suggest that the age-related reduction in BER capacity in the synaptosomal fraction might contribute to mitochondrial and synaptic dysfunction during aging. The development of AD-like pathology in the 3xTgAD mouse model was, however, not associated with deficiencies of the BER mechanisms......Brain aging is associated with synaptic decline and synaptic function is highly dependent on mitochondria. Increased levels of oxidative DNA base damage and accumulation of mitochondrial DNA (mtDNA) mutations or deletions lead to mitochondrial dysfunction, playing an important role in the aging...... process and the pathogenesis of several neurodegenerative diseases. Here we have investigated the repair of oxidative base damage, in synaptosomes of mouse brain during normal aging and in an AD model. During normal aging, a reduction in the base excision repair (BER) capacity was observed...

  7. Cre/lox-mediated marker gene excision in transgenic maize (Zea mays L.) plants.

    Science.gov (United States)

    Zhang, W; Subbarao, S; Addae, P; Shen, A; Armstrong, C; Peschke, V; Gilbertson, L

    2003-11-01

    After the initial transformation and tissue culture process is complete, selectable marker genes, which are used in virtually all transformation approaches, are not required for the expression of the gene of interest in the transgenic plants. There are several advantages to removing the selectable marker gene after it is no longer needed, such as enabling the reuse of selectable markers and simplifying transgene arrays. We have tested the Cre/ lox system from bacteriophage P1 for its ability to precisely excise stably integrated marker genes from chromosomes in transgenic maize plants. Two strategies, crossing and autoexcision, have been tested and demonstrated. In the crossing strategy, plants expressing the Cre recombinase are crossed with plants bearing a transgene construct in which the selectable marker gene is flanked by directly repeated lox sites. Unlike previous reports in which incomplete somatic and germline excision were common, in our experiments complete somatic and germline marker gene excision occurred in the F(1) plants from most crosses with multiple independent Cre and lox lines. In the autoexcision strategy, the cre gene, under the control of a heat shock-inducible promoter, is excised along with the nptII marker gene. Our results show that a transient heat shock treatment of primary transgenic callus is sufficient for inducing cre and excising the cre and nptII genes. Genetic segregation and molecular analysis confirmed that marker gene removal is precise, complete and stable. The autoexcision strategy provides a way of removing the selectable marker gene from callus or other tissues such as embryos and kernels.

  8. Oxidative stress alters base excision repair pathway and increases apoptotic response in apurinic/apyrimidinic endonuclease 1/redox factor-1 haploinsufficient mice.

    Science.gov (United States)

    Unnikrishnan, Archana; Raffoul, Julian J; Patel, Hiral V; Prychitko, Thomas M; Anyangwe, Njwen; Meira, Lisiane B; Friedberg, Errol C; Cabelof, Diane C; Heydari, Ahmad R

    2009-06-01

    Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is the redox regulator of multiple stress-inducible transcription factors, such as NF-kappaB, and the major 5'-endonuclease in base excision repair (BER). We utilized mice containing a heterozygous gene-targeted deletion of APE1/Ref-1 (Apex(+/-)) to determine the impact of APE1/Ref-1 haploinsufficiency on the processing of oxidative DNA damage induced by 2-nitropropane (2-NP) in the liver tissue of mice. APE1/Ref-1 haploinsufficiency results in a significant decline in NF-kappaB DNA-binding activity in response to oxidative stress in liver. In addition, loss of APE1/Ref-1 increases the apoptotic response to oxidative stress, in which significant increases in GADD45g expression, p53 protein stability, and caspase activity are observed. Oxidative stress displays a differential impact on monofunctional (UNG) and bifunctional (OGG1) DNA glycosylase-initiated BER in the liver of Apex(+/-) mice. APE1/Ref-1 haploinsufficiency results in a significant decline in the repair of oxidized bases (e.g., 8-OHdG), whereas removal of uracil is increased in liver nuclear extracts of mice using an in vitro BER assay. Apex(+/-) mice exposed to 2-NP displayed a significant decline in 3'-OH-containing single-strand breaks and an increase in aldehydic lesions in their liver DNA, suggesting an accumulation of repair intermediates of failed bifunctional DNA glycosylase-initiated BER.

  9. Erythrosine B and quinoline yellow dyes regulate DNA repair gene expression in human HepG2 cells.

    Science.gov (United States)

    Chequer, Farah Md; Venancio, Vinicius P; Almeida, Mara R; Aissa, Alexandre F; Bianchi, Maria Lourdes P; Antunes, Lusânia Mg

    2017-10-01

    Erythrosine B (ErB) is a cherry pink food colorant and is widely used in foods, drugs, and cosmetics. Quinoline yellow (QY) is a chinophthalon derivative used in cosmetic compositions for application to the skin, lips, and/or body surface. Previously, ErB and QY synthetic dyes were found to induce DNA damage in HepG2 cells. The aim of this study was to investigate the molecular basis underlying the genotoxicity attributed to ErB and QY using the RT2 Profiler polymerase chain reaction array and by analyzing the expression profile of 84 genes involved in cell cycle arrest, apoptosis, and DNA repair in HepG2 cells. ErB (70 mg/L) significantly decreased the expression of two genes ( FEN1 and REV1) related to DNA base repair. One gene ( LIG1) was downregulated and 20 genes related to ATR/ATM signaling ( ATR, RBBP8, RAD1, CHEK1, CHEK2, TOPB1), nucleotide excision repair ( ERCC1, XPA), base excision repair ( FEN1, MBD4), mismatch repair ( MLH1, MSH3, TP73), double strand break repair ( BLM), other DNA repair genes ( BRIP1, FANCA, GADD45A, REV1), and apoptosis ( BAX, PPP1R15A) were significantly increased after treatment with QY (20 mg/L). In conclusion, our data suggest that the genotoxic mechanism of ErB and QY dyes involves the modulation of genes related to the DNA repair system and cell cycle.

  10. The C-terminal Region and SUMOylation of Cockayne Syndrome Group B Protein Play Critical Roles in Transcription-coupled Nucleotide Excision Repair.

    Science.gov (United States)

    Sin, Yooksil; Tanaka, Kiyoji; Saijo, Masafumi

    2016-01-15

    Cockayne syndrome (CS) is a recessive disorder that results in deficiencies in transcription-coupled nucleotide excision repair (TC-NER), a subpathway of nucleotide excision repair, and cells from CS patients exhibit hypersensitivity to UV light. CS group B protein (CSB), which is the gene product of one of the genes responsible for CS, belongs to the SWI2/SNF2 DNA-dependent ATPase family and has an ATPase domain and an ubiquitin-binding domain (UBD) in the central region and the C-terminal region, respectively. The C-terminal region containing the UBD is essential for the functions of CSB. In this study, we generated several CSB deletion mutants and analyzed the functions of the C-terminal region of CSB in TC-NER. Not only the UBD but also the C-terminal 30-amino acid residues were required for UV light resistance and TC-NER. This region was needed for the interaction of CSB with RNA polymerase II, the translocation of CS group A protein to the nuclear matrix, and the association of CSB with chromatin after UV irradiation. CSB was modified by small ubiquitin-like modifier 2/3 in a UV light-dependent manner. This modification was abolished in a CSB mutant lacking the C-terminal 30 amino acid residues. However, the substitution of lysine residues in this region with arginine did not affect SUMOylation or TC-NER. By contrast, substitution of a lysine residue in the N-terminal region with arginine decreased SUMOylation and resulted in cells with defects in TC-NER. These results indicate that both the most C-terminal region and SUMOylation are important for the functions of CSB in TC-NER. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. The C-terminal Region and SUMOylation of Cockayne Syndrome Group B Protein Play Critical Roles in Transcription-coupled Nucleotide Excision Repair*

    Science.gov (United States)

    Sin, Yooksil; Tanaka, Kiyoji; Saijo, Masafumi

    2016-01-01

    Cockayne syndrome (CS) is a recessive disorder that results in deficiencies in transcription-coupled nucleotide excision repair (TC-NER), a subpathway of nucleotide excision repair, and cells from CS patients exhibit hypersensitivity to UV light. CS group B protein (CSB), which is the gene product of one of the genes responsible for CS, belongs to the SWI2/SNF2 DNA-dependent ATPase family and has an ATPase domain and an ubiquitin-binding domain (UBD) in the central region and the C-terminal region, respectively. The C-terminal region containing the UBD is essential for the functions of CSB. In this study, we generated several CSB deletion mutants and analyzed the functions of the C-terminal region of CSB in TC-NER. Not only the UBD but also the C-terminal 30-amino acid residues were required for UV light resistance and TC-NER. This region was needed for the interaction of CSB with RNA polymerase II, the translocation of CS group A protein to the nuclear matrix, and the association of CSB with chromatin after UV irradiation. CSB was modified by small ubiquitin-like modifier 2/3 in a UV light-dependent manner. This modification was abolished in a CSB mutant lacking the C-terminal 30 amino acid residues. However, the substitution of lysine residues in this region with arginine did not affect SUMOylation or TC-NER. By contrast, substitution of a lysine residue in the N-terminal region with arginine decreased SUMOylation and resulted in cells with defects in TC-NER. These results indicate that both the most C-terminal region and SUMOylation are important for the functions of CSB in TC-NER. PMID:26620705

  12. Overexpression of DNA ligase III in mitochondria protects cells against oxidative stress and improves mitochondrial DNA base excision repair.

    Science.gov (United States)

    Akbari, Mansour; Keijzers, Guido; Maynard, Scott; Scheibye-Knudsen, Morten; Desler, Claus; Hickson, Ian D; Bohr, Vilhelm A

    2014-04-01

    Base excision repair (BER) is the most prominent DNA repair pathway in human mitochondria. BER also results in a temporary generation of AP-sites, single-strand breaks and nucleotide gaps. Thus, incomplete BER can result in the generation of DNA repair intermediates that can disrupt mitochondrial DNA replication and transcription and generate mutations. We carried out BER analysis in highly purified mitochondrial extracts from human cell lines U2OS and HeLa, and mouse brain using a circular DNA substrate containing a lesion at a specific position. We found that DNA ligation is significantly slower than the preceding mitochondrial BER steps. Overexpression of DNA ligase III in mitochondria improved the rate of overall BER, increased cell survival after menadione induced oxidative stress and reduced autophagy following the inhibition of the mitochondrial electron transport chain complex I by rotenone. Our results suggest that the amount of DNA ligase III in mitochondria may be critical for cell survival following prolonged oxidative stress, and demonstrate a functional link between mitochondrial DNA damage and repair, cell survival upon oxidative stress, and removal of dysfunctional mitochondria by autophagy. Copyright © 2014. Published by Elsevier B.V.

  13. Human longevity and variation in DNA damage response and repair: Study of the contribution of sub-processes using competitive gene-set analysis

    DEFF Research Database (Denmark)

    Debrabant, Birgit; Sørensen, Mette; Flachsbart, Friederike

    2014-01-01

    others. Data were applied on 592 SNPs from 77 genes involved in nine sub-processes: DNA-damage response, base excision repair (BER), nucleotide excision repair, mismatch repair, non-homologous end-joining, homologous recombinational repair (HRR), RecQ helicase activities (RECQ), telomere functioning...... and mitochondrial DNA processes. The study population was 1089 long-lived and 736 middle-aged Danes. A self-contained set-based test of all SNPs displayed association with longevity (P-value=9.9 × 10-5), supporting that the overall pathway could affect longevity. Investigation of the nine sub-processes using...

  14. Nucleotide sequence, DNA damage location and protein stoichiometry influence base excision repair outcome at CAG/CTG repeats

    Science.gov (United States)

    Goula, Agathi-Vasiliki; Pearson, Christopher E.; Della Maria, Julie; Trottier, Yvon; Tomkinson, Alan E.; Wilson, David M.; Merienne, Karine

    2012-01-01

    Expansion of CAG/CTG repeats is the underlying cause of >fourteen genetic disorders, including Huntington’s disease (HD) and myotonic dystrophy. The mutational process is ongoing, with increases in repeat size enhancing the toxicity of the expansion in specific tissues. In many repeat diseases the repeats exhibit high instability in the striatum, whereas instability is minimal in the cerebellum. We provide molecular insights as to how base excision repair (BER) protein stoichiometry may contribute to the tissue-selective instability of CAG/CTG repeats by using specific repair assays. Oligonucleotide substrates with an abasic site were mixed with either reconstituted BER protein stoichiometries mimicking the levels present in HD mouse striatum or cerebellum, or with protein extracts prepared from HD mouse striatum or cerebellum. In both cases, repair efficiency at CAG/CTG repeats and at control DNA sequences was markedly reduced under the striatal conditions, likely due to the lower level of APE1, FEN1 and LIG1. Damage located towards the 5’ end of the repeat tract was poorly repaired accumulating incompletely processed intermediates as compared to an AP lesion in the centre or at the 3’ end of the repeats or within a control sequences. Moreover, repair of lesions at the 5’ end of CAG or CTG repeats involved multinucleotide synthesis, particularly under the cerebellar stoichiometry, suggesting that long-patch BER processes lesions at sequences susceptible to hairpin formation. Our results show that BER stoichiometry, nucleotide sequence and DNA damage position modulate repair outcome, and suggest that a suboptimal LP-BER activity promotes CAG/CTG repeat instability. PMID:22497302

  15. The nucleotide sequence, DNA damage location, and protein stoichiometry influence the base excision repair outcome at CAG/CTG repeats.

    Science.gov (United States)

    Goula, Agathi-Vasiliki; Pearson, Christopher E; Della Maria, Julie; Trottier, Yvon; Tomkinson, Alan E; Wilson, David M; Merienne, Karine

    2012-05-08

    Expansion of CAG/CTG repeats is the underlying cause of >14 genetic disorders, including Huntington's disease (HD) and myotonic dystrophy. The mutational process is ongoing, with increases in repeat size enhancing the toxicity of the expansion in specific tissues. In many repeat diseases, the repeats exhibit high instability in the striatum, whereas instability is minimal in the cerebellum. We provide molecular insights into how base excision repair (BER) protein stoichiometry may contribute to the tissue-selective instability of CAG/CTG repeats by using specific repair assays. Oligonucleotide substrates with an abasic site were mixed with either reconstituted BER protein stoichiometries mimicking the levels present in HD mouse striatum or cerebellum, or with protein extracts prepared from HD mouse striatum or cerebellum. In both cases, the repair efficiency at CAG/CTG repeats and at control DNA sequences was markedly reduced under the striatal conditions, likely because of the lower level of APE1, FEN1, and LIG1. Damage located toward the 5' end of the repeat tract was poorly repaired, with the accumulation of incompletely processed intermediates as compared to an AP lesion in the center or at the 3' end of the repeats or within control sequences. Moreover, repair of lesions at the 5' end of CAG or CTG repeats involved multinucleotide synthesis, particularly at the cerebellar stoichiometry, suggesting that long-patch BER processes lesions at sequences susceptible to hairpin formation. Our results show that the BER stoichiometry, nucleotide sequence, and DNA damage position modulate repair outcome and suggest that a suboptimal long-patch BER activity promotes CAG/CTG repeat instability.

  16. The amino-terminal tails of histones H2A and H3 coordinate efficient base excision repair, DNA damage signaling and postreplication repair in Saccharomyces cerevisiae

    Science.gov (United States)

    Meas, Rithy; Smerdon, Michael J.; Wyrick, John J.

    2015-01-01

    Histone amino-terminal tails (N-tails) are required for cellular resistance to DNA damaging agents; therefore, we examined the role of histone N-tails in regulating DNA damage response pathways in Saccharomyces cerevisiae. Combinatorial deletions reveal that the H2A and H3 N-tails are important for the removal of MMS-induced DNA lesions due to their role in regulating the basal and MMS-induced expression of DNA glycosylase Mag1. Furthermore, overexpression of Mag1 in a mutant lacking the H2A and H3 N-tails rescues base excision repair (BER) activity but not MMS sensitivity. We further show that the H3 N-tail functions in the Rad9/Rad53 DNA damage signaling pathway, but this function does not appear to be the primary cause of MMS sensitivity of the double tailless mutants. Instead, epistasis analyses demonstrate that the tailless H2A/H3 phenotypes are in the RAD18 epistasis group, which regulates postreplication repair. We observed increased levels of ubiquitylated PCNA and significantly lower mutation frequency in the tailless H2A/H3 mutant, indicating a defect in postreplication repair. In summary, our data identify novel roles of the histone H2A and H3 N-tails in (i) regulating the expression of a critical BER enzyme (Mag1), (ii) supporting efficient DNA damage signaling and (iii) facilitating postreplication repair. PMID:25897129

  17. Expression of DNA repair genes in burned skin exposed to low-level red laser.

    Science.gov (United States)

    Trajano, Eduardo Tavares Lima; Mencalha, Andre Luiz; Monte-Alto-Costa, Andréa; Pôrto, Luís Cristóvão; de Souza da Fonseca, Adenilson

    2014-11-01

    Although red laser lights lie in the region of non-ionizing radiations in the electromagnetic spectrum, there are doubts whether absorption of these radiations causes lesions in the DNA molecule. Our aim was to investigate the expression of the genes involved with base excision and nucleotide excision repair pathways in skin tissue submitted to burn injury and exposed to low-level red laser. Wistar rats were divided as follows: control group-rats burned and not irradiated, laser group-rats burned and irradiated 1 day after injury for five consecutive days, and later laser group-rats injured and treated 4 days after injury for five consecutive days. Irradiation was performed according to a clinical protocol (20 J/cm(2), 100 mW, continuous wave emission mode). The animals were sacrificed on day 10, and scarred tissue samples were withdrawn for total RNA extraction, complementary DNA (cDNA) synthesis, and evaluation of gene expression by quantitative polymerase chain reaction. Low-level red laser exposure (1) reduces the expression of APE1 messenger (mRNA), (2) increases the expression of OGG1 mRNA, (3) reduces the expression of XPC mRNA, and (4) increases the expression of XPA mRNA both in laser and later laser groups. Red laser exposure at therapeutic fluences alters the expression of genes related to base excision and nucleotide excision pathways of DNA repair during wound healing of burned skin.

  18. Laxity of the elbow after experimental excision of the radial head and division of the medial collateral ligament. Efficacy of ligament repair and radial head prosthetic replacement

    DEFF Research Database (Denmark)

    Jensen, Steen Lund; Deutch, Søren R; Olsen, Bo Sanderhoff

    2003-01-01

    We studied the stabilising effect of prosthetic replacement of the radial head and repair of the medial collateral ligament (MCL) after excision of the radial head and section of the MCL in five cadaver elbows. Division of the MCL increased valgus angulation (mean 3.9 +/- 1.5 degrees) and internal...... that the radial head is a constraint secondary to the MCL for both valgus displacement and internal rotation. Isolated repair of the ligament is superior to isolated prosthetic replacement and may be sufficient to restore valgus and internal rotatory stability after excision of the radial head in MCL...

  19. An inverse switch in DNA base excision and strand break repair contributes to melphalan resistance in multiple myeloma cells.

    Directory of Open Access Journals (Sweden)

    Mirta M L Sousa

    Full Text Available Alterations in checkpoint and DNA repair pathways may provide adaptive mechanisms contributing to acquired drug resistance. Here, we investigated the levels of proteins mediating DNA damage signaling and -repair in RPMI8226 multiple myeloma cells and its Melphalan-resistant derivative 8226-LR5. We observed markedly reduced steady-state levels of DNA glycosylases UNG2, NEIL1 and MPG in the resistant cells and cross-resistance to agents inducing their respective DNA base lesions. Conversely, repair of alkali-labile sites was apparently enhanced in the resistant cells, as substantiated by alkaline comet assay, autoribosylation of PARP-1, and increased sensitivity to PARP-1 inhibition by 4-AN or KU58684. Reduced base-excision and enhanced single-strand break repair would both contribute to the observed reduction in genomic alkali-labile sites, which could jeopardize productive processing of the more cytotoxic Melphalan-induced interstrand DNA crosslinks (ICLs. Furthermore, we found a marked upregulation of proteins in the non-homologous end-joining (NHEJ pathway of double-strand break (DSB repair, likely contributing to the observed increase in DSB repair kinetics in the resistant cells. Finally, we observed apparent upregulation of ATR-signaling and downregulation of ATM-signaling in the resistant cells. This was accompanied by markedly increased sensitivity towards Melphalan in the presence of ATR-, DNA-PK, or CHK1/2 inhibitors whereas no sensitizing effect was observed subsequent to ATM inhibition, suggesting that replication blocking lesions are primary triggers of the DNA damage response in the Melphalan resistant cells. In conclusion, Melphalan resistance is apparently contributed by modulation of the DNA damage response at multiple levels, including downregulation of specific repair pathways to avoid repair intermediates that could impair efficient processing of cytotoxic ICLs and ICL-induced DSBs. This study has revealed several novel

  20. Stabilization of Ultraviolet (UV)-stimulated Scaffold Protein A by Interaction with Ubiquitin-specific Peptidase 7 Is Essential for Transcription-coupled Nucleotide Excision Repair.

    Science.gov (United States)

    Higa, Mitsuru; Zhang, Xue; Tanaka, Kiyoji; Saijo, Masafumi

    2016-06-24

    UV-sensitive syndrome is an autosomal recessive disorder characterized by hypersensitivity to UV light and deficiency in transcription-coupled nucleotide excision repair (TC-NER), a subpathway of nucleotide excision repair that rapidly removes transcription-blocking DNA damage. UV-sensitive syndrome consists of three genetic complementation groups caused by mutations in the CSA, CSB, and UVSSA genes. UV-stimulated scaffold protein A (UVSSA), the product of UVSSA, which is required for stabilization of Cockayne syndrome group B (CSB) protein and reappearance of the hypophosphorylated form of RNA polymerase II after UV irradiation, forms a complex with ubiquitin-specific peptidase 7 (USP7). In this study, we demonstrated that the deubiquitination activity of USP7 is suppressed by its interaction with UVSSA. The interaction required the tumor necrosis factor receptor-associated factor domain of USP7 and the central region of UVSSA and was disrupted by an amino acid substitution in the tumor necrosis factor receptor-associated factor-binding motif of UVSSA. Cells expressing mutant UVSSA were highly sensitive to UV irradiation and defective in recovery of RNA synthesis after UV irradiation. These results indicate that the interaction between UVSSA and USP7 is important for TC-NER. Furthermore, the mutant UVSSA was rapidly degraded by the proteasome, and CSB was also degraded after UV irradiation as observed in UVSSA-deficient cells. Thus, stabilization of UVSSA by interaction with USP7 is essential for TC-NER. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. A UV-Induced Genetic Network Links the RSC Complex to Nucleotide Excision Repair and Shows Dose-Dependent Rewiring

    Directory of Open Access Journals (Sweden)

    Rohith Srivas

    2013-12-01

    Full Text Available Efficient repair of UV-induced DNA damage requires the precise coordination of nucleotide excision repair (NER with numerous other biological processes. To map this crosstalk, we generated a differential genetic interaction map centered on quantitative growth measurements of >45,000 double mutants before and after different doses of UV radiation. Integration of genetic data with physical interaction networks identified a global map of 89 UV-induced functional interactions among 62 protein complexes, including a number of links between the RSC complex and several NER factors. We show that RSC is recruited to both silenced and transcribed loci following UV damage where it facilitates efficient repair by promoting nucleosome remodeling. Finally, a comparison of the response to high versus low levels of UV shows that the degree of genetic rewiring correlates with dose of UV and reveals a network of dose-specific interactions. This study makes available a large resource of UV-induced interactions, and it illustrates a methodology for identifying dose-dependent interactions based on quantitative shifts in genetic networks.

  2. Mutations in UVSSA cause UV-sensitive syndrome and impair RNA polymerase IIo processing in transcription-coupled nucleotide-excision repair.

    Science.gov (United States)

    Nakazawa, Yuka; Sasaki, Kensaku; Mitsutake, Norisato; Matsuse, Michiko; Shimada, Mayuko; Nardo, Tiziana; Takahashi, Yoshito; Ohyama, Kaname; Ito, Kosei; Mishima, Hiroyuki; Nomura, Masayo; Kinoshita, Akira; Ono, Shinji; Takenaka, Katsuya; Masuyama, Ritsuko; Kudo, Takashi; Slor, Hanoch; Utani, Atsushi; Tateishi, Satoshi; Yamashita, Shunichi; Stefanini, Miria; Lehmann, Alan R; Yoshiura, Koh-ichiro; Ogi, Tomoo

    2012-05-01

    UV-sensitive syndrome (UV(S)S) is a genodermatosis characterized by cutaneous photosensitivity without skin carcinoma. Despite mild clinical features, cells from individuals with UV(S)S, like Cockayne syndrome cells, are very UV sensitive and are deficient in transcription-coupled nucleotide-excision repair (TC-NER), which removes DNA damage in actively transcribed genes. Three of the seven known UV(S)S cases carry mutations in the Cockayne syndrome genes ERCC8 or ERCC6 (also known as CSA and CSB, respectively). The remaining four individuals with UVSS , one of whom is described for the first time here, formed a separate UV(S)S-A complementation group; however, the responsible gene was unknown. Using exome sequencing, we determine that mutations in the UVSSA gene (formerly known as KIAA1530) cause UV(S)S-A. The UVSSA protein interacts with TC-NER machinery and stabilizes the ERCC6 complex; it also facilitates ubiquitination of RNA polymerase IIo stalled at DNA damage sites. Our findings provide mechanistic insights into the processing of stalled RNA polymerase and explain the different clinical features across these TC-NER–deficient disorders.

  3. Quantitation of intracellular NAD(P)H can monitor an imbalance of DNA single strand break repair in base excision repair deficient cells in real time

    Science.gov (United States)

    Nakamura, Jun; Asakura, Shoji; Hester, Susan D.; de Murcia, Gilbert; Caldecott, Keith W.; Swenberg, James A.

    2003-01-01

    DNA single strand breaks (SSBs) are one of the most frequent DNA lesions in genomic DNA generated either by oxidative stress or during the base excision repair pathways. Here we established a new real-time assay to assess an imbalance of DNA SSB repair by indirectly measuring PARP-1 activation through the depletion of intracellular NAD(P)H. A water-soluble tetrazolium salt is used to monitor the amount of NAD(P)H in living cells through its reduction to a yellow colored water-soluble formazan dye. While this assay is not a direct method, it does not require DNA extraction or alkaline treatment, both of which could potentially cause an artifactual induction of SSBs. In addition, it takes only 4 h and requires less than a half million cells to perform this measurement. Using this assay, we demonstrated that the dose- and time-dependent depletion of NAD(P)H in XRCC1-deficient CHO cells exposed to methyl methanesulfonate. This decrease was almost completely blocked by a PARP inhibitor. Furthermore, methyl methanesulfonate reduced NAD(P)H in PARP-1+/+cells, whereas PARP-1–/– cells were more resistant to the decrease in NAD(P)H. These results indicate that the analysis of intracellular NAD(P)H level using water-soluble tetrazolium salt can assess an imbalance of SSB repair in living cells in real time. PMID:12930978

  4. Laparoscopic Partial Cystectomy With Excision of Mesh Migration Into the Bladder Following Repair of Inguinal Hernia

    Directory of Open Access Journals (Sweden)

    Satoshi Funada

    2016-09-01

    Full Text Available Migration of hernia mesh into the bladder is a rare complication of inguinal hernioplasty. We present the case of an 85-year-old man who complained of hematuria and fever some 20 years after right hernioplasty. Cystoscopy and computed tomography revealed mesh migration into the right anterior wall of the bladder. Laparoscopic partial cystectomy with excision of the migrated mesh was performed successfully. To our knowledge, this is the first case of mesh migration into the bladder treated by laparoscopic partial cystectomy.

  5. 1-beta-D-Arabinofuranosylcytosine is Cytotoxic in Quiescent Normal Lymphocytes Undergoing DNA Excision Repair

    OpenAIRE

    Yamauchi, Takahiro; Kawai, Yasukazu; Ueda, Takanori

    2002-01-01

    We have sought to clarify the potential activity of S-phase specific antileukemic agent 1--D-arabinofuranosylcytosine (ara-C), an inhibitor of DNA synthesis, in quiescent cells that are substantially non-sensitive to nucleoside analogues. It was hypothesized that the combination of ara-C with DNA damaging agents that initiate DNA repair will expand ara-C cytotoxicity to non-cycling cells. The repair kinetics, which included incision of damaged DNA, gap filling by DNA synthesis and rejoinin...

  6. The Challenges of Validating in Precision Medicine: The Case of Excision Repair Cross-Complement Group 1 Diagnostic Testing.

    Science.gov (United States)

    Barsanti-Innes, Brianna; Hey, Spencer Phillips; Kimmelman, Jonathan

    2017-01-01

    Personalized medicine relies upon the successful identification and translation of predictive biomarkers. Unfortunately, biomarker development has often fallen short of expectations. To better understand the obstacles to successful biomarker development, we systematically mapped research activities for a biomarker that has been in development for at least 12 years: excision repair cross-complement group 1 protein (ERCC1) as a biomarker for predicting clinical benefit with platinum-based chemotherapy in non-small cell lung cancer. We found that although research activities explored a wide range of approaches to ERCC1 testing, there was little replication or validation of techniques, and design and reporting of results were generally poor. Our analysis points to problems with coordinating and standardizing research in biomarker development. Clinically meaningful progress in personalized medicine will require concerted efforts to address these problems. In the interim, health care providers should be aware of the complexity involved in biomarker development, cautious about their near-term clinical value, and conscious of applying only validated diagnostics in the clinic. 2017;22:89-96 IMPLICATIONS FOR PRACTICE: : Many hospitals, policy makers, and scientists have made ambitious claims about the promise of personalizing cancer care. When one uses a case example of excision repair cross-complement group 1 protein-a biomarker that has a strong biological rationale and that has been researched for 12 years-the current research environment seems poorly suited for efficient development of biomarker tests. The findings provide grounds for tempering expectations about personalized cancer care-at least in the near term-and shed light on the current gap between the promise and practice of personalized medicine. © AlphaMed Press 2016.

  7. Acetylation regulates WRN catalytic activities and affects base excision DNA repair

    DEFF Research Database (Denmark)

    Muftuoglu, Meltem; Kusumoto, Rika; Speina, Elzbieta

    2008-01-01

    The Werner protein (WRN), defective in the premature aging disorder Werner syndrome, participates in a number of DNA metabolic processes, and we have been interested in the possible regulation of its function in DNA repair by post-translational modifications. Acetylation mediated by histone...

  8. First reported patient with human ERCC1 deficiency has cerebro-oculo-facio- skeletal syndrome with a mild defect in nucleotide excision repair and severe developmental failure

    NARCIS (Netherlands)

    N.G.J. Jaspers (Nicolaas); A. Raams (Anja); M.C. Silengo; N. Wijgers (Nils); L.J. Niedernhofer (Laura); A.R. Robinson (Andria Rasile); G. Giglia-Mari (Giuseppina); D. Hoogstraten (Deborah); W.J. Kleijer (Wim); J.H.J. Hoeijmakers (Jan); W. Vermeulen (Wim)

    2007-01-01

    textabstractNucleotide excision repair (NER) is a genome caretaker mechanism responsible for removing helix-distorting DNA lesions, most notably ultraviolet photodimers. Inherited defects in NER result in profound photosensitivity and the cancer-prone syndrome xeroderma pigmentosum (XP) or two

  9. The role of Schizosaccharomyces pombe DNA repair enzymes Apn1p and Uve1p in the base excision repair of apurinic/apyrimidinic sites.

    Science.gov (United States)

    Tanihigashi, Haruna; Yamada, Ayako; Igawa, Emi; Ikeda, Shogo

    2006-09-08

    In Schizosaccharomyces pombe the repair of apurinic/apyrimidinic (AP) sites is mainly initiated by AP lyase activity of DNA glycosylase Nth1p. In contrast, the major AP endonuclease Apn2p functions by removing 3'-alpha,beta-unsaturated aldehyde ends induced by Nth1p, rather than by incising the AP sites. S. pombe possesses other minor AP endonuclease activities derived from Apn1p and Uve1p. In this study, we investigated the function of these two enzymes in base excision repair (BER) for methyl methanesulfonate (MMS) damage using the nth1 and apn2 mutants. Deletion of apn1 or uve1 from nth1Delta cells did not affect sensitivity to MMS. Exogenous expression of Apn1p failed to suppress the MMS sensitivity of nth1Delta cells. Although Apn1p and Uve1p incised the oligonucleotide containing an AP site analogue, these enzymes could not initiate repair of the AP sites in vivo. Despite this, expression of Apn1p partially restored the MMS sensitivity of apn2Delta cells, indicating that the enzyme functions as a 3'-phosphodiesterase to remove 3'-blocked ends. Localization of Apn1p in the nucleus and cytoplasm hints at an additional function of the enzyme other than nuclear DNA repair. Heterologous expression of Saccharomyces cerevisiae homologue of Apn1p completely restored the MMS resistance of the nth1Delta and apn2Delta cells. This result confirms a difference in the major pathway for processing the AP site between S. pombe and S. cerevisiae cells.

  10. The amino-terminal tails of histones H2A and H3 coordinate efficient base excision repair, DNA damage signaling and postreplication repair in Saccharomyces cerevisiae.

    Science.gov (United States)

    Meas, Rithy; Smerdon, Michael J; Wyrick, John J

    2015-05-26

    Histone amino-terminal tails (N-tails) are required for cellular resistance to DNA damaging agents; therefore, we examined the role of histone N-tails in regulating DNA damage response pathways in Saccharomyces cerevisiae. Combinatorial deletions reveal that the H2A and H3 N-tails are important for the removal of MMS-induced DNA lesions due to their role in regulating the basal and MMS-induced expression of DNA glycosylase Mag1. Furthermore, overexpression of Mag1 in a mutant lacking the H2A and H3 N-tails rescues base excision repair (BER) activity but not MMS sensitivity. We further show that the H3 N-tail functions in the Rad9/Rad53 DNA damage signaling pathway, but this function does not appear to be the primary cause of MMS sensitivity of the double tailless mutants. Instead, epistasis analyses demonstrate that the tailless H2A/H3 phenotypes are in the RAD18 epistasis group, which regulates postreplication repair. We observed increased levels of ubiquitylated PCNA and significantly lower mutation frequency in the tailless H2A/H3 mutant, indicating a defect in postreplication repair. In summary, our data identify novel roles of the histone H2A and H3 N-tails in (i) regulating the expression of a critical BER enzyme (Mag1), (ii) supporting efficient DNA damage signaling and (iii) facilitating postreplication repair. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  11. Genomic survey and expression analysis of DNA repair genes in the genus Leptospira.

    Science.gov (United States)

    Martins-Pinheiro, Marinalva; Schons-Fonseca, Luciane; da Silva, Josefa B; Domingos, Renan H; Momo, Leonardo Hiroyuki Santos; Simões, Ana Carolina Quirino; Ho, Paulo Lee; da Costa, Renata M A

    2016-04-01

    Leptospirosis is an emerging zoonosis with important economic and public health consequences and is caused by pathogenic leptospires. The genus Leptospira belongs to the order Spirochaetales and comprises saprophytic (L. biflexa), pathogenic (L. interrogans) and host-dependent (L. borgpetersenii) members. Here, we present an in silico search for DNA repair pathways in Leptospira spp. The relevance of such DNA repair pathways was assessed through the identification of mRNA levels of some genes during infection in animal model and after exposition to spleen cells. The search was performed by comparison of available Leptospira spp. genomes in public databases with known DNA repair-related genes. Leptospires exhibit some distinct and unexpected characteristics, for instance the existence of a redundant mechanism for repairing a chemically diverse spectrum of alkylated nucleobases, a new mutS-like gene and a new shorter version of uvrD. Leptospira spp. shares some characteristics from Gram-positive, as the presence of PcrA, two RecQ paralogs and two SSB proteins; the latter is considered a feature shared by naturally competent bacteria. We did not find a significant reduction in the number of DNA repair-related genes in both pathogenic and host-dependent species. Pathogenic leptospires were enriched for genes dedicated to base excision repair and non-homologous end joining. Their evolutionary history reveals a remarkable importance of lateral gene transfer events for the evolution of the genus. Up-regulation of specific DNA repair genes, including components of SOS regulon, during infection in animal model validates the critical role of DNA repair mechanisms for the complex interplay between host/pathogen.

  12. Calcium-binding capacity of centrin2 is required for linear POC5 assembly but not for nucleotide excision repair.

    Directory of Open Access Journals (Sweden)

    Tiago J Dantas

    Full Text Available Centrosomes, the principal microtubule-organising centres in animal cells, contain centrins, small, conserved calcium-binding proteins unique to eukaryotes. Centrin2 binds to xeroderma pigmentosum group C protein (XPC, stabilising it, and its presence slightly increases nucleotide excision repair (NER activity in vitro. In previous work, we deleted all three centrin isoforms present in chicken DT40 cells and observed delayed repair of UV-induced DNA lesions, but no centrosome abnormalities. Here, we explore how centrin2 controls NER. In the centrin null cells, we expressed centrin2 mutants that cannot bind calcium or that lack sites for phosphorylation by regulatory kinases. Expression of any of these mutants restored the UV sensitivity of centrin null cells to normal as effectively as expression of wild-type centrin. However, calcium-binding-deficient and T118A mutants showed greatly compromised localisation to centrosomes. XPC recruitment to laser-induced UV-like lesions was only slightly slower in centrin-deficient cells than in controls, and levels of XPC and its partner HRAD23B were unaffected by centrin deficiency. Interestingly, we found that overexpression of the centrin interactor POC5 leads to the assembly of linear, centrin-dependent structures that recruit other centrosomal proteins such as PCM-1 and NEDD1. Together, these observations suggest that assembly of centrins into complex structures requires calcium binding capacity, but that such assembly is not required for centrin activity in NER.

  13. Modulation of proteostasis counteracts oxidative stress and affects DNA base excision repair capacity in ATM-deficient cells.

    Science.gov (United States)

    Poletto, Mattia; Yang, Di; Fletcher, Sally C; Vendrell, Iolanda; Fischer, Roman; Legrand, Arnaud J; Dianov, Grigory L

    2017-09-29

    Ataxia telangiectasia (A-T) is a syndrome associated with loss of ATM protein function. Neurodegeneration and cancer predisposition, both hallmarks of A-T, are likely to emerge as a consequence of the persistent oxidative stress and DNA damage observed in this disease. Surprisingly however, despite these severe features, a lack of functional ATM is still compatible with early life, suggesting that adaptation mechanisms contributing to cell survival must be in place. Here we address this gap in our knowledge by analysing the process of human fibroblast adaptation to the lack of ATM. We identify profound rearrangement in cellular proteostasis occurring very early on after loss of ATM in order to counter protein damage originating from oxidative stress. Change in proteostasis, however, is not without repercussions. Modulating protein turnover in ATM-depleted cells also has an adverse effect on the DNA base excision repair pathway, the major DNA repair system that deals with oxidative DNA damage. As a consequence, the burden of unrepaired endogenous DNA lesions intensifies, progressively leading to genomic instability. Our study provides a glimpse at the cellular consequences of loss of ATM and highlights a previously overlooked role for proteostasis in maintaining cell survival in the absence of ATM function. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  14. Base excision repair activities differ in human lung cancer cells and corresponding normal controls

    DEFF Research Database (Denmark)

    Karahalil, Bensu; Bohr, Vilhelm A; De Souza-Pinto, Nadja C

    2010-01-01

    for the repair of oxidized modifications both in nuclear and mitochondrial DNA. In order to ascertain whether diminished BER capacity might account for increased levels of oxidative DNA damage in cancer cells, the activities of BER enzymes in three different lung cancer cell lines and their non......-cancerous counterparts were measured using oligonucleotide substrates with single DNA lesions to assess specific BER enzymes. The activities of four BER enzymes, OGG1, NTH1, UDG and APE1, were compared in mitochondrial and nuclear extracts. For each specific lesion, the repair activities were similar among the three...... cell lines used. However, the specific activities and cancer versus control comparison differed significantly between the nuclear and mitochondrial compartments. OGG1 activity, as measured by 8-oxodA incision, was up-regulated in cancer cell mitochondria but down-regulated in the nucleus when compared...

  15. DNA glycosylases involved in base excision repair may be associated with cancer risk in BRCA1 and BRCA2 mutation carriers.

    Directory of Open Access Journals (Sweden)

    Ana Osorio

    2014-04-01

    Full Text Available Single Nucleotide Polymorphisms (SNPs in genes involved in the DNA Base Excision Repair (BER pathway could be associated with cancer risk in carriers of mutations in the high-penetrance susceptibility genes BRCA1 and BRCA2, given the relation of synthetic lethality that exists between one of the components of the BER pathway, PARP1 (poly ADP ribose polymerase, and both BRCA1 and BRCA2. In the present study, we have performed a comprehensive analysis of 18 genes involved in BER using a tagging SNP approach in a large series of BRCA1 and BRCA2 mutation carriers. 144 SNPs were analyzed in a two stage study involving 23,463 carriers from the CIMBA consortium (the Consortium of Investigators of Modifiers of BRCA1 and BRCA2. Eleven SNPs showed evidence of association with breast and/or ovarian cancer at p<0.05 in the combined analysis. Four of the five genes for which strongest evidence of association was observed were DNA glycosylases. The strongest evidence was for rs1466785 in the NEIL2 (endonuclease VIII-like 2 gene (HR: 1.09, 95% CI (1.03-1.16, p = 2.7 × 10(-3 for association with breast cancer risk in BRCA2 mutation carriers, and rs2304277 in the OGG1 (8-guanine DNA glycosylase gene, with ovarian cancer risk in BRCA1 mutation carriers (HR: 1.12 95%CI: 1.03-1.21, p = 4.8 × 10(-3. DNA glycosylases involved in the first steps of the BER pathway may be associated with cancer risk in BRCA1/2 mutation carriers and should be more comprehensively studied.

  16. DNA glycosylases involved in base excision repair may be associated with cancer risk in BRCA1 and BRCA2 mutation carriers.

    Science.gov (United States)

    Osorio, Ana; Milne, Roger L; Kuchenbaecker, Karoline; Vaclová, Tereza; Pita, Guillermo; Alonso, Rosario; Peterlongo, Paolo; Blanco, Ignacio; de la Hoya, Miguel; Duran, Mercedes; Díez, Orland; Ramón Y Cajal, Teresa; Konstantopoulou, Irene; Martínez-Bouzas, Cristina; Andrés Conejero, Raquel; Soucy, Penny; McGuffog, Lesley; Barrowdale, Daniel; Lee, Andrew; Swe-Brca; Arver, Brita; Rantala, Johanna; Loman, Niklas; Ehrencrona, Hans; Olopade, Olufunmilayo I; Beattie, Mary S; Domchek, Susan M; Nathanson, Katherine; Rebbeck, Timothy R; Arun, Banu K; Karlan, Beth Y; Walsh, Christine; Lester, Jenny; John, Esther M; Whittemore, Alice S; Daly, Mary B; Southey, Melissa; Hopper, John; Terry, Mary B; Buys, Saundra S; Janavicius, Ramunas; Dorfling, Cecilia M; van Rensburg, Elizabeth J; Steele, Linda; Neuhausen, Susan L; Ding, Yuan Chun; Hansen, Thomas V O; Jønson, Lars; Ejlertsen, Bent; Gerdes, Anne-Marie; Infante, Mar; Herráez, Belén; Moreno, Leticia Thais; Weitzel, Jeffrey N; Herzog, Josef; Weeman, Kisa; Manoukian, Siranoush; Peissel, Bernard; Zaffaroni, Daniela; Scuvera, Giulietta; Bonanni, Bernardo; Mariette, Frederique; Volorio, Sara; Viel, Alessandra; Varesco, Liliana; Papi, Laura; Ottini, Laura; Tibiletti, Maria Grazia; Radice, Paolo; Yannoukakos, Drakoulis; Garber, Judy; Ellis, Steve; Frost, Debra; Platte, Radka; Fineberg, Elena; Evans, Gareth; Lalloo, Fiona; Izatt, Louise; Eeles, Ros; Adlard, Julian; Davidson, Rosemarie; Cole, Trevor; Eccles, Diana; Cook, Jackie; Hodgson, Shirley; Brewer, Carole; Tischkowitz, Marc; Douglas, Fiona; Porteous, Mary; Side, Lucy; Walker, Lisa; Morrison, Patrick; Donaldson, Alan; Kennedy, John; Foo, Claire; Godwin, Andrew K; Schmutzler, Rita Katharina; Wappenschmidt, Barbara; Rhiem, Kerstin; Engel, Christoph; Meindl, Alfons; Ditsch, Nina; Arnold, Norbert; Plendl, Hans Jörg; Niederacher, Dieter; Sutter, Christian; Wang-Gohrke, Shan; Steinemann, Doris; Preisler-Adams, Sabine; Kast, Karin; Varon-Mateeva, Raymonda; Gehrig, Andrea; Stoppa-Lyonnet, Dominique; Sinilnikova, Olga M; Mazoyer, Sylvie; Damiola, Francesca; Poppe, Bruce; Claes, Kathleen; Piedmonte, Marion; Tucker, Kathy; Backes, Floor; Rodríguez, Gustavo; Brewster, Wendy; Wakeley, Katie; Rutherford, Thomas; Caldés, Trinidad; Nevanlinna, Heli; Aittomäki, Kristiina; Rookus, Matti A; van Os, Theo A M; van der Kolk, Lizet; de Lange, J L; Meijers-Heijboer, Hanne E J; van der Hout, A H; van Asperen, Christi J; Gómez Garcia, Encarna B; Hoogerbrugge, Nicoline; Collée, J Margriet; van Deurzen, Carolien H M; van der Luijt, Rob B; Devilee, Peter; Hebon; Olah, Edith; Lázaro, Conxi; Teulé, Alex; Menéndez, Mireia; Jakubowska, Anna; Cybulski, Cezary; Gronwald, Jacek; Lubinski, Jan; Durda, Katarzyna; Jaworska-Bieniek, Katarzyna; Johannsson, Oskar Th; Maugard, Christine; Montagna, Marco; Tognazzo, Silvia; Teixeira, Manuel R; Healey, Sue; Investigators, Kconfab; Olswold, Curtis; Guidugli, Lucia; Lindor, Noralane; Slager, Susan; Szabo, Csilla I; Vijai, Joseph; Robson, Mark; Kauff, Noah; Zhang, Liying; Rau-Murthy, Rohini; Fink-Retter, Anneliese; Singer, Christian F; Rappaport, Christine; Geschwantler Kaulich, Daphne; Pfeiler, Georg; Tea, Muy-Kheng; Berger, Andreas; Phelan, Catherine M; Greene, Mark H; Mai, Phuong L; Lejbkowicz, Flavio; Andrulis, Irene; Mulligan, Anna Marie; Glendon, Gord; Toland, Amanda Ewart; Bojesen, Anders; Pedersen, Inge Sokilde; Sunde, Lone; Thomassen, Mads; Kruse, Torben A; Jensen, Uffe Birk; Friedman, Eitan; Laitman, Yael; Shimon, Shani Paluch; Simard, Jacques; Easton, Douglas F; Offit, Kenneth; Couch, Fergus J; Chenevix-Trench, Georgia; Antoniou, Antonis C; Benitez, Javier

    2014-04-01

    Single Nucleotide Polymorphisms (SNPs) in genes involved in the DNA Base Excision Repair (BER) pathway could be associated with cancer risk in carriers of mutations in the high-penetrance susceptibility genes BRCA1 and BRCA2, given the relation of synthetic lethality that exists between one of the components of the BER pathway, PARP1 (poly ADP ribose polymerase), and both BRCA1 and BRCA2. In the present study, we have performed a comprehensive analysis of 18 genes involved in BER using a tagging SNP approach in a large series of BRCA1 and BRCA2 mutation carriers. 144 SNPs were analyzed in a two stage study involving 23,463 carriers from the CIMBA consortium (the Consortium of Investigators of Modifiers of BRCA1 and BRCA2). Eleven SNPs showed evidence of association with breast and/or ovarian cancer at p<0.05 in the combined analysis. Four of the five genes for which strongest evidence of association was observed were DNA glycosylases. The strongest evidence was for rs1466785 in the NEIL2 (endonuclease VIII-like 2) gene (HR: 1.09, 95% CI (1.03-1.16), p = 2.7 × 10(-3)) for association with breast cancer risk in BRCA2 mutation carriers, and rs2304277 in the OGG1 (8-guanine DNA glycosylase) gene, with ovarian cancer risk in BRCA1 mutation carriers (HR: 1.12 95%CI: 1.03-1.21, p = 4.8 × 10(-3)). DNA glycosylases involved in the first steps of the BER pathway may be associated with cancer risk in BRCA1/2 mutation carriers and should be more comprehensively studied.

  17. Differential participation of homologous recombination and nucleotide excision repair in yeast survival to ultraviolet light radiation.

    Science.gov (United States)

    Toussaint, Martin; Wellinger, Raymund J; Conconi, Antonio

    2010-04-30

    The purpose of this research was to assess the ultraviolet light (UV) phenotype of yeast sirDelta cells vs. WT cells, and to determine whether de-silenced chromatin or the intrinsic pseudoploidy of sirDelta mutants contributes to their response to UV. Additional aims were to study the participation of HR and NER in promoting UV survival during the cell cycle, and to define the extent of the co-participation for both repair pathways. The sensitivity of yeast Saccharomyces cerevisiae to UV light was determined using a method based on automatic measurements of optical densities of very small (100mul) liquid cell cultures. We show that pseudo-diploidy of sirDelta strains promotes resistance to UV irradiation and that HR is the main mechanism that is responsible for this phenotype. In addition, HR together with GG-NER renders cells in the G2-phase of the cell cycle more resistant to UV irradiation than cells in the G1-phase, which underscore the importance of HR when two copies of the chromosomes are present. Nevertheless, in asynchronously growing cells NER is the main repair pathway that responds to UV induced DNA damage. This study provides detailed and quantitative information on the co-participation of HR and NER in UV survival of yeast cells. Crown Copyright 2010. Published by Elsevier B.V. All rights reserved.

  18. DNA Glycosylases Involved in Base Excision Repair May Be Associated with Cancer Risk in BRCA1 and BRCA2 Mutation Carriers

    Science.gov (United States)

    Osorio, Ana; Milne, Roger L.; Kuchenbaecker, Karoline; Vaclová, Tereza; Pita, Guillermo; Alonso, Rosario; Peterlongo, Paolo; Blanco, Ignacio; de la Hoya, Miguel; Duran, Mercedes; Díez, Orland; Ramón y Cajal, Teresa; Konstantopoulou, Irene; Martínez-Bouzas, Cristina; Andrés Conejero, Raquel; Soucy, Penny; McGuffog, Lesley; Barrowdale, Daniel; Lee, Andrew; SWE-BRCA; Arver, Brita; Rantala, Johanna; Loman, Niklas; Ehrencrona, Hans; Olopade, Olufunmilayo I.; Beattie, Mary S.; Domchek, Susan M.; Nathanson, Katherine; Rebbeck, Timothy R.; Arun, Banu K.; Karlan, Beth Y.; Walsh, Christine; Lester, Jenny; John, Esther M.; Whittemore, Alice S.; Daly, Mary B.; Southey, Melissa; Hopper, John; Terry, Mary B.; Buys, Saundra S.; Janavicius, Ramunas; Dorfling, Cecilia M.; van Rensburg, Elizabeth J.; Steele, Linda; Neuhausen, Susan L.; Ding, Yuan Chun; Hansen, Thomas v. O.; Jønson, Lars; Ejlertsen, Bent; Gerdes, Anne-Marie; Infante, Mar; Herráez, Belén; Moreno, Leticia Thais; Weitzel, Jeffrey N.; Herzog, Josef; Weeman, Kisa; Manoukian, Siranoush; Peissel, Bernard; Zaffaroni, Daniela; Scuvera, Giulietta; Bonanni, Bernardo; Mariette, Frederique; Volorio, Sara; Viel, Alessandra; Varesco, Liliana; Papi, Laura; Ottini, Laura; Tibiletti, Maria Grazia; Radice, Paolo; Yannoukakos, Drakoulis; Garber, Judy; Ellis, Steve; Frost, Debra; Platte, Radka; Fineberg, Elena; Evans, Gareth; Lalloo, Fiona; Izatt, Louise; Eeles, Ros; Adlard, Julian; Davidson, Rosemarie; Cole, Trevor; Eccles, Diana; Cook, Jackie; Hodgson, Shirley; Brewer, Carole; Tischkowitz, Marc; Douglas, Fiona; Porteous, Mary; Side, Lucy; Walker, Lisa; Morrison, Patrick; Donaldson, Alan; Kennedy, John; Foo, Claire; Godwin, Andrew K.; Schmutzler, Rita Katharina; Wappenschmidt, Barbara; Rhiem, Kerstin; Engel, Christoph; Meindl, Alfons; Ditsch, Nina; Arnold, Norbert; Plendl, Hans Jörg; Niederacher, Dieter; Sutter, Christian; Wang-Gohrke, Shan; Steinemann, Doris; Preisler-Adams, Sabine; Kast, Karin; Varon-Mateeva, Raymonda; Gehrig, Andrea; Stoppa-Lyonnet, Dominique; Sinilnikova, Olga M.; Mazoyer, Sylvie; Damiola, Francesca; Poppe, Bruce; Claes, Kathleen; Piedmonte, Marion; Tucker, Kathy; Backes, Floor; Rodríguez, Gustavo; Brewster, Wendy; Wakeley, Katie; Rutherford, Thomas; Caldés, Trinidad; Nevanlinna, Heli; Aittomäki, Kristiina; Rookus, Matti A.; van Os, Theo A. M.; van der Kolk, Lizet; de Lange, J. L.; Meijers-Heijboer, Hanne E. J.; van der Hout, A. H.; van Asperen, Christi J.; Gómez Garcia, Encarna B.; Hoogerbrugge, Nicoline; Collée, J. Margriet; van Deurzen, Carolien H. M.; van der Luijt, Rob B.; Devilee, Peter; HEBON; Olah, Edith; Lázaro, Conxi; Teulé, Alex; Menéndez, Mireia; Jakubowska, Anna; Cybulski, Cezary; Gronwald, Jacek; Lubinski, Jan; Durda, Katarzyna; Jaworska-Bieniek, Katarzyna; Johannsson, Oskar Th.; Maugard, Christine; Montagna, Marco; Tognazzo, Silvia; Teixeira, Manuel R.; Healey, Sue; Investigators, kConFab; Olswold, Curtis; Guidugli, Lucia; Lindor, Noralane; Slager, Susan; Szabo, Csilla I.; Vijai, Joseph; Robson, Mark; Kauff, Noah; Zhang, Liying; Rau-Murthy, Rohini; Fink-Retter, Anneliese; Singer, Christian F.; Rappaport, Christine; Geschwantler Kaulich, Daphne; Pfeiler, Georg; Tea, Muy-Kheng; Berger, Andreas; Phelan, Catherine M.; Greene, Mark H.; Mai, Phuong L.; Lejbkowicz, Flavio; Andrulis, Irene; Mulligan, Anna Marie; Glendon, Gord; Toland, Amanda Ewart; Bojesen, Anders; Pedersen, Inge Sokilde; Sunde, Lone; Thomassen, Mads; Kruse, Torben A.; Jensen, Uffe Birk; Friedman, Eitan; Laitman, Yael; Shimon, Shani Paluch; Simard, Jacques; Easton, Douglas F.; Offit, Kenneth; Couch, Fergus J.; Chenevix-Trench, Georgia; Antoniou, Antonis C.; Benitez, Javier

    2014-01-01

    Single Nucleotide Polymorphisms (SNPs) in genes involved in the DNA Base Excision Repair (BER) pathway could be associated with cancer risk in carriers of mutations in the high-penetrance susceptibility genes BRCA1 and BRCA2, given the relation of synthetic lethality that exists between one of the components of the BER pathway, PARP1 (poly ADP ribose polymerase), and both BRCA1 and BRCA2. In the present study, we have performed a comprehensive analysis of 18 genes involved in BER using a tagging SNP approach in a large series of BRCA1 and BRCA2 mutation carriers. 144 SNPs were analyzed in a two stage study involving 23,463 carriers from the CIMBA consortium (the Consortium of Investigators of Modifiers of BRCA1 and BRCA2). Eleven SNPs showed evidence of association with breast and/or ovarian cancer at pgenes for which strongest evidence of association was observed were DNA glycosylases. The strongest evidence was for rs1466785 in the NEIL2 (endonuclease VIII-like 2) gene (HR: 1.09, 95% CI (1.03–1.16), p = 2.7×10−3) for association with breast cancer risk in BRCA2 mutation carriers, and rs2304277 in the OGG1 (8-guanine DNA glycosylase) gene, with ovarian cancer risk in BRCA1 mutation carriers (HR: 1.12 95%CI: 1.03–1.21, p = 4.8×10−3). DNA glycosylases involved in the first steps of the BER pathway may be associated with cancer risk in BRCA1/2 mutation carriers and should be more comprehensively studied. PMID:24698998

  19. UvrD Participation in Nucleotide Excision Repair Is Required for the Recovery of DNA Synthesis following UV-Induced Damage in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Kelley N. Newton

    2012-01-01

    Full Text Available UvrD is a DNA helicase that participates in nucleotide excision repair and several replication-associated processes, including methyl-directed mismatch repair and recombination. UvrD is capable of displacing oligonucleotides from synthetic forked DNA structures in vitro and is essential for viability in the absence of Rep, a helicase associated with processing replication forks. These observations have led others to propose that UvrD may promote fork regression and facilitate resetting of the replication fork following arrest. However, the molecular activity of UvrD at replication forks in vivo has not been directly examined. In this study, we characterized the role UvrD has in processing and restoring replication forks following arrest by UV-induced DNA damage. We show that UvrD is required for DNA synthesis to recover. However, in the absence of UvrD, the displacement and partial degradation of the nascent DNA at the arrested fork occur normally. In addition, damage-induced replication intermediates persist and accumulate in uvrD mutants in a manner that is similar to that observed in other nucleotide excision repair mutants. These data indicate that, following arrest by DNA damage, UvrD is not required to catalyze fork regression in vivo and suggest that the failure of uvrD mutants to restore DNA synthesis following UV-induced arrest relates to its role in nucleotide excision repair.

  20. Initial steps of the base excision repair pathway within the nuclear architecture; Les etapes initiales du mecanisme de reparation par excision de bases au sein de l'architecture nucleaire

    Energy Technology Data Exchange (ETDEWEB)

    Amouroux, R

    2009-09-15

    Oxidative stress induced lesions threaten aerobic organisms by representing a major cause of genomic instability. A common product of guanine oxidation, 8-oxo-guanine (8- oxoG) is particularly mutagenic by provoking G to T transversions. Removal of oxidised bases from DNA is initiated by the recognition and excision of the damaged base by a DNA glycosylase, initiating the base excision repair (BER) pathway. In mammals, 8-oxoG is processed by the 8-oxoG-DNA-glycosylase I (OGG1), which biochemical mechanisms has been well characterised in vitro. However how and where this enzyme finds the modified base within the complex chromatin architecture is not yet understood. We show that upon induction of 8-oxoG, OGG1, together with at least two other proteins involved in BER, is recruited from a soluble fraction to chromatin. Formation kinetics of this patches correlates with 8-oxoG excision, suggesting a direct link between presence of this chromatin-associated complexes and 8-oxoG repair. More precisely, these repair patches are specifically directed to euchromatin regions, and completely excluded from heterochromatin regions. Inducing of artificial chromatin compaction results in a complete inhibition of the in vivo repair of 8-oxoG, probably by impeding the access of OGG1 to the lesion. Using OGG1 mutants, we show that OGG1 direct recognition of 8-oxoG did not trigger its re-localisation to the chromatin. We conclude that in response to the induction of oxidative DNA damage, the DNA glycosylase is actively recruited to regions of open chromatin allowing the access of the BER machinery to the lesions. (author)

  1. Novel organization of genes involved in prophage excision identified in the temperate lactococcal bacteriophage TP901-1

    DEFF Research Database (Denmark)

    Breuner, Anne; Brøndsted, Lone; Hammer, Karin

    1999-01-01

    In this work, the phage-encoded proteins involved in site-specific excision of the prophage genome of the temperate lactococcal bacteriophage TP901-1 were identified. The phage integrase is required for the process, and a low but significant frequency of excision is observed when the integrase...... of extended resolvases. Orf7 is a basic protein of 64 amino acids, and the corresponding gene (orf7) is the third gene in the early lytic operon. This location of an excisionase gene of a temperate bacteriophage has never been described before. The experiments are based on in vivo excision of specifically...... of genetic material based upon the upp gene (encoding uracil phosphoribosyltransferase) was designed, since upp mutants are resistant to fluorouracil. By using this system, frequencies of excision on the order of 10(-5) per cell could easily be measured. The described selection principle may be of general...

  2. The role of the PHP domain associated with DNA polymerase X from Thermus thermophilus HB8 in base excision repair.

    Science.gov (United States)

    Nakane, Shuhei; Nakagawa, Noriko; Kuramitsu, Seiki; Masui, Ryoji

    2012-11-01

    Base excision repair (BER) is one of the most commonly used DNA repair pathways involved in genome stability. X-family DNA polymerases (PolXs) play critical roles in BER, especially in filling single-nucleotide gaps. In addition to a polymerase core domain, bacterial PolXs have a polymerase and histidinol phosphatase (PHP) domain with phosphoesterase activity which is also required for BER. However, the role of the PHP domain of PolX in bacterial BER remains unresolved. We found that the PHP domain of Thermus thermophilus HB8 PolX (ttPolX) functions as two types of phosphoesterase in BER, including a 3'-phosphatase and an apurinic/apyrimidinic (AP) endonuclease. Experiments using T. thermophilus HB8 cell lysates revealed that the majority of the 3'-phosphatase and AP endonuclease activities are attributable to the another phosphoesterase in T. thermophilus HB8, endonuclease IV (ttEndoIV). However, ttPolX possesses significant 3'-phosphatase activity in ΔttendoIV cell lysate, indicating possible complementation. Our experiments also reveal that there are only two enzymes that display the 3'-phosphatase activity in the T. thermophilus HB8 cell, ttPolX and ttEndoIV. Furthermore, phenotypic analysis of ΔttpolX, ΔttendoIV, and ΔttpolX/ΔttendoIV using hydrogen peroxide and sodium nitrite supports the hypothesis that ttPolX functions as a backup for ttEndoIV in BER. Copyright © 2012 Elsevier B.V. All rights reserved.

  3. C. elegans lifespan extension by osmotic stress requires FUdR, base excision repair, FOXO, and sirtuins

    Science.gov (United States)

    Anderson, Edward N; Corkins, Mark E; Li, Jia-Cheng; Singh, Komudi; Parsons, Sadé; Tucey, Tim M; Sorkaç, Altar; Huang, Huiyan; Dimitriadi, Maria; Sinclair, David A

    2016-01-01

    Moderate stress can increase lifespan by hormesis, a beneficial low-level induction of stress response pathways. 5’-fluorodeoxyuridine (FUdR) is commonly used to sterilize Caenorhabditis elegans in aging experiments. However, FUdR alters lifespan in some genotypes and induces resistance to thermal and proteotoxic stress. We report that hypertonic stress in combination with FUdR treatment or inhibition of the FUdR target thymidylate synthase, TYMS-1, extends C. elegans lifespan by up to 30%. By contrast, in the absence of FUdR, hypertonic stress decreases lifespan. Adaptation to hypertonic stress requires diminished Notch signaling and loss of Notch co-ligands leads to lifespan extension only in combination with FUdR. Either FUdR treatment or TYMS-1 loss induced resistance to acute hypertonic stress, anoxia, and thermal stress. FUdR treatment increased expression of DAF-16 FOXO and the osmolyte biosynthesis enzyme GPDH-1. FUdR-induced hypertonic stress resistance was partially dependent on sirtuins and base excision repair (BER) pathways, while FUdR-induced lifespan extension under hypertonic stress conditions requires DAF-16, BER, and sirtuin function. Combined, these results demonstrate that FUdR, through inhibition of TYMS-1, activates stress response pathways in somatic tissues to confer hormetic resistance to acute and chronic stress. C. elegans lifespan studies using FUdR may need re-interpretation in light of this work. PMID:26854551

  4. Instability of CTG Repeats is Governed by the Position of a DNA Base Lesion through Base Excision Repair

    Science.gov (United States)

    Zhang, Zunzhen; Liu, Yuan

    2013-01-01

    Trinucleotide repeat (TNR) expansions and deletions are associated with human neurodegeneration and cancer. However, their underlying mechanisms remain to be elucidated. Recent studies have demonstrated that CAG repeat expansions can be initiated by oxidative DNA base damage and fulfilled by base excision repair (BER), suggesting active roles for oxidative DNA damage and BER in TNR instability. Here, we provide the first evidence that oxidative DNA damage can induce CTG repeat deletions along with limited expansions in human cells. Biochemical characterization of BER in the context of (CTG)20 repeats further revealed that repeat instability correlated with the position of a base lesion in the repeat tract. A lesion located at the 5′-end of CTG repeats resulted in expansion, whereas a lesion located either in the middle or the 3′-end of the repeats led to deletions only. The positioning effects appeared to be determined by the formation of hairpins at various locations on the template and the damaged strands that were bypassed by DNA polymerase β and processed by flap endonuclease 1 with different efficiency. Our study indicates that the position of a DNA base lesion governs whether TNR is expanded or deleted through BER. PMID:23468897

  5. Role of the DNA Base Excision Repair Protein, APE1 in Cisplatin, Oxaliplatin, or Carboplatin Induced Sensory Neuropathy

    Science.gov (United States)

    Kelley, Mark R.; Jiang, Yanlin; Guo, Chunlu; Reed, April; Meng, Hongdi; Vasko, Michael R.

    2014-01-01

    Although chemotherapy-induced peripheral neuropathy (CIPN) is a dose-limiting side effect of platinum drugs, the mechanisms of this toxicity remain unknown. Previous work in our laboratory suggests that cisplatin-induced CIPN is secondary to DNA damage which is susceptible to base excision repair (BER). To further examine this hypothesis, we studied the effects of cisplatin, oxaliplatin, and carboplatin on cell survival, DNA damage, ROS production, and functional endpoints in rat sensory neurons in culture in the absence or presence of reduced expression of the BER protein AP endonuclease/redox factor-1 (APE1). Using an in situ model of peptidergic sensory neuron function, we examined the effects of the platinum drugs on hind limb capsaicin-evoked vasodilatation. Exposing sensory neurons in culture to the three platinum drugs caused a concentration-dependent increase in apoptosis and cell death, although the concentrations of carboplatin were 10 fold higher than cisplatin. As previously observed with cisplatin, oxaliplatin and carboplatin also increased DNA damage as indicated by an increase in phospho-H2AX and reduced the capsaicin-evoked release of CGRP from neuronal cultures. Both cisplatin and oxaliplatin increased the production of ROS as well as 8-oxoguanine DNA adduct levels, whereas carboplatin did not. Reducing levels of APE1 in neuronal cultures augmented the cisplatin and oxaliplatin induced toxicity, but did not alter the effects of carboplatin. Using an in vivo model, systemic injection of cisplatin (3 mg/kg), oxaliplatin (3 mg/kg), or carboplatin (30 mg/kg) once a week for three weeks caused a decrease in capsaicin-evoked vasodilatation, which was delayed in onset. The effects of cisplatin on capsaicin-evoked vasodilatation were attenuated by chronic administration of E3330, a redox inhibitor of APE1 that serendipitously enhances APE1 DNA repair activity in sensory neurons. These outcomes support the importance of the BER pathway, and particularly APE

  6. Polymorphisms in human DNA repair genes and head and neck ...

    Indian Academy of Sciences (India)

    Genetic polymorphisms in some DNA repair proteins are associated with a number of malignant transformations like head and neck squamous cell carcinoma (HNSCC). Xeroderma pigmentosum group D (XPD) and X-ray repair cross-complementing proteins 1 (XRCC1) and 3 (XRCC3) genes are involved in DNA repair ...

  7. Graphene oxide nanosheets induce DNA damage and activate the base excision repair (BER) signaling pathway both in vitro and in vivo.

    Science.gov (United States)

    Lu, Chun-Jiao; Jiang, Xue-Feng; Junaid, Muhammad; Ma, Yan-Bo; Jia, Pan-Pan; Wang, Hua-Bin; Pei, De-Sheng

    2017-10-01

    Graphene oxide (GO) has widespread concerns in the fields of biological sciences and medical applications. Currently, studies have reported that excessive GO exposure can cause cellular DNA damage through reactive oxygen species (ROS) generation. However, DNA damage mediated response of the base excision repair (BER) pathway due to GO exposure is not elucidated yet. Therefore, we exposed HEK293T cells and zebrafish embryos to different concentrations of GO for 24 h, and transcriptional profiles of BER pathway genes, DNA damage, and cell viability were analyzed both in vitro and in vivo. Moreover, the deformation of HEK293T cells before and after GO exposure was also investigated using atomic force microscopy (AFM) to identify the physical changes occurred in the cells' structure. CCK-8 and Comet assay revealed the significant decrease in cell viability and increase in DNA damage in HEK293T cells at higher GO doses (25 and 50 μg/mL). Among the investigated genetic markers in HEK293T cells, BER pathway genes (APEX1, OGG1, CREB1, UNG) were significantly up-regulated upon exposure to higher GO dose (50 μg/mL), however, low exposure concentration (5, 25 μg/mL) failed to induce significant genetic induction except for CREB1 at 25 μg/mL. Additionally, the viscosity of HEK293T cells decreased upon GO exposure. In zebrafish, the results of up-regulated gene expressions (apex1, ogg1, polb, creb1) were consistent with those in the HEK293T cells. Taken all together, the exposure to elevated GO concentration could cause DNA damage to HEK293T cells and zebrafish embryos; BER pathway could be proposed as the possible inner response mechanism. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Evidence that the Rad1 and Rad10 proteins of Saccharomyces cerevisiae participate as a complex in nucleotide excision repair of UV radiation damage.

    OpenAIRE

    Siede, W.; Friedberg, A S; Friedberg, E C

    1993-01-01

    A newly characterized rad1 missense mutation (rad1-20) in the yeast Saccharomyces cerevisiae maps to a region of the Rad1 polypeptide known to be required for Rad1-Rad10 complex formation. The UV sensitivity of the rad1-20 mutant can be partially and specifically corrected by overexpression of wild-type Rad10 protein. These results suggest that complex formation between the Rad1 and Rad10 proteins is required for nucleotide excision repair.

  9. First Reported Patient with Human ERCC1 Deficiency Has Cerebro-Oculo-Facio-Skeletal Syndrome with a Mild Defect in Nucleotide Excision Repair and Severe Developmental Failure

    OpenAIRE

    Jaspers, Nicolaas G.J.; Raams, Anja; Silengo, Margherita Cirillo; Wijgers, Nils; Niedernhofer, Laura J; Robinson, Andria Rasile; Giglia-Mari, Giuseppina; Hoogstraten, Deborah; Kleijer, Wim J.; Hoeijmakers, Jan H.J.; Vermeulen, Wim

    2007-01-01

    Nucleotide excision repair (NER) is a genome caretaker mechanism responsible for removing helix-distorting DNA lesions, most notably ultraviolet photodimers. Inherited defects in NER result in profound photosensitivity and the cancer-prone syndrome xeroderma pigmentosum (XP) or two progeroid syndromes: Cockayne and trichothiodystrophy syndromes. The heterodimer ERCC1-XPF is one of two endonucleases required for NER. Mutations in XPF are associated with mild XP and rarely with progeria. Mutati...

  10. The NR4A2 nuclear receptor is recruited to novel nuclear foci in response to UV irradiation and participates in nucleotide excision repair.

    Directory of Open Access Journals (Sweden)

    Kasturee Jagirdar

    Full Text Available Ultraviolet radiation (UVR is one of the most common mutagens encountered by humans and induces the formation of cyclobutane pyrimidine dimers (CPDs and pyrimidine-(6-4-pyrimidone photoproduct (6-4PP lesions in the genomic DNA. To prevent the accumulation of deleterious mutations these lesions must be efficiently repaired, primarily by nucleotide excision repair. We have previously demonstrated that the NR4A family of nuclear receptors are crucial mediators of the DNA repair function of the MC1R signalling pathway in melanocytes. Here we explore the role of the NR4A2 protein in the DNA repair process further. Using EYFP tagged-NR4A2 we have demonstrated a UVR induced recruitment to distinct nuclear foci where they co-localise with known DNA repair proteins. We reveal that the N-terminal domain of the receptor is required for this translocation and identify a role for p38 and PARP signalling in this process. Moreover disruption of the functional integrity of the Ligand Binding Domain of the receptor by deleting the terminal helix 12 effectively blocks co-localisation of the receptor with DNA repair factors. Restored co-localisation of the mutant receptor with DNA repair proteins in the presence of a Histone Deacetylase Inhibitor suggests that impaired chromatin accessibility underpins the mis-localisation observed. Finally NR4A2 over-expression facilitated a more efficient clearance of UVR induced CPD and 6-4PP lesions. Taken together these data uncover a novel role for the NR4A nuclear receptors as direct facilitators of nucleotide excision repair.

  11. Endonuclease IV Is the Main Base Excision Repair Enzyme Involved in DNA Damage Induced by UVA Radiation and Stannous Chloride

    Directory of Open Access Journals (Sweden)

    Ellen S. Motta

    2010-01-01

    Full Text Available Stannous chloride (SnCl2 and UVA induce DNA lesions through ROS. The aim of this work was to study the toxicity induced by UVA preillumination, followed by SnCl2 treatment. E. coli BER mutants were used to identify genes which could play a role in DNA lesion repair generated by these agents. The survival assays showed (i The nfo mutant was the most sensitive to SnCl2; (ii lethal synergistic effect was observed after UVA pre-illumination, plus SnCl2 incubation, the nfo mutant being the most sensitive; (iii wild type and nfo mutants, transformed with pBW21 plasmid (nfo+ had their survival increased following treatments. The alkaline agarose gel electrophoresis assays pointed that (i UVA induced DNA breaks and fpg mutant was the most sensitive; (ii SnCl2-induced DNA strand breaks were higher than those from UVA and nfo mutant had the slowest repair kinetics; (iii UVA+SnCl2 promoted an increase in DNA breaks than SnCl2 and, again, nfo mutant displayed the slowest repair kinetics. In summary, Nfo protects E. coli cells against damage induced by SnCl2 and UVA+ SnCl2.

  12. Rapid assessment of repair of ultraviolet DNA damage with a modified host-cell reactivation assay using a luciferase reporter gene and correlation with polymorphisms of DNA repair genes in normal human lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Qiao Yawei; Spitz, Margaret R.; Guo Zhaozheng; Hadeyati, Mohammad; Grossman, Lawrence; Kraemer, Kenneth H.; Wei Qingyi

    2002-11-30

    As DNA repair plays an important role in genetic susceptibility to cancer, assessment of the DNA repair phenotype is critical for molecular epidemiological studies of cancer. In this report, we compared use of the luciferase (luc) reporter gene in a host-cell reactivation (HCR) (LUC) assay of repair of ultraviolet (UV) damage to DNA to use of the chloramphenicol (cat) gene-based HCR (CAT) assay we used previously for case-control studies. We performed both the assays on cryopreserved lymphocytes from 102 healthy non-Hispanic white subjects. There was a close correlation between DNA repair capacity (DRC) as measured by the LUC and CAT assays. Although these two assays had similar variation, the LUC assay was faster and more sensitive. We also analyzed the relationship between DRC and the subjects' previously determined genotypes for four polymorphisms of two nucleotide-excision repair (NER) genes (in intron 9 of xeroderma pigmentosum (XP) C and exons 6, 10 and 23 of XPD) and one polymorphism of a base-excision repair gene in exon 10 of X-ray complementing group 1 (XRCC1). The DRC was significantly lower in subjects homozygous for one or more polymorphisms of the two NER genes than in subjects with other genotypes (P=0.010). In contrast, the polymorphic XRCC1 allele had no significant effect on DRC. These results suggest that the post-UV LUC assay measures NER phenotype and that polymorphisms of XPC and XPD genes modulate DRC. For population studies of the DNA repair phenotype, many samples need to be evaluated, and so the LUC assay has several advantages over the CAT assay: the LUC assay was more sensitive, had less variation, was not radioactive, was easier to perform, and required fewer cryopreserved cells. These features make the LUC-based HCR assay suitable for molecular epidemiological studies.

  13. Poly(ADP-ribose) polymerase 1 regulates activity of DNA polymerase {beta} in long patch base excision repair

    Energy Technology Data Exchange (ETDEWEB)

    Sukhanova, Maria; Khodyreva, Svetlana [Institute of Chemical Biology and Fundamental Medicine SB RAS, Novosibirsk (Russian Federation); Lavrik, Olga, E-mail: lavrik@niboch.nsc.ru [Institute of Chemical Biology and Fundamental Medicine SB RAS, Novosibirsk (Russian Federation)

    2010-03-01

    Poly(ADP-ribose)polymerase 1 (PARP1), functioning as DNA nick-sensor, interacts with base excision repair (BER) DNA intermediates containing single-strand breaks. When bound to DNA breaks, PARP1 catalyzes synthesis of poly(ADP-ribose) covalently attached to itself and some nuclear proteins. Autopoly(ADP-ribosyl)ation of PARP1 facilitates its dissociation from DNA breaks and is considered as a factor regulating DNA repair. In the study, using system reconstituted from purified BER proteins, bovine testis nuclear extract and model BER DNA intermediates, we examined the influence of PARP1 and its autopoly(ADP-ribosyl)ation on DNA polymerase {beta} (Pol {beta})-mediated long patch (LP) BER DNA synthesis that is accomplished through a cooperation between Pol {beta} and apurinic/apyrimidinic endonuclease1 (APE1) or flap endonuclease 1 (FEN1) and gap-filling activity of Pol {beta}. PARP1 upon interaction with nicked LP BER DNA intermediated, formed after gap-filling, was shown to suppress the subsequent steps in LP pathway. PARP1 interferes with APE1-dependent stimulation of DNA synthesis by Pol {beta} via strand-displacement mechanism. PARP1 also represses Pol {beta}/FEN1-mediated LP BER DNA synthesis via a 'gap translation' mechanism inhibiting FEN1 activity on the nicked DNA intermediate. Poly(ADP-ribosyl)ation of PARP1 abolishes its inhibitory influence on LP BER DNA synthesis catalyzed by Pol {beta} both via APE1-mediated strand-displacement and FEN1-mediated 'gap translation' mechanism. Thus PARP1 may act as a negative regulator of Pol {beta} activity in LP BER pathway and poly(ADP-ribosyl)ation of PARP1 seems to play a critical role in enablement of Pol {beta}-mediated DNA synthesis in this process. In contrast, interaction of PARP1 with one nucleotide gapped DNA mimicking the intermediate of short patch (SP) BER slightly inhibits the gap-filling activity of Pol {beta} and the overall efficiency of SP BER is practically unaffected by PARP1. Thus

  14. Allele and Genotype Distributions of DNA Repair Gene Polymorphisms in South Indian Healthy Population

    Directory of Open Access Journals (Sweden)

    Katiboina Srinivasa Rao

    2014-01-01

    Full Text Available Various DNA repair pathways protect the structural and chemical integrity of the human genome from environmental and endogenous threats. Polymorphisms of genes encoding the proteins involved in DNA repair have been found to be associated with cancer risk and chemotherapeutic response. In this study, we aim to establish the normative frequencies of DNA repair genes in South Indian healthy population and compare with HapMap populations. Genotyping was done on 128 healthy volunteers from South India, and the allele and genotype distributions were established. The minor allele frequency of Xeroderma pigmentosum group A ( XPA G23A, Excision repair cross-complementing 2 ( ERCC2 /Xeroderma pigmentosum group D ( XPD Lys751Gln, Xeroderma pigmentosum group G ( XPG His46His, XPG Asp1104His, and X-ray repair cross-complementing group 1 ( XRCC1 Arg399Gln polymorphisms were 49.2%, 36.3%, 48.0%, 23.0%, and 34.0% respectively. Ethnic variations were observed in the frequency distribution of these polymorphisms between the South Indians and other HapMap populations. The present work forms the groundwork for cancer association studies and biomarker identification for treatment response and prognosis.

  15. Epidermal Growth Factor Receptor, Excision-Repair Cross-Complementation Group 1 Protein, and Thymidylate Synthase Expression in Penile Cancer.

    Science.gov (United States)

    Dorff, Tanya B; Schuckman, Anne K; Schwartz, Rachel; Rashad, Sadaf; Bulbul, Ajaz; Cai, Jie; Pinski, Jacek; Ma, Yanling; Danenberg, Kathleen; Skinner, Eila; Quinn, David I

    2016-10-01

    To describe the expression of tissue epidermal growth factor receptor (EGFR), excision-repair cross-complementation group 1 protein (ERCC1), and thymidylate synthase (TS) in patients with penile cancer and explore their association with stage and outcome. A total of 52 patients with penile squamous cell cancer who were treated at the University of Southern California from 1995 to 2010 were identified. Paraffin-embedded tissue underwent mRNA quantitation and immunohistochemistry for expression of EGFR, ERCC1, and TS. KRAS mutations were evaluated using polymerase chain reaction-based sequencing. EGFR overexpression was common by mRNA (median, 5.09; range, 1.92-104.5) and immunohistochemistry. EGFR expression > 7 was associated with advanced stage and poor differentiation (P = .01 and .034 respectively) but not with survival in multivariate analysis. ERCC1 mRNA expression was a median of 0.65 (range, 0.21-1.87). TS expression was a median of 1.88 (range, 0.54-6.47). ERCC1 and TS expression were not associated with grade, stage, or survival. There were no KRAS mutations identified. A total of 17 men received chemotherapy; 8 (47%) had an objective response, including 1 with a pathologic complete response. There was a trend for lower expression of EGFR corresponding to a higher likelihood of response (response rate [RR]) to chemotherapy: 67% RR in EGFR mRNA  7 (P = .31). High expression of EGFR mRNA in squamous cell carcinoma of the penis is associated with advanced stage and poor differentiation, but not survival. In our small heterogeneous subset, molecular marker expression did not show a correlation with the likelihood of chemotherapy response. A prospective evaluation of the role of the EGFR pathway and its regulatory environment in penile cancer is warranted. Given the rarity of this cancer, collaborative prospective cohort evaluations and trials need to be encouraged. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. Cytogenetic Response to Ionizing Radiation Exposure in Human Fibroblasts with Suppressed Expression of Non-DSB Repair Genes

    Science.gov (United States)

    Zhang, Ye; Rohde, Larry H.; Emami, Kamal; Hammond, Dianne; Mehta, Satish K.; Jeevarajan, Antony S.; Pierson, Duane L.; Wu, Honglu

    2009-01-01

    Changes of gene expression profile are one of the most important biological responses in living cells after ionizing radiation (IR) exposure. Although some studies have shown that genes up-regulated by IR may play important roles in DNA damage repair, the relationship between the regulation of gene expression by IR, particularly genes not known for their roles in double-strand break (DSB) repair, and its impact on cytogenetic responses has not been well studied. The purpose of this study is to identify new roles of IR inducible genes in radiation-induced chromosome aberrations and micronuclei formation. In the study, the expression of 25 genes selected on the basis of their transcriptional changes in response to IR was individually knocked down by small interfering RNA in human fibroblast cells. Frequencies of micronuclei (MN) formation and chromosome aberrations were measured to determine the efficiency of cytogenetic repair, and the fraction of bi-nucleated cells in the MN analysis was used as a marker for cell cycle progression. In response to gamma radiation, the formation of MN was significantly increased by suppressed expression of five genes: Ku70 (DSB repair pathway), XPA (nucleotide excision repair pathway), RPA1 (mismatch repair pathway), RAD17 and RBBP8 (cell cycle control). Knocked-down expression of four genes (MRE11A, RAD51 in the DSB pathway, SESN1, and SUMO1) significantly inhibited cell cycle progression, possibly because of severe impairment of DNA damage repair. Moreover, decreased XPA, p21, or MLH1 expression resulted in both significantly enhanced cell cycle progression and increased yields of chromosome aberrations, indicating that these gene products modulate both cell cycle control and DNA damage repair. Nine of these eleven genes, whose knock-down expression affected cytogenetic repair, were up-regulated in cells exposed to gamma radiation, suggesting that genes transcriptionally modulated by IR were critical to regulate IR

  17. DNA damage and gene therapy of xeroderma pigmentosum, a human DNA repair-deficient disease

    Energy Technology Data Exchange (ETDEWEB)

    Dupuy, Aurélie [Laboratory of Genetic Instability and Oncogenesis UMR8200CNRS, Institut Gustave Roussy and University Paris-Sud, Villejuif (France); Sarasin, Alain, E-mail: alain.sarasin@gustaveroussy.fr [Laboratory of Genetic Instability and Oncogenesis UMR8200CNRS, Institut Gustave Roussy and University Paris-Sud, Villejuif (France); Service de Génétique, Institut Gustave Roussy (France)

    2015-06-15

    Graphical abstract: - Highlights: • Full correction of mutation in the XPC gene by engineered nucleases. • Meganucleases and TALENs are inhibited by 5-MeC for inducing double strand breaks. • Gene therapy of XP cells is possible using homologous recombination for DSB repair. - Abstract: Xeroderma pigmentosum (XP) is a genetic disease characterized by hypersensitivity to ultra-violet and a very high risk of skin cancer induction on exposed body sites. This syndrome is caused by germinal mutations on nucleotide excision repair genes. No cure is available for these patients except a complete protection from all types of UV radiations. We reviewed the various techniques to complement or to correct the genetic defect in XP cells. We, particularly, developed the correction of XP-C skin cells using the fidelity of the homologous recombination pathway during repair of double-strand break (DSB) in the presence of XPC wild type sequences. We used engineered nucleases (meganuclease or TALE nuclease) to induce a DSB located at 90 bp of the mutation to be corrected. Expression of specific TALE nuclease in the presence of a repair matrix containing a long stretch of homologous wild type XPC sequences allowed us a successful gene correction of the original TG deletion found in numerous North African XP patients. Some engineered nucleases are sensitive to epigenetic modifications, such as cytosine methylation. In case of methylated sequences to be corrected, modified nucleases or demethylation of the whole genome should be envisaged. Overall, we showed that specifically-designed TALE-nuclease allowed us to correct a 2 bp deletion in the XPC gene leading to patient's cells proficient for DNA repair and showing normal UV-sensitivity. The corrected gene is still in the same position in the human genome and under the regulation of its physiological promoter. This result is a first step toward gene therapy in XP patients.

  18. The repair of melphalan-induced DNA adducts in the transcribed strand of active genes is subject to a strong polarity effect.

    OpenAIRE

    Episkopou, Hara; Kyrtopoulos, Soterios A.; Sfikakis, Petros P.; Meletios A Dimopoulos; Souliotis, Vassilis L

    2011-01-01

    To investigate the mechanisms of the therapeutic action and drug resistance to the nitrogen mustard melphalan, melphalan-induced DNA damage repair and chromatin structure were examined along the p53, N-ras and d-globin gene loci in cells carrying different repair activities. In nucleotide excision repair-deficient XP-A cells, similar levels of adducts were found in all fragments examined, indicating uniform distribution of DNA damage. In both, repair-proficient CS-B and XP-C cells, faster rep...

  19. Developmentally-Regulated Excision of the SPβ Prophage Reconstitutes a Gene Required for Spore Envelope Maturation in Bacillus subtilis

    Science.gov (United States)

    Abe, Kimihiro; Kawano, Yuta; Iwamoto, Keito; Arai, Kenji; Maruyama, Yuki; Eichenberger, Patrick; Sato, Tsutomu

    2014-01-01

    Temperate phages infect bacteria by injecting their DNA into bacterial cells, where it becomes incorporated into the host genome as a prophage. In the genome of Bacillus subtilis 168, an active prophage, SPβ, is inserted into a polysaccharide synthesis gene, spsM. Here, we show that a rearrangement occurs during sporulation to reconstitute a functional composite spsM gene by precise excision of SPβ from the chromosome. SPβ excision requires a putative site-specific recombinase, SprA, and an accessory protein, SprB. A minimized SPβ, where all the SPβ genes were deleted, except sprA and sprB, retained the SPβ excision activity during sporulation, demonstrating that sprA and sprB are necessary and sufficient for the excision. While expression of sprA was observed during vegetative growth, sprB was induced during sporulation and upon mitomycin C treatment, which triggers the phage lytic cycle. We also demonstrated that overexpression of sprB (but not of sprA) resulted in SPβ prophage excision without triggering the lytic cycle. These results suggest that sprB is the factor that controls the timing of phage excision. Furthermore, we provide evidence that spsM is essential for the addition of polysaccharides to the spore envelope. The presence of polysaccharides on the spore surface renders the spore hydrophilic in water. This property may be beneficial in allowing spores to disperse in natural environments via water flow. A similar rearrangement occurs in Bacillus amyloliquefaciens FZB42, where a SPβ-like element is excised during sporulation to reconstitute a polysaccharide synthesis gene, suggesting that this type of gene rearrangement is common in spore-forming bacteria because it can be spread by phage infection. PMID:25299644

  20. Base excision repair of chemotherapeutically-induced alkylated DNA damage predominantly causes contractions of expanded GAA repeats associated with Friedreich's ataxia.

    Directory of Open Access Journals (Sweden)

    Yanhao Lai

    Full Text Available Expansion of GAA·TTC repeats within the first intron of the frataxin gene is the cause of Friedreich's ataxia (FRDA, an autosomal recessive neurodegenerative disorder. However, no effective treatment for the disease has been developed as yet. In this study, we explored a possibility of shortening expanded GAA repeats associated with FRDA through chemotherapeutically-induced DNA base lesions and subsequent base excision repair (BER. We provide the first evidence that alkylated DNA damage induced by temozolomide, a chemotherapeutic DNA damaging agent can induce massive GAA repeat contractions/deletions, but only limited expansions in FRDA patient lymphoblasts. We showed that temozolomide-induced GAA repeat instability was mediated by BER. Further characterization of BER of an abasic site in the context of (GAA20 repeats indicates that the lesion mainly resulted in a large deletion of 8 repeats along with small expansions. This was because temozolomide-induced single-stranded breaks initially led to DNA slippage and the formation of a small GAA repeat loop in the upstream region of the damaged strand and a small TTC loop on the template strand. This allowed limited pol β DNA synthesis and the formation of a short 5'-GAA repeat flap that was cleaved by FEN1, thereby leading to small repeat expansions. At a later stage of BER, the small template loop expanded into a large template loop that resulted in the formation of a long 5'-GAA repeat flap. Pol β then performed limited DNA synthesis to bypass the loop, and FEN1 removed the long repeat flap ultimately causing a large repeat deletion. Our study indicates that chemotherapeutically-induced alkylated DNA damage can induce large contractions/deletions of expanded GAA repeats through BER in FRDA patient cells. This further suggests the potential of developing chemotherapeutic alkylating agents to shorten expanded GAA repeats for treatment of FRDA.

  1. Involvement of two endonuclease III homologs in the base excision repair pathway for the processing of DNA alkylation damage in Saccharomyces cerevisiae.

    Science.gov (United States)

    Hanna, Michelle; Chow, Barbara L; Morey, Natalie J; Jinks-Robertson, Sue; Doetsch, Paul W; Xiao, Wei

    2004-01-05

    DNA base excision repair (BER) is initiated by DNA glycosylases that recognize and remove damaged bases. The phosphate backbone adjacent to the resulting apurinic/apyrimidinic (AP) site is then cleaved by an AP endonuclease or glycosylase-associated AP lyase to invoke subsequent BER steps. We have used a genetic approach in Saccharomyces cerevisiae to determine whether or not AP sites are blocks to DNA replication and the biological consequences if AP sites persist in the genome. We previously reported that yeast cells deficient in the two AP endonucleases (apn1 apn2 double mutant) are extremely sensitive to killing by a model DNA alkylating agent methyl methanesulfonate (MMS) and that this sensitivity can be reduced by deleting the MAG1 3-methyladenine DNA glycosylase gene. Here we report that in the absence of the AP endonucleases, deletion of two Escherichia coli endonuclease III homologs, NTG1 and NTG2, partially suppresses MMS-induced killing, which indicates that the AP lyase products are deleterious unless they are further processed by an AP endonuclease. The severe MMS sensitivity seen in AP endonuclease deficient strains can also be rescued by treatment of cells with the AP lyase inhibitor methoxyamine, which suggests that the product of AP lyase action on an AP site is indeed an extremely toxic lesion. In addition to the AP endonuclease interactions, deletion of NTG1 and NTG2 enhances the mag1 mutant sensitivity to MMS, whereas overexpression of MAG1 in either the ntg1 or ntg2 mutant severely affects cell growth. These results help to delineate alkylation base lesion flow within the BER pathway.

  2. Base excision repair of chemotherapeutically-induced alkylated DNA damage predominantly causes contractions of expanded GAA repeats associated with Friedreich's ataxia.

    Science.gov (United States)

    Lai, Yanhao; Beaver, Jill M; Lorente, Karla; Melo, Jonathan; Ramjagsingh, Shyama; Agoulnik, Irina U; Zhang, Zunzhen; Liu, Yuan

    2014-01-01

    Expansion of GAA·TTC repeats within the first intron of the frataxin gene is the cause of Friedreich's ataxia (FRDA), an autosomal recessive neurodegenerative disorder. However, no effective treatment for the disease has been developed as yet. In this study, we explored a possibility of shortening expanded GAA repeats associated with FRDA through chemotherapeutically-induced DNA base lesions and subsequent base excision repair (BER). We provide the first evidence that alkylated DNA damage induced by temozolomide, a chemotherapeutic DNA damaging agent can induce massive GAA repeat contractions/deletions, but only limited expansions in FRDA patient lymphoblasts. We showed that temozolomide-induced GAA repeat instability was mediated by BER. Further characterization of BER of an abasic site in the context of (GAA)20 repeats indicates that the lesion mainly resulted in a large deletion of 8 repeats along with small expansions. This was because temozolomide-induced single-stranded breaks initially led to DNA slippage and the formation of a small GAA repeat loop in the upstream region of the damaged strand and a small TTC loop on the template strand. This allowed limited pol β DNA synthesis and the formation of a short 5'-GAA repeat flap that was cleaved by FEN1, thereby leading to small repeat expansions. At a later stage of BER, the small template loop expanded into a large template loop that resulted in the formation of a long 5'-GAA repeat flap. Pol β then performed limited DNA synthesis to bypass the loop, and FEN1 removed the long repeat flap ultimately causing a large repeat deletion. Our study indicates that chemotherapeutically-induced alkylated DNA damage can induce large contractions/deletions of expanded GAA repeats through BER in FRDA patient cells. This further suggests the potential of developing chemotherapeutic alkylating agents to shorten expanded GAA repeats for treatment of FRDA.

  3. Distant neighbor base sequence context effects in human nucleotide excision repair of a benzo[a]pyrene-derived DNA lesion.

    Science.gov (United States)

    Cai, Yuqin; Kropachev, Konstantin; Xu, Rong; Tang, Yijin; Kolbanovskii, Marina; Kolbanovskii, Alexander; Amin, Shantu; Patel, Dinshaw J; Broyde, Suse; Geacintov, Nicholas E

    2010-06-11

    The effects of non-nearest base sequences, beyond the nucleotides flanking a DNA lesion on either side, on nucleotide excision repair (NER) in extracts from human cells were investigated. We constructed two duplexes containing the same minor groove-aligned 10S (+)-trans-anti-B[a]P-N(2)-dG (G*) DNA adduct, derived from the environmental carcinogen benzo[a]pyrene (B[a]P): 5'-C-C-A-T-C-G*-C-T-A-C-C-3' (CG*C-I), and 5'-C-A-C3-A4-C5-G*-C-A-C-A-C-3' (CG*C-II). We used polyacrylamide gel electrophoresis to compare the extent of DNA bending, and molecular dynamics simulations to analyze the structural characteristics of these two DNA duplexes. The NER efficiencies are 1.6(+/-0.2)-fold greater in the case of the CG*C-II than the CG*C-I sequence context in 135-mer duplexes. Gel electrophoresis and self-ligation circularization experiments revealed that the CG*C-II duplex is more bent than the CG*C-I duplex, while molecular dynamics simulations showed that the unique -C3-A4-C5- segment in the CG*C-II duplex plays a key role. The presence of a minor groove-positioned guanine amino group, the Watson-Crick partner to C3, acts as a wedge; facilitated by a highly deformable local -C3-A4- base step, this amino group allows the B[a]P ring system to produce a more enlarged minor groove in CG*C-II than in CG*C-I, as well as a local untwisting and enlarged and flexible Roll only in the CG*C-II sequence. These structural properties fit well with our earlier findings that in the case of the family of minor groove 10S (+)-trans-anti-B[a]P-N(2)-dG lesions, flexible bends and enlarged minor groove widths constitute NER recognition signals, and extend our understanding of sequence context effects on NER to the neighbors that are distant to the lesion. Copyright 2010 Elsevier Ltd. All rights reserved.

  4. Recognition of Damaged DNA for Nucleotide Excision Repair: A Correlated Motion Mechanism with a Mismatched cis-syn Thymine Dimer Lesion.

    Science.gov (United States)

    Mu, Hong; Geacintov, Nicholas E; Zhang, Yingkai; Broyde, Suse

    2015-09-01

    Mammalian global genomic nucleotide excision repair requires lesion recognition by XPC, whose detailed binding mechanism remains to be elucidated. Here we have delineated the dynamic molecular pathway and energetics of lesion-specific and productive binding by the Rad4/yeast XPC lesion recognition factor, as it forms the open complex [Min, J. H., and Pavletich, N. P. (2007) Nature 449, 570-575; Chen, X., et al. (2015) Nat. Commun. 6, 5849] that is required for excision. We investigated extensively a cis-syn cyclobutane pyrimidine dimer in mismatched duplex DNA, using high-level computational approaches. Our results delineate a preferred correlated motion mechanism, which provides for the first time an atomistic description of the sequence of events as Rad4 productively binds to the damaged DNA.

  5. Faulty DNA-polymerase {delta}/{epsilon}-mediated excision-repair in response to gamma-radiation or ultraviolet-light in P53-deficient fibroblast strains from affected members of a cancer-prone family with Li-Fraumeni syndrome

    Energy Technology Data Exchange (ETDEWEB)

    Mirzayans, R.; Enns, L.; Dietrich, K.; Barley, R.D.C.; Paterson, M.C. [Alberta Univ., Edmonton, AB (Canada). Cross Cancer Inst.]|[Alberta Univ., Edmonton, AB (Canada). Dept. of Oncology]|[Alberta Univ., Edmonton, AB (Canada). Dept. of Biological Science

    1996-04-01

    Dermal fibroblast strains cultured from affected members of a cancer-prone family with Li-Fraumeni syndrome (LFS) harbor a point mutation in one allele of the p53 tumor suppressor gene, resulting in loss of normal p53-deficient strains to carry out the long-patch mode of excision repair, mediated by DNA polymerases delta and epsilon, after exposure to Co-60 gamma radiation or far ultraviolet (UV) (chiefly 254 mm) light. Repair was monitored by incubation of the irradiated cultures in the presence of aphidicolin (ape) or 1-beta-D-arabinofuranosylcytosine (araC), each a specific inhibitor of long-patch repair, followed by measurement of drug-induced DNA strand breaks (reflecting non-ligated strand incision events) by alkaline surcrose velocity sedimentation. The LFS strains displayed deficient repair capacity in response to both gamma rays and UV light. The repair anomaly in UV-irradiated LFS cultures was manifested not only in the overall genome, but also in the transcriptionally active, preferentially repaired c-myc gene. Using autoradiography we also assessed unscheduled DNA synthesis (UDS) after UV irradiation and found this conventional measure of repair replication to be deficient in LFS strains. Moreover, both ape and araC decreased the level of UV-induced UDS by similar to 75% in normal cells, but each had only a marginal effect on LFS cells. We further demonstrated that the LFS strains are impaired in the recovery of both RNA and replicative DNA syntheses after UV treatment, two molecular anomalies of the DNA repair deficiency disorders xeroderma pigmentosum and Cockayne`s syndrome. Together these results imply a critical role for wild-type p53 protein in DNA polymerase delta/epsilon-mediated excision repair, both the mechanism operating on the entire genome and that acting on expressed genes. (Author).

  6. Yeast DNA-repair gene RAD14 encodes a zinc metalloprotein with affinity for ultraviolet-damaged DNA.

    Science.gov (United States)

    Guzder, S N; Sung, P; Prakash, L; Prakash, S

    1993-06-15

    Xeroderma pigmentosum (XP) patients suffer from a high incidence of skin cancers due to a defect in excision repair of UV light-damaged DNA. Of the seven XP complementation groups, A-G, group A represents a severe and frequent form of the disease. The Saccharomyces cerevisiae RAD14 gene is a homolog of the XP-A correcting (XPAC) gene. Like XP-A cells, rad14-null mutants are defective in the incision step of excision repair of UV-damaged DNA. We have purified RAD14 protein to homogeneity from extract of a yeast strain genetically tailored to overexpress RAD14. As determined by atomic emission spectroscopy, RAD14 contains one zinc atom. We also show in vitro that RAD14 binds zinc but does not bind other divalent metal ions. In DNA mobility-shift assays, RAD14 binds specifically to UV-damaged DNA. Removal of cyclobutane pyrimidine dimers from damaged DNA by enzymatic photoreactivation has no effect on binding, strongly suggesting that RAD14 recognizes pyrimidine(6-4)pyrimidone photoproduct sites. These findings indicate that RAD14 functions in damage recognition during excision repair.

  7. Sensitivity of excision repair in normal human, xeroderma pigmentosum variant and Cockayne's syndrome fibroblasts to inhibition by cytosine arabinoside

    Energy Technology Data Exchange (ETDEWEB)

    Cleaver, J.E.

    1981-08-01

    Inhibition of the gap-filling, polymerizing step of excision repair by 1-..beta..-D-arabinofuranosylcytosine (ara-C) after irradiation with ultraviolet light in human diploid fibroblasts resulted in the formation of persistent DNA strand breaks in G/sub 1/, G/sub 2/, and plateau phase cells, but not in S phase cells. Addition of hydroxyurea to ara-C resulted in partial inhibition of repair in S phase cells. These observations can be explained either in terms of changing roles in repair for different DNA polymerases throughout the cell cycle or by the presence of a pool of deoxycytidine nucleotides during S phase equivalent to an external source of deoxycytidine at 50 ..mu..M concentration. A similar concentration dependence on ara-C was observed for inhibition of repair in normal human, xeroderma pigmentosum (XP) variant, and Cockayne's syndrome cells. Ara-C produced a similar number of breaks in normal and Cockayne's syndrome cells. Ara-C produced a similar number of breaks in normal and Cockayne's syndrome cells but slightly more in XP variant cells. Exonuclease III and S1 nuclease independently both degraded about 50% of the /sup 3/H-thymidine incorporated into repaired regions in the presence of ara-C. Sequential digestion with both enzymes degraded nearly 90% of the repaired regions. These observations can be explained if excision repair proceeds by displacing the damaged strand so that both the /sup 3/H-labeled patch and the damaged region are still ligated to high molecular weight DNA and compete for the same complementary strand during in vitro incubation with the nucleases. The amount of /sup 3/H-thymidine incorporated in DNA by repair decreased with increasing concentrations of ara-C and hydroxyurea, suggesting that the incomplete patches became shorter under these conditions. Extrapolation of the digestion kinetics with exonuclease III permits an estimate of the normal patch size of about 100 nucleotides, consistent with previous estimates.

  8. The Association of Low-Penetrance Variants in DNA Repair Genes with Colorectal Cancer: A Systematic Review and Meta-Analysis.

    Science.gov (United States)

    Aggarwal, Nikhil; Donald, Neil D; Malik, Salim; Selvendran, Subothini S; McPhail, Mark Jw; Monahan, Kevin J

    2017-07-27

    Approximately 35% of colorectal cancer (CRC) risk is attributable to heritable factors known hereditary syndromes, accounting for 6%. The remainder may be due to lower penetrance polymorphisms particularly of DNA repair genes. DNA repair pathways, including base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), direct reversal repair (DRR), and double-strand break repair are complex, evolutionarily conserved, and critical in carcinogenesis. Germline mutations in these genes are associated with high-penetrance CRC syndromes such as Lynch syndrome. However, the association of low-penetrance polymorphisms of DNA repair genes with CRC risk remains unclear. A systematic literature review of PubMed, Embase, and HuGENet databases was conducted. Pre-specified criteria determined study inclusion/exclusion. Per-allele, pooled odds ratios disclosed the risk attributed to each variant. Heterogeneity was investigated by subgroup analyses for ethnicity and tumor location; funnel plots and Egger's test assessed publication bias. Sixty-one polymorphisms in 26 different DNA repair genes were identified. Meta-analyses for 22 polymorphisms in 17 genes revealed that six polymorphisms were significantly associated with CRC risk within BER (APE1, PARP1), NER (ERCC5, XPC), double-strand break (RAD18), and DRR (MGMT), but none within MMR. Subgroup analyses revealed significant association of OGG1 rs1052133 with rectal cancer risk. Egger's test revealed no publication bias. Low-penetrance polymorphisms in DNA repair genes alter susceptibility to CRC. Future studies should therefore analyze whole-genome polymorphisms and any synergistic effects on CRC risk.Translational impact:This knowledge may enhance CRC risk assessment and facilitate a more personalized approach to cancer prevention.

  9. Decreased transcription-coupled nucleotide excision repair capacity is associated with increased p53- and MLH1-independent apoptosis in response to cisplatin

    Directory of Open Access Journals (Sweden)

    Smith Jennifer M

    2010-05-01

    Full Text Available Abstract Background One of the most commonly used classes of anti-cancer drugs presently in clinical practice is the platinum-based drugs, including cisplatin. The efficacy of cisplatin therapy is often limited by the emergence of resistant tumours following treatment. Cisplatin resistance is multi-factorial but can be associated with increased DNA repair capacity, mutations in p53 or loss of DNA mismatch repair capacity. Methods RNA interference (RNAi was used to reduce the transcription-coupled nucleotide excision repair (TC-NER capacity of several prostate and colorectal carcinoma cell lines with specific defects in p53 and/or DNA mismatch repair. The effect of small inhibitory RNAs designed to target the CSB (Cockayne syndrome group B transcript on TC-NER and the sensitivity of cells to cisplatin-induced apoptosis was determined. Results These prostate and colon cancer cell lines were initially TC-NER proficient and RNAi against CSB significantly reduced their DNA repair capacity. Decreased TC-NER capacity was associated with an increase in the sensitivity of tumour cells to cisplatin-induced apoptosis, even in p53 null and DNA mismatch repair-deficient cell lines. Conclusion The present work indicates that CSB and TC-NER play a prominent role in determining the sensitivity of tumour cells to cisplatin even in the absence of p53 and DNA mismatch repair. These results further suggest that CSB represents a potential target for cancer therapy that may be important to overcome resistance to cisplatin in the clinic.

  10. DNA repair gene ERCC2 polymorphisms and associations with breast and ovarian cancer risk.

    Science.gov (United States)

    Bernard-Gallon, Dominique; Bosviel, Rémy; Delort, Laetitia; Fontana, Luc; Chamoux, Alain; Rabiau, Nadège; Kwiatkowski, Fabrice; Chalabi, Nasséra; Satih, Samir; Bignon, Yves-Jean

    2008-05-02

    Breast and ovarian cancers increased in the last decades. Except rare cases with a genetic predisposition and high penetrance, these pathologies are viewed as a polygenic disease. In this concept, association studies look for genetic variations such as polymorphisms in low penetrance genes, i.e. genes in interaction with environmental factors. DNA repair systems that protect the genome from deleterious endogenous and exogenous damages have been shown to have significantly reduced. In particular, enzymes of the nucleotide excision repair pathway are suspected to be implicated in cancer. In this study, 2 functional polymorphisms in a DNA repair gene ERCC2 were analyzed. The population included 911 breast cancer cases, 51 ovarian cancer cases and 1000 controls. The genotyping of 2 SNP (Single Nucleotide Polymorphism) was carried out on the population with the MGB (Minor Groove Binder) probe technique which consists of the use of the allelic discrimination with the Taqman method. This study enabled us to show an increase in risk of breast cancer with no oral contraceptive users and with women exhibiting a waist-to-hip ratio (WHR) > 0.85 for Asn homozygous for ERCC2 312.

  11. DNA repair gene ERCC2 polymorphisms and associations with breast and ovarian cancer risk

    Directory of Open Access Journals (Sweden)

    Rabiau Nadège

    2008-05-01

    Full Text Available Abstract Breast and ovarian cancers increased in the last decades. Except rare cases with a genetic predisposition and high penetrance, these pathologies are viewed as a polygenic disease. In this concept, association studies look for genetic variations such as polymorphisms in low penetrance genes, i.e. genes in interaction with environmental factors. DNA repair systems that protect the genome from deleterious endogenous and exogenous damages have been shown to have significantly reduced. In particular, enzymes of the nucleotide excision repair pathway are suspected to be implicated in cancer. In this study, 2 functional polymorphisms in a DNA repair gene ERCC2 were analyzed. The population included 911 breast cancer cases, 51 ovarian cancer cases and 1000 controls. The genotyping of 2 SNP (Single Nucleotide Polymorphism was carried out on the population with the MGB (Minor Groove Binder probe technique which consists of the use of the allelic discrimination with the Taqman® method. This study enabled us to show an increase in risk of breast cancer with no oral contraceptive users and with women exhibiting a waist-to-hip ratio (WHR > 0.85 for Asn homozygous for ERCC2 312.

  12. First reported patient with human ERCC1 deficiency has cerebro-oculo-facio-skeletal syndrome with a mild defect in nucleotide excision repair and severe developmental failure.

    Science.gov (United States)

    Jaspers, Nicolaas G J; Raams, Anja; Silengo, Margherita Cirillo; Wijgers, Nils; Niedernhofer, Laura J; Robinson, Andria Rasile; Giglia-Mari, Giuseppina; Hoogstraten, Deborah; Kleijer, Wim J; Hoeijmakers, Jan H J; Vermeulen, Wim

    2007-03-01

    Nucleotide excision repair (NER) is a genome caretaker mechanism responsible for removing helix-distorting DNA lesions, most notably ultraviolet photodimers. Inherited defects in NER result in profound photosensitivity and the cancer-prone syndrome xeroderma pigmentosum (XP) or two progeroid syndromes: Cockayne and trichothiodystrophy syndromes. The heterodimer ERCC1-XPF is one of two endonucleases required for NER. Mutations in XPF are associated with mild XP and rarely with progeria. Mutations in ERCC1 have not been reported. Here, we describe the first case of human inherited ERCC1 deficiency. Patient cells showed moderate hypersensitivity to ultraviolet rays and mitomycin C, yet the clinical features were very severe and, unexpectedly, were compatible with a diagnosis of cerebro-oculo-facio-skeletal syndrome. This discovery represents a novel complementation group of patients with defective NER. Further, the clinical severity, coupled with a relatively mild repair defect, suggests novel functions for ERCC1.

  13. Comparative study of the application of microcurrent and AsGa 904 nm laser radiation in the process of repair after calvaria bone excision in rats

    Science.gov (United States)

    Mendonça, J. S.; Neves, L. M. G.; Esquisatto, M. A. M.; Mendonça, F. A. S.; Santos, G. M. T.

    2013-03-01

    This study evaluated the effects of microcurrent stimulation (10 μA/5 min) and 904 nm GaAs laser irradiation (3 J cm-2 for 69 s/day) on excisional lesions created in the calvaria bone of Wistar rats. The results showed significant responses in the reduction of inflammatory cells and an increase in the number of new blood vessels, number of fibroblasts and deposition of birefringent collagen fibers when these data were compared with those of samples of the untreated lesions. Both applications, microcurrent and laser at 904 nm, favored tissue repair in the region of bone excisions during the study period and these techniques can be used as coadjuvantes in the repair of bone tissue.

  14. [Polymorphism of genes encoding proteins of DNA repair vs. occupational and environmental exposure to lead, arsenic and pesticides].

    Science.gov (United States)

    Bukowski, Karol; Woźniak, Katarzyna

    2017-10-12

    Genetic polymorphism is associated with the occurrence of at least 2 different alleles in the locus with a frequency higher than 1% in the population. Among polymorphisms we can find single nucleotide polymorphism (SNP) and polymorphism of variable number of tandem repeats. The presence of certain polymorphisms in genes encoding DNA repair enzymes is associated with the speed and efficiency of DNA repair and can protect or expose humans to the effects provoked by xenobiotics. Chemicals, such as lead, arsenic pesticides are considered to exhibit strong toxicity. There are many different polymorphisms in genes encoding DNA repair enzymes, which determine the speed and efficiency of DNA damage repair induced by these xenobiotics. In the case of lead, the influence of various polymorphisms, such as APE1 (apurinic/apyrimidinic endonuclease 1) (rs1130409), hOGG1 (human 8-oxoguanine glycosylase) (rs1052133), XRCC1 (X-ray repair cross-complementing protein group 1) (rs25487), XRCC1 (rs1799782) and XRCC3 (X-ray repair cross-complementing protein group 3) (rs861539) were described. For arsenic polymorphisms, such as ERCC2 (excision repair cross-complementing) (rs13181), XRCC3 (rs861539), APE1 (rs1130409) and hOGG1 (rs1052133) were examined. As to pesticides, separate and combined effects of polymorphisms in genes encoding DNA repair enzymes, such as XRCC1 (rs1799782), hOGG1 (rs1052133), XRCC4 (X-ray repair cross-complementing protein group 4) (rs28360135) and the gene encoding the detoxification enzyme PON1 paraoxonase (rs662) were reported. Med Pr 2018;69(1). This work is available in Open Access model and licensed under a CC BY-NC 3.0 PL license.

  15. DNA Repair Gene Polymorphisms in Relation to Non-Small Cell Lung Cancer Survival

    Directory of Open Access Journals (Sweden)

    Yuliang Su

    2015-07-01

    Full Text Available Background: Single nucleotide polymorphisms (SNPs in the DNA repair genes are suspected to be related to the survival of lung cancer patients due to their possible influence on DNA repair capacity (DRC. However, the study results are inconsistent. Methods: A follow-up study of 610 non-small cell lung cancer (NSCLC patients was conducted to investigate genetic polymorphisms associated with the DNA repair genes in relation to NSCLC survival; 6 SNPs were genotyped, including XRCC1 (rs25487 G>A, hOGG1 (rs1052133 C>G, MUTYH (rs3219489 G>C, XPA (rs1800975 G>A, ERCC2 (rs1799793 G>A and XRCC3 (rs861539 C>T. Kaplan-Meier survival curve and Cox proportional hazards regression analyses were performed. SNP-SNP interaction was also examined using the survival tree analysis. Results: Advanced disease stage and older age at diagnosis were associated with poor prognosis of NSCLC. Patients with the variant ‘G' allele of hOGG1 rs1052133 had poor overall survival compared with those with the homozygous wild ‘CC' genotype, especially in female patients, adenocarcinoma histology, early stage, light smokers and without family history of cancer. For never smoking female lung cancer patients, individuals carrying homozygous variant ‘AA' genotype of XPA had shorter survival time compared to those with wild ‘G' alleles. Furthermore, females carrying homozygous variant XPA and hOGG1 genotypes simultaneously had 2.78-fold increased risk for death. Among all 6 polymorphisms, the homozygous variant ‘AA' of XPA carriers had poor prognosis compared to the carriers of wild ‘G' alleles of XPA together with other base excision repair (BER polymorphisms. Conclusions: Besides disease stage and age, the study found DNA repair gene polymorphisms were associated with lung cancer survival.

  16. [Removal of laryngeal cancer with thyroid cartilage membrane excision and repair of laryngeal cavity with outside thyroid cartilage membrane flap of healthy side: oncologic and functional outcomes].

    Science.gov (United States)

    Zhong, Z T; Liang, M Z; Chen, Z

    2016-08-07

    To study the efficacy and feasibility of removal of laryngeal cancer with thyroid cartilage membrane excision and repair of laryngeal cavity by the outside thyroid cartilage membrane flap of healthy side. A total of 28 patients were reviewed who underwent the removal of laryngeal cancer with thyroid cartilage membrane excision combined with the repair of laryngeal cavity by the outside thyroid cartilage membrane flap in our hospital between 2005 and 2011. Respiratory function, swallowing function, and voice quality of patients after surgery were evaluated. Survival and recurrence were observed with the follow up of five years. The decannulation rate was 96.4%. Aspiration rate was 10.7%, but aspiration was completely revolved by swallowing training in the patients. All patients had the voice quality required for communication although they complained of hoarseness after surgery. Tumor recurrence was found in one patient and cervical lymph node metastasis in 2 patients. The three-year and five-year survival rates were 89.3% and 85.7% respectively. This surgical procedure was applicable in some of patients with T2 laryngeal cancer, with good laryngeal functions after surgery.

  17. The Effect of Msh2 Knockdown on Toxicity Induced by tert-Butyl-hydroperoxide, Potassium Bromate, and Hydrogen Peroxide in Base Excision Repair Proficient and Deficient Cells

    Directory of Open Access Journals (Sweden)

    N. Cooley

    2013-01-01

    Full Text Available The DNA mismatch repair (MMR and base excision repair (BER systems are important determinants of cellular toxicity following exposure to agents that cause oxidative DNA damage. To examine the interactions between these different repair systems, we examined whether toxicity, induced by t-BOOH and KBrO3, differs in BER proficient (Mpg+/+, Nth1+/+ and deficient (Mpg−/−, Nth1−/− mouse embryonic fibroblasts (MEFs following Msh2 knockdown of between 79 and 88% using an shRNA expression vector. Msh2 knockdown in Nth1+/+ cells had no effect on t-BOOH and KBrO3 induced toxicity as assessed by an MTT assay; knockdown in Nth1−/− cells resulted in increased resistance to t-BOOH and KBrO3, a result consistent with Nth1 removing oxidised pyrimidines. Msh2 knockdown in Mpg+/+ cells had no effect on t-BOOH toxicity but increased resistance to KBrO3; in Mpg−/− cells, Msh2 knockdown increased cellular sensitivity to KBrO3 but increased resistance to t-BOOH, suggesting a role for Mpg in removing DNA damage induced by these agents. MSH2 dependent and independent pathways then determine cellular toxicity induced by oxidising agents. A complex interaction between MMR and BER repair systems, that is, exposure dependent, also exists to determine cellular toxicity.

  18. The Effect of Msh2 Knockdown on Toxicity Induced by tert-Butyl-hydroperoxide, Potassium Bromate, and Hydrogen Peroxide in Base Excision Repair Proficient and Deficient Cells

    Science.gov (United States)

    Cooley, N.; Elder, R. H.; Povey, A. C.

    2013-01-01

    The DNA mismatch repair (MMR) and base excision repair (BER) systems are important determinants of cellular toxicity following exposure to agents that cause oxidative DNA damage. To examine the interactions between these different repair systems, we examined whether toxicity, induced by t-BOOH and KBrO3, differs in BER proficient (Mpg +/+, Nth1 +/+) and deficient (Mpg −/−, Nth1 −/−) mouse embryonic fibroblasts (MEFs) following Msh2 knockdown of between 79 and 88% using an shRNA expression vector. Msh2 knockdown in Nth1 +/+ cells had no effect on t-BOOH and KBrO3 induced toxicity as assessed by an MTT assay; knockdown in Nth1 −/− cells resulted in increased resistance to t-BOOH and KBrO3, a result consistent with Nth1 removing oxidised pyrimidines. Msh2 knockdown in Mpg +/+ cells had no effect on t-BOOH toxicity but increased resistance to KBrO3; in Mpg −/− cells, Msh2 knockdown increased cellular sensitivity to KBrO3 but increased resistance to t-BOOH, suggesting a role for Mpg in removing DNA damage induced by these agents. MSH2 dependent and independent pathways then determine cellular toxicity induced by oxidising agents. A complex interaction between MMR and BER repair systems, that is, exposure dependent, also exists to determine cellular toxicity. PMID:23984319

  19. Pathological assessment of mismatch repair gene variants in Lynch syndrome

    DEFF Research Database (Denmark)

    Rasmussen, Lene Juel; Heinen, Christopher D; Royer-Pokora, Brigitte

    2012-01-01

    Lynch syndrome (LS) is caused by germline mutations in DNA mismatch repair (MMR) genes and is the most prevalent hereditary colorectal cancer syndrome. A significant proportion of variants identified in MMR and other common cancer susceptibility genes are missense or noncoding changes whose conse...

  20. Assessment of single nucleotide polymorphisms in screening 52 DNA repair and cell cycle control genes in Fanconi anemia patients

    Directory of Open Access Journals (Sweden)

    Petrović Sandra

    2015-01-01

    Full Text Available Fanconi anemia (FA is a rare genetically heterogeneous disorder associated with bone marrow failure, birth defects and cancer susceptibility. Apart from the disease- causing mutations in FANC genes, the identification of specific DNA variations, such as single nucleotide polymorphisms (SNPs, in other candidate genes may lead to a better clinical description of this condition enabling individualized treatment with improvement of the prognosis. In this study, we have assessed 95 SNPs located in 52 key genes involved in base excision repair (BER, nucleotide excision repair (NER, mismatch repair (MMR, double strand break (DSB repair and cell cycle control using a DNA repair chip (Asper Biotech, Estonia which includes most of the common variants for the candidate genes. The SNP genotyping was performed in five FA-D2 patients and in one FA-A patient. The polymorphisms studied were synonymous (n=10, nonsynonymous (missense (n=52 and in non-coding regions of the genome (introns and 5 ‘and 3’ untranslated regions (UTR (n=33. Polymorphisms found at the homozygous state are selected for further analysis. Our results have shown a significant inter-individual variability among patients in the type and the frequency of SNPs and also elucidate the need for further studies of polymorphisms located in ATM, APEX APE 1, XRCC1, ERCC2, MSH3, PARP4, NBS1, BARD1, CDKN1B, TP53 and TP53BP1 which may be of great importance for better clinical description of FA. In addition, the present report recommends the use of SNPs as predictive and prognostic genetic markers to individualize therapy of FA patients. [Projekat Ministarstva nauke Republike Srbije, br. 173046

  1. ATP-Dependent Chromatin Remodeling Is Required for Base Excision Repair in Conventional but Not in Variant H2A.Bbd Nucleosomes▿

    Science.gov (United States)

    Menoni, Hervé; Gasparutto, Didier; Hamiche, Ali; Cadet, Jean; Dimitrov, Stefan; Bouvet, Philippe; Angelov, Dimitar

    2007-01-01

    In eukaryotes, base excision repair (BER) is responsible for the repair of oxidatively generated lesions. The mechanism of BER on naked DNA substrates has been studied in detail, but how it operates on chromatin remains unclear. Here we have studied the mechanism of BER by introducing a single 8-oxo-7,8-dihydroguanine (8-oxoG) lesion in the DNA of reconstituted positioned conventional and histone variant H2A.Bbd nucleosomes. We found that 8-oxoguanine DNA glycosylase, apurinic/apyrimidinic endonuclease, and polymerase β activities were strongly reduced in both types of nucleosomes. In conventional nucleosomes SWI/SNF stimulated the processing of 8-oxoG by each one of the three BER repair factors to efficiencies similar to those for naked DNA. Interestingly, SWI/SNF-induced remodeling, but not mobilization of conventional nucleosomes, was required to achieve this effect. A very weak effect of SWI/SNF on the 8-oxoG BER removal in H2A.Bbd histone variant nucleosomes was observed. The possible implications of our data for the understanding of in vivo mechanisms of BER are discussed. PMID:17591702

  2. DICER- and MMSET-catalyzed H4K20me2 recruits the nucleotide excision repair factor XPA to DNA damage sites.

    Science.gov (United States)

    Chitale, Shalaka; Richly, Holger

    2017-12-12

    Ultraviolet (UV) irradiation triggers the recruitment of DNA repair factors to the lesion sites and the deposition of histone marks as part of the DNA damage response. The major DNA repair pathway removing DNA lesions caused by exposure to UV light is nucleotide excision repair (NER). We have previously demonstrated that the endoribonuclease DICER facilitates chromatin decondensation during lesion recognition in the global-genomic branch of NER. Here, we report that DICER mediates the recruitment of the methyltransferase MMSET to the DNA damage site. We show that MMSET is required for efficient NER and that it catalyzes the dimethylation of histone H4 at lysine 20 (H4K20me2). H4K20me2 at DNA damage sites facilitates the recruitment of the NER factor XPA. Our work thus provides evidence for an H4K20me2-dependent mechanism of XPA recruitment during lesion recognition in the global-genomic branch of NER. © 2018 Chitale and Richly.

  3. Self-excising Cre/mutant lox marker recycling system for multiple gene integrations and consecutive gene deletions in Aspergillus oryzae.

    Science.gov (United States)

    Zhang, Silai; Ban, Akihiko; Ebara, Naoki; Mizutani, Osamu; Tanaka, Mizuki; Shintani, Takahiro; Gomi, Katsuya

    2017-04-01

    In this study, we developed a self-excising Cre/loxP-mediated marker recycling system with mutated lox sequences to introduce a number of biosynthetic genes into Aspergillus oryzae. To construct the self-excising marker cassette, both the selectable marker, the Aspergillus nidulans adeA gene, and the Cre recombinase gene (cre), conditionally expressed by the xylanase-encoding gene promoter, were designed to be located between the mutant lox sequences, lox66 and lox71. However, construction of the plasmid failed, possibly owing to a slight expression of cre downstream of the fungal gene promoter in Escherichia coli. Hence, to avoid the excision of the cassette in E. coli, a 71-bp intron of the A. oryzae xynG2 gene was inserted into the cre gene. The A. oryzae adeA deletion mutant was transformed with the resulting plasmid in the presence of glucose, and the transformants were cultured in medium containing xylose as the sole carbon source. PCR analysis of genomic DNA from resultant colonies revealed the excision of both the marker and Cre expression construct, indicating that the self-excising marker cassette was efficient at removing the selectable marker. Using the marker recycling system, hyperproduction of kojic acid could be achieved in A. oryzae by the introduction of two genes that encode oxidoreductase and transporter. Furthermore, we also constructed an alternative marker recycling cassette bearing the A. nidulans pyrithiamine resistant gene (ptrA) as a dominant selectable marker. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  4. Mismatch repair genes in Lynch syndrome: a review

    OpenAIRE

    Silva,Felipe Cavalcanti Carneiro da; Valentin, Mev Dominguez; Ferreira, Fábio de Oliveira; Carraro, Dirce Maria; Rossi, Benedito Mauro

    2009-01-01

    Lynch syndrome represents 1-7% of all cases of colorectal cancer and is an autosomal-dominant inherited cancer predisposition syndrome caused by germline mutations in deoxyribonucleic acid (DNA) mismatch repair genes. Since the discovery of the major human genes with DNA mismatch repair function, mutations in five of them have been correlated with susceptibility to Lynch syndrome: mutS homolog 2 (MSH2); mutL homolog 1 (MLH1); mutS homolog 6 (MSH6); postmeiotic segregation increased 2 (PMS2); ...

  5. Base excision repair efficiency and mechanism in nuclear extracts are influenced by the ratio between volume of nuclear extraction buffer and nuclei-Implications for comparative studies

    DEFF Research Database (Denmark)

    Akbari, Mansour; Krokan, Hans E

    2012-01-01

    attention. Here we have examined BER activity of nuclear cell extracts from HeLa cells, using as substrate a circular DNA molecule with either uracil or an AP-site in a defined position. We show that BER activity of nuclear extracts from the same batch of cells varies inversely with the volume of nuclear......The base excision repair (BER) pathway corrects many different DNA base lesions and is important for genomic stability. The mechanism of BER cannot easily be investigated in intact cells and therefore in vitro methods that reflect the in vivo processes are in high demand. Reconstitution of BER...... using purified proteins essentially mirror properties of the proteins used, and does not necessarily reflect the mechanism as it occurs in the cell. Nuclear extracts from cultured cells have the capacity to carry out complete BER and can give important information on the mechanism. Furthermore...

  6. Cockayne Syndrome exhibits dysregulation of p21 and other gene products that may be independent of transcription coupled repair

    Science.gov (United States)

    Cleaver, J.E.; Hefner, E.; Laposa, R.R.; Karentz, D.

    2007-01-01

    Cockayne syndrome (CS) is a progressive childhood neurodegenerative disorder associated with a DNA repair defect caused by mutations in either of two genes, CSA and CSB. These genes are involved in nucleotide excision repair (NER) of DNA damage from ultraviolet (UV) light, other bulky chemical adducts and reactive oxygen in transcriptionally active genes (transcription coupled repair, TCR). For a long period it has been assumed that the symptoms of CS patients are all due to reduced TCR of endogenous DNA damage in the brain, together with unexplained unique sensitivity of specific neural cells in the cerebellum. Not all the symptoms of CS patients are however easily related to repair deficiencies, so we hypothesize that there are additional pathways relevant to the disease, particularly those that are downstream consequences of a common defect in the E3 ubiquitin ligase associated with the CSA and CSB gene products. We have found that the CSB defect results in altered expression of anti-angiogenic and cell cycle genes and proteins at the level of both gene expression and protein lifetime. We find an over-abundance of p21 due to reduced protein turnover, possibly due to the loss of activity of the CSA/CSB E3 ubiquitylation pathway. Increased levels of p21 can result in growth inhibition, reduced repair from the p21-PCNA interaction, and increased generation of reactive oxygen. Consistent with increased reactive ozygen levels we find that CS-A and -B cells grown under ambient oxygen show increased DNA breakage, as compared to xeroderma pigmentosum cells. Thus the complex symptoms of CS may be due to multiple, independent downstream targets of the E3 ubiquitylation system that results in increased DNA damage, reduced transcription coupled repair, and inhibition of cell cycle progression and growth. PMID:17055654

  7. Alcohol-induced one-carbon metabolism impairment promotes dysfunction of DNA base excision repair in adult brain.

    Science.gov (United States)

    Fowler, Anna-Kate; Hewetson, Aveline; Agrawal, Rajiv G; Dagda, Marisela; Dagda, Raul; Moaddel, Ruin; Balbo, Silvia; Sanghvi, Mitesh; Chen, Yukun; Hogue, Ryan J; Bergeson, Susan E; Henderson, George I; Kruman, Inna I

    2012-12-21

    The brain is one of the major targets of chronic alcohol abuse. Yet the fundamental mechanisms underlying alcohol-mediated brain damage remain unclear. The products of alcohol metabolism cause DNA damage, which in conditions of DNA repair dysfunction leads to genomic instability and neural death. We propose that one-carbon metabolism (OCM) impairment associated with long term chronic ethanol intake is a key factor in ethanol-induced neurotoxicity, because OCM provides cells with DNA precursors for DNA repair and methyl groups for DNA methylation, both critical for genomic stability. Using histological (immunohistochemistry and stereological counting) and biochemical assays, we show that 3-week chronic exposure of adult mice to 5% ethanol (Lieber-Decarli diet) results in increased DNA damage, reduced DNA repair, and neuronal death in the brain. These were concomitant with compromised OCM, as evidenced by elevated homocysteine, a marker of OCM dysfunction. We conclude that OCM dysfunction plays a causal role in alcohol-induced genomic instability in the brain because OCM status determines the alcohol effect on DNA damage/repair and genomic stability. Short ethanol exposure, which did not disturb OCM, also did not affect the response to DNA damage, whereas additional OCM disturbance induced by deficiency in a key OCM enzyme, methylenetetrahydrofolate reductase (MTHFR) in Mthfr(+/-) mice, exaggerated the ethanol effect on DNA repair. Thus, the impact of long term ethanol exposure on DNA repair and genomic stability in the brain results from OCM dysfunction, and MTHFR mutations such as Mthfr 677C→T, common in human population, may exaggerate the adverse effects of ethanol on the brain.

  8. Base-Excision-Repair-Induced Construction of a Single Quantum-Dot-Based Sensor for Sensitive Detection of DNA Glycosylase Activity.

    Science.gov (United States)

    Wang, Li-Juan; Ma, Fei; Tang, Bo; Zhang, Chun-Yang

    2016-08-02

    DNA glycosylase is an initiating enzyme of cellular base excision repair pathway which is responsible for the repair of various DNA lesions and the maintenance of genomic stability, and the dysregulation of DNA glycosylase activity is associated with a variety of human pathology. Accurate detection of DNA glycosylase activity is critical to both clinical diagnosis and therapeutics, but conventional methods for the DNA glycosylase assay are usually time-consuming with poor sensitivity. Here, we demonstrate the base-excision-repair-induced construction of a single quantum dot (QD)-based sensor for highly sensitive measurement of DNA glycosylase activity. We use human 8-oxoguanine-DNA glycosylase 1 (hOGG1), which is responsible for specifically repairing the damaged 8-hydroxyguanine (8-oxoG, one of the most abundant and widely studied DNA damage products), as a model DNA glycosylase. In the presence of biotin-labeled DNA substrate, the hOGG1 may catalyze the removal of 8-oxo G from 8-oxoG·C base pairs to generate an apurinic/apyrimidinic (AP) site. With the assistance of apurinic/apyrimidinic endonuclease (APE1), the cleavage of the AP site results in the generation of a single-nucleotide gap. Subsequently, DNA polymerase β incorporates a Cy5-labeled dGTP into the DNA substrate to fill the gap. With the addition of streptavidin-coated QDs, a QD-DNA-Cy5 nanostructure is formed via specific biotin-streptavidin binding, inducing the occurrence of fluorescence resonance energy transfer (FRET) from the QD to Cy5. The resulting Cy5 signal can be simply monitored by total internal reflection fluorescence (TIRF) imaging. The proposed method enables highly sensitive measurement of hOGG1 activity with a detection limit of 1.8 × 10(-6) U/μL. Moreover, it can be used to measure the enzyme kinetic parameters and detect the hOGG1 activity in crude cell extracts, offering a powerful tool for biomedical research and clinical diagnosis.

  9. Bypass of a 5',8-cyclopurine-2'-deoxynucleoside by DNA polymerase β during DNA replication and base excision repair leads to nucleotide misinsertions and DNA strand breaks.

    Science.gov (United States)

    Jiang, Zhongliang; Xu, Meng; Lai, Yanhao; Laverde, Eduardo E; Terzidis, Michael A; Masi, Annalisa; Chatgilialoglu, Chryssostomos; Liu, Yuan

    2015-09-01

    5',8-Cyclopurine-2'-deoxynucleosides including 5',8-cyclo-dA (cdA) and 5',8-cyclo-dG (cdG) are induced by hydroxyl radicals resulting from oxidative stress such as ionizing radiation. 5',8-cyclopurine-2'-deoxynucleoside lesions are repaired by nucleotide excision repair with low efficiency, thereby leading to their accumulation in the human genome and lesion bypass by DNA polymerases during DNA replication and base excision repair (BER). In this study, for the first time, we discovered that DNA polymerase β (pol β) efficiently bypassed a 5'R-cdA, but inefficiently bypassed a 5'S-cdA during DNA replication and BER. We found that cell extracts from pol β wild-type mouse embryonic fibroblasts exhibited significant DNA synthesis activity in bypassing a cdA lesion located in replication and BER intermediates. However, pol β knock-out cell extracts exhibited little DNA synthesis to bypass the lesion. This indicates that pol β plays an important role in bypassing a cdA lesion during DNA replication and BER. Furthermore, we demonstrated that pol β inserted both a correct and incorrect nucleotide to bypass a cdA at a low concentration. Nucleotide misinsertion was significantly stimulated by a high concentration of pol β, indicating a mutagenic effect induced by pol β lesion bypass synthesis of a 5',8-cyclopurine-2'-deoxynucleoside. Moreover, we found that bypass of a 5'S-cdA by pol β generated an intermediate that failed to be extended by pol β, resulting in accumulation of single-strand DNA breaks. Our study provides the first evidence that pol β plays an important role in bypassing a 5',8-cyclo-dA during DNA replication and repair, as well as new insight into mutagenic effects and genome instability resulting from pol β bypassing of a cdA lesion. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Probing for DNA damage with β-hairpins: Similarities in incision efficiencies of bulky DNA adducts by prokaryotic and human nucleotide excision repair systems in vitro

    Science.gov (United States)

    Liu, Yang; Reeves, Dara; Kropachev, Konstantin; Cai, Yuqin; Ding, Shuang; Kolbanovskiy, Marina; Kolbanovskiy, Alexander; Bolton, Judith L.; Broyde, Suse; Van Houten, Bennett; Geacintov, Nicholas E.

    2011-01-01

    Nucleotide excision repair (NER) is an important prokaryotic and eukaryotic defense mechanism that removes a large variety of structurally distinct lesions in cellular DNA. While the proteins involved are completely different, the mode of action of these two repair systems is similar, involving a cut-and-patch mechanism in which an oligonucleotide sequence containing the lesion is excised. The prokaryotic and eukaryotic NER damage-recognition factors have common structural features of β-hairpin intrusion between the two DNA strands at the site of the lesion. In the present study, we explored the hypothesis that this common β-hairpin intrusion motif is mirrored in parallel NER incision efficiencies in the two systems. We have utilized human HeLa cell extracts and the prokaryotic UvrABC proteins to determine their relative NER incision efficiencies. We report here comparisons of relative NER efficiencies with a set of stereoisomeric DNA lesions derived from metabolites of benzo[a]pyrene and equine estrogens in different sequence contexts, utilizing 21 samples. We found a general qualitative trend towards similar relative NER incision efficiencies for ~ 65% of these substrates; the other cases deviate mostly by ~ 30% or less from a perfect correlation, although several more distant outliers are also evident. This resemblance is consistent with the hypothesis that lesion recognition through β-hairpin insertion, a common feature of the two systems, is facilitated by local thermodynamic destabilization induced by the lesions in both cases. In the case of the UvrABC system, varying the nature of the UvrC endonuclease, while maintaining the same UvrA/B proteins, can markedly affect the relative incision efficiencies. These observations suggest that, in addition to recognition involving the initial modified duplexes, downstream events involving UvrC can also play a role in distinguishing and processing different lesions in prokaryotic NER. PMID:21741328

  11. Preferential repair of ionizing radiation-induced damage in the transcribed strand of an active human gene is defective in Cockayne syndrome

    Energy Technology Data Exchange (ETDEWEB)

    Leadon, S.A. (Univ. of North Carolina, Chapel Hill, NC (United States)); Copper, P.K. (Lawrence Berkeley Lab., CA (United States))

    1993-11-15

    Cells from patients with Cockayne syndrome (CS), which are sensitive to killing by UV although overall damage removal appears normal, are specifically defective in repair of UV damage in actively transcribe genes. Because several CS strains display cross-sensitivity to killing by ionizing radiation, the authors examined whether ionizing radiation-induced damage in active genes is preferentially repaired by normal cells and whether the radiosensitivity of CS cells can be explained by a defect in this process. They found that ionizing radiation-induced damage was repaired more rapidly in the transcriptionally active metallothionein IIA (MTIIA) gene than in the inactive MTIIB gene or in the genome overall in normal cells as a result of faster repair on the transcribed strand of MTIIA. Cells of the radiosensitive CS strain CS1AN are completely defective in this strand-selective repair of ionizing radiation-induced damage, although their overall repair rate appears normal. CS3BE cells, which are intermediate in radiosensitivity, do exhibit more rapid repair of the transcribed strand but at a reduced rate compared to normal cells. Xeroderma pigmentosum complementation group A cells, which are hypersensitive to UV light because of a defect in the nucleotide excision repair pathway but do not show increased sensitivity to ionizing radiation, preferentially repair ionizing radiation-induced damage on the transcribed strand of MTIIA. Thus, the ability to rapidly repair ionizing radiation-induced damage in actively transcribing genes correlates with cell survival. The results extend the generality of preferential repair in active genes to include damage other than bulky lesions.

  12. Preferential repair of ionizing radiation-induced damage in the transcribed strand of an active human gene is defective in Cockayne syndrome.

    Science.gov (United States)

    Leadon, S A; Cooper, P K

    1993-11-15

    Cells from patients with Cockayne syndrome (CS), which are sensitive to killing by UV although overall damage removal appears normal, are specifically defective in repair of UV damage in actively transcribed genes. Because several CS strains display cross-sensitivity to killing by ionizing radiation, we examined whether ionizing radiation-induced damage in active genes is preferentially repaired by normal cells and whether the radiosensitivity of CS cells can be explained by a defect in this process. We found that ionizing radiation-induced damage was repaired more rapidly in the transcriptionally active metallothionein IIA (MTIIA) gene than in the inactive MTIIB gene or in the genome overall in normal cells as a result of faster repair on the transcribed strand of MTIIA. Cells of the radiosensitive CS strain CS1AN are completely defective in this strand-selective repair of ionizing radiation-induced damage, although their overall repair rate appears normal. CS3BE cells, which are intermediate in radiosensitivity, do exhibit more rapid repair of the transcribed strand but at a reduced rate compared to normal cells. Xeroderma pigmentosum complementation group A cells, which are hypersensitive to UV light because of a defect in the nucleotide excision repair pathway but do not show increased sensitivity to ionizing radiation, preferentially repair ionizing radiation-induced damage on the transcribed strand of MTIIA. Thus, the ability to rapidly repair ionizing radiation-induced damage in actively transcribing genes correlates with cell survival. Our results extend the generality of preferential repair in active genes to include damage other than bulky lesions.

  13. ATR- and ATM-Mediated DNA Damage Response Is Dependent on Excision Repair Assembly during G1 but Not in S Phase of Cell Cycle.

    Science.gov (United States)

    Ray, Alo; Blevins, Chessica; Wani, Gulzar; Wani, Altaf A

    2016-01-01

    Cell cycle checkpoint is mediated by ATR and ATM kinases, as a prompt early response to a variety of DNA insults, and culminates in a highly orchestrated signal transduction cascade. Previously, we defined the regulatory role of nucleotide excision repair (NER) factors, DDB2 and XPC, in checkpoint and ATR/ATM-dependent repair pathway via ATR and ATM phosphorylation and recruitment to ultraviolet radiation (UVR)-induced damage sites. Here, we have dissected the molecular mechanisms of DDB2- and XPC- mediated regulation of ATR and ATM recruitment and activation upon UVR exposures. We show that the ATR and ATM activation and accumulation to UVR-induced damage not only depends on DDB2 and XPC, but also on the NER protein XPA, suggesting that the assembly of an active NER complex is essential for ATR and ATM recruitment. ATR and ATM localization and H2AX phosphorylation at the lesion sites occur as early as ten minutes in asynchronous as well as G1 arrested cells, showing that repair and checkpoint-mediated by ATR and ATM starts early upon UV irradiation. Moreover, our results demonstrated that ATR and ATM recruitment and H2AX phosphorylation are dependent on NER proteins in G1 phase, but not in S phase. We reasoned that in G1 the UVR-induced ssDNA gaps or processed ssDNA, and the bound NER complex promote ATR and ATM recruitment. In S phase, when the UV lesions result in stalled replication forks with long single-stranded DNA, ATR and ATM recruitment to these sites is regulated by different sets of proteins. Taken together, these results provide evidence that UVR-induced ATR and ATM recruitment and activation differ in G1 and S phases due to the existence of distinct types of DNA lesions, which promote assembly of different proteins involved in the process of DNA repair and checkpoint activation.

  14. A general role of the DNA glycosylase Nth1 in the abasic sites cleavage step of base excision repair in Schizosaccharomyces pombe.

    Science.gov (United States)

    Alseth, Ingrun; Korvald, Hanne; Osman, Fekret; Seeberg, Erling; Bjørås, Magnar

    2004-01-01

    One of the most frequent lesions formed in cellular DNA are abasic (apurinic/apyrimidinic, AP) sites that are both cytotoxic and mutagenic, and must be removed efficiently to maintain genetic stability. It is generally believed that the repair of AP sites is initiated by the AP endonucleases; however, an alternative pathway seems to prevail in Schizosaccharomyces pombe. A mutant lacking the DNA glycosylase/AP lyase Nth1 is very sensitive to the alkylating agent methyl methanesulfonate (MMS), suggesting a role for Nth1 in base excision repair (BER) of alkylation damage. Here, we have further evaluated the role of Nth1 and the second putative S.pombe AP endonuclease Apn2, in abasic site repair. The deletion of the apn2 open reading frame dramatically increased the sensitivity of the yeast cells to MMS, also demonstrating that the Apn2 has an important function in the BER pathway. The deletion of nth1 in the apn2 mutant strain partially relieves the MMS sensitivity of the apn2 single mutant, indicating that the Apn2 and Nth1 act in the same pathway for the repair of abasic sites. Analysis of the AP site cleavage in whole cell extracts of wild-type and mutant strains showed that the AP lyase activity of Nth1 represents the major AP site incision activity in vitro. Assays with DNA substrates containing base lesions removed by monofunctional DNA glycosylases Udg and MutY showed that Nth1 will also cleave the abasic sites formed by these enzymes and thus act downstream of these enzymes in the BER pathway. We suggest that the main function of Apn2 in BER is to remove the resulting 3'-blocking termini following AP lyase cleavage by Nth1.

  15. Importance of excision repair cross-complementation group 1 and ribonucleotide reductase M1 as prognostic biomarkers in malignant pleural mesothelioma treated with platinum-based induction chemotherapy followed by surgery.

    Science.gov (United States)

    Frischknecht, Lukas; Meerang, Mayura; Soltermann, Alex; Stahel, Rolf; Moch, Holger; Seifert, Burkhardt; Weder, Walter; Opitz, Isabelle

    2015-06-01

    Survival and response to platinum-based induction chemotherapy are heterogeneous among patients with malignant pleural mesothelioma. The aim of the present study was to assess the prognostic role of DNA repair markers, such as excision repair cross-complementation group 1 and ribonucleotide reductase M1, in multimodally treated patients with malignant pleural mesothelioma. Tumor tissue of a malignant pleural mesothelioma cohort (n = 107) treated with platinum/gemcitabine (n = 46) or platinum/pemetrexed (n = 61) induction chemotherapy followed by extrapleural pneumonectomy was assembled on a tissue microarray. Immunohistochemical expression of excision repair cross-complementation group 1 (nuclear) and ribonucleotide reductase M1 (nuclear and cytoplasmic) was assessed for its prognostic impact (association with overall survival or freedom from recurrence). Patients with high nuclear ribonucleotide reductase M1 expression before chemotherapy showed significantly longer freedom from recurrence (P = .03). When specifically analyzed in the subgroup of patients receiving platinum/gemcitabine followed by extrapleural pneumonectomy, high nuclear ribonucleotide reductase M1 was associated with prolonged freedom from recurrence (P = .03) and overall survival (P = .02). Low excision repair cross-complementation group 1 expression in prechemotherapy tumor tissues was associated with significantly longer freedom from recurrence (P = .04). Nuclear ribonucleotide reductase M1 and excision repair cross-complementation group 1 were independent prognosticators of freedom from recurrence in addition to pT stage in multivariate analysis. In the present study, nuclear ribonucleotide reductase M1 and excision repair cross-complementation group 1 expression were identified as independent prognosticators for freedom from recurrence of malignant pleural mesothelioma in patients undergoing induction chemotherapy followed by extrapleural pneumonectomy. Copyright © 2015 The American

  16. Clinical heterogeneity within xeroderma pigmentosum associated with mutations in the DNA repair and transcription gene ERCC3

    Energy Technology Data Exchange (ETDEWEB)

    Vermeulen, W.; Kleijer, W.J.; Bootsma, D.; Hoeijmakers, J.H.J.; Weeda, G. (Erasmus Univ., Rotterdam (Netherlands)); Scott, R.J.; Rodgers, S.; Mueller, H.J. (Univ. Hospital, Basel (Switzerland)); Cole, J.; Arlett, C.F. (Univ. of Sussex, Brighton (United Kingdom))

    1994-02-01

    The human DNA excision repair gene ERCC3 specifically corrects the nucleotide excision repair (NER) defect of xeroderma pigmentosum (XP) complementation group B. In addition to its function in NER, the ERCC3 DNA helicase was recently identified as one of the components of the human BTF2/TFIIH transcription factor complex, which is required for initiation of transcription of class II genes. To date, a single patient (XP11BE) has been assigned to this XP group B (XP-B), with the remarkable conjunction of two autosomal recessive DNA repair deficiency disorders: XP and Cockayne syndrome (CS). The intriguing involvement of the ERCC3 protein in the vital process of transcription may provide an explanation for the rarity, severity, and wide spectrum of clinical features in this complementation group. Here the authors report the identification of two new XP-B patients: XPCS1BA and XPCS2BA (siblings), by microneedle injection of the cloned ERCC3 repair gene as well as by cell hybridization. Molecular analysis of the ERCC3 gene in both patients revealed a single base substitution causing a missense mutation in a region that is completely conserved in yeast, Drosophila, mouse, and human ERCC3. As in patient XP11BE, the expression of only one allele (paternal) is detected. The mutation causes a virtually complete inactivation of the NER function of the protein. Despite this severe NER defect, both patients display a late onset of neurologic impairment, mild cutaneous symptoms, and a striking absence of skin tumors even at an age of >40 years. Analysis of the frequency of hprt[sup [minus

  17. DNA repair gene ERCC2, XPC, XRCC1, XRCC3 polymorphisms and associations with bladder cancer risk in a French cohort.

    Science.gov (United States)

    Fontana, Luc; Bosviel, Rémy; Delort, Laetitia; Guy, Laurent; Chalabi, Nasséra; Kwiatkowski, Fabrice; Satih, Samir; Rabiau, Nadège; Boiteux, Jean-Paul; Chamoux, Alain; Bignon, Yves-Jean; Bernard-Gallon, Dominique J

    2008-01-01

    In polygenic diseases, association studies look for genetic variation such as polymorphisms in low penetrance genes, i.e. genes in interaction with environmental factors. DNA repair systems that protect the genome from deleterious endogenous and exogenous damage have been shown to significantly reduce activity. In particular, enzymes of the nucleotide excision repair pathway are suspected to be implicated in cancer. In this study bladder cancer which is viewed as a polygenic disease was investigated. The functional polymorphisms of four DNA repair genes, excision repair cross-complementing group 2 (ERCC2), Xeroderma Pigmentosum group C (XPC), and Xray repair cross-complementing groups 1 and 3 (XRCC1 and XRCC3) were analyzed. The studied population included 51 bladder cancer cases and 45 controls. The genotyping of six SNP (single nucleotide polymorphism) was carried out on these populations with the MGB (Minor Groove Binder) probe technique which uses allelic discrimination with the Taqman method. The Gln allele of the XPC 939 polymorphism was found to be associated with an increase in bladder cancer risk.

  18. Polymorphisms in human DNA repair genes and head and neck ...

    Indian Academy of Sciences (India)

    Sci. USA 97, 9886–9891. Viswanathan H. and Wilson J. A. 2004 Alcohol—the neglected factor in head and neck cancer. Clin. Otolaryngol. 29, 295–300. Vogel U., Hedayati M., Dybdahl M., Grossman L. and Nexo B. A.. 2001 Polymorphisms of the DNA repair gene XPD, correlations with risk of basal cell carcinoma revisited.

  19. Dynamic regulation of cerebral DNA repair genes by psychological stress

    DEFF Research Database (Denmark)

    Forsberg, Kristin; Aalling, Nadia; Wörtwein, Gitta

    2015-01-01

    was seen in HC, but with overall smaller effects and without the induction after acute stress. Nuclear DNA damage from oxidation as measured by the comet assay was unaffected by stress in both regions. We conclude that psychological stress have a dynamic influence on brain DNA repair gene expression...

  20. Review: Clinical aspects of hereditary DNA Mismatch repair gene mutations

    NARCIS (Netherlands)

    Sijmons, Rolf H.; Hofstra, Robert M. W.

    Inherited mutations of the DNA Mismatch repair genes MLH1, MSH2, MSH6 and PMS2 can result in two hereditary tumor syndromes: the adult-onset autosomal dominant Lynch syndrome, previously referred to as Hereditary Non-Polyposis Colorectal Cancer (HNPCC) and the childhood-onset autosomal recessive

  1. Cockayne syndrome: varied requirement of transcription-coupled nucleotide excision repair for the removal of three structurally different adducts from transcribed DNA.

    Directory of Open Access Journals (Sweden)

    Nataliya Kitsera

    Full Text Available Hereditary defects in the transcription-coupled nucleotide excision repair (TC-NER pathway of damaged DNA cause severe neurodegenerative disease Cockayne syndrome (CS, however the origin and chemical nature of the underlying DNA damage had remained unknown. To find out, to which degree the structural properties of DNA lesions determine the extent of transcription arrest in human CS cells, we performed quantitative host cell reactivation analyses of expression vectors containing various synthetic adducts. We found that a single 3-(deoxyguanosin-N2-yl-2-acetylaminofluorene adduct (dG(N2-AAF constitutes an unsurmountable obstacle to transcription in both CS-A and CS-B cells and is removed exclusively by the CSA- and CSB-dependent pathway. In contrast, contribution of the CS proteins to the removal of two other transcription-blocking DNA lesions - N-(deoxyguanosin-8-yl-2-acetylaminofluorene (dG(C8-AAF and cyclobutane thymine-thymine (TT dimer - is only minor (TT dimer or none (dG(C8-AAF. The unique properties of dG(N2-AAF identify this adduct as a prototype for a new class of DNA lesions that escape the alternative global genome repair and could be critical for the CS pathogenesis.

  2. Is the Oxidative DNA Damage Level of Human Lymphocyte Correlated with the Antioxidant Capacity of Serum or the Base Excision Repair Activity of Lymphocyte?

    Directory of Open Access Journals (Sweden)

    Yi-Chih Tsai

    2013-01-01

    Full Text Available A random screening of human blood samples from 24 individuals of nonsmoker was conducted to examine the correlation between the oxidative DNA damage level of lymphocytes and the antioxidant capacity of serum or the base excision repair (BER activity of lymphocytes. The oxidative DNA damage level was measured with comet assay containing Fpg/Endo III cleavage, and the BER activity was estimated with a modified comet assay including nuclear extract of lymphocytes for enzymatic cleavage. Antioxidant capacity was determined with trolox equivalent antioxidant capacity assay. We found that though the endogenous DNA oxidation levels varied among the individuals, each individual level appeared to be steady for at least 1 month. Our results indicate that the oxidative DNA damage level is insignificantly or weakly correlated with antioxidant capacity or BER activity, respectively. However, lymphocytes from carriers of Helicobacter pylori (HP or Hepatitis B virus (HBV tend to give higher levels of oxidative DNA damage (P<0.05. Though sera of this group of individuals show no particular tendency with reduced antioxidant capacity, the respective BER activities of lymphocytes are lower in average (P<0.05. Thus, reduction of repair activity may be associated with the genotoxic effect of HP or HBV infection.

  3. Mitotic regulator Nlp interacts with XPA/ERCC1 complexes and regulates nucleotide excision repair (NER) in response to UV radiation.

    Science.gov (United States)

    Ma, Xiao-Juan; Shang, Li; Zhang, Wei-Min; Wang, Ming-Rong; Zhan, Qi-Min

    2016-04-10

    Cellular response to DNA damage, including ionizing radiation (IR) and UV radiation, is critical for the maintenance of genomic fidelity. Defects of DNA repair often result in genomic instability and malignant cell transformation. Centrosomal protein Nlp (ninein-like protein) has been characterized as an important cell cycle regulator that is required for proper mitotic progression. In this study, we demonstrate that Nlp is able to improve nucleotide excision repair (NER) activity and protects cells against UV radiation. Upon exposure of cells to UVC, Nlp is translocated into the nucleus. The C-terminus (1030-1382) of Nlp is necessary and sufficient for its nuclear import. Upon UVC radiation, Nlp interacts with XPA and ERCC1, and enhances their association. Interestingly, down-regulated expression of Nlp is found to be associated with human skin cancers, indicating that dysregulated Nlp might be related to the development of human skin cancers. Taken together, this study identifies mitotic protein Nlp as a new and important member of NER pathway and thus provides novel insights into understanding of regulatory machinery involved in NER. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  4. Risk of colorectal cancer for people with a mutation in both a MUTYH and a DNA mismatch repair gene

    Science.gov (United States)

    Win, Aung Ko; Reece, Jeanette C.; Buchanan, Daniel D.; Clendenning, Mark; Young, Joanne P.; Cleary, Sean P.; Kim, Hyeja; Cotterchio, Michelle; Dowty, James G.; MacInnis, Robert J.; Tucker, Katherine M.; Winship, Ingrid M.; Macrae, Finlay A.; Burnett, Terrilea; Le Marchand, Loïc; Casey, Graham; Haile, Robert W.; Newcomb, Polly A.; Thibodeau, Stephen N.; Lindor, Noralane M.; Hopper, John L.; Gallinger, Steven; Jenkins, Mark A.

    2015-01-01

    The base excision repair protein, MUTYH, functionally interacts with the DNA mismatch repair (MMR) system. As genetic testing moves from testing one gene at a time, to gene panel and whole exome next generation sequencing approaches, understanding the risk associated with co-existence of germline mutations in these genes will be important for clinical interpretation and management. From the Colon Cancer Family Registry, we identified 10 carriers who had both a MUTYH mutation (6 with c.1187G>A p.(Gly396Asp), 3 with c.821G>A p.(Arg274Gln), and 1 with c.536A>G p.(Tyr179Cys)) and a MMR gene mutation (3 in MLH1, 6 in MSH2, and 1 in PMS2), 375 carriers of a single (monoallelic) MUTYH mutation alone, and 469 carriers of a MMR gene mutation alone. Of the 10 carriers of both gene mutations, 8 were diagnosed with colorectal cancer. Using a weighted cohort analysis, we estimated that risk of colorectal cancer for carriers of both a MUTYH and a MMR gene mutation was substantially higher than that for carriers of a MUTYH mutation alone [hazard ratio (HR) 21.5, 95 % confidence interval (CI) 9.19–50.1; p colorectal cancer for carriers of a MMR gene mutation alone. Our finding suggests MUTYH mutation testing in MMR gene mutation carriers is not clinically informative. PMID:26202870

  5. Resisting the Resistance in Cancer: Cheminformatics Studies on Short- Path Base Excision Repair Pathway Antagonists Using Supervised Learning Approaches.

    Science.gov (United States)

    Jain, Ritu; Jamal, Salma; Goyal, Sukriti; Wahi, Divya; Singh, Aditi; Grover, Abhinav

    2015-01-01

    Survival of cells and maintenance of genome depend on detection and repair of damaged DNA through intricate mechanisms. Cancer treatment relies on chemotherapy or radiation therapy that kills neoplastic cells by causing immense damage to the DNA. In many cases, escalated DNA repair mechanism leads to resistance against these therapies and therefore, there is a need to expand the interest in developing drugs that can sensitize the cells to such therapies by interfering with the DNA repair mechanism. Several studies have suggested a link between over expression of the primary mammalian enzyme, Apurinic/Apyrimidinic Endonuclease (APE1), responsible for abasic (or AP) site removal in the DNA and resistance of these cells to cancer therapy, whereas APE1 down-regulation sensitizes the cells to DNA damaging agents. Thus, the current treatment efficacy can be improved by aiding to selective sensitization of cancer cells and protection of normal cells. In the present study, we have used machine learning based approach by selecting assorted compounds with known activity for APE1 and constructed a range of in silico predictive classification models to discriminate between the inhibitors and non-inhibitors. These models can be applied to numerous other unscreened compounds to select the ones which are more likely to be the inhibitors for APE1. We have further found the common molecular substructures which were associated with the molecular activity of the compounds using a substructure search approach.

  6. Polymorphisms in DNA Repair Gene and Susceptibility to Glioma: A Systematic Review and Meta-Analysis Based on 33 Studies with 15 SNPs in 9 Genes.

    Science.gov (United States)

    Liu, Kun; Jiang, Yugang

    2017-03-01

    At present, many publications have evaluated the correlation between the DNA repair gene polymorphisms and glioma susceptibility. However, the results remain inconclusive. The aim of this research is to exhaustively assess the association of genetic polymorphisms in DNA repair genes with glioma risk in human. Meta-analysis method was conducted, and 33 studies with 15 SNPs in 9 genes were included (12553 glioma cases and 17178 controls). Correlation strength was evaluated by odds ratio with a 95 % confidence interval. Rs1799782 T allele and rs25487A allele might bring about higher risk of glioma in Asian population. Rs1805377 G allele was an increased risk genetic factor of glioma. Asian carried with rs3212986 A allele was more likely to have glioma. Rs1800067 G allele was a risk factor of developing glioma. Carriers with rs12917 CC genotype in MGMT gene had higher risk of glioma in Caucasian than other non-CC genotype carriers. Carriers with rs1136410 T allele in PARP1 gene could more likely to develop glioma in Caucasian. This meta-analysis suggests that glioma susceptibility is associated with rs1799782 and rs25487 of X-ray repair complementing defective repair in Chinese hamster cells 1 (XRCC1), rs1805377 of XRCC4, rs1800067 of excision repair cross-complementing rodent repair deficiency complementation group 4 (ERCC4) and rs3212986 of ERCC1 in Asian population, and rs12917 of O-6-methylguanine-DNA methyltransferase (MGMT) and rs1136410 of poly(ADP-ribose) polymerase 1 (PARP1) in Caucasian population.

  7. Monte Carlo simulation of base and nucleotide excision repair of clustered DNA damage sites. I. Model properties and predicted trends

    Energy Technology Data Exchange (ETDEWEB)

    Semenenko, Vladimir; Stewart, Robert D.; Ackerman, Eric J.

    2005-12-31

    Single-cell irradiators and new experimental assays are rapidly expanding our ability to quantify the molecular mechanisms responsible for phenomena such as toxicant-induced adaptations in DNA repair and signal-mediated changes to the genome stability of cells not directly damaged by radiation (i.e., bystander cells). To advance our understanding of, and ability to predict and mitigate, the potentially harmful effects of radiological agents, effective strategies must be devised to incorporate information from molecular and cellular studies into mechanism-based, hierarchical models. A key advantage of the hierarchical modeling approach is that information from DNA repair and other in vitro assays can be systematically integrated into higher-level cell transformation and, eventually, carcinogenesis models. This presentation will outline the hierarchical modeling strategy used to integrate information from in vitro studies into the Virtual Cell (VC) radiobiology software (see Endnote). A new multi-path genomic instability model will be introduced and used to link biochemical processing of double strand breaks (DSBs) to neoplastic cell transformation. Bystander and directly damaged cells are treated explicitly in the model using a microdosimetric approach, although many of the details of the bystander response model are of a necessarily preliminary nature. The new model will be tested against several published radiobiological datasets. Results illustrating how hypothesized bystander mechanisms affect the shape of dose-response curves for neoplastic transformation as a function of Linear Energy Transfer (LET) will be presented. EndNote: R.D. Stewart, Virtual Cell (VC) Radiobiology Software. PNNL-13579, July 2001. Available at http://www.pnl.gov/berc/kbem/vc/ The DNA repair model used in the VC computer program is based on the Two-Lesion Kinetic (TLK) model [Radiat. Res. 156(4), 365-378 October 2001].

  8. Xeroderma pigmentosum-Cockayne syndrome complex in two patients: absence of skin tumors despite severe deficiency of DNA excision repair.

    Science.gov (United States)

    Scott, R J; Itin, P; Kleijer, W J; Kolb, K; Arlett, C; Muller, H

    1993-11-01

    Two brothers had a complex combination of two DNA repair disorders: Cockayne syndrome and xeroderma pigmentosum. This rare combination has previously been observed in only two other patients. The clinical signs shared by these two brothers and the two other previously described patients include severe sun sensitivity, freckling, diminished stature, hearing and movement impairment, and neurologic degeneration. Although defective UV-induced unscheduled DNA synthesis has been demonstrated (5% of normal), no skin cancers have appeared in these 38- and 41-year-old brothers, whereas skin cancers developed at a relatively early age in the two previously described patients who also had defective UV-induced unscheduled DNA synthesis.

  9. Overexpression of DNA ligase III in mitochondria protects cells against oxidative stress and improves mitochondrial DNA base excision repair

    DEFF Research Database (Denmark)

    Akbari, Mansour; Keijzers, Guido; Maynard, Scott

    2014-01-01

    slower than the preceding mitochondrial BER steps. Overexpression of DNA ligase III in mitochondria improved the rate of overall BER, increased cell survival after menadione induced oxidative stress and reduced autophagy following the inhibition of the mitochondrial electron transport chain complex I...... by rotenone. Our results suggest that the amount of DNA ligase III in mitochondria may be critical for cell survival following prolonged oxidative stress, and demonstrate a functional link between mitochondrial DNA damage and repair, cell survival upon oxidative stress, and removal of dysfunctional...

  10. Induction of base excision repair enzymes NTH1 and APE1 in rat spleen following aniline exposure.

    Science.gov (United States)

    Ma, Huaxian; Wang, Jianling; Abdel-Rahman, Sherif Z; Boor, Paul J; Khan, M Firoze

    2013-03-15

    Mechanisms by which aniline exposure elicits splenotoxicity, especially a tumorigenic response, are not well-understood. Earlier, we have shown that aniline exposure leads to oxidative DNA damage and up-regulation of OGG1 and NEIL1/2 DNA glycosylases in rat spleen. However, the contribution of endonuclease III homolog 1 (NTH1) and apurinic/apyrimidinic endonuclease 1 (APE1) in the repair of aniline-induced oxidative DNA damage in the spleen is not known. This study was, therefore, focused on examining whether NTH1 and APE1 contribute to the repair of oxidative DNA lesions in the spleen, in an experimental condition preceding tumorigenesis. To achieve this, male SD rats were subchronically exposed to aniline (0.5 mmol/kg/day via drinking water for 30 days), while controls received drinking water only. By quantitating the cleavage products, the activities of NTH1 and APE1 were assayed using substrates containing thymine glycol (Tg) and tetrahydrofuran, respectively. Aniline treatment led to significant increases in NTH1- and APE1-mediated BER activity in the nuclear extracts of spleen of aniline-treated rats compared to the controls. NTH1 and APE1 mRNA expression in the spleen showed 2.9- and 3.2-fold increases, respectively, in aniline-treated rats compared to the controls. Likewise, Western blot analysis showed that protein expression of NTH1 and APE1 in the nuclear extracts of spleen from aniline-treated rats was 1.9- and 2.7-fold higher than the controls, respectively. Immunohistochemistry indicated that aniline treatment also led to stronger immunoreactivity for both NTH1 and APE1 in the spleens, confined to the red pulp areas. These results, thus, show that aniline exposure is associated with induction of NTH1 and APE1 in the spleen. The increased repair activity of NTH1 and APE1 could be an important mechanism for the removal of oxidative DNA lesions. These findings thus identify a novel mechanism through which NTH1 and APE1 may regulate the repair of

  11. Tricuspid valve mycetoma in an infant successfully treated by excision and complex tricuspid valve repair followed by fluconazole therapy.

    Science.gov (United States)

    Anil Kumar, V; Francis, Edwin; Sreehari, Sreekala; Raj, Benedict

    2014-04-01

    Fungal valve endocarditis in children is an uncommon and lethal disease. The risk increases with use of central venous catheters (CVC), total parenteral nutrition (TPN), and use of broad-spectrum antibiotics during the neonatal period. Due to high mortality, a combination of surgery and antifungal therapy is usually recommended for treatment. Case report and review of the literature. We present a case of an asymptomatic infant with multiple Candida tricuspid valve mycetomas. Complete cure was achieved by combined tricuspid valve repair and fluconazole therapy. We also review 26 cases of tricuspid valve Candida endocarditis in children published in the literature. From being uniformly fatal five decades ago to a current survival rate of 64% to 100%, the prognosis of Candida endocarditis has changed dramatically with the use of antifungal therapy alone or in combination with surgery. Our case re-emphasizes the role of valve-sparing debridement with repair of the native valve using autologous pericardium in combination with long-term antifungal therapy as a feasible option in managing tricuspid valve Candida endocarditis.

  12. Germline Mutations in DNA Repair Genes in Lung Adenocarcinoma.

    Science.gov (United States)

    Parry, Erin M; Gable, Dustin L; Stanley, Susan E; Khalil, Sara E; Antonescu, Valentin; Florea, Liliana; Armanios, Mary

    2017-11-01

    Although lung cancer is generally thought to be environmentally provoked, anecdotal familial clustering has been reported, suggesting that there may be genetic susceptibility factors. We systematically tested whether germline mutations in eight candidate genes may be risk factors for lung adenocarcinoma. We studied lung adenocarcinoma cases for which germline sequence data had been generated as part of The Cancer Genome Atlas project but had not been previously analyzed. We selected eight genes, ATM serine/threonine kinase gene (ATM), BRCA2, DNA repair associated gene (BRCA2), checkpoint kinase 2 gene (CHEK2), EGFR, parkin RBR E3 ubiquitin protein ligase gene (PARK2), telomerase reverse transcriptase gene (TERT), tumor protein p53 gene (TP53), and Yes associated protein 1 gene (YAP1), on the basis of prior anecdotal association with lung cancer or genome-wide association studies. Among 555 lung adenocarcinoma cases, we detected 14 pathogenic mutations in five genes; they occurred at a frequency of 2.5% and represented an OR of 66 (95% confidence interval: 33-125, p mutations fell most commonly in ATM (50%), followed by TP53, BRCA2, EGFR, and PARK2. Most (86%) of these variants had been reported in other familial cancer syndromes. Another 12 cases (2%) carried ultrarare variants that were predicted to be deleterious by three protein prediction programs; these most frequently involved ATM and BRCA2. A subset of patients with lung adenocarcinoma, at least 2.5% to 4.5%, carry germline variants that have been linked to cancer risk in Mendelian syndromes. The genes fall most frequently in DNA repair pathways. Our data indicate that patients with lung adenocarcinoma, similar to other solid tumors, include a subset of patients with inherited susceptibility. Copyright © 2017 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

  13. Gene therapy and peripheral nerve repair: a perspective

    Directory of Open Access Journals (Sweden)

    Stefan A. Hoyng

    2015-07-01

    Full Text Available Clinical phase I/II studies have demonstrated the safety of gene therapy for a variety of central nervous system disorders, including Canavan’s, Parkinson’s and Alzheimer’s disease, retinal diseases and pain. The majority of gene therapy studies in the CNS have used adeno-associated viral vectors (AAV and the first AAV-based therapeutic, a vector encoding lipoprotein lipase, is now marketed in Europe under the name Glybera. These remarkable advances may become relevant to translational research on gene therapy to promote peripheral nervous system (PNS repair. This short review first summarizes the results of gene therapy in animal models for peripheral nerve repair. Secondly, we identify key areas of future research in the domain of PNS-gene therapy. Finally, a perspective is provided on the path to clinical translation of PNS gene therapy for traumatic nerve injuries. In the latter section we discuss the route and mode of delivery of the vector to human patients, the efficacy and safety of the vector, and the choice of the patient population for a first possible proof-of-concept clinical study.

  14. Genomic approaches to DNA repair and mutagenesis.

    Science.gov (United States)

    Wyrick, John J; Roberts, Steven A

    2015-12-01

    DNA damage is a constant threat to cells, causing cytotoxicity as well as inducing genetic alterations. The steady-state abundance of DNA lesions in a cell is minimized by a variety of DNA repair mechanisms, including DNA strand break repair, mismatch repair, nucleotide excision repair, base excision repair, and ribonucleotide excision repair. The efficiencies and mechanisms by which these pathways remove damage from chromosomes have been primarily characterized by investigating the processing of lesions at defined genomic loci, among bulk genomic DNA, on episomal DNA constructs, or using in vitro substrates. However, the structure of a chromosome is heterogeneous, consisting of heavily protein-bound heterochromatic regions, open regulatory regions, actively transcribed genes, and even areas of transient single stranded DNA. Consequently, DNA repair pathways function in a much more diverse set of chromosomal contexts than can be readily assessed using previous methods. Recent efforts to develop whole genome maps of DNA damage, repair processes, and even mutations promise to greatly expand our understanding of DNA repair and mutagenesis. Here we review the current efforts to utilize whole genome maps of DNA damage and mutation to understand how different chromosomal contexts affect DNA excision repair pathways. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. [The expression of thymidylate synthase (TS) and excision repair complementing-1 (ERCC-1) protein in patients with unresectable colorectal cancer treated with mFOLFOX6 therapy].

    Science.gov (United States)

    Ishibashi, Keiichiro; Okada, Norimichi; Ishiguro, Toru; Kuwabara, Kouki; Ohsawa, Tomonori; Yokoyama, Masaru; Kumamoto, Kensuke; Haga, Norihiro; Mori, Takashi; Yamada, Hirofumi; Miura, Ichiro; Tamaru, Junichi; Itoyama, Shinji; Ishida, Hideyuki

    2010-11-01

    Thymidylate synthase (TS) and excision repair complementing-1 (ERCC-1) were known to be important biomarkers to predict a tumor response to 5-fluorouracil (5-FU) and oxaliplatin, but the relationship between these expressions and tumor response were still unclear. The aim of this study was to determine whether the expression of TS and ERCC-1 protein predict a tumor response in patients with unresectable colorectal cancer treated with mFOLFOX6 therapy as first-line treatment. Fifty patients with unresectable colorectal cancer treated with mFOLFOX6 therapy were enrolled in this study. The expression of TS and ERCC-1 protein in primary cancer cells were examined using immunohistochemistry. There were no significant differences between response rate and the expression of TS or ERCC-1 protein (TS: p>0.99, ERCC-1: p= 0.50). There were no significant differences between progression-free survival time and the expression of TS or ERCC-1 protein (TS: p=0.60, ERCC-1: p=0.60). In this study, the expression TS and ERCC-1 protein may not be useful for the prediction of tumor response in patients with unresectable colorectal cancer treated with mFOLFOX6 therapy.

  16. Microsatellite Instability Use in Mismatch Repair Gene Sequence Variant Classification

    Directory of Open Access Journals (Sweden)

    Bryony A. Thompson

    2015-03-01

    Full Text Available Inherited mutations in the DNA mismatch repair genes (MMR can cause MMR deficiency and increased susceptibility to colorectal and endometrial cancer. Microsatellite instability (MSI is the defining molecular signature of MMR deficiency. The clinical classification of identified MMR gene sequence variants has a direct impact on the management of patients and their families. For a significant proportion of cases sequence variants of uncertain clinical significance (also known as unclassified variants are identified, constituting a challenge for genetic counselling and clinical management of families. The effect on protein function of these variants is difficult to interpret. The presence or absence of MSI in tumours can aid in determining the pathogenicity of associated unclassified MMR gene variants. However, there are some considerations that need to be taken into account when using MSI for variant interpretation. The use of MSI and other tumour characteristics in MMR gene sequence variant classification will be explored in this review.

  17. Non-DBS DNA Repair Genes Regulate Radiation-induced Cytogenetic Damage Repair and Cell Cycle Progression

    Science.gov (United States)

    Zhang, Ye; Rohde, Larry H.; Emami, Kamal; Casey, Rachael; Wu, Honglu

    2008-01-01

    Changes of gene expression profile are one of the most important biological responses in living cells after ionizing radiation (IR) exposure. Although some studies have shown that genes up-regulated by IR may play important roles in DNA damage repair, the relationship between the regulation of gene expression by IR, particularly genes not known for their roles in DSB repair, and its impact on cytogenetic responses has not been systematically studied. In the present study, the expression of 25 genes selected on the basis of their transcriptional changes in response to IR was individually knocked down by transfection with small interfering RNA in human fibroblast cells. The purpose of this study is to identify new roles of these selected genes on regulating DSB repair and cell cycle progression , as measured in the micronuclei formation and chromosome aberration. In response to IR, the formation of MN was significantly increased by suppressed expression of 5 genes: Ku70 in the DSB repair pathway, XPA in the NER pathway, RPA1 in the MMR pathway, and RAD17 and RBBP8 in cell cycle control. Knocked-down expression of 4 genes (MRE11A, RAD51 in the DSB pathway, SESN1, and SUMO1) significantly inhibited cell cycle progression, possibly because of severe impairment of DNA damage repair. Furthermore, loss of XPA, P21, or MLH1 expression resulted in both significantly enhanced cell cycle progression and increased yields of chromosome aberrations, indicating that these gene products modulate both cell cycle control and DNA damage repair. Most of the 11 genes that affected cytogenetic responses are not known to have clear roles influencing DBS repair. Nine of these 11 genes were up-regulated in cells exposed to gamma radiation, suggesting that genes transcriptionally modulated by IR were critical to regulate the biological consequences after IR.

  18. Energy and Technology Review: Unlocking the mysteries of DNA repair

    Energy Technology Data Exchange (ETDEWEB)

    Quirk, W.A.

    1993-04-01

    DNA, the genetic blueprint, has the remarkable property of encoding its own repair following diverse types of structural damage induced by external agents or normal metabolism. We are studying the interplay of DNA damaging agents, repair genes, and their protein products to decipher the complex biochemical pathways that mediate such repair. Our research focuses on repair processes that correct DNA damage produced by chemical mutagens and radiation, both ionizing and ultraviolet. The most important type of DNA repair in human cells is called excision repair. This multistep process removes damaged or inappropriate pieces of DNA -- often as a string of 29 nucleotides containing the damage -- and replaces them with intact ones. We have isolated, cloned, and mapped several human repair genes associated with the nucleotide excision repair pathway and involved in the repair of DNA damage after exposure to ultraviolet light or mutagens in cooked food. We have shown that a defect in one of these repair genes, ERCC2, is responsible for the repair deficiency in one of the groups of patients with the recessive genetic disorder xeroderma pigmentosum (XP group D). We are exploring ways to purify sufficient quantities (milligrams) of the protein products of these and other repair genes so that we can understand their functions. Our long-term goals are to link defective repair proteins to human DNA repair disorders that predispose to cancer, and to produce DNA-repair-deficient mice that can serve as models for the human disorders.

  19. Transient expression of a plasmid gene, a tool to study DNA repair in human cells: defect of DNA repair in Cockayne syndrome; one thymine cyclobutane dimer is sufficient to block transcription.

    Science.gov (United States)

    Klocker, H; Schneider, R; Burtscher, H J; Auer, B; Hirsch-Kauffmann, M; Schweiger, M

    1986-01-01

    Transfected recombinant DNA with regulatory elements such as eukaryotic promoter and termination sites is transiently expressed in human fibroblast cells. Utilizing an expression vector containing the simian virus 40 (SV 40) early control region followed by the E. coli chloramphenicol acetyltransferase (CAT) gene, we investigated the ability of normal, Xeroderma pigmentosum and Cockayne Syndrome cells to repair UV lesions in transfected DNA. Fibroblasts from Xeroderma pigmentosum patients which cannot excise pyrimidine cyclobutane dimers were unable to restore expression of UV irradiated CAT gene. An UV dose inducing one thymine cyclobutane dimer in the transcribed strand of the CAT gene blocked its transcription in these repair deficient cells. Normal cell were able to repair the lesions in transfected DNA during an incubation period of about 40 h and in this way could overcome the UV block. In several fibroblast cell lines from patients suffering from Cockayne Syndrome expression of UV damaged CAT gene was restored significantly less than in normal fibroblasts, indicating that Cockayne Syndrome is associated with a UV repair defect.

  20. The stealth episome: suppression of gene expression on the excised genomic island PPHGI-1 from Pseudomonas syringae pv. phaseolicola.

    Directory of Open Access Journals (Sweden)

    Scott A C Godfrey

    2011-03-01

    Full Text Available Pseudomonas syringae pv. phaseolicola is the causative agent of halo blight in the common bean, Phaseolus vulgaris. P. syringae pv. phaseolicola race 4 strain 1302A contains the avirulence gene avrPphB (syn. hopAR1, which resides on PPHGI-1, a 106 kb genomic island. Loss of PPHGI-1 from P. syringae pv. phaseolicola 1302A following exposure to the hypersensitive resistance response (HR leads to the evolution of strains with altered virulence. Here we have used fluorescent protein reporter systems to gain insight into the mobility of PPHGI-1. Confocal imaging of dual-labelled P. syringae pv. phaseolicola 1302A strain, F532 (dsRFP in chromosome and eGFP in PPHGI-1, revealed loss of PPHGI-1::eGFP encoded fluorescence during plant infection and when grown in vitro on extracted leaf apoplastic fluids. Fluorescence-activated cell sorting (FACS of fluorescent and non-fluorescent PPHGI-1::eGFP F532 populations showed that cells lost fluorescence not only when the GI was deleted, but also when it had excised and was present as a circular episome. In addition to reduced expression of eGFP, quantitative PCR on sub-populations separated by FACS showed that transcription of other genes on PPHGI-1 (avrPphB and xerC was also greatly reduced in F532 cells harbouring the excised PPHGI-1::eGFP episome. Our results show how virulence determinants located on mobile pathogenicity islands may be hidden from detection by host surveillance systems through the suppression of gene expression in the episomal state.

  1. Approaches to diagnose DNA mismatch repair gene defects in cancer.

    Science.gov (United States)

    Peña-Diaz, Javier; Rasmussen, Lene Juel

    2016-02-01

    The DNA repair pathway mismatch repair (MMR) is responsible for the recognition and correction of DNA biosynthetic errors caused by inaccurate nucleotide incorporation during replication. Faulty MMR leads to failure to address the mispairs or insertion deletion loops (IDLs) left behind by the replicative polymerases and results in increased mutation load at the genome. The realization that defective MMR leads to a hypermutation phenotype and increased risk of tumorigenesis highlights the relevance of this pathway for human disease. The association of MMR defects with increased risk of cancer development was first observed in colorectal cancer patients that carried inactivating germline mutations in MMR genes and the disease was named as hereditary non-polyposis colorectal cancer (HNPCC). Currently, a growing list of cancers is found to be MMR defective and HNPCC has been renamed Lynch syndrome (LS) partly to include the associated risk of developing extra-colonic cancers. In addition, a number of non-hereditary, mostly epigenetic, alterations of MMR genes have been described in sporadic tumors. Besides conferring a strong cancer predisposition, genetic or epigenetic inactivation of MMR genes also renders cells resistant to some chemotherapeutic agents. Therefore, diagnosis of MMR deficiency has important implications for the management of the patients, the surveillance of their relatives in the case of LS and for the choice of treatment. Some of the alterations found in MMR genes have already been well defined and their pathogenicity assessed. Despite this substantial wealth of knowledge, the effects of a large number of alterations remain uncharacterized (variants of uncertain significance, VUSs). The advent of personalized genomics is likely to increase the list of VUSs found in MMR genes and anticipates the need of diagnostic tools for rapid assessment of their pathogenicity. This review describes current tools and future strategies for addressing the relevance

  2. Lys98 substitution in human AP endonuclease 1 affects the kinetic mechanism of enzyme action in base excision and nucleotide incision repair pathways.

    Directory of Open Access Journals (Sweden)

    Nadezhda A Timofeyeva

    Full Text Available Human apurinic/apyrimidinic endonuclease 1 (APE1 is a key enzyme in the base excision repair (BER and nucleotide incision repair (NIR pathways. We recently analyzed the conformational dynamics and kinetic mechanism of wild-type (wt protein, in a stopped-flow fluorescence study. In this study, we investigated the mutant enzyme APE1K98A using the same approach. Lys98 was known to hydrogen bond to the carboxyl group of Asp70, a residue implicated in binding the divalent metal ion. Our data suggested that the conformational selection and induced fit occur during the enzyme action. We expanded upon the evidence that APE1 can pre-exist in two conformations. The isomerization of an enzyme-product complex in the BER process and the additional isomerization stage of enzyme-substrate complex in the NIR process were established for APE1K98A. These stages had not been registered for the wtAPE1. We found that the K98A substitution resulted in a 12-fold reduction of catalytic constant of 5'-phosphodiester bond hydrolysis in (3-hydroxytetrahydrofuran-2-ylmethyl phosphate (F, tetrahydrofuran containing substrate, and in 200-fold reduction in 5,6-dihydrouridine (DHU containing substrate. Thus, the K98A substitution influenced NIR more than BER. We demonstrated that the K98A mutation influenced the formation of primary unspecific enzyme-substrate complex in a complicated manner, depending on the Mg(2+ concentration and pH. This mutation obstructed the induced fit of enzyme in the complex with undamaged DNA and F-containing DNA and appreciably decreased the stability of primary complex upon interaction of enzyme with DNA, containing the natural apurinic/apyrimidinic (AP site. Furthermore, it significantly delayed the activation of the less active form of enzyme during NIR and slowed down the conformational conversion of the complex of enzyme with the cleavage product of DHU-substrate. Our data revealed that APE1 uses the same active site to catalyze the cleavage

  3. Nucleotide excision repair of oxidised genomic DNA is not a source of urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine.

    Science.gov (United States)

    Evans, Mark D; Mistry, Vilas; Singh, Rajinder; Gackowski, Daniel; Różalski, Rafał; Siomek-Gorecka, Agnieszka; Phillips, David H; Zuo, Jie; Mullenders, Leon; Pines, Alex; Nakabeppu, Yusaku; Sakumi, Kunihiko; Sekiguchi, Mutsuo; Tsuzuki, Teruhisa; Bignami, Margherita; Oliński, Ryszard; Cooke, Marcus S

    2016-10-01

    Urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) is a widely measured biomarker of oxidative stress. It has been commonly assumed to be a product of DNA repair, and therefore reflective of DNA oxidation. However, the source of urinary 8-oxodGuo is not understood, although potential confounding contributions from cell turnover and diet have been ruled out. Clearly it is critical to understand the precise biological origins of this important biomarker, so that the target molecule that is oxidised can be identified, and the significance of its excretion can be interpreted fully. In the present study we aimed to assess the contributions of nucleotide excision repair (NER), by both the global genome NER (GG-NER) and transcription-coupled NER (TC-NER) pathways, and sanitisation of the dGTP pool (e.g. via the activity of the MTH1 protein), on the production of 8-oxodGuo, using selected genetically-modified mice. In xeroderma pigmentosum A (XPA) mice, in which GG-NER and TC-NER are both defective, the urinary 8-oxodGuo data were unequivocal in ruling out a contribution from NER. In line with the XPA data, the production of urinary 8-oxodGuo was not affected in the xeroderma pigmentosum C mice, specifically excluding a role of the GG-NER pathway. The bulk of the literature supports the mechanism that the NER proteins are responsible for removing damage to the transcribed strand of DNA via TC-NER, and on this basis we also examined Cockayne Syndrome mice, which have a functional loss of TC-NER. These mice showed no difference in urinary 8-oxodGuo excretion, compared to wild type, demonstrating that TC-NER does not contribute to urinary 8-oxodGuo levels. These findings call into question whether genomic DNA is the primary source of urinary 8-oxodGuo, which would largely exclude it as a biomarker of DNA oxidation. The urinary 8-oxodGuo levels from the MTH1 mice (both knock-out and hMTH1-Tg) were not significantly different to the wild-type mice. We suggest that these

  4. Excision repair cross-complementation group 1 protein expression predicts survival in patients with high-grade, non-metastatic osteosarcoma treated with neoadjuvant chemotherapy.

    Science.gov (United States)

    Hattinger, Claudia Maria; Michelacci, Francesca; Sella, Federica; Magagnoli, Giovanna; Benini, Stefania; Gambarotti, Marco; Palmerini, Emanuela; Picci, Piero; Serra, Massimo; Ferrari, Stefano

    2015-09-01

    To evaluate the clinical impact of excision repair cross-complementation group 1 (ERCC1) expression in high-grade osteosarcoma (OS). Immunohistochemistry was performed on biopsies from 99 OS patients enrolled in the ISG/OS-Oss training set or ISG/SSG1 validation set neoadjuvant chemotherapy protocols, based on the use of cisplatin, adriamycin, methotrexate, and ifosfamide. In the training set, ERCC1 positivity was found in eight of 31 (26%) patients, and was significantly associated with worse event-free survival (EFS) (P = 0.042) and overall survival (OVS) (P = 0.001). In the validation set, ERCC1 positivity was found in 22 of 68 (32%) patients, and its significant associations with poorer EFS (P = 0.028) and OVS (P = 0.022) were confirmed. Multivariate analyses performed on the whole patient series indicated that ERCC1 positivity was the only marker that was significantly associated with a higher risk of worse prognosis, in terms of both EFS and OVS (P = 0.013). Co-evaluation of ERCC1 and ABCB1 expression showed that patients who were positive for both markers had a significantly worse prognosis. The ERCC1 level at diagnosis is predictive for the outcome of patients with non-metastatic, high-grade OS treated with neoadjuvant chemotherapy, and co-evaluation with ABCB1 can identify high-risk groups of OS patients who are refractory to standard regimens. © 2015 John Wiley & Sons Ltd.

  5. Heat shock induced excision of selectable marker genes in transgenic banana by the Cre-lox site-specific recombination system.

    Science.gov (United States)

    Chong-Pérez, Borys; Kosky, Rafael G; Reyes, Maritza; Rojas, Luis; Ocaña, Bárbara; Tejeda, Marisol; Pérez, Blanca; Angenon, Geert

    2012-06-30

    Selectable marker genes are indispensable for efficient production of transgenic events, but are no longer needed after the selection process and may cause public concern and technological problems. Although several gene excision systems exist, few have been optimized for vegetatively propagated crops. Using a Cre-loxP auto-excision strategy, we obtained transgenic banana plants cv. Grande Naine (Musa AAA) devoid of the marker gene used for selection. We used T-DNA vectors with the cre recombinase gene under control of a heat shock promoter and selectable marker gene cassettes placed between two loxP sites in direct orientation, and a gene of interest inserted outside of the loxP sites. Heat shock promoters pGmHSP17.6-L and pHSP18.2, from soybean and Arabidopsis respectively, were tested. A transient heat shock treatment of primary transgenic embryos was sufficient for inducing cre and excising cre and the marker genes. Excision efficiency, as determined by PCR and Southern hybridization was 59.7 and 40.0% for the GmHSP17.6-L and HSP18.2 promoters, respectively. Spontaneous excision was not observed in 50 plants derived from untreated transgenic embryos. To our knowledge this is the first report describing an efficient marker gene removal system for banana. The method described is simple and might be generally applicable for the production of marker-free transgenic plants of many crop species. Copyright © 2011 Elsevier B.V. All rights reserved.

  6. Single-Nucleotide Polymorphisms of Genes Involved in Repair of Oxidative DNA Damage and the Risk of Recurrent Depressive Disorder

    Science.gov (United States)

    Czarny, Piotr; Kwiatkowski, Dominik; Toma, Monika; Gałecki, Piotr; Orzechowska, Agata; Bobińska, Kinga; Bielecka-Kowalska, Anna; Szemraj, Janusz; Berk, Michael; Anderson, George; Śliwiński, Tomasz

    2016-01-01

    Background Depressive disorder, including recurrent type (rDD), is accompanied by increased oxidative stress and activation of inflammatory pathways, which may induce DNA damage. This thesis is supported by the presence of increased levels of DNA damage in depressed patients. Such DNA damage is repaired by the base excision repair (BER) pathway. BER efficiency may be influenced by polymorphisms in BER-related genes. Therefore, we genotyped nine single-nucleotide polymorphisms (SNPs) in six genes encoding BER proteins. Material/Methods Using TaqMan, we selected and genotyped the following SNPs: c.-441G>A (rs174538) of FEN1, c.2285T>C (rs1136410) of PARP1, c.580C>T (rs1799782) and c.1196A>G (rs25487) of XRCC1, c.*83A>C (rs4796030) and c.*50C>T (rs1052536) of LIG3, c.-7C>T (rs20579) of LIG1, and c.-468T>G (rs1760944) and c.444T>G (rs1130409) of APEX1 in 599 samples (288 rDD patients and 311 controls). Results We found a strong correlation between rDD and both SNPs of LIG3, their haplotypes, as well as a weaker association with the c.-468T>G of APEXI which diminished after Nyholt correction. Polymorphisms of LIG3 were also associated with early onset versus late onset depression, whereas the c.-468T>G polymorphism showed the opposite association. Conclusions The SNPs of genes involved in the repair of oxidative DNA damage may modulate rDD risk. Since this is an exploratory study, the results should to be treated with caution and further work needs to be done to elucidate the exact involvement of DNA damage and repair mechanisms in the development of this disease. PMID:27866211

  7. Single-Nucleotide Polymorphisms of Genes Involved in Repair of Oxidative DNA Damage and the Risk of Recurrent Depressive Disorder.

    Science.gov (United States)

    Czarny, Piotr; Kwiatkowski, Dominik; Toma, Monika; Gałecki, Piotr; Orzechowska, Agata; Bobińska, Kinga; Bielecka-Kowalska, Anna; Szemraj, Janusz; Berk, Michael; Anderson, George; Śliwiński, Tomasz

    2016-11-20

    BACKGROUND Depressive disorder, including recurrent type (rDD), is accompanied by increased oxidative stress and activation of inflammatory pathways, which may induce DNA damage. This thesis is supported by the presence of increased levels of DNA damage in depressed patients. Such DNA damage is repaired by the base excision repair (BER) pathway. BER efficiency may be influenced by polymorphisms in BER-related genes. Therefore, we genotyped nine single-nucleotide polymorphisms (SNPs) in six genes encoding BER proteins. MATERIAL AND METHODS Using TaqMan, we selected and genotyped the following SNPs: c.-441G>A (rs174538) of FEN1, c.2285T>C (rs1136410) of PARP1, c.580C>T (rs1799782) and c.1196A>G (rs25487) of XRCC1, c.*83A>C (rs4796030) and c.*50C>T (rs1052536) of LIG3, c.-7C>T (rs20579) of LIG1, and c.-468T>G (rs1760944) and c.444T>G (rs1130409) of APEX1 in 599 samples (288 rDD patients and 311 controls). RESULTS We found a strong correlation between rDD and both SNPs of LIG3, their haplotypes, as well as a weaker association with the c.-468T>G of APEXI which diminished after Nyholt correction. Polymorphisms of LIG3 were also associated with early onset versus late onset depression, whereas the c.-468T>G polymorphism showed the opposite association. CONCLUSIONS The SNPs of genes involved in the repair of oxidative DNA damage may modulate rDD risk. Since this is an exploratory study, the results should to be treated with caution and further work needs to be done to elucidate the exact involvement of DNA damage and repair mechanisms in the development of this disease.

  8. Gene specific damage and repair after treatment of cells with UV and chemotherapeutical agents

    Energy Technology Data Exchange (ETDEWEB)

    Bohr, V.A. (Division of Cancer Treatment, National Cancer Institute, NIH, Bethesda, MD (USA))

    1991-01-01

    The authors have previously demonstrated preferential DNA repair of active genes in mammalian cells. The methodology involves the use of a specific endonuclease or other more direct approaches to create nicks at sites of damage followed by quantitative Southern analysis and probing for specific genes. Initially, they used pyrimidine dimer specific endonuclease to detect pyrimidine dimers after UV irradiation. They now also use the bacterial enzyme ABC excinuclease to examine the DNA damage and repair of a number of adducts other than pyrimidine dimers in specific genes. They can detect gene specific alkylation damage by creating nicks via depurination and alkaline hydrolysis. In our assay for preferential repair, they compare the efficiency of repair in the DHFR gene to that in the 3{prime} flanking, non-coding region to the gene. In CHO cells, UV induced pyrimidine dimers are efficiently repaired from the active DHFR gene, but not from the inactive region. They have demonstrated that the 6-4 photoproducts are also preferentially repaired and that they are removed faster from the regions studied than pyrimidine dimers. Using similar approaches, they find that DNA adducts and crosslinks caused by cisplatinum are preferentially repaired in the active gene compared to the inactive regions and to the inactive c-fos oncogene. Also, nitrogen mustard and methylnitrosurea damage is preferentially repaired whereas dimethylsulphate damage is not. NAAAF adducts do not appear to be preferentially repaired in this system. 32 refs.

  9. Stripped-down DNA repair in a highly reduced parasite

    Directory of Open Access Journals (Sweden)

    Fast Naomi M

    2007-03-01

    Full Text Available Abstract Background Encephalitozoon cuniculi is a member of a distinctive group of single-celled parasitic eukaryotes called microsporidia, which are closely related to fungi. Some of these organisms, including E. cuniculi, also have uniquely small genomes that are within the prokaryotic range. Thus, E. cuniculi has undergone a massive genome reduction which has resulted in a loss of genes from diverse biological pathways, including those that act in DNA repair. DNA repair is essential to any living cell. A loss of these mechanisms invariably results in accumulation of mutations and/or cell death. Six major pathways of DNA repair in eukaryotes include: non-homologous end joining (NHEJ, homologous recombination repair (HRR, mismatch repair (MMR, nucleotide excision repair (NER, base excision repair (BER and methyltransferase repair. DNA polymerases are also critical players in DNA repair processes. Given the close relationship between microsporidia and fungi, the repair mechanisms present in E. cuniculi were compared to those of the yeast Saccharomyces cerevisiae to ascertain how the process of genome reduction has affected the DNA repair pathways. Results E. cuniculi lacks 16 (plus another 6 potential absences of the 56 DNA repair genes sought via BLASTP and PSI-BLAST searches. Six of 14 DNA polymerases or polymerase subunits are also absent in E. cuniculi. All of these genes are relatively well conserved within eukaryotes. The absence of genes is not distributed equally among the different repair pathways; some pathways lack only one protein, while there is a striking absence of many proteins that are components of both double strand break repair pathways. All specialized repair polymerases are also absent. Conclusion Given the large number of DNA repair genes that are absent from the double strand break repair pathways, E. cuniculi is a prime candidate for the study of double strand break repair with minimal machinery. Strikingly, all of the

  10. Pathological complete response after cisplatin neoadjuvant therapy is associated with the downregulation of DNA repair genes in BRCA1-associated triple-negative breast cancers.

    Science.gov (United States)

    Domagala, Pawel; Hybiak, Jolanta; Rys, Janusz; Byrski, Tomasz; Cybulski, Cezary; Lubinski, Jan

    2016-10-18

    Pathologic complete response (pCR) after neoadjuvant chemotherapy is considered a suitable surrogate marker of treatment efficacy in patients with triple-negative breast cancers (TNBCs). However, the molecular mechanisms underlying pCR as a result of such treatment remain obscure. Using real-time PCR arrays we compared the expression levels of 120 genes involved in the main mechanisms of DNA repair in 43 pretreatment biopsies of BRCA1-associated TNBCs exhibiting pCR and no pathological complete response (non-pCR) after neoadjuvant chemotherapy with cisplatin. Altogether, 25 genes were significantly differentially expressed between tumors exhibiting pCR and non-pCR, and these genes were downregulated in the pCR group compared to the non-pCR group. A difference in expression level greater than 1.5-fold was detected for nine genes: MGMT, ERCC4, FANCB, UBA1, XRCC5, XPA, XPC, PARP3, and RPA1. The non-homologous end joining and nucleotide excision repair pathways of DNA repair showed the most significant relevance. Expression profile of DNA repair genes associated with pCR was different in the node-positive (20 genes with fold change >1.5) and node-negative (only 3 genes) subgroups. Although BRCA1 germline mutations are the principal defects in BRCA1-associated TNBC, our results indicate that the additional downregulation of other genes engaged in major pathways of DNA repair may play a decisive role in the pathological response of these tumors to cisplatin neoadjuvant chemotherapy. The results suggest that patients with node-positive BRCA1-associated TNBCs that do not exhibit pCR after cisplatin neoadjuvant chemotherapy may be candidates for subsequent therapy with PARP inhibitors, whereas UBA1 may be a potential therapeutic target in node-negative subgroup.

  11. Effects of top excision on the potassium accumulation and expression of potassium channel genes in tobacco.

    Science.gov (United States)

    Dai, Xiao Yan; Su, Yi Rong; Wei, Wen Xue; Wu, Jin Shui; Fan, Ye Kuan

    2009-01-01

    The effects of the removal of the shoot apex of tobacco on the relative transcript levels of potassium channel genes, determined by real-time PCR, and on the relationship between the expression of genes encoding potassium channels and potassium concentration, were studied. The results from the study indicated that comparatively more assimilates of photosynthesis were allocated to the apex in control plants than in both decapitated and IAA-treated decapitated plants. By contrast, dry matter in the upper leaves, roots, and stems in both decapitated and IAA-treated plants was significantly increased relative to control plants. The potassium level in whole plants decreased post-decapitation compared with control plants, and so did the potassium concentration in middle and upper leaves, stem, and roots. Expression of NKT1, NtKC1, NTORK1, and NKT2 was inhibited by decapitation in tobacco leaves with a gradual reduction after decapitation, but was induced in roots. The relative expression of NKT1, NTORK1, and NKT2 in tobacco leaves was higher than that in roots, whereas the expression of NtKC1 was higher in roots. The levels of inhibition and induction of NKT1, NtKC1, NTORK1, and NKT2 in leaves and roots, respectively, associated with decapitation were reduced by the application of IAA on the cut surface of the decapitated stem. Further results showed that the level of endogenous auxin IAA in decapitated plants, which dropped in leaves and increased in roots by 140.7% at 14 d compared with the control plant, might be attributed to the change in the expression of potassium channel genes. The results suggest that there is a reciprocal relationship among endogenous auxin IAA, expression of potassium channel genes and potassium accumulation. They further imply that the endogenous IAA probably plays a role in regulating the expression of potassium channel genes, and that variations in expression of these genes affected the accumulation and distribution of potassium in tobacco.

  12. In vivo effects of UV radiation on multiple endpoints and expression profiles of DNA repair and heat shock protein (Hsp) genes in the cycloid copepod Paracyclopina nana

    Energy Technology Data Exchange (ETDEWEB)

    Won, Eun-Ji; Han, Jeonghoon [Department of Biological Science, College of Science, Sungkyunkwan University, Suwon 440-746 (Korea, Republic of); Lee, Yeonjung; Kumar, K. Suresh; Shin, Kyung-Hoon [Department of Marine Sciences and Convergent Technology, College of Science and Technology, Hanyang University, Ansan 426-791 (Korea, Republic of); Lee, Su-Jae [Department of Life Sciences, College of Natural Sciences, Hanyang University, Seoul 133-791 (Korea, Republic of); Park, Heum Gi, E-mail: hgpark@gwnu.ac.kr [Department of Marine Resource Development, College of Life Sciences, Gangneung-Wonju National University, Gangneung 210-702 (Korea, Republic of); Lee, Jae-Seong, E-mail: jslee2@skku.edu [Department of Biological Science, College of Science, Sungkyunkwan University, Suwon 440-746 (Korea, Republic of)

    2015-08-15

    Highlights: • UV-B radiation induced a significant reduction of the re-brooding rate of ovigerous females. • A dose-dependent decrease in food ingestion and the rate of assimilation to the body upon UV radiation. • Expression of base excision repair-associated and hsp chaperoning genes was significantly increased upon UV radiation in P. nana. - Abstract: To evaluate the effects of ultraviolet (UV) radiation on energy acquisition and consumption, the copepod Paracyclopina nana was irradiated with several doses (0–3 kJ/m{sup 2}) of UV. After UV radiation, we measured the re-brooding success, growth pattern of newly hatched nauplii, ingestion rate, and assimilation of diet. In addition, we checked the modulated patterns of DNA repair and heat shock protein (hsp) chaperoning genes of P. nana. UV-B radiation induced a significant reduction (7–87%) of the re-brooding rate of ovigerous females, indicating that UV-induced egg sac damage is closely correlated with a reduction in the hatching rate of UV-irradiated ovigerous female offspring. Using chlorophyll a and stable carbon isotope incubation experiments, we found a dose-dependent decrease (P < 0.05) in food ingestion and the rate of assimilation to the body in response to UV radiation, implying that P. nana has an underlying ability to shift its balanced-energy status from growth and reproduction to DNA repair and adaptation. Also, expression of P. nana base excision repair (BER)-associated genes and hsp chaperoning genes was significantly increased in response to UV radiation in P. nana. These findings indicate that even 1 kJ/m{sup 2} of UV radiation induces a reduction in reproduction and growth patterns, alters the physiological balance and inhibits the ability to cope with UV-induced damage in P. nana.

  13. DNA mismatch repair preferentially protects genes from mutation.

    Science.gov (United States)

    Belfield, Eric J; Ding, Zhong Jie; Jamieson, Fiona J C; Visscher, Anne M; Zheng, Shao Jian; Mithani, Aziz; Harberd, Nicholas P

    2017-12-12

    Mutation is the source of genetic variation and fuels biological evolution. Many mutations first arise as DNA replication errors. These errors subsequently evade correction by cellular DNA repair, for example, by the well-known DNA mismatch repair (MMR) mechanism. Here, we determine the genome-wide effects of MMR on mutation. We first identify almost 9000 mutations accumulated over five generations in eight MMR-deficient mutation accumulation (MA) lines of the model plant species, Arabidopsis thaliana We then show that MMR deficiency greatly increases the frequency of both smaller-scale insertions and deletions (indels) and of single-nucleotide variant (SNV) mutations. Most indels involve A or T nucleotides and occur preferentially in homopolymeric (poly A or poly T) genomic stretches. In addition, we find that the likelihood of occurrence of indels in homopolymeric stretches is strongly related to stretch length, and that this relationship causes ultrahigh localized mutation rates in specific homopolymeric stretch regions. For SNVs, we show that MMR deficiency both increases their frequency and changes their molecular mutational spectrum, causing further enhancement of the GC to AT bias characteristic of organisms with normal MMR function. Our final genome-wide analyses show that MMR deficiency disproportionately increases the numbers of SNVs in genes, rather than in nongenic regions of the genome. This latter observation indicates that MMR preferentially protects genes from mutation and has important consequences for understanding the evolution of genomes during both natural selection and human tumor growth. © 2018 Belfield et al.; Published by Cold Spring Harbor Laboratory Press.

  14. Retraction: 'Dose-dependent dual effect of HTLV-1 tax oncoprotein on p53-dependent nucleotide excision repair in human T-cells' by Yana Schavinsky-Khrapunsky, Esther Priel and Mordechai Aboud.

    Science.gov (United States)

    2017-06-15

    The above article, published online on 4 October 2007 in Wiley Online Library (wileyonlinelibrary.com), and in Volume 122, pp. 305-316, has been retracted by agreement between the journal Editor in Chief, Professor Peter Lichter, and John Wiley & Sons Ltd. The retraction has been agreed as the bands in Figs 1, 2, 5 and 6 appear to have been manipulated. Schavinsky-Khrapunsky, Y., Priel, E. and Aboud, M. (2008), Dose-dependent dual effect of HTLV-1 tax oncoprotein on p53-dependent nucleotide excision repair in human T-cells. Int. J. Cancer, 122: 305-316. doi:10.1002/ijc.23091. © 2017 UICC.

  15. Reverted glutathione S-transferase-like genes that influence flower color intensity of carnation (Dianthus caryophyllus L.) originated from excision of a transposable element

    OpenAIRE

    Momose, Masaki; Itoh, Yoshio; Umemoto, Naoyuki; Nakayama, Masayoshi; Ozeki, Yoshihiro

    2013-01-01

    A glutathione S-transferase-like gene, DcGSTF2, is responsible for carnation (Dianthus caryophyllus L.) flower color intensity. Two defective genes, DcGSTF2mu with a nonsense mutation and DcGSTF2-dTac1 containing a transposable element dTac1, have been characterized in detail in this report. dTac1 is an active element that produces reverted functional genes by excision of the element. A pale-pink cultivar ‘Daisy’ carries both defective genes, whereas a spontaneous deep-colored mutant ‘Daisy-V...

  16. Interactive effects of ultraviolet-B radiation and pesticide exposure on DNA photo-adduct accumulation and expression of DNA damage and repair genes in Xenopus laevis embryos.

    Science.gov (United States)

    Yu, Shuangying; Tang, Song; Mayer, Gregory D; Cobb, George P; Maul, Jonathan D

    2015-02-01

    Pesticide use and ultraviolet-B (UVB) radiation have both been suggested to adversely affect amphibians; however, little is known about their interactive effects. One potential adverse interaction could involve pesticide-induced dysregulation of DNA repair pathways, resulting in greater numbers of DNA photo-adducts from UVB exposure. In the present study, we investigated the interactive effects of UVB radiation and two common pesticides (endosulfan and α-cypermethrin) on induction of DNA photo-adducts and expression of DNA damage and repair related genes in African clawed frog (Xenopus laevis) embryos. We examined 13 genes that are, collectively, involved in stress defense, cell cycle arrest, nucleotide excision repair (NER), base excision repair, mismatch repair, DNA repair regulation, and apoptosis. We exposed X. laevis embryos to 0, 25, and 50 μg/L endosulfan or 0, 2.5, and 5.0 μg/L α-cypermethrin for 96 h, with environmentally relevant exposures of UVB radiation during the last 7 h of the 96 h exposure. We measured the amount of cyclobutane pyrimidine dimers (CPDs) and mRNA abundance of the 13 genes among treatments including control, pesticide only, UVB only, and UVB and pesticide co-exposures. Each of the co-exposure scenarios resulted in elevated CPD levels compared to UVB exposure alone, suggesting an inhibitory effect of endosulfan and α-cypermethrin on CPD repair. This is attributed to results indicating that α-cypermethrin and endosulfan reduced mRNA abundance of XPA and HR23B, respectively, to levels that may affect the initial recognition of DNA lesions. In contrast, both pesticides increased transcript abundance of CSA and MUTL. In addition, mRNA abundance of HSP70 and GADD45α were increased by endosulfan and mRNA abundance of XPG was increased by α-cypermethrin. XPC, HR23B, XPG, and GADD45α exhibited elevated mRNA concentrations whereas there was a reduction in MUTL transcript concentrations in UVB-alone treatments. It appeared that even

  17. Polymorphisms of Selected DNA Repair Genes and Lung Cancer in Chromium Exposure.

    Science.gov (United States)

    Halasova, E; Matakova, T; Skerenova, M; Krutakova, M; Slovakova, P; Dzian, A; Javorkova, S; Pec, M; Kypusova, K; Hamzik, J

    2016-01-01

    Chromium is a well-known mutagen and carcinogen involved in lung cancer development. DNA repair genes play an important role in the elimination of genetic changes caused by chromium exposure. In the present study, we investigated the polymorphisms of the following DNA repair genes: XRCC3, participating in the homologous recombination repair, and hMLH1 and hMSH2, functioning in the mismatch repair. We focused on the risk the polymorphisms present in the development of lung cancer regarding the exposure to chromium. We analyzed 106 individuals; 45 patients exposed to chromium with diagnosed lung cancer and 61 healthy controls. Genotypes were determined by a PCR-RFLP method. We unravelled a potential for increased risk of lung cancer development in the hMLH1 (rs1800734) AA genotype in the recessive model. In conclusion, gene polymorphisms in the DNA repair genes underscores the risk of lung cancer development in chromium exposed individuals.

  18. Gene expression of the mismatch repair gene MSH2 in primary colorectal cancer

    DEFF Research Database (Denmark)

    Jensen, Lars Henrik; Kuramochi, Hidekazu; Crüger, Dorthe Gylling

    2011-01-01

    Microsatellite instability (MSI) is caused by defective mismatch repair (MMR) and is one of the very few molecular markers with proven clinical importance in colorectal cancer with respect to heredity, prognosis, and treatment effect. The gene expression of the MMR gene MSH2 may be a quantitative...... marker for the level of MMR and a potential molecular marker with clinical relevance. The aim was to investigate the gene expression of MSH2 in primary operable colorectal cancer in correlation with MSI, protein expression, and promoter hypermethylation. In a cohort of 210 patients, the primary tumor...... and lymphnode metastases were analyzed with immunohistochemistry, methylation and MSI analyses, and quantitative polymerase chain reaction (PCR). The median gene expression of MSH2 was 1.00 (range 0.16-11.2, quartiles 0.70-1.51) and there was good agreement between the gene expression in primary tumor and lymph...

  19. DNA Mismatch Repair and Oxidative DNA Damage: Implications for Cancer Biology and Treatment

    Energy Technology Data Exchange (ETDEWEB)

    Bridge, Gemma; Rashid, Sukaina; Martin, Sarah A., E-mail: sarah.martin@qmul.ac.uk [Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, Charterhouse Square, London EC1M 6BQ (United Kingdom)

    2014-08-05

    Many components of the cell, including lipids, proteins and both nuclear and mitochondrial DNA, are vulnerable to deleterious modifications caused by reactive oxygen species. If not repaired, oxidative DNA damage can lead to disease-causing mutations, such as in cancer. Base excision repair and nucleotide excision repair are the two DNA repair pathways believed to orchestrate the removal of oxidative lesions. However, recent findings suggest that the mismatch repair pathway may also be important for the response to oxidative DNA damage. This is particularly relevant in cancer where mismatch repair genes are frequently mutated or epigenetically silenced. In this review we explore how the regulation of oxidative DNA damage by mismatch repair proteins may impact on carcinogenesis. We discuss recent studies that identify potential new treatments for mismatch repair deficient tumours, which exploit this non-canonical role of mismatch repair using synthetic lethal targeting.

  20. Excision repair cross-complementation group 1 (ERCC1) in platinum-based treatment of non-small cell lung cancer with special emphasis on carboplatin: a review of current literature

    DEFF Research Database (Denmark)

    Vilmar, A.; Sorensen, J.B.

    2009-01-01

    BACKGROUND: Patients diagnosed with advanced non-small cell lung cancer have a dismal prognosis and are often relative resistant to chemotherapy. A need for markers has emerged based on tumour biology in order to predict which patients will respond to treatment. Excision repair cross-complementat......BACKGROUND: Patients diagnosed with advanced non-small cell lung cancer have a dismal prognosis and are often relative resistant to chemotherapy. A need for markers has emerged based on tumour biology in order to predict which patients will respond to treatment. Excision repair cross......-complementation group 1 (ERCC1) has shown potential as a predictive marker in patients with NSCLC treated with cisplatin-based chemotherapy. Carboplatin has gained widespread use in the treatment of advanced NSCLC and its mechanisms of action are likely similar to that of cisplatin. MATERIALS AND METHODS: A literature...... articles and 1 clinical abstract were identified. Laboratory methods were mainly RT-PCR (reverse transcriptase polymerase chain reaction) or immunohistochemistry (IHC) for expression of ERCC1. Preclinical studies pointed towards similar mechanisms of chemotherapy-resistance among platinum compounds...

  1. Excision of a selectable marker gene in transgenic banana using a Cre/lox system controlled by an embryo specific promoter.

    Science.gov (United States)

    Chong-Pérez, Borys; Reyes, Maritza; Rojas, Luis; Ocaña, Bárbara; Ramos, Adolfo; Kosky, Rafael G; Angenon, Geert

    2013-09-01

    Antibiotic and herbicide resistance genes have been used in transgene technology as powerful selection tools. Nonetheless, once transgenic events have been obtained their presence is no longer needed and can even be undesirable. In this work, we have developed a system to excise the selectable marker and the cre recombinase genes from transgenic banana cv. 'Grande Naine' (Musa AAA). To achieve this, the embryo specific REG-2 promoter was isolated from rice and its expression pattern in banana cell clumps, somatic embryos and regenerated plantlets was characterized by using a pREG2::uidA fusion construct. Subsequently, the REG-2 promoter was placed upstream of the cre gene, conferring Cre functionality in somatic embryos and recombination of lox sites resulting in excision of the selectable marker and cre genes. PCR analysis revealed that 41.7 % of the analysed transgenic plants were completely marker free, results that were thereafter confirmed by Southern blot hybridization. These results demonstrate the feasibility of using developmentally controlled promoters to mediate marker excision in banana. This system does not require any extra handling compared to the conventional transformation procedure and might be useful in other species regenerating through somatic embryogenesis.

  2. Interactive effects of ultraviolet-B radiation and pesticide exposure on DNA photo-adduct accumulation and expression of DNA damage and repair genes in Xenopus laevis embryos

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Shuangying, E-mail: shuangying.yu@ttu.edu [Department of Environmental Toxicology, The Institute of Environmental and Human Health, Texas Tech University, 1207 S. Gilbert Dr., Lubbock, TX 79416 (United States); Tang, Song, E-mail: song.tang@usask.ca [Department of Environmental Toxicology, The Institute of Environmental and Human Health, Texas Tech University, 1207 S. Gilbert Dr., Lubbock, TX 79416 (United States); Mayer, Gregory D., E-mail: greg.mayer@ttu.edu [Department of Environmental Toxicology, The Institute of Environmental and Human Health, Texas Tech University, 1207 S. Gilbert Dr., Lubbock, TX 79416 (United States); Cobb, George P., E-mail: george_cobb@baylor.edu [Department of Environmental Science, Baylor University, One Bear Place #97266, Waco, TX 76798 (United States); Maul, Jonathan D., E-mail: jonathan.maul@ttu.edu [Department of Environmental Toxicology, The Institute of Environmental and Human Health, Texas Tech University, 1207 S. Gilbert Dr., Lubbock, TX 79416 (United States)

    2015-02-15

    Highlights: • Interactive effects of UVB radiation-pesticide co-exposures were examined in frogs. • Responses included induction of DNA photo-adducts and DNA damage and repair genes. • Elevated DNA adduct levels occurred for co-exposures compared to UVB alone. • One mechanism is that pesticides may alter nuclear excision repair gene expression. - Abstract: Pesticide use and ultraviolet-B (UVB) radiation have both been suggested to adversely affect amphibians; however, little is known about their interactive effects. One potential adverse interaction could involve pesticide-induced dysregulation of DNA repair pathways, resulting in greater numbers of DNA photo-adducts from UVB exposure. In the present study, we investigated the interactive effects of UVB radiation and two common pesticides (endosulfan and α-cypermethrin) on induction of DNA photo-adducts and expression of DNA damage and repair related genes in African clawed frog (Xenopus laevis) embryos. We examined 13 genes that are, collectively, involved in stress defense, cell cycle arrest, nucleotide excision repair (NER), base excision repair, mismatch repair, DNA repair regulation, and apoptosis. We exposed X. laevis embryos to 0, 25, and 50 μg/L endosulfan or 0, 2.5, and 5.0 μg/L α-cypermethrin for 96 h, with environmentally relevant exposures of UVB radiation during the last 7 h of the 96 h exposure. We measured the amount of cyclobutane pyrimidine dimers (CPDs) and mRNA abundance of the 13 genes among treatments including control, pesticide only, UVB only, and UVB and pesticide co-exposures. Each of the co-exposure scenarios resulted in elevated CPD levels compared to UVB exposure alone, suggesting an inhibitory effect of endosulfan and α-cypermethrin on CPD repair. This is attributed to results indicating that α-cypermethrin and endosulfan reduced mRNA abundance of XPA and HR23B, respectively, to levels that may affect the initial recognition of DNA lesions. In contrast, both pesticides

  3. Repair of some active genes in Cockayne syndrome cells is at the genome overall rate.

    Science.gov (United States)

    Kantor, G J; Bastin, S A

    1995-05-01

    Repair of UV (254 nm)-induced DNA damage in cells from patients with the genetic disease Cockayne syndrome (CS; CS3BE, CS2BE) has been examined in several different genomic regions. These regions include those that contain the actively transcribed beta-actin and adenosine deaminase (ADA) genes and the inactive insulin and 754 loci. The beta-actin, ADA and insulin regions are repaired at about the same rate, one which is equal to the genome overall repair rate. The 754 locus is repaired considerably more slowly. The insulin region is repaired at the same rate in both CS and normal cells as is the 754 locus. The only difference from normal is that the active genes, while repaired well, are not preferentially repaired relative to the genome overall. Our results are consistent with the hypothesis that the repair defect in CS is due to an inactive transcription-repair coupling factor (TRCF). However, the results also indicate that factors other than TRCF and active transcription must also promote repair of some regions relative to others in both normal and CS cells.

  4. Evidence for a repair enzyme complex involving ERCC1, and the correcting activities of ERCC4, ERCC11 and the xeroderma pigmentosum group F.

    NARCIS (Netherlands)

    A.J. van Vuuren (Hanneke); E. Appeldoorn (Esther); H. Odijk (Hanny); A. Yasui (Akira); N.G.J. Jaspers (Nicolaas); D. Bootsma (Dirk); J.H.J. Hoeijmakers (Jan)

    1993-01-01

    textabstractNucleotide excision repair (NER), one of the major cellular DNA repair systems, removes a wide range of lesions in a multi-enzyme reaction. In man, a NER defect due to a mutation in one of at least 11 distinct genes, can give rise to the inherited repair disorders xeroderma pigmentosum

  5. DNA repair in human cells: from genetic complementation to isolation of genes.

    NARCIS (Netherlands)

    D. Bootsma (Dirk); A. Westerveld (Andries); J.H.J. Hoeijmakers (Jan)

    1988-01-01

    textabstractThe genetic disease xeroderma pigmentosum (XP) demonstrates the association between defective repair of DNA lesions and cancer. Complementation analysis performed on XP cell strains and on repair deficient rodent cell lines has revealed that at least nine and possibly more than 13 genes

  6. Genomic Approaches to DNA repair and Mutagenesis

    OpenAIRE

    Wyrick, John J.; Roberts, Steven A.

    2015-01-01

    DNA damage is a constant threat to cells, causing cytotoxicity as well as inducing genetic alterations. The steady-state abundance of DNA lesions in a cell is minimized by a variety of DNA repair mechanisms, including DNA strand break repair, mismatch repair, nucleotide excision repair, base excision repair, and ribonucleotide excision repair. The efficiencies and mechanisms by which these pathways remove damage from chromosomes have been primarily characterized by investigating the processin...

  7. Nucleotide Excision Repair Lesion-Recognition Protein Rad4 Captures a Pre-Flipped Partner Base in a Benzo[a]pyrene-Derived DNA Lesion: How Structure Impacts the Binding Pathway.

    Science.gov (United States)

    Mu, Hong; Geacintov, Nicholas E; Min, Jung-Hyun; Zhang, Yingkai; Broyde, Suse

    2017-06-19

    The xeroderma pigmentosum C protein complex (XPC) recognizes a variety of environmentally induced DNA lesions and is the key in initiating their repair by the nucleotide excision repair (NER) pathway. When bound to a lesion, XPC flips two nucleotide pairs that include the lesion out of the DNA duplex, yielding a productively bound complex that can lead to successful lesion excision. Interestingly, the efficiencies of NER vary greatly among different lesions, influencing their toxicity and mutagenicity in cells. Though differences in XPC binding may influence NER efficiency, it is not understood whether XPC utilizes different mechanisms to achieve productive binding with different lesions. Here, we investigated the well-repaired 10R-(+)-cis-anti-benzo[a]pyrene-N 2 -dG (cis-B[a]P-dG) DNA adduct in a duplex containing normal partner C opposite the lesion. This adduct is derived from the environmental pro-carcinogen benzo[a]pyrene and is likely to be encountered by NER in the cell. We have extensively investigated its binding to the yeast XPC orthologue, Rad4, using umbrella sampling with restrained molecular dynamics simulations and free energy calculations. The NMR solution structure of this lesion in duplex DNA has shown that the dC complementary to the adducted dG is flipped out of the DNA duplex in the absence of XPC. However, it is not known whether the "pre-flipped" base would play a role in its recognition by XPC. Our results show that Rad4 first captures the displaced dC, which is followed by a tightly coupled lesion-extruding pathway for productive binding. This binding path differs significantly from the one deduced for the small cis-syn cyclobutane pyrimidine dimer lesion opposite mismatched thymines [ Mu , H. , ( 2015 ) Biochemistry , 54 ( 34 ), 5263 - 7 ]. The possibility of multiple paths that lead to productive binding to XPC is consistent with the versatile lesion recognition by XPC that is required for successful NER.

  8. DNA-repair gene variants are associated with glioblastoma survival

    DEFF Research Database (Denmark)

    Wibom, Carl; Sjöström, Sara; Henriksson, Roger

    2012-01-01

    Abstract Patient outcome from glioma may be influenced by germline variation. Considering the importance of DNA repair in cancer biology as well as in response to treatment, we studied the relationship between 1458 SNPs, which captured the majority of the common genetic variation in 136 DNA repair...

  9. DNA repair gene XRCC1 polymorphisms, smoking, and bladder cancer risk: a meta-analysis.

    Directory of Open Access Journals (Sweden)

    Shan Li

    Full Text Available BACKGROUND AND OBJECTIVE: The X-ray repair cross-complementing group 1 (XRCC1 protein plays a crucial role in base excision repair (BER pathway by acting as a scaffold for other BER enzymes. Variants in the XRCC1 gene might alter protein structure or function or create alternatively spliced proteins which may influence BER efficiency and hence affect individual susceptibility to bladder cancer. Recent epidemiological studies have shown inconsistent associations between these polymorphisms and bladder cancer. To clarify the situation, a comprehensive meta-analysis of all available studies was performed in this study. METHODS: PubMed, EMBASE, and Chinese Biomedical Literature database (CBM databases have been systematically searched to identify all relevant studies for the period up to February 2013. Data were abstracted independently by two reviewers and Odds ratios (ORs and 95% confidence intervals (CIs were calculated. Subgroup analyses were performed mainly by ethnicity and smoking status. RESULTS: A total of 26 case-control studies, including 24 studies for R399Q polymorphism, 15 studies for R194W polymorphism, and 7 studies for R280H polymorphism met the inclusion criteria and were selected. With respect to R399Q polymorphism, significantly decreased bladder cancer risk was found among smokers (AA vs. GG: OR=0.693, 95%CI= 0.515-0.932, P=0.015 and recessive model AA vs. GA+GG: OR=0.680, 95%CI= 0.515-0.898, P=0.007, respectively. With respect to R194W and R280H polymorphism, significantly increased bladder cancer risk were observed among Asians (TT+CT vs. CC:OR = 1.327, 95% CI 1.086-1.622, P=0.006 for R194W, and AA+GA vs. GG: OR=2.094, 95% CI 1.211-3.621, P=0.008 for R280H, respectively. CONCLUSIONS: This meta-analysis suggests that the XRCC1 R399Q polymorphism may play a protective role against bladder cancer among smokers. However, the XRCC1 R194W and R280H polymorphisms were both associated with increased bladder cancer risk among Asians

  10. Developmental defects and male sterility in mice lacking the ubiquitin-like DNA repair gene mHR23B.

    NARCIS (Netherlands)

    J.M.Y. Ng (Jessica); H. Vrieling (Harry); K. Sugasawa (Kaoru); M.P. Ooms (Marja); J.A. Grootegoed (Anton); J.T.M. Vreeburg (Jan); P. Visser (Pim); R.B. Beems (Rudolf); T.G.M.F. Gorgels (Theo); F. Hanaoka (Fumio); J.H.J. Hoeijmakers (Jan); G.T.J. van der Horst (Gijsbertus)

    2002-01-01

    textabstractmHR23B encodes one of the two mammalian homologs of Saccharomyces cerevisiae RAD23, a ubiquitin-like fusion protein involved in nucleotide excision repair (NER). Part of mHR23B is complexed with the XPC protein, and this heterodimer functions as the main damage detector and initiator of

  11. SELECTIVE-INHIBITION OF REPAIR OF ACTIVE GENES BY HYPERTHERMIA IS DUE TO INHIBITION OF GLOBAL AND TRANSCRIPTION COUPLED REPAIR PATHWAYS

    NARCIS (Netherlands)

    SAKKERS, RJ; FILON, AR; BRUNSTING, JF; KAMPINGA, HH; KONINGS, AWT; MULLENDERS, LHF

    Hyperthermia specifically inhibits the repair of UV-induced DNA photolesions in transcriptionally active genes, To define more precisely which mechanisms underlie the heat-induced inhibition of repair of active genes, removal of cyclobutane pyrimidine dimers (CPDs) was studied in human fibroblasts

  12. PAH-DNA adducts in environmentally exposed population in relation to metabolic and DNA repair gene polymorphisms

    Energy Technology Data Exchange (ETDEWEB)

    Binkova, Blanka [Laboratory of Genetic Ecotoxicology, Institute of Experimental Medicine AS CR and Health Institute of Central Bohemia, Videnska 1083, 14220 Prague (Czech Republic); Chvatalova, Irena [Laboratory of Genetic Ecotoxicology, Institute of Experimental Medicine AS CR and Health Institute of Central Bohemia, Videnska 1083, 14220 Prague (Czech Republic); Lnenickova, Zdena [Laboratory of Genetic Ecotoxicology, Institute of Experimental Medicine AS CR and Health Institute of Central Bohemia, Videnska 1083, 14220 Prague (Czech Republic); Milcova, Alena [Laboratory of Genetic Ecotoxicology, Institute of Experimental Medicine AS CR and Health Institute of Central Bohemia, Videnska 1083, 14220 Prague (Czech Republic); Tulupova, Elena [Laboratory of Genetic Ecotoxicology, Institute of Experimental Medicine AS CR and Health Institute of Central Bohemia, Videnska 1083, 14220 Prague (Czech Republic); Cancer Biomarkers and Prevention Group, Biocentre, University of Leicester (United Kingdom); Farmer, Peter B. [Cancer Biomarkers and Prevention Group, Biocentre, University of Leicester (United Kingdom); Sram, Radim J. [Laboratory of Genetic Ecotoxicology, Institute of Experimental Medicine AS CR and Health Institute of Central Bohemia, Videnska 1083, 14220 Prague (Czech Republic)]. E-mail: sram@biomed.cas.cz

    2007-07-01

    .003). A significant difference in both the total (P < 0.05) and the B[a]P-'like' DNA adducts (P < 0.01) between smokers and nonsmokers within both groups was observed. A significant positive association between DNA adduct and cotinine levels (r = 0.368, P < 0.001) and negative association between DNA adduct and vitamin C levels (r = -0.290, P = 0.004) was found. The results of multivariate regression analysis showed smoking, vitamin C, polymorphisms of XPD repair gene in exon 23 and GSTM1 gene as significant predictors for total DNA adduct levels. Exposure to ambient air pollution, smoking, and polymorphisms of XPD repair gene in exon 6 were significant predictors for B[a]P-'like' DNA adduct. To sum up, this study suggests that polymorphisms of DNA repair genes involved in nucleotide excision repair may modify aromatic DNA adduct levels and may be useful biomarkers to identify individuals susceptible to DNA damage resulting from c-PAHs exposure.

  13. DNA repair in human fibroblasts, as reflected by host-cell reactivation of a transfected UV-irradiated luciferase gene, is not related to donor age

    Energy Technology Data Exchange (ETDEWEB)

    Merkle, Thomas J.; O' Brien, Katherine; Brooks, Philip J.; Tarone, Robert E.; Robbins, Jay H

    2004-10-04

    The effect of donor age on the ability of mammalian cells to repair ultraviolet (UV)-induced DNA damage has been studied using several approaches, most recently via assays that measure the host-cell reactivation (HCR) of UV-irradiated reporter gene-containing plasmid vectors following their transfection into cells. Plasmid HCR assays indirectly quantify a cell line's ability to perform nucleotide excision repair (NER) by measuring the enzyme activity of the repaired reporter gene, e.g., chloramphenical acetyltransferase (cat) or luciferase (luc), and are useful in studies investigating whether increasing age may be a risk factor for the deficient repair of potentially cancer-causing, sunlight-induced, DNA lesions in skin cells. In our study, we quantified the DNA repair ability of cultured, nontransformed, human skin fibroblast lines through their HCR of a transfected UV-C-irradiated plasmid containing luc. HCR was measured at various times after transfection in five lines from normal donors of ages 21-96 years, and from one donor who had xeroderma pigmentosum (XP). The normal lines displayed increasing HCR at successive post-transfection time points and showed no significant correlation between HCR and donor age. The XP-A line, known to be markedly deficient in NER of UV-induced DNA damage, showed minimal evidence of HCR compared to the normal lines. To further assess potential variation in HCR with donor age, fibroblast lines from five old donors, ages 84-94 years, were compared with lines from five young donors, ages 17-26 years. While significant differences in HCR were found between some lines, no significant difference was found between the young and old age groups (P=0.44). Our study provides no indication that the higher incidence of skin cancer observed with increasing age is due to an age-related decrease in the ability to repair UV-induced DNA damage.

  14. [Interaction of the HSM3 gene with genes initiating homologous recombination repair in yeast Saccharomyces cerevisiae].

    Science.gov (United States)

    Chernenkov, A Iu; Fedorov, D V; Gracheva, L M; Evstukhina, T A; Koval'tsova, S V; Peshekhonov, V T; Fedorova, I V; Korolev, V G

    2012-03-01

    It was assumed previously that the mutator phenotype of the hms3 mutant was determined by processes taking place in the D-loop. As a next step, genetic analysis was performed to study the interactions between the hsm3 mutation and mutations of the genes that control the initial steps of the D-loop formation. The mutations of the MMS4 and XRS2 genes, which initiate the double-strand break formation and subsequent repair, were shown to completely block HSM3-dependent UV-induced mutagenesis. Mutations of the RAD51, RAD52, and RAD54 genes, which are also involved in the D-loop formation, only slightly decreased the level of UV-induced mutagenesis in the hsm3 mutant. Similar results were observed for the interaction of hsm3 with the mph1 mutation, which stabilizes the D-loop. In contrast, the shu1 mutation, which destabilizes the D-loop structure, led to an extremely high level of UV-induced mutagenesis and displayed epistatic interactions with the hsm3 mutation. The results made it possible to assume that the hsm3 mutation destabilizes the D-loop, which is a key substrate of both Rad5- and Rad52-dependent postreplicative repair pathways.

  15. Xeroderma pigmentosum group F caused by a defect in a structure-specific DNA repair endonuclease.

    NARCIS (Netherlands)

    A.M. Sijbers (Anneke); W.L. de Laat (Wouter); R.A. Ariza (Rafael); M. Biggerstaff (Maureen); Y-F. Wei; J.G. Moggs (Jonathan); K.C. Carter (Kenneth); B.K. Shell (Brenda); E. Evans (Elizabeth); M.C. de Jong (Mariska); S. Rademakers (Suzanne); J.D. de Rooij (Johan); N.G.J. Jaspers (Nicolaas); J.H.J. Hoeijmakers (Jan); R.D. Wood (Richard)

    1996-01-01

    textabstractNucleotide excision repair, which is defective in xeroderma pigmentosum (XP), involves incision of a DNA strand on each side of a lesion. We isolated a human gene homologous to yeast Rad1 and found that it corrects the repair defects of XP group F as well as rodent groups 4 and 11.

  16. Defective transcription-coupled repair in Cockayne syndrome B mice is associated with skin cancer predisposition.

    NARCIS (Netherlands)

    G.T.J. van der Horst (Gijsbertus); H. van Steeg (Harry); R.J.W. Berg (Rob); A.J. van Gool (Alain); J. de Wit (Jan); G. Weeda (Geert); H. Morreau (Hans); R.B. Beems (Rudolf); C.F. van Kreijl (Coen); F.R. de Gruijl (Frank); D. Bootsma (Dirk); J.H.J. Hoeijmakers (Jan)

    1997-01-01

    textabstractA mouse model for the nucleotide excision repair disorder Cockayne syndrome (CS) was generated by mimicking a truncation in the CSB(ERCC6) gene of a CS-B patient. CSB-deficient mice exhibit all of the CS repair characteristics: ultraviolet (UV) sensitivity, inactivation of

  17. DNA Repair Gene Polymorphisms in Hereditary and Sporadic Breast Cancer

    National Research Council Canada - National Science Library

    Ricks-Santi, Luisel

    2006-01-01

    .... There is variable penetrance for breast cancer among women in families with known BRCA1 mutations, and we hypothesize that this might be due to genetic variants in wild-type BRCA1 or other DNA repair...

  18. Transcript RNA supports precise repair of its own DNA gene.

    Science.gov (United States)

    Keskin, Havva; Meers, Chance; Storici, Francesca

    2016-01-01

    The transfer of genetic information from RNA to DNA is considered an extraordinary process in molecular biology. Despite the fact that cells transcribe abundant amount of RNA with a wide range of functions, it has been difficult to uncover whether RNA can serve as a template for DNA repair and recombination. An increasing number of experimental evidences suggest a direct role of RNA in DNA modification. Recently, we demonstrated that endogenous transcript RNA can serve as a template to repair a DNA double-strand break (DSB), the most harmful DNA lesion, not only indirectly via formation of a DNA copy (cDNA) intermediate, but also directly in a homology driven mechanism in budding yeast. These results point out that the transfer of genetic information from RNA to DNA is more general than previously thought. We found that transcript RNA is more efficient in repairing a DSB in its own DNA (in cis) than in a homologous but ectopic locus (in trans). Here, we summarize current knowledge about the process of RNA-driven DNA repair and recombination, and provide further data in support of our model of DSB repair by transcript RNA in cis. We show that a DSB is precisely repaired predominately by transcript RNA and not by residual cDNA in conditions in which formation of cDNA by reverse transcription is inhibited. Additionally, we demonstrate that defects in ribonuclease (RNase) H stimulate precise DSB repair by homologous RNA or cDNA sequence, and not by homologous DNA sequence carried on a plasmid. These results highlight an antagonistic role of RNase H in RNA-DNA recombination. Ultimately, we discuss several questions that should be addressed to better understand mechanisms and implications of RNA-templated DNA repair and recombination.

  19. A mutation in the XPB/ERCC3 DNA repair transcription gene, associated with trichothiodystrophy

    Energy Technology Data Exchange (ETDEWEB)

    Weeda, G.; Donker, I.; Vermeulen, W. [Erasmus Univ., Rotterdam (Netherlands)] [and others

    1997-02-01

    Trichothiodystrophy (TTD) is a rare, autosomal recessive disorder characterized by sulfur-deficient brittle hair and nails, mental retardation, impaired sexual development, and ichthyosis. Photosensitivity has been reported in {approximately}50% of the cases, but no skin cancer is associated with TTD. Virtually all photosensitive TTD patients have a deficiency in the nucleotide excision repair (NER) of UV-induced DNA damage that is indistinguishable from that of xeroderma pigmentosum (XP) complementation group D (XP-D) patients. DNA repair defects in XP-D are associated with two additional, quite different diseases; XP, a sun-sensitive and cancer-prone repair disorder, and Cockayne syndrome (CS), a photosensitive condition characterized by physical and mental retardation and wizened facial appearance. One photosensitive TTD case constitutes a new repair-deficient complementation group, TTD-A. Remarkably, both TTD-A and XP-D defects are associated with subunits of TFIIH, a basal transcription factor with a second function in DNA repair. Thus, mutations in TFIIH components may, on top of a repair defect, also cause transcriptional insufficiency, which may explain part of the non-XP clinical features of TTD. To date, three patients with the remarkable conjunction of XP and CS but not TM have been assigned to XP complementation group B (XP-B). Here we present the characterization of the NER defect in two mild TTD patients (TTD6VI and TTD4VI) and confirm the assignment to X-PB. The causative mutation was found to be a single base substitution resulting in a missense mutation (T119P) in a region of the XPB protein. These findings define a third TTD complementation group, extend the clinical heterogeneity associated with XP-B, stress the exclusive relationship between TTD and mutations in subunits of repair/transcription factor TFIIH, and strongly support the concept of {open_quotes}transcription syndromes.{close_quotes} 46 refs., 6 figs., 2 tabs.

  20. Different impact of excision repair cross-complementation group 1 on survival in male and female patients with inoperable non-small-cell lung cancer treated with carboplatin and gemcitabine

    DEFF Research Database (Denmark)

    Holm, Bente; Mellemgaard, Anders; Skov, Torsten

    2009-01-01

    PURPOSE: The excision repair cross-complementation group 1 (ERCC1) status was assessed in patients receiving carboplatin and gemcitabine for inoperable non-small-cell lung cancer (NSCLC). We analyzed the association between the ERCC1 status and the overall survival after the chemotherapy. PATIENTS...... AND METHODS: We retrospectively identified 163 patients with inoperable NSCLC and sufficient tumor tissue for ERCC1 analysis, who had received carboplatin and gemcitabine as first-line treatment. Immunohistochemistry was used to assess the expression of ERCC1. RESULTS: One hundred sixty-three patients were...... included. Seventy (42%) were ERCC1 positive. Patients treated with carboplatin and gemcitabine and having ERCC1-negative tumors had a significantly increased survival when compared to patients with ERCC1-positive tumors (median survival, 12.0 months v 8.2 months; P = .02). This difference was mainly seen...

  1. DNA Repair Systems

    Indian Academy of Sciences (India)

    nal factors such as UV radiation, high energy radiation such as X-. Keywords. DNA repair, DNA damage, base excision repair, nucleotide exci- sion repair, methlyl-directed mis- match repair, Nobel Prize. rays and gamma rays, mutagenic chemicals and viruses. Different types of DNA ... be especially important in plants.

  2. Genome analysis of DNA repair genes in the alpha proteobacterium Caulobacter crescentus

    Directory of Open Access Journals (Sweden)

    Menck Carlos FM

    2007-03-01

    Full Text Available Abstract Background The integrity of DNA molecules is fundamental for maintaining life. The DNA repair proteins protect organisms against genetic damage, by removal of DNA lesions or helping to tolerate them. DNA repair genes are best known from the gamma-proteobacterium Escherichia coli, which is the most understood bacterial model. However, genome sequencing raises questions regarding uniformity and ubiquity of these DNA repair genes and pathways, reinforcing the need for identifying genes and proteins, which may respond to DNA damage in other bacteria. Results In this study, we employed a bioinformatic approach, to analyse and describe the open reading frames potentially related to DNA repair from the genome of the alpha-proteobacterium Caulobacter crescentus. This was performed by comparison with known DNA repair related genes found in public databases. As expected, although C. crescentus and E. coli bacteria belong to separate phylogenetic groups, many of their DNA repair genes are very similar. However, some important DNA repair genes are absent in the C. crescentus genome and other interesting functionally related gene duplications are present, which do not occur in E. coli. These include DNA ligases, exonuclease III (xthA, endonuclease III (nth, O6-methylguanine-DNA methyltransferase (ada gene, photolyase-like genes, and uracil-DNA-glycosylases. On the other hand, the genes imuA and imuB, which are involved in DNA damage induced mutagenesis, have recently been described in C. crescentus, but are absent in E. coli. Particularly interesting are the potential atypical phylogeny of one of the photolyase genes in alpha-proteobacteria, indicating an origin by horizontal transfer, and the duplication of the Ada orthologs, which have diverse structural configurations, including one that is still unique for C. crescentus. Conclusion The absence and the presence of certain genes are discussed and predictions are made considering the particular

  3. Bypass of a 5′,8-cyclopurine-2′-deoxynucleoside by DNA polymerase β during DNA replication and base excision repair leads to nucleotide misinsertions and DNA strand breaks

    Science.gov (United States)

    Jiang, Zhongliang; Xu, Meng; Lai, Yanhao; Laverde, Eduardo E.; Terzidis, Michael A.; Masi, Annalisa; Chatgilialoglu, Chryssostomos; Liu, Yuan

    2015-01-01

    5′,8-cyclopurine-2′-deoxynucleosides including 5′,8-cyclo-dA (cdA) and 5′,8-cyclo-dG (cdG) are induced by hydroxyl radicals resulting from oxidative stress such as ionizing radiation. 5′,8-cyclopurine-2′-deoxynucleoside lesions are repaired by nucleotide excision repair with low efficiency, thereby leading to their accumulation in the human genome and lesion bypass by DNA polymerases during DNA replication and base excision repair (BER). In this study, for the first time, we discovered that DNA polymerase β (pol β) efficiently bypassed a 5′R-cdA, but inefficiently bypassed a 5′S-cdA during DNA replication and BER. We found that cell extracts from pol β wild-type mouse embryonic fibroblasts exhibited significant DNA synthesis activity in bypassing a cdA lesion located in replication and BER intermediates. However, pol β knock-out cell extracts exhibited little DNA synthesis to bypass the lesion. This indicates that pol β plays an important role in bypassing a cdA lesion during DNA replication and BER. Furthermore, we demonstrated that pol β inserted both a correct and incorrect nucleotide to bypass a cdA at a low concentration. Nucleotide misinsertion was significantly stimulated by a high concentration of pol β, indicating a mutagenic effect induced by pol β lesion bypass synthesis of a 5′,8-cyclopurine-2′-deoxynucleoside. Moreover, we found that bypass of a 5′S-cdA by pol β generated an intermediate that failed to be extended by pol β, resulting in accumulation of single-strand DNA breaks. Our study provides the first evidence that pol β plays an important role in bypassing a 5′,8-cyclo-dA during DNA replication and repair, as well as new insight into mutagenic effects and genome instability resulting from pol β bypassing of a cdA lesion. PMID:26123757

  4. Direct and Inverted Repeat stimulated excision (DIRex: Simple, single-step, and scar-free mutagenesis of bacterial genes.

    Directory of Open Access Journals (Sweden)

    Joakim Näsvall

    Full Text Available The need for generating precisely designed mutations is common in genetics, biochemistry, and molecular biology. Here, I describe a new λ Red recombineering method (Direct and Inverted Repeat stimulated excision; DIRex for fast and easy generation of single point mutations, small insertions or replacements as well as deletions of any size, in bacterial genes. The method does not leave any resistance marker or scar sequence and requires only one transformation to generate a semi-stable intermediate insertion mutant. Spontaneous excision of the intermediate efficiently and accurately generates the final mutant. In addition, the intermediate is transferable between strains by generalized transductions, enabling transfer of the mutation into multiple strains without repeating the recombineering step. Existing methods that can be used to accomplish similar results are either (i more complicated to design, (ii more limited in what mutation types can be made, or (iii require expression of extrinsic factors in addition to λ Red. I demonstrate the utility of the method by generating several deletions, small insertions/replacements, and single nucleotide exchanges in Escherichia coli and Salmonella enterica. Furthermore, the design parameters that influence the excision frequency and the success rate of generating desired point mutations have been examined to determine design guidelines for optimal efficiency.

  5. Germline mutations in DNA repair genes may predict neoadjuvant therapy response in triple negative breast patients.

    Science.gov (United States)

    Spugnesi, Laura; Gabriele, Michele; Scarpitta, Rosa; Tancredi, Mariella; Maresca, Luisa; Gambino, Gaetana; Collavoli, Anita; Aretini, Paolo; Bertolini, Ilaria; Salvadori, Barbara; Landucci, Elisabetta; Fontana, Andrea; Rossetti, Elena; Roncella, Manuela; Naccarato, Giuseppe Antonio; Caligo, Maria Adelaide

    2016-12-01

    Triple negative breast cancers (TNBCs) represent about 15-20% of all breast cancer cases and are characterized by a complex molecular heterogeneity. Some TNBCs exhibit clinical and pathological properties similar to BRCA-mutated tumors, without actually bearing a mutation in BRCA genes. This "BRCAness" phenotype may be explained by germline mutations in other genes involved in DNA repair. Although respond to chemotherapy with alkylating agents, they have a high risk of recurrence and progression. Some studies have shown the efficacy of neoadjuvant therapy in TNBC patients with DNA repair defects, but proper biomarkers of DNA repair deficiency are still needed. Here, we investigated if mutations in DNA repair genes may be correlated with anthracyclines/taxanes neoadjuvant therapy response. DNA from 19 TNBC patients undergoing neoadjuvant therapy were subjected to next generation sequencing of a panel of 24 genes in DNA repair and breast cancer predisposition. In this study, 5 of 19 patients (26%) carried a pathogenic mutation in BRCA1, PALB2, RAD51C and two patients carried a probable pathogenic missense variant. Moreover, VUS (Variants of Unknown Significance) in other genes, predicted to be deleterious by in silico tools, were detected in five patients. Germline mutations in DNA repair genes were found to be associated with the group of TNBC patients who responded to therapy. We conclude that a subgroup of TNBC patients have defects in DNA repair genes, other than BRCA1, and such patients respond favourably to neoadjuvant anthracyclines/taxanes therapy. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  6. DNA repair gene expression level in peripheral blood and tumour tissue from non-small cell lung cancer and head and neck squamous cell cancer patients.

    Science.gov (United States)

    Schena, Marina; Guarrera, Simonetta; Buffoni, Lucio; Salvadori, Angelica; Voglino, Floriana; Allione, Alessandra; Pecorari, Giancarlo; Ruffini, Enrico; Garzino-Demo, Paolo; Bustreo, Sara; Consito, Lorena; Bironzo, Paolo; Matullo, Giuseppe

    2012-04-01

    The nucleotide excision repair pathway is crucial for cellular DNA integrity and the ERCC1 helicase is also potentially involved in resistance to platinum-based chemotherapy, and high levels of ERCC1 mRNA in tumours have been associated with cisplatin resistance in different human cancers. The aim of this work was to investigate the correlation between DNA repair gene expression levels in tumour tissue, normal tissue and peripheral blood samples from patients with two common human cancers, non-small cell lung cancer (NSCLC) and squamous cell carcinoma of the head and neck (HNSCC), to test if blood gene expression could be a proxy for tumour tissue gene expression to predict response to platinum-based chemotherapy. Using RT-qPCR we determined ERCC1, ERCC2, ERCC4, XPA, XPC, XRCC1, XRCC3, APEX, OGG1, MGMT mRNA levels in fresh NSCLC, normal lung and HNSCC tissue, as well as blood, from NSCLC and HNSCC patients who were treated surgically. Target gene expression in NSCLC and HNSCC tissue was higher than in blood. A statistically significant correlation (pAPEX, ERCC1, ERCC2, ERCC4, XRCC1 and XRCC3 in HNSCC. The existence of a significant correlation between blood and tumour tissue expression of some genes of clinical interest, such as ERCC1 in NSCLC and HNSCC, could allow the introduction in clinical practice of a simple test that would measure mRNA levels of DNA repair genes in peripheral blood samples instead of tissue samples to determine prognostic and predictive factors in NSCLC and HNSCC patients. Copyright © 2012 Elsevier B.V. All rights reserved.

  7. Assignment of ten DNA repair genes from Schizosaccharomyces pombe to chromosomal NotI restriction fragments

    NARCIS (Netherlands)

    B.C. Broughton; N.C. Barbet; J. Murray (Johanne); F.Z. Watts (Felicity); M.H.M. Koken (Marcel); A.R. Lehmann (Alan); A.M. Carr (Anthony)

    1991-01-01

    textabstractTen DNA repair (rad) genes from the fission yeast, Schizosaccharomyces pombe were mapped to the 17 NotI fragments of the three chromosomes. Nine of the genes map to chromosome I, but there is no evidence for significant clustering.

  8. Different organization of base excision repair of uracil in DNA in nuclei and mitochondria and selective upregulation of mitochondrial uracil-DNA glycosylase after oxidative stress

    DEFF Research Database (Denmark)

    Akbari, M; Otterlei, M; Pena Diaz, Javier

    2007-01-01

    , indicating regulatory effects of oxidative stress on mitochondrial BER. To examine the overall organization of uracil-BER in nuclei and mitochondria, we constructed cell lines expressing EYFP (enhanced yellow fluorescent protein) fused to UNG1 or UNG2. These were used to investigate the possible presence...... BER processes are differently organized. Furthermore, the upregulation of mRNA for mitochondrial UNG1 after oxidative stress indicates that it may have an important role in repair of oxidized pyrimidines....

  9. A general role of the DNA glycosylase Nth1 in the abasic sites cleavage step of base excision repair in Schizosaccharomyces pombe

    OpenAIRE

    Alseth, Ingrun; Korvald, Hanne; Osman, Fekret; Seeberg, Erling; Bjørås, Magnar

    2004-01-01

    One of the most frequent lesions formed in cellular DNA are abasic (apurinic/apyrimidinic, AP) sites that are both cytotoxic and mutagenic, and must be removed efficiently to maintain genetic stability. It is generally believed that the repair of AP sites is initiated by the AP endonucleases; however, an alternative pathway seems to prevail in Schizosaccharomyces pombe. A mutant lacking the DNA glycosylase/AP lyase Nth1 is very sensitive to the alkylating agent methyl methanesulfonate (MMS), ...

  10. Expression signatures of DNA repair genes correlate with survival prognosis of astrocytoma patients.

    Science.gov (United States)

    de Sousa, Juliana Ferreira; Torrieri, Raul; Serafim, Rodolfo Bortolozo; Di Cristofaro, Luis Fernando Macedo; Escanfella, Fábio Dalbon; Ribeiro, Rodrigo; Zanette, Dalila Lucíola; Paçó-Larson, Maria Luisa; da Silva, Wilson Araujo; Tirapelli, Daniela Pretti da Cunha; Neder, Luciano; Carlotti, Carlos Gilberto; Valente, Valeria

    2017-04-01

    Astrocytomas are the most common primary brain tumors. They are very resistant to therapies and usually progress rapidly to high-grade lesions. Here, we investigated the potential role of DNA repair genes in astrocytoma progression and resistance. To this aim, we performed a polymerase chain reaction array-based analysis focused on DNA repair genes and searched for correlations between expression patters and survival prognoses. We found 19 genes significantly altered. Combining these genes in all possible arrangements, we found 421 expression signatures strongly associated with poor survival. Importantly, five genes (DDB2, EXO1, NEIL3, BRCA2, and BRIP1) were independently correlated with worse prognoses, revealing single-gene signatures. Moreover, silencing of EXO1, which is remarkably overexpressed, promoted faster restoration of double-strand breaks, while NEIL3 knockdown, also highly overexpressed, caused an increment in DNA damage and cell death after irradiation of glioblastoma cells. These results disclose the importance of DNA repair pathways for the maintenance of genomic stability of high-grade astrocytomas and suggest that EXO1 and NEIL3 overexpression confers more efficiency for double-strand break repair and resistance to reactive oxygen species, respectively. Thereby, we highlight these two genes as potentially related with tumor aggressiveness and promising candidates as novel therapeutic targets.

  11. Pan-cancer analysis of bi-allelic alterations in homologous recombination DNA repair genes.

    Science.gov (United States)

    Riaz, Nadeem; Blecua, Pedro; Lim, Raymond S; Shen, Ronglai; Higginson, Daniel S; Weinhold, Nils; Norton, Larry; Weigelt, Britta; Powell, Simon N; Reis-Filho, Jorge S

    2017-10-11

    BRCA1 and BRCA2 are involved in homologous recombination (HR) DNA repair and are germ-line cancer pre-disposition genes that result in a syndrome of hereditary breast and ovarian cancer (HBOC). Whether germ-line or somatic alterations in these genes or other members of the HR pathway and if mono- or bi-allelic alterations of HR-related genes have a phenotypic impact on other cancers remains to be fully elucidated. Here, we perform a pan-cancer analysis of The Cancer Genome Atlas (TCGA) data set and observe that bi-allelic pathogenic alterations in homologous recombination (HR) DNA repair-related genes are prevalent across many malignancies. These bi-allelic alterations often associate with genomic features of HR deficiency. Further, in ovarian, breast and prostate cancers, bi-allelic alterations are mutually exclusive of each other. The combination of these two properties facilitates reclassification of variants of unknown significance affecting DNA repair genes, and may help personalize HR directed therapies in the clinic.Germline mutations in homologous recombination (HR) DNA repair genes are linked to breast and ovarian cancer. Here, the authors show that mutually exclusive bi-allelic inactivation of HR genes are present in other cancer types and associated with genomic features of HR deficiency, expanding the potential use of HR-directed therapies.

  12. Genetic variants of the DNA repair genes from Exome Aggregation Consortium (EXAC) database: significance in cancer.

    Science.gov (United States)

    Das, Raima; Ghosh, Sankar Kumar

    2017-04-01

    DNA repair pathway is a primary defense system that eliminates wide varieties of DNA damage. Any deficiencies in them are likely to cause the chromosomal instability that leads to cell malfunctioning and tumorigenesis. Genetic polymorphisms in DNA repair genes have demonstrated a significant association with cancer risk. Our study attempts to give a glimpse of the overall scenario of the germline polymorphisms in the DNA repair genes by taking into account of the Exome Aggregation Consortium (ExAC) database as well as the Human Gene Mutation Database (HGMD) for evaluating the disease link, particularly in cancer. It has been found that ExAC DNA repair dataset (which consists of 228 DNA repair genes) comprises 30.4% missense, 12.5% dbSNP reported and 3.2% ClinVar significant variants. 27% of all the missense variants has the deleterious SIFT score of 0.00 and 6% variants carrying the most damaging Polyphen-2 score of 1.00, thus affecting the protein structure and function. However, as per HGMD, only a fraction (1.2%) of ExAC DNA repair variants was found to be cancer-related, indicating remaining variants reported in both the databases to be further analyzed. This, in turn, may provide an increased spectrum of the reported cancer linked variants in the DNA repair genes present in ExAC database. Moreover, further in silico functional assay of the identified vital cancer-associated variants, which is essential to get their actual biological significance, may shed some lights in the field of targeted drug development in near future. Copyright © 2017. Published by Elsevier B.V.

  13. The Cockayne syndrome B protein, involved in transcription-coupled repair resides in a RNA polymerase II-containing complex.

    NARCIS (Netherlands)

    A.J. van Gool (Alain); E. Citterio (Elisabetta); S. Rademakers (Suzanne); R. van Os; W. Vermeulen (Wim); A. Constantinou; J-M. Egly (Jean-Marc); D. Bootsma (Dirk); J.H.J. Hoeijmakers (Jan)

    1997-01-01

    textabstractTranscription-coupled repair (TCR), a subpathway of nucleotide excision repair (NER) defective in Cockayne syndrome A and B (CSA and CSB), is responsible for the preferential removal of DNA lesions from the transcribed strand of active genes, permitting rapid resumption of blocked

  14. Analysis of gene repair tracts from Cas9/gRNA double-stranded breaks in the human CFTR gene

    OpenAIRE

    Hollywood, Jennifer A.; Lee, Ciaran M.; Scallan, Martina F.; Harrison, Patrick T.

    2016-01-01

    To maximise the efficiency of template-dependent gene editing, most studies describe programmable and/or RNA-guided endonucleases that make a double-stranded break at, or close to, the target sequence to be modified. The rationale for this design strategy is that most gene repair tracts will be very short. Here, we describe a CRISPR Cas9/gRNA selection-free strategy which uses deep sequencing to characterise repair tracts from a donor plasmid containing seven nucleotide differences across a 2...

  15. Approaches to diagnose DNA mismatch repair gene defects in cancer

    DEFF Research Database (Denmark)

    Peña-Diaz, Javier; Rasmussen, Lene Juel

    2016-01-01

    genes also renders cells resistant to some chemotherapeutic agents. Therefore, diagnosis of MMR deficiency has important implications for the management of the patients, the surveillance of their relatives in the case of LS and for the choice of treatment. Some of the alterations found in MMR genes have...... genes and anticipates the need of diagnostic tools for rapid assessment of their pathogenicity. This review describes current tools and future strategies for addressing the relevance of MMR gene alterations in human disease....

  16. Prevalence and prognostic role of mismatch repair gene defect in endometrial cancer patients.

    Science.gov (United States)

    Tangjitgamol, Siriwan; Kittisiam, Thannaporn; Tanvanich, Sujitra

    2017-09-01

    The study was to evaluate the prevalence of mismatch repair gene defect among Thai patients with endometrial cancer and its association with clinico-pathological features and survivals. The formalin fixed paraffin-embedded blocks of EMC tissue from hysterectomy specimens of patients having surgery in our institution between 1 Jan 1995 and 31 December 2016 were assessed for the immunohistochemical expression of 4 mismatch repair proteins (MLH1, PMS, MSH2, MSH 6). Mismatch repair gene defect was determined by a negative expression of at least 1 protein. Among 385 EMC patients included in the study, mean age was 57.3 ± 10.8 years with 62.3% aged ⩽ 60 years. The most frequent mismatch repair gene defect was MSH6 (38.7%), followed by PMS2 (34.3%), MLH1 (33.2%), and MSH2 (16.4%). Overall, 55.1% showed negative expression of at least one protein. We found significantly higher mismatch repair gene defect in patients aged ⩽ 60 years, with early stage disease, and negative lymph node status than the other comparative groups: 59.2% vs 48.3% for age (p = 0.037), 58.2% vs 45.2% (p = 0.027) for stage, and 58.1% vs 44.6% (p = 0.048) for nodal status. The 5-year progression-free survival, overall survival, and endometrial cancer-specific survival of patients with mismatch repair gene defect was higher than those without gene defect. The differences were statistically significant for only progression-free survival and endometrial cancer-specific survival: 87.7% (95% confidence interval = 83.0%-92.4%) vs 81.5% (95% confidence interval = 75.4%-87.6%) (p = 0.049) for progression-free survival and 91.0% (95% confidence interval = 86.9%-95.1%) vs 85.5% (95% confidence interval = 80.0%-91.0%) (p = 0.044) for endometrial cancer-specific survival, respectively. In conclusion, more than half of Thai endometrial cancer patients had mismatch repair gene defect. The patients with mismatch repair gene defect had significantly younger age (⩽ 60 years) and better prognosis in terms of

  17. Archaeal DNA Polymerase-B as a DNA Template Guardian: Links between Polymerases and Base/Alternative Excision Repair Enzymes in Handling the Deaminated Bases Uracil and Hypoxanthine

    Directory of Open Access Journals (Sweden)

    Javier Abellón-Ruiz

    2016-01-01

    Full Text Available In Archaea repair of uracil and hypoxanthine, which arise by deamination of cytosine and adenine, respectively, is initiated by three enzymes: Uracil-DNA-glycosylase (UDG, which recognises uracil; Endonuclease V (EndoV, which recognises hypoxanthine; and Endonuclease Q (EndoQ, (which recognises both uracil and hypoxanthine. Two archaeal DNA polymerases, Pol-B and Pol-D, are inhibited by deaminated bases in template strands, a feature unique to this domain. Thus the three repair enzymes and the two polymerases show overlapping specificity for uracil and hypoxanthine. Here it is demonstrated that binding of Pol-D to primer-templates containing deaminated bases inhibits the activity of UDG, EndoV, and EndoQ. Similarly Pol-B almost completely turns off EndoQ, extending earlier work that demonstrated that Pol-B reduces catalysis by UDG and EndoV. Pol-B was observed to be a more potent inhibitor of the enzymes compared to Pol-D. Although Pol-D is directly inhibited by template strand uracil, the presence of Pol-B further suppresses any residual activity of Pol-D, to near-zero levels. The results are compatible with Pol-D acting as the replicative polymerase and Pol-B functioning primarily as a guardian preventing deaminated base-induced DNA mutations.

  18. Roles of base excision repair enzymes Nth1p and Apn2p from Schizosaccharomyces pombe in processing alkylation and oxidative DNA damage.

    Science.gov (United States)

    Sugimoto, Takanori; Igawa, Emi; Tanihigashi, Haruna; Matsubara, Mayumi; Ide, Hiroshi; Ikeda, Shogo

    2005-11-21

    Schizosaccharomyces pombe Nthpl, an ortholog of the endonuclease III family, is the sole bifunctional DNA glycosylase encoded in its genome. The enzyme removes oxidative pyrimidine and incises 3' to the apurinic/apyrimidinic (AP) site, leaving 3'-alpha,beta-unsaturated aldehyde. Analysis of nth1 cDNA revealed an intronless structure including 5'- and 3'-untranslated regions. An Nth1p-green fluorescent fusion protein was predominantly localized in the nuclei of yeast cells, indicating a nuclear function. Deletion of nth1 confirmed that Nth1p is responsible for the majority of activity for thymine glycol and AP site incision in the absence of metal ions, while nth1 mutants exhibit hypersensitivity to methylmethanesulfonate (MMS). Complementation of sensitivity by heterologous expression of various DNA glycosylases showed that the methyl-formamidopyrimidine (me-fapy) and/or AP sites are plausible substrates for Nth1p in repairing MMS damage. Apn2p, the major AP endonuclease in S. pombe, also greatly contributes to the repair of MMS damage. Deletion of nth1 from an apn2 mutant resulted in tolerance to MMS damage, indicating that Nth1p-induced 3'-blocks are responsible for MMS sensitivity in apn2 mutants. Overexpression of Apn2p in nth1 mutants failed to suppress MMS sensitivity. These results indicate that Nth1p, not Apn2p, primarily incises AP sites and that the resultant 3'-blocks are removed by the 3'-phosphodiesterase activity of Apn2p. Nth1p is dispensable for cell survival against low levels of oxidative stress, but wild-type yeast became more sensitive than the nth1 mutant at high levels. Overexpression of Nth1p in heavily damaged cells probably induced cell death via the formation of 3'-blocked single-strand breaks.

  19. Gene therapy and peripheral nerve repair : a perspective

    NARCIS (Netherlands)

    Hoyng, Stefan A; de Winter, Fred; Tannemaat, Martijn R; Blits, Bas; Malessy, Martijn J A; Verhaagen, J.

    2015-01-01

    Clinical phase I/II studies have demonstrated the safety of gene therapy for a variety of central nervous system disorders, including Canavan's, Parkinson's (PD) and Alzheimer's disease (AD), retinal diseases and pain. The majority of gene therapy studies in the CNS have used adeno-associated viral

  20. Cloning of human and mouse genes homologous to RAD52, a yeast gene involved in DNA repair and recombination.

    NARCIS (Netherlands)

    D.F.R. Muris; O.Y. Bezzubova (Olga); J-M. Buerstedde; K. Vreeken; A.S. Balajee; C.J. Osgood; C. Troelstra (Christine); J.H.J. Hoeijmakers (Jan); K. Ostermann; H. Schmidt (Henning); A.T. Natarajan; J.C.J. Eeken; P.H.M. Lohmann (Paul); A. Pastink (Albert)

    1994-01-01

    textabstractThe RAD52 gene of Saccharomyces cerevisiae is required for recombinational repair of double-strand breaks. Using degenerate oligonucleotides based on conserved amino acid sequences of RAD52 and rad22, its counterpart from Schizosaccharomyces pombe, RAD52 homologs from man and mouse were

  1. Hypermutation of Immunoglobulin Genes in Memory B Cells of DNA Repair–deficient Mice

    Science.gov (United States)

    Jacobs, Heinz; Fukita, Yosho; van der Horst, Gijsbertus T.J.; de Boer, Jan; Weeda, Geert; Essers, Jeroen; de Wind, Niels; Engelward, Bevin P.; Samson, Leona; Verbeek, Sjef; de Murcia, Josiane Ménissier; de Murcia, Gilbert; e Riele, Hein t; Rajewsky, Klaus

    1998-01-01

    To investigate the possible involvement of DNA repair in the process of somatic hypermutation of rearranged immunoglobulin variable (V) region genes, we have analyzed the occurrence, frequency, distribution, and pattern of mutations in rearranged Vλ1 light chain genes from naive and memory B cells in DNA repair–deficient mutant mouse strains. Hypermutation was found unaffected in mice carrying mutations in either of the following DNA repair genes: xeroderma pigmentosum complementation group (XP)A and XPD, Cockayne syndrome complementation group B (CSB), mutS homologue 2 (MSH2), radiation sensitivity 54 (RAD54), poly (ADP-ribose) polymerase (PARP), and 3-alkyladenine DNA-glycosylase (AAG). These results indicate that both subpathways of nucleotide excision repair, global genome repair, and transcription-coupled repair are not required for somatic hypermutation. This appears also to be true for mismatch repair, RAD54-dependent double-strand–break repair, and AAG-mediated base excision repair. PMID:9607915

  2. An update on targeted gene repair in mammalian cells: methods and mechanisms

    Directory of Open Access Journals (Sweden)

    Bolund Lars

    2011-02-01

    Full Text Available Abstract Transfer of full-length genes including regulatory elements has been the preferred gene therapy strategy for clinical applications. However, with significant drawbacks emerging, targeted gene alteration (TGA has recently become a promising alternative to this method. By means of TGA, endogenous DNA repair pathways of the cell are activated leading to specific genetic correction of single-base mutations in the genome. This strategy can be implemented using single-stranded oligodeoxyribonucleotides (ssODNs, small DNA fragments (SDFs, triplex-forming oligonucleotides (TFOs, adeno-associated virus vectors (AAVs and zinc-finger nucleases (ZFNs. Despite difficulties in the use of TGA, including lack of knowledge on the repair mechanisms stimulated by the individual methods, the field holds great promise for the future. The objective of this review is to summarize and evaluate the different methods that exist within this particular area of human gene therapy research.

  3. Inactivation of RAD52 and HDF1 DNA repair genes leads to ...

    Indian Academy of Sciences (India)

    Home; Journals; Journal of Biosciences; Volume 42; Issue 2. Inactivation of RAD52 and HDF1 DNA repair genes leads to premature chronological aging and cellular instability. SILVIA MERCADO-SÁENZ BEATRIZ LÓPEZ-DÍAZ FRANCISCO SENDRA-PORTERO MANUEL MARTÍNEZ-MORILLO MIGUEL J RUIZ-GÓMEZ.

  4. DHR6, a Drosophila homolog of the yeast DNA repair gene RAD6.

    NARCIS (Netherlands)

    M.H.M. Koken (Marcel); P. Reynolds (Paul); D. Bootsma (Dirk); J.H.J. Hoeijmakers (Jan); S. Prakash; L. Prakash

    1991-01-01

    textabstractThe RAD6 gene of the yeast Saccharomyces cerevisiae is required for DNA repair, for DNA damage-induced mutagenesis, and for sporulation, and it encodes a ubiquitin-conjugating enzyme. We have cloned the RAD6 homolog from Drosophila melanogaster and find that its encoded protein displays

  5. Selecting patients with young-onset colorectal cancer for mismatch repair gene analysis

    DEFF Research Database (Denmark)

    Walker, M; O'Sullivan, B; Perakath, B

    2007-01-01

    BACKGROUND: Young patients with colorectal cancer are at increased risk of carrying a germline mutation in mismatch repair (MMR) genes. This study investigated the role of clinical criteria and immunohistochemistry for MMR proteins in selecting young patients for mutation testing. METHODS: A cohort...

  6. Patients with systemic sclerosis present increased DNA damage differentially associated with DNA repair gene polymorphisms.

    Science.gov (United States)

    Palomino, Gustavo Martelli; Bassi, Carmen L; Wastowski, Isabela J; Xavier, Danilo J; Lucisano-Valim, Yara M; Crispim, Janaina C O; Rassi, Diane M; Marques-Neto, Joao F; Sakamoto-Hojo, Elza T; Moreau, Philippe; Sampaio-Barros, Percival D; Donadi, Eduardo A

    2014-03-01

    Patients with systemic sclerosis (SSc) exhibit increased toxicity when exposed to genotoxic agents. In our study, we evaluated DNA damage and polymorphic sites in 2 DNA repair genes (XRCC1 Arg399Gln and XRCC4 Ile401Thr) in patients with SSc. A total of 177 patients were studied for DNA repair gene polymorphisms. Fifty-six of them were also evaluated for DNA damage in peripheral blood cells using the comet assay. Compared to controls, the patients as a whole or stratified into major clinical variants (limited or diffuse skin involvement), irrespective of the underlying treatment schedule, exhibited increased DNA damage. XRCC1 (rs: 25487) and XRCC4 (rs: 28360135) allele and genotype frequencies observed in patients with SSc were not significantly different from those observed in controls; however, the XRCC1 Arg399Gln allele was associated with increased DNA damage only in healthy controls and the XRCC4 Ile401Thr allele was associated with increased DNA damage in both patients and controls. Further, the XRCC1 Arg399Gln allele was associated with the presence of antinuclear antibody and anticentromere antibody. No association was observed between these DNA repair gene polymorphic sites and clinical features of patients with SSc. These results corroborate the presence of genomic instability in SSc peripheral blood cells, as evaluated by increased DNA damage, and show that polymorphic sites of the XRCC1 and XRCC4 DNA repair genes may differentially influence DNA damage and the development of autoantibodies.

  7. DNA repair gene XRCC7 G6721T variant and susceptibility to ...

    African Journals Online (AJOL)

    Mostafa Saadat

    2016-02-20

    Feb 20, 2016 ... Abstract Background: The human XRCC7 (MIM: 600899) is a DNA double-strand break repair gene, involved in non-homologous end joining (NHEJ). Polymorphism G6721T (rs7003908) is located in the intron 8 of the XRCC7. This polymorphism may regulate splicing and cause mRNA instability. Aim: The ...

  8. Analysis of DNA repair gene polymorphisms and survival in low-grade and anaplastic gliomas

    DEFF Research Database (Denmark)

    Berntsson, Shala Ghaderi; Wibom, Carl; Sjöström, Sara

    2011-01-01

    different DNA repair genes (ATM, NEIL1, NEIL2, ERCC6 and RPA4) which were associated with survival. Finally, these eight genetic variants were adjusted for treatment, malignancy grade, patient age and gender, leaving one variant, rs4253079, mapped to ERCC6, with a significant association to survival (OR 0...

  9. DNA repair gene XRCC7 G6721T variant and susceptibility to ...

    African Journals Online (AJOL)

    Background: The human XRCC7 (MIM: 600899) is a DNA double-strand break repair gene, involved in non-homologous end joining (NHEJ). Polymorphism G6721T (rs7003908) is located in the intron 8 of the XRCC7. This polymorphism may regulate splicing and cause mRNA instability. Aim: The aim of the present study ...

  10. Germline stem cell gene PIWIL2 mediates DNA repair through relaxation of chromatin.

    Directory of Open Access Journals (Sweden)

    De-Tao Yin

    Full Text Available DNA damage response (DDR is an intrinsic barrier of cell to tumorigenesis initiated by genotoxic agents. However, the mechanisms underlying the DDR are not completely understood despite of extensive investigation. Recently, we have reported that ectopic expression of germline stem cell gene PIWIL2 is associated with tumor stem cell development, although the underlying mechanisms are largely unknown. Here we show that PIWIL2 is required for the repair of DNA-damage induced by various types of genotoxic agents. Upon ultraviolet (UV irradiation, silenced PIWIL2 gene in normal human fibroblasts was transiently activated after treatment with UV light. This activation was associated with DNA repair, because Piwil2-deficienct mouse embryonic fibroblasts (mili(-/- MEFs were defective in cyclobutane pyrimidine dimers (CPD repair after UV treatment. As a result, the UV-treated mili(-/- MEFs were more susceptible to apoptosis, as characterized by increased levels of DNA damage-associated apoptotic proteins, such as active caspase-3, cleaved Poly (ADP-ribose polymerase (PARP and Bik. The impaired DNA repair in the mili(-/- MEFs was associated with the reductions of histone H3 acetylation and chromatin relaxation, although the DDR pathway downstream chromatin relaxation appeared not to be directly affected by Piwil2. Moreover, guanine-guanine (Pt-[GG] and double strand break (DSB repair were also defective in the mili(-/- MEFs treated by genotoxic chemicals Cisplatin and ionizing radiation (IR, respectively. The results indicate that Piwil2 can mediate DNA repair through an axis of Piwil2 → histone acetylation → chromatin relaxation upstream DDR pathways. The findings reveal a new role for Piwil2 in DNA repair and suggest that Piwil2 may act as a gatekeeper against DNA damage-mediated tumorigenesis.

  11. Multigene panel analysis identified germline mutations of DNA repair genes in breast and ovarian cancer.

    Science.gov (United States)

    Hirotsu, Yosuke; Nakagomi, Hiroshi; Sakamoto, Ikuko; Amemiya, Kenji; Oyama, Toshio; Mochizuki, Hitoshi; Omata, Masao

    2015-09-01

    Approximately 5-10% of all breast and/or ovarian cancer cases are considered as inherited. BRCA1 and BRCA2 tumor suppressor genes account for a high penetrance of hereditary cases, but familial cases without mutations in these genes can also occur. Despite their low penetrance, other hereditary cancer-related genes are known to be associated with breast and ovarian cancer risk. However, the extent to which these genes prevail in breast and ovarian cancer remains to be elucidated. To estimate the frequency of mutations in these predisposition genes, we analyzed the germline mutations of 25 hereditary cancer-related genes in 155 patients using targeted next-generation sequencing. These subjects included 11 BRCA1/2 mutation-positive cases and 144 negative cases. Of these, three patients (1.9%) had pathogenic mutations in ATM, MRE11A, or MSH6, all of which have a central role in DNA repair and the mismatch repair pathway. The MSH6 splice-site mutation (IVS6+1G>T) was predicted to be pathogenic, as demonstrated by in vitro and immunohistochemical analyses. These results suggested deficiencies in cellular DNA repair functions result in the development of breast and ovarian cancer.

  12. Polymorphisms in DNA Repair Genes and MDR1 and the Risk for Non-Hodgkin Lymphoma

    Directory of Open Access Journals (Sweden)

    Hee Nam Kim

    2014-04-01

    Full Text Available The damage caused by oxidative stress and exposure to cigarette smoke and alcohol necessitate DNA damage repair and transport by multidrug resistance-1 (MDR1. To explore the association between polymorphisms in these genes and non-Hodgkin lymphoma risk, we analyzed 15 polymorphisms of 12 genes in a population-based study in Korea (694 cases and 1700 controls. Four genotypes of DNA repair pathway genes (XRCC1 399 GA, OGG1 326 GG, BRCA1 871 TT, and WRN 787 TT were associated with a decreased risk for NHL [odds ratio (ORXRCC1 GA = 0.80, p = 0.02; OROGG1 GG = 0.70, p = 0.008; ORBRCA1 TT = 0.71, p = 0.048; ORWRN TT = 0.68, p = 0.01]. Conversely, the MGMT 115 CT genotype was associated with an increased risk for NHL (OR = 1.25, p = 0.04. In the MDR1 gene, the 1236 CC genotype was associated with a decreased risk for NHL (OR = 0.74, p = 0.04, and the 3435 CT and TT genotypes were associated with an increased risk (OR3435CT = 1.50, p < 0.0001; OR3435TT = 1.43, p = 0.02. These results suggest that polymorphisms in the DNA repair genes XRCC1, OGG1, BRCA1, WRN1, and MGMT and in the MDR1 gene may affect the risk for NHL in Korean patients.

  13. Loss of transcription factor early growth response gene 1 results in impaired endochondral bone repair.

    Science.gov (United States)

    Reumann, Marie K; Strachna, Olga; Yagerman, Sarah; Torrecilla, Daniel; Kim, Jihye; Doty, Stephen B; Lukashova, Lyudmila; Boskey, Adele L; Mayer-Kuckuk, Philipp

    2011-10-01

    Transcription factors that play a role in ossification during development are expected to participate in postnatal fracture repair since the endochondral bone formation that occurs in embryos is recapitulated during fracture repair. However, inherent differences exist between bone development and fracture repair, including a sudden disruption of tissue integrity followed by an inflammatory response. This raises the possibility that repair-specific transcription factors participate in bone healing. Here, we assessed the consequence of loss of early growth response gene 1 (EGR-1) on endochondral bone healing because this transcription factor has been shown to modulate repair in vascularized tissues. Model fractures were created in ribs of wild type (wt) and EGR-1(-/-) mice. Differences in tissue morphology and composition between these two animal groups were followed over 28 post fracture days (PFDs). In wt mice, bone healing occurred in healing phases characteristic of endochondral bone repair. A similar healing sequence was observed in EGR-1(-/-) mice but was impaired by alterations. A persistent accumulation of fibrin between the disconnected bones was observed on PFD7 and remained pronounced in the callus on PFD14. Additionally, the PFD14 callus was abnormally enlarged and showed increased deposition of mineralized tissue. Cartilage ossification in the callus was associated with hyper-vascularity and -proliferation. Moreover, cell deposits located in proximity to the callus within skeletal muscle were detected on PFD14. Despite these impairments, repair in EGR-1(-/-) callus advanced on PFD28, suggesting EGR-1 is not essential for healing. Together, this study provides genetic evidence that EGR-1 is a pleiotropic regulator of endochondral fracture repair. Copyright © 2011 Elsevier Inc. All rights reserved.

  14. Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity.

    Science.gov (United States)

    Komor, Alexis C; Zhao, Kevin T; Packer, Michael S; Gaudelli, Nicole M; Waterbury, Amanda L; Koblan, Luke W; Kim, Y Bill; Badran, Ahmed H; Liu, David R

    2017-08-01

    We recently developed base editing, the programmable conversion of target C:G base pairs to T:A without inducing double-stranded DNA breaks (DSBs) or requiring homology-directed repair using engineered fusions of Cas9 variants and cytidine deaminases. Over the past year, the third-generation base editor (BE3) and related technologies have been successfully used by many researchers in a wide range of organisms. The product distribution of base editing-the frequency with which the target C:G is converted to mixtures of undesired by-products, along with the desired T:A product-varies in a target site-dependent manner. We characterize determinants of base editing outcomes in human cells and establish that the formation of undesired products is dependent on uracil N-glycosylase (UNG) and is more likely to occur at target sites containing only a single C within the base editing activity window. We engineered CDA1-BE3 and AID-BE3, which use cytidine deaminase homologs that increase base editing efficiency for some sequences. On the basis of these observations, we engineered fourth-generation base editors (BE4 and SaBE4) that increase the efficiency of C:G to T:A base editing by approximately 50%, while halving the frequency of undesired by-products compared to BE3. Fusing BE3, BE4, SaBE3, or SaBE4 to Gam, a bacteriophage Mu protein that binds DSBs greatly reduces indel formation during base editing, in most cases to below 1.5%, and further improves product purity. BE4, SaBE4, BE4-Gam, and SaBE4-Gam represent the state of the art in C:G-to-T:A base editing, and we recommend their use in future efforts.

  15. Determining the functional significance of mismatch repair gene missense variants using biochemical and cellular assays

    DEFF Research Database (Denmark)

    Heinen, Christopher D; Juel Rasmussen, Lene

    2012-01-01

    provided an important experimental tool for studying the functional consequences of VUS. However, beyond this repair assay, a number of other experimental methods have been developed that allow us to test the effect of a VUS on discrete biochemical steps or other aspects of MMR function. Here, we describe......ABSTRACT: With the discovery that the hereditary cancer susceptibility disease Lynch syndrome (LS) is caused by deleterious germline mutations in the DNA mismatch repair (MMR) genes nearly 20 years ago, genetic testing can now be used to diagnose this disorder in patients. A definitive diagnosis...

  16. Comprehensive analysis of DNA repair gene variants and risk of meningioma

    DEFF Research Database (Denmark)

    Bethke, L.; Murray, A.; Webb, E.

    2008-01-01

    BACKGROUND: Meningiomas account for up to 37% of all primary brain tumors. Genetic susceptibility to meningioma is well established, with the risk among relatives of meningioma patients being approximately threefold higher than that in the general population. A relationship between risk...... of meningioma and exposure to ionizing radiation is also well known and led us to examine whether variants in DNA repair genes contribute to disease susceptibility. METHODS: We analyzed 1127 tagging single-nucleotide polymorphisms (SNPs) that were selected to capture most of the common variation in 136 DNA...... repair genes in five case-control series (631 case patients and 637 control subjects) from four countries in Europe. We also analyzed 388 putative functional SNPs in these genes for their association with meningioma. All statistical tests were two-sided. RESULTS: The SNP rs4968451, which maps to intron 4...

  17. Mismatch repair gene mutation spectrum in the Swedish Lynch syndrome population

    DEFF Research Database (Denmark)

    Lagerstedt-Robinson, Kristina; Rohlin, Anna; Aravidis, Christos

    2016-01-01

    Lynch syndrome caused by constitutional mismatch‑repair defects is one of the most common hereditary cancer syndromes with a high risk for colorectal, endometrial, ovarian and urothelial cancer. Lynch syndrome is caused by mutations in the mismatch repair (MMR) genes i.e., MLH1, MSH2, MSH6 and PMS2....... After 20 years of genetic counseling and genetic testing for Lynch syndrome, we have compiled the mutation spectrum in Sweden with the aim to provide a population-based perspective on the contribution from the different MMR genes, the various types of mutations and the influence from founder mutations....... Mutation data were collected on a national basis from all laboratories involved in genetic testing. Mutation analyses were performed using mainly Sanger sequencing and multiplex ligation-dependent probe amplification. A total of 201 unique disease-predisposing MMR gene mutations were identified in 369...

  18. Treating Duchenne Cardiomyopathy in the Mouse Model by Gene Repair

    Science.gov (United States)

    2017-08-01

    Lipid nanoparticles and liposomes have been used to deliver exon-skipping AONs for DMD gene therapy. In the former, cationic lipids are used to coat ...the delivery tool. AAV is a bio- nanoparticle . We published a review article on the current state-of-art on nanotherapy (both viral and noviral) for...bearing binding domains for various materials including gold nanoparticles and carbon nanotubes for testing (at the LaBean lab) the assembly of

  19. Epigenetic Studies Point to DNA Replication/Repair Genes as a Basis for the Heritable Nature of Long Term Complications in Diabetes

    Directory of Open Access Journals (Sweden)

    Alexey A. Leontovich

    2016-01-01

    Full Text Available Metabolic memory (MM is defined as the persistence of diabetic (DM complications even after glycemic control is pharmacologically achieved. Using a zebrafish diabetic model that induces a MM state, we previously reported that, in this model, tissue dysfunction was of a heritable nature based on cell proliferation studies in limb tissue and this correlated with epigenetic DNA methylation changes that paralleled alterations in gene expression. In the current study, control, DM, and MM excised fin tissues were further analyzed by MeDIP sequencing and microarray techniques. Bioinformatics analysis of the data found that genes of the DNA replication/DNA metabolism process group (with upregulation of the apex1, mcm2, mcm4, orc3, lig1, and dnmt1 genes were altered in the DM state and these molecular changes continued into MM. Interestingly, DNA methylation changes could be found as far as 6–13 kb upstream of the transcription start site for these genes suggesting potential higher levels of epigenetic control. In conclusion, DNA methylation changes in members of the DNA replication/repair process group best explain the heritable nature of cell proliferation impairment found in the zebrafish DM/MM model. These results are consistent with human diabetic epigenetic studies and provide one explanation for the persistence of long term tissue complications as seen in diabetes.

  20. A data mining approach for classifying DNA repair genes into ageing-related or non-ageing-related.

    Science.gov (United States)

    Freitas, Alex A; Vasieva, Olga; de Magalhães, João Pedro

    2011-01-12

    The ageing of the worldwide population means there is a growing need for research on the biology of ageing. DNA damage is likely a key contributor to the ageing process and elucidating the role of different DNA repair systems in ageing is of great interest. In this paper we propose a data mining approach, based on classification methods (decision trees and Naive Bayes), for analysing data about human DNA repair genes. The goal is to build classification models that allow us to discriminate between ageing-related and non-ageing-related DNA repair genes, in order to better understand their different properties. The main patterns discovered by the classification methods are as follows: (a) the number of protein-protein interactions was a predictor of DNA repair proteins being ageing-related; (b) the use of predictor attributes based on protein-protein interactions considerably increased predictive accuracy of attributes based on Gene Ontology (GO) annotations; (c) GO terms related to "response to stimulus" seem reasonably good predictors of ageing-relatedness for DNA repair genes; (d) interaction with the XRCC5 (Ku80) protein is a strong predictor of ageing-relatedness for DNA repair genes; and (e) DNA repair genes with a high expression in T lymphocytes are more likely to be ageing-related. The above patterns are broadly integrated in an analysis discussing relations between Ku, the non-homologous end joining DNA repair pathway, ageing and lymphocyte development. These patterns and their analysis support non-homologous end joining double strand break repair as central to the ageing-relatedness of DNA repair genes. Our work also showcases the use of protein interaction partners to improve accuracy in data mining methods and our approach could be applied to other ageing-related pathways.

  1. A data mining approach for classifying DNA repair genes into ageing-related or non-ageing-related

    Directory of Open Access Journals (Sweden)

    Vasieva Olga

    2011-01-01

    Full Text Available Abstract Background The ageing of the worldwide population means there is a growing need for research on the biology of ageing. DNA damage is likely a key contributor to the ageing process and elucidating the role of different DNA repair systems in ageing is of great interest. In this paper we propose a data mining approach, based on classification methods (decision trees and Naive Bayes, for analysing data about human DNA repair genes. The goal is to build classification models that allow us to discriminate between ageing-related and non-ageing-related DNA repair genes, in order to better understand their different properties. Results The main patterns discovered by the classification methods are as follows: (a the number of protein-protein interactions was a predictor of DNA repair proteins being ageing-related; (b the use of predictor attributes based on protein-protein interactions considerably increased predictive accuracy of attributes based on Gene Ontology (GO annotations; (c GO terms related to "response to stimulus" seem reasonably good predictors of ageing-relatedness for DNA repair genes; (d interaction with the XRCC5 (Ku80 protein is a strong predictor of ageing-relatedness for DNA repair genes; and (e DNA repair genes with a high expression in T lymphocytes are more likely to be ageing-related. Conclusions The above patterns are broadly integrated in an analysis discussing relations between Ku, the non-homologous end joining DNA repair pathway, ageing and lymphocyte development. These patterns and their analysis support non-homologous end joining double strand break repair as central to the ageing-relatedness of DNA repair genes. Our work also showcases the use of protein interaction partners to improve accuracy in data mining methods and our approach could be applied to other ageing-related pathways.

  2. Germline mutations in DNA repair genes predispose asbestos-exposed patients to malignant pleural mesothelioma.

    Science.gov (United States)

    Betti, Marta; Casalone, Elisabetta; Ferrante, Daniela; Aspesi, Anna; Morleo, Giulia; Biasi, Alessandra; Sculco, Marika; Mancuso, Giuseppe; Guarrera, Simonetta; Righi, Luisella; Grosso, Federica; Libener, Roberta; Pavesi, Mansueto; Mariani, Narciso; Casadio, Caterina; Boldorini, Renzo; Mirabelli, Dario; Pasini, Barbara; Magnani, Corrado; Matullo, Giuseppe; Dianzani, Irma

    2017-10-01

    Malignant pleural mesothelioma (MPM) is a rare, aggressive cancer caused by asbestos exposure. An inherited predisposition has been suggested to explain multiple cases in the same family and the observation that not all individuals highly exposed to asbestos develop the tumor. Germline mutations in BAP1 are responsible for a rare cancer predisposition syndrome that includes predisposition to mesothelioma. We hypothesized that other genes involved in hereditary cancer syndromes could be responsible for the inherited mesothelioma predisposition. We investigated the prevalence of germline variants in 94 cancer-predisposing genes in 93 MPM patients with a quantified asbestos exposure. Ten pathogenic truncating variants (PTVs) were identified in PALB2, BRCA1, FANCI, ATM, SLX4, BRCA2, FANCC, FANCF, PMS1 and XPC. All these genes are involved in DNA repair pathways, mostly in homologous recombination repair. Patients carrying PTVs represented 9.7% of the panel and showed lower asbestos exposure than did all the other patients (p = 0.0015). This suggests that they did not efficiently repair the DNA damage induced by asbestos and leading to carcinogenesis. This study shows that germline variants in several genes may increase MPM susceptibility in the presence of asbestos exposure and may be important for specific treatment. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  3. Glial kon/NG2 gene network for central nervous system repair

    Directory of Open Access Journals (Sweden)

    Maria Losada-Perez

    2017-01-01

    Full Text Available The glial regenerative response to central nervous system (CNS injury, although limited, can be harnessed to promote regeneration and repair. Injury provokes the proliferation of ensheathing glial cells, which can differentiate to remyelinate axons, and partially restore function. This response is evolutionarily conserved, strongly implying an underlying genetic mechanism. In mammals, it is elicited by NG2 glia, but most often newly generated cells fail to differentiate. Thus an important goal had been to find out how to promote glial differentiation following the proliferative response. A gene network involving Notch and prospero (pros controls the balance between glial proliferation and differentiation in flies and mice, and promotes CNS repair at least in fruit-flies. A key missing link had been how to relate the function of NG2 to this gene network. Recent findings by Losada-Perez et al., published in JCB, demonstrated that the Drosophila NG2 homologue kon-tiki (kon is functionally linked to Notch and pros in glia. By engaging in two feedback loops with Notch and Pros, in response to injury, Kon can regulate both glial cell number and glial shape homeostasis, essential for repair. Drosophila offers powerful genetics to unravel the control of stem and progenitor cells for regeneration and repair.

  4. Repair of oxidatively generated DNA damage in Cockayne syndrome.

    Science.gov (United States)

    Khobta, Andriy; Epe, Bernd

    2013-01-01

    Defects in the repair of endogenously (especially oxidatively) generated DNA modifications and the resulting genetic instability can potentially explain the clinical symptoms of Cockayne syndrome (CS), a hereditary disease characterized by developmental defects and neurological degeneration. In this review, we describe the evidence for the involvement of CSA and CSB proteins, which are mutated in most of the CS patients, in the repair and processing of DNA damage induced by reactive oxygen species and the implications for the induction of cell death and mutations. Taken together, the data demonstrate that CSA and CSB, in addition to their established role in transcription-coupled nucleotide excision repair, can modulate the base excision repair (BER) of oxidized DNA bases both directly (by interaction with BER proteins) and indirectly (by modulating the expression of the DNA repair genes). Both nuclear and mitochondrial DNA repair is affected by mutations in CSA and CSB genes. However, the observed retardations of repair and the resulting accumulation of unrepaired endogenously generated DNA lesions are often mild, thus pointing to the relevance of additional roles of the CS proteins, e.g. in the mitochondrial response to oxidatively generated DNA damage and in the maintenance of gene transcription. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  5. Immunohistochemical and DNA sequencing analysis on human mismatch repair gene MLH1 in cervical squamous cell carcinoma with LOH of this gene

    NARCIS (Netherlands)

    Hu, X.; Guo, Z.; Pang, T.; Li, Q.; Afink, G.; Pontén, J.

    2000-01-01

    BACKGROUND: The human MLH1 gene (hMLH1) is one of the DNA mismatch repair genes. Defects in these genes are believed to be the underlying cause of microsatellite instability (MSI). MSI has been demonstrated in many human cancers such as colon cancer and some female-specific tumors. The hMLH1 gene

  6. U. V. induces long-lived DNA breaks in Cockayne's syndrome and cells from an immunodeficient individual (46BR): defects and disturbance in post incision steps of excision repair

    Energy Technology Data Exchange (ETDEWEB)

    Squires, S.; Johnson, R.T.

    1983-01-01

    In normal cells exposed to low U.V. doses the several enzymic steps of the excision repair process are closely coupled with the result that DNA gaps are transient and present at such low frequency that it is very difficult to detect them. Cells from a U.V.-sensitive human genetic disorder, Cockayne's Syndrome (CS) and from an immunodeficient individual 46BR, have been examined with respect to their incision capacity after U.V. in the presence and absence of inhibitors of DNA synthesis. We have measured the initial rates of DNA break accumulation in the presence of hydroxyurea and 1-beta-D arabinofuranosylcytosine and find that in both these groups the rate is only slightly lower than in normal cells. However, there is a marked difference between U.V. sensitive cells and normal in the accumulation of long-lived DNA breaks in the absence of inhibitors. While in normal cells practically no breaks could be detected, the U.V. sensitive cells accumulated significant numbers of DNA breaks within 15 min of incubation; 46BR cells showed almost the same level of DNA breaks without the inhibitors as with them. In CS break accumulation can be detected in the absence of inhibitors for only a short time after irradiation (approximately 30 min), but less so when deoxyribonucleosides are provided. The spontaneous break accumulation is related to the time elapsed since proteolytic detachment of the cells from monolayer; 24 h after replating CS breaks no longer accumulate in response to U.V. 46BR cells, on the other hand, accumulate breaks even 1 day after replating and express unligated gaps 2 h after irradiation with a relatively low U.V. dose such as 4 Jm-2. Provision of DNA precursors does not greatly reduce break accumulation. The extremely slow rate of gap sealing in 46BR cells is consistent with the hypothesis that a ligase defect is expressed in these cells.

  7. RAD25(SSL2), a yeast homolog of the human xeroderma pigmentosum group B DNA repair gene, is essential for viability.

    NARCIS (Netherlands)

    E. Park (Robert); S.N. Guzder; M.H.M. Koken (Marcel); I. Jaspers-Dekker (Iris); G. Weeda (Geert); J.H.J. Hoeijmakers (Jan); S. Prakash; L. Prakash

    1992-01-01

    textabstractXeroderma pigmentosum (XP) patients are extremely sensitive to ultraviolet (UV) light and suffer from a high incidence of skin cancers, due to a defect in nucleotide excision repair. The disease is genetically heterogeneous, and seven complementation groups, A-G, have been identified.

  8. A comparison of synthetic oligodeoxynucleotides, DNA fragments and AAV-1 for targeted episomal and chromosomal gene repair

    Directory of Open Access Journals (Sweden)

    Leclerc Xavier

    2009-04-01

    Full Text Available Abstract Background Current strategies for gene therapy of inherited diseases consist in adding functional copies of the gene that is defective. An attractive alternative to these approaches would be to correct the endogenous mutated gene in the affected individual. This study presents a quantitative comparison of the repair efficiency using different forms of donor nucleic acids, including synthetic DNA oligonucleotides, double stranded DNA fragments with sizes ranging from 200 to 2200 bp and sequences carried by a recombinant adeno-associated virus (rAAV-1. Evaluation of each gene repair strategy was carried out using two different reporter systems, a mutated eGFP gene or a dual construct with a functional eGFP and an inactive luciferase gene, in several different cell systems. Gene targeting events were scored either following transient co-transfection of reporter plasmids and donor DNAs, or in a system where a reporter construct was stably integrated into the chromosome. Results In both episomal and chromosomal assays, DNA fragments were more efficient at gene repair than oligonucleotides or rAAV-1. Furthermore, the gene targeting frequency could be significantly increased by using DNA repair stimulating drugs such as doxorubicin and phleomycin. Conclusion Our results show that it is possible to obtain repair frequencies of 1% of the transfected cell population under optimized transfection protocols when cells were pretreated with phleomycin using rAAV-1 and dsDNA fragments.

  9. Repair of Staphylococcus aureus-infected wound with gene-modified C3H10T1/2 cells expressing BPI-BD3 fusion antibiotic peptide

    Directory of Open Access Journals (Sweden)

    Xin-ran ZHANG

    2015-10-01

    Full Text Available Objective To study the antibacterial and tissue reparative effect of BPI-BD3 gene-modified mesenchymal stem cells in a mouse model of wound infection. Methods C3H10T1/2 cells were transfected with recombinant adenovirus vector pAdxsi-BPI-BD3, the expression of BPI-BD3 fusion protein was verified by RT-PCR and Western blotting. Excision wound with a diameter of 1cm was inoculated with Staphylococcus aureuswas made on the back of 30 mice. The mice were randomly divided into 3 groups (10 each. Mice in group T were injected with BPI-BD3 gene-modified C3H10T1/2 cells through caudal vein, those in group C were injected with unmodified C3H10T1/2 cells, and in group N were injected with PBS as control. The wound repair result was evaluated by estimation of the percentage of remaining wound area and the amount of wound bacteria under the scar, followed by observation of pathological changes. Inflammatory reactions of the wounds were assessed accordingly. Results The amount of bacteria under the scar was less in group T than in the other two groups (P<0.05. It was also found that the wound healing process was faster in group T than in group C and group N. Pathological observation showed that the inflammatory reaction in group T was also significantly milder than in the other two groups. Conclusion BPI-BD3 gene-modified mesenchymal stem cells may enhance wound repair by controlling infection and promoting tissue regeneration, thus it may be promising in clinical application. DOI: 10.11855/j.issn.0577-7402.2015.09.07

  10. A genome-scale DNA repair RNAi screen identifies SPG48 as a novel gene associated with hereditary spastic paraplegia.

    Directory of Open Access Journals (Sweden)

    Mikołaj Słabicki

    Full Text Available DNA repair is essential to maintain genome integrity, and genes with roles in DNA repair are frequently mutated in a variety of human diseases. Repair via homologous recombination typically restores the original DNA sequence without introducing mutations, and a number of genes that are required for homologous recombination DNA double-strand break repair (HR-DSBR have been identified. However, a systematic analysis of this important DNA repair pathway in mammalian cells has not been reported. Here, we describe a genome-scale endoribonuclease-prepared short interfering RNA (esiRNA screen for genes involved in DNA double strand break repair. We report 61 genes that influenced the frequency of HR-DSBR and characterize in detail one of the genes that decreased the frequency of HR-DSBR. We show that the gene KIAA0415 encodes a putative helicase that interacts with SPG11 and SPG15, two proteins mutated in hereditary spastic paraplegia (HSP. We identify mutations in HSP patients, discovering KIAA0415/SPG48 as a novel HSP-associated gene, and show that a KIAA0415/SPG48 mutant cell line is more sensitive to DNA damaging drugs. We present the first genome-scale survey of HR-DSBR in mammalian cells providing a dataset that should accelerate the discovery of novel genes with roles in DNA repair and associated medical conditions. The discovery that proteins forming a novel protein complex are required for efficient HR-DSBR and are mutated in patients suffering from HSP suggests a link between HSP and DNA repair.

  11. Thyroid nodules, polymorphic variants in DNA repair and RET-related genes, and interaction with ionizing radiation exposure from nuclear tests in Kazakhstan.

    Science.gov (United States)

    Sigurdson, Alice J; Land, Charles E; Bhatti, Parveen; Pineda, Marbin; Brenner, Alina; Carr, Zhanat; Gusev, Boris I; Zhumadilov, Zhaxibay; Simon, Steven L; Bouville, Andre; Rutter, Joni L; Ron, Elaine; Struewing, Jeffery P

    2009-01-01

    Risk factors for thyroid cancer remain largely unknown except for ionizing radiation exposure during childhood and a history of benign thyroid nodules. Because thyroid nodules are more common than thyroid cancers and are associated with thyroid cancer risk, we evaluated several polymorphisms potentially relevant to thyroid tumors and assessed interaction with ionizing radiation exposure to the thyroid gland. Thyroid nodules were detected in 1998 by ultrasound screening of 2997 persons who lived near the Semipalatinsk nuclear test site in Kazakhstan when they were children (1949-1962). Cases with thyroid nodules (n = 907) were frequency matched (1:1) to those without nodules by ethnicity (Kazakh or Russian), gender and age at screening. Thyroid gland radiation doses were estimated from fallout deposition patterns, residence history and diet. We analyzed 23 polymorphisms in 13 genes and assessed interaction with ionizing radiation exposure using likelihood ratio tests (LRT). Elevated thyroid nodule risks were associated with the minor alleles of RET S836S (rs1800862, P = 0.03) and GFRA1 -193C>G (rs not assigned, P = 0.05) and decreased risk with XRCC1 R194W (rs1799782, P trend = 0.03) and TGFB1 T263I (rs1800472, P = 0.009). Similar patterns of association were observed for a small number of papillary thyroid cancers (n = 25). Ionizing radiation exposure to the thyroid gland was associated with significantly increased risk of thyroid nodules (age and gender adjusted excess odds ratio/Gy = 0.30, 95% CI 0.05-0.56), with evidence for interaction by genotype found for XRCC1 R194W (LRT P value = 0.02). Polymorphisms in RET signaling, DNA repair and proliferation genes may be related to risk of thyroid nodules, consistent with some previous reports on thyroid cancer. Borderline support for gene-radiation interaction was found for a variant in XRCC1, a key base excision repair protein. Other pathways such as genes in double-strand break repair, apoptosis and genes related to

  12. Methylation profile analysis of DNA repair genes in hepatocellular carcinoma with MS-MLPA.

    Science.gov (United States)

    Ozer, Ozge; Bilezikci, Banu; Aktas, Sema; Sahin, Feride I

    2013-12-01

    Hepatocellular carcinoma (HCC) is one of the rare tumors with well-defined risk factors. The multifactorial etiology of HCC can be explained by its complex molecular pathogenesis. In the current study, the methylation status of 7 genes involved in DNA repair mechanisms, namely MLH1, PMS2, MSH6, MSH2, MGMT, MSH3, and MLH3, was investigated in tumor samples from HCC patients, using the methylation-specific-multiplex ligated probe amplification method and the results were correlated with available clinical findings. The most common etiological factor in these cases was the presence of hepatitis B alone (47.2%). Among the 56 cases that were studied, promoter methylation was detected in at least one of the genes in 27 (48.2%) cases, only in 1 gene in 13 (23.2%) cases, and in >1 gene in 14 (25%) cases. Of the 7 genes investigated, methylation was most frequently observed in MSH3, in 14 (25%) cases. Methylation of at least 1 gene was significantly more frequent in patients with single tumors than multifocal tumors. There were significant differences regarding hepatitis B status, Child Class, tumor number, grade, and TNM stage in cases where PMS2 methylation was detected. Our results suggest that methylation of genes involved in mismatch repair may be responsible in the pathogenesis of HCC, and evaluating changes in multiple genes in these pathways simultaneously would be more informative. Despite being a robust and relatively inexpensive method, the methylation-specific-multiplex ligated probe amplification assay could be more extensively applied with improvements in the currently intricate data analysis component.

  13. Bone fragility and decline in stem cells in prematurely aging DNA repair deficient trichothiodystrophy mice

    NARCIS (Netherlands)

    K.E.M. Diderich (Karin); C. Nicolaije (Claudia); M. Priemel (Matthias); J.H. Waarsing (Jan); J.S. Day (Judd); R.M.C. Brandt (Renata); A.F. Schilling (Arndt); S.M. Botter (Sander); H.H. Weinans (Harrie); G.T.J. van der Horst (Gijsbertus); J.H.J. Hoeijmakers (Jan); J.P.T.M. van Leeuwen (Hans)

    2012-01-01

    textabstractTrichothiodystrophy (TTD) is a rare, autosomal recessive nucleotide excision repair (NER) disorder caused by mutations in components of the dual functional NER/basal transcription factor TFIIH. TTD mice, carrying a patient-based point mutation in the Xpd gene, strikingly resemble many

  14. Mechanisms of skeletal muscle injury and repair revealed by gene expression studies in mouse models.

    Science.gov (United States)

    Warren, Gordon L; Summan, Mukesh; Gao, Xin; Chapman, Rebecca; Hulderman, Tracy; Simeonova, Petia P

    2007-07-15

    Common acute injuries to skeletal muscle can lead to significant pain and disability. The current therapeutic approaches for treating muscle injuries are dependent on the clinical severity but not on the type of injury. In the present studies, the pathophysiology and molecular pathways associated with two different types of skeletal muscle injury, one induced by direct destruction of muscle tissue (i.e. FI) and the other induced by a contractile overload (more specifically high-force eccentric contractions, i.e. CI) were compared side by side. Histopathological evaluation and measurements of muscle strength were accompanied by analyses of expression for 12 488 known genes at four time points ranging from 6 h to 7 days after injury. Real-time RT-PCR was used to confirm some of the injury type differences in the temporal profiles of gene expression. Our data revealed several pools of genes, including early induction of transcription, myogenic and stress-responsive factors, common for both types of injury as well as pools of genes expressed specifically with one of the injury types. Only CI activated a set of genes associated with the repair of impaired proteins and structures including genes related to apoptosis, whereas FI uniquely activated gene sets involved in extensive inflammatory responses, tissue remodelling, angiogenesis and myofibre/extracellular matrix synthesis. In conclusion, knowledge of the sets of genes associated specifically with the nature of the injury may have application for development of new strategies for acceleration of the recovery process in injured skeletal muscle.

  15. Pms2 and uracil-DNA glycosylases act jointly in the mismatch repair pathway to generate Ig gene mutations at A-T base pairs.

    Science.gov (United States)

    Girelli Zubani, Giulia; Zivojnovic, Marija; De Smet, Annie; Albagli-Curiel, Olivier; Huetz, François; Weill, Jean-Claude; Reynaud, Claude-Agnès; Storck, Sébastien

    2017-04-03

    During somatic hypermutation (SHM) of immunoglobulin genes, uracils introduced by activation-induced cytidine deaminase are processed by uracil-DNA glycosylase (UNG) and mismatch repair (MMR) pathways to generate mutations at G-C and A-T base pairs, respectively. Paradoxically, the MMR-nicking complex Pms2/Mlh1 is apparently dispensable for A-T mutagenesis. Thus, how detection of U:G mismatches is translated into the single-strand nick required for error-prone synthesis is an open question. One model proposed that UNG could cooperate with MMR by excising a second uracil in the vicinity of the U:G mismatch, but it failed to explain the low impact of UNG inactivation on A-T mutagenesis. In this study, we show that uracils generated in the G1 phase in B cells can generate equal proportions of A-T and G-C mutations, which suggests that UNG and MMR can operate within the same time frame during SHM. Furthermore, we show that Ung-/-Pms2-/- mice display a 50% reduction in mutations at A-T base pairs and that most remaining mutations at A-T bases depend on two additional uracil glycosylases, thymine-DNA glycosylase and SMUG1. These results demonstrate that Pms2/Mlh1 and multiple uracil glycosylases act jointly, each one with a distinct strand bias, to enlarge the immunoglobulin gene mutation spectrum from G-C to A-T bases. © 2017 Girelli Zubani et al.

  16. Altered Gene Expressions and Cytogenetic Repair Efficiency in Cells with Suppressed Expression of XPA after Proton Exposure

    Science.gov (United States)

    Zhang, Ye; Rohde, Larry H.; Gridley, Daila S.; Mehta, Satish K.; Pierson, Duane L.; Wu, Honglu

    2009-01-01

    Cellular responses to damages from ionizing radiation (IR) exposure are influenced not only by the genes involved in DNA double strand break (DSB) repair, but also by non- DSB repair genes. We demonstrated previously that suppressed expression of several non-DSB repair genes, such as XPA, elevated IR-induced cytogenetic damages. In the present study, we exposed human fibroblasts that were treated with control or XPA targeting siRNA to 250 MeV protons (0 to 4 Gy), and analyzed chromosome aberrations and expressions of genes involved in DNA repair. As expected, after proton irradiation, cells with suppressed expression of XPA showed a significantly elevated frequency of chromosome aberrations compared with control siRNA treated (CS) cells. Protons caused more severe DNA damages in XPA knock-down cells, as 36% cells contained multiple aberrations compared to 25% in CS cells after 4Gy proton irradiation. Comparison of gene expressions using the real-time PCR array technique revealed that expressions of p53 and its regulated genes in irradiated XPA suppressed cells were altered similarly as in CS cells, suggesting that the impairment of IR induced DNA repair in XPA suppressed cells is p53-independent. Except for XPA, which was more than 2 fold down regulated in XPA suppressed cells, several other DNA damage sensing and repair genes (GTSE1, RBBP8, RAD51, UNG and XRCC2) were shown a more than 1.5 fold difference between XPA knock-down cells and CS cells after proton exposure. The possible involvement of these genes in the impairment of DNA repair in XPA suppressed cells will be further investigated.

  17. RAD26, the functional S. cerevisiae homolog of the Cockayne syndrome B gene ERCC6.

    NARCIS (Netherlands)

    A.J. van Gool (Alain); R. Verhage; S.M.A. Swagemakers (Sigrid); P. van de Putte (Pieter); J. Brouwer (Jaap); C. Troelstra (Christine); D. Bootsma (Dirk); J.H.J. Hoeijmakers (Jan)

    1994-01-01

    textabstractTranscription-coupled repair (TCR) is a universal sub-pathway of the nucleotide excision repair (NER) system that is limited to the transcribed strand of active structural genes. It accomplishes the preferential elimination of transcription-blocking DNA lesions and permits rapid

  18. Whole transcriptome analysis reveals an 8-oxoguanine DNA glycosylase-1-driven DNA repair-dependent gene expression linked to essential biological processes.

    Science.gov (United States)

    Aguilera-Aguirre, Leopoldo; Hosoki, Koa; Bacsi, Attila; Radák, Zsolt; Wood, Thomas G; Widen, Steven G; Sur, Sanjiv; Ameredes, Bill T; Saavedra-Molina, Alfredo; Brasier, Allan R; Ba, Xueqing; Boldogh, Istvan

    2015-04-01

    Reactive oxygen species inflict oxidative modifications on various biological molecules, including DNA. One of the most abundant DNA base lesions, 8-oxo-7,8-dihydroguanine (8-oxoG) is repaired by 8-oxoguanine DNA glycosylase-1 (OGG1) during DNA base excision repair (OGG1-BER). 8-OxoG accumulation in DNA has been associated with various pathological and aging processes, although its role is unclear. The lack of OGG1-BER in Ogg1(-/-) mice resulted in decreased inflammatory responses and increased susceptibility to infections and metabolic disorders. Therefore, we proposed that OGG1 and/or 8-oxoG base may have a role in immune and homeostatic processes. To test our hypothesis, we challenged mouse lungs with OGG1-BER product 8-oxoG base and changes in gene expression were determined by RNA sequencing and data were analyzed by Gene Ontology and statistical tools. RNA-Seq analysis identified 1592 differentially expressed (≥ 3-fold change) transcripts. The upregulated mRNAs were related to biological processes, including homeostatic, immune-system, macrophage activation, regulation of liquid-surface tension, and response to stimulus. These processes were mediated by chemokines, cytokines, gonadotropin-releasing hormone receptor, integrin, and interleukin signaling pathways. Taken together, these findings point to a new paradigm showing that OGG1-BER plays a role in various biological processes that may benefit the host, but when in excess could be implicated in disease and/or aging processes. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. The democratization of gene editing: Insights from site-specific cleavage and double-strand break repair.

    Science.gov (United States)

    Jasin, Maria; Haber, James E

    2016-08-01

    DNA double-strand breaks (DSBs) are dangerous lesions that if not properly repaired can lead to genomic change or cell death. Organisms have developed several pathways and have many factors devoted to repairing DSBs, which broadly occurs by homologous recombination, which relies on an identical or homologous sequence to template repair, or nonhomologous end-joining. Much of our understanding of these repair mechanisms has come from the study of induced DNA cleavage by site-specific endonucleases. In addition to their biological role, these cellular pathways can be co-opted for gene editing to study gene function or for gene therapy or other applications. While the first gene editing experiments were done more than 20 years ago, the recent discovery of RNA-guided endonucleases has simplified approaches developed over the years to make gene editing an approach that is available to the entire biomedical research community. Here, we review DSB repair mechanisms and site-specific cleavage systems that have provided insight into these mechanisms and led to the current gene editing revolution. Copyright © 2016. Published by Elsevier B.V.

  20. The Democratization of Gene Editing: Insights from site-specific cleavage and double-strand break repair

    Science.gov (United States)

    Jasin, Maria; Haber, James E.

    2017-01-01

    DNA double-strand breaks (DSBs) are dangerous lesions that if not properly repaired can lead to genomic change or cell death. Organisms have developed several pathways and have many factors devoted to repairing DSBs, which broadly occur by homologous recombination that relies on an identical or homologous sequence to template repair, or nonhomologous end-joining. Much of our understanding of these repair mechanisms has come from the study of induced DNA cleavage by site-specific endonucleases. In addition to their biological role, these cellular pathways can be co-opted for gene editing to study gene function or for gene therapy or other applications. While the first gene editing experiments were done more than 20 years ago, the recent discovery of RNA-guided endonucleases has simplified approaches developed over the years to make gene editing an approach that is available to the entire biomedical research community. Here, we review DSB repair mechanisms and site-specific cleavage systems that have provided insight into these mechanisms and led to the current gene editing revolution. PMID:27261202

  1. Defective transcription-coupled repair in Cockayne syndrome B mice is associated with skin cancer predisposition.

    Science.gov (United States)

    van der Horst, G T; van Steeg, H; Berg, R J; van Gool, A J; de Wit, J; Weeda, G; Morreau, H; Beems, R B; van Kreijl, C F; de Gruijl, F R; Bootsma, D; Hoeijmakers, J H

    1997-05-02

    A mouse model for the nucleotide excision repair disorder Cockayne syndrome (CS) was generated by mimicking a truncation in the CSB(ERCC6) gene of a CS-B patient. CSB-deficient mice exhibit all of the CS repair characteristics: ultraviolet (UV) sensitivity, inactivation of transcription-coupled repair, unaffected global genome repair, and inability to resume RNA synthesis after UV exposure. Other CS features thought to involve the functioning of basal transcription/repair factor TFIIH, such as growth failure and neurologic dysfunction, are present in mild form. In contrast to the human syndrome, CSB-deficient mice show increased susceptibility to skin cancer. Our results demonstrate that transcription-coupled repair of UV-induced cyclobutane pyrimidine dimers contributes to the prevention of carcinogenesis in mice. Further, they suggest that the lack of cancer predisposition in CS patients is attributable to a global genome repair process that in humans is more effective than in rodents.

  2. Human DNA repair genes possess potential G-quadruplex sequences in their promoters and 5`-untranslated regions.

    Science.gov (United States)

    Fleming, Aaron M; Zhu, Judy; Ding, Yun; Visser, Joshua A; Zhu, Julia; Burrows, Cynthia J

    2018-01-10

    The cellular response to oxidative stress includes transcriptional changes, particularly for genes involved in DNA repair. Recently, our laboratory demonstrated that oxidation of 2`-deoxyguanosine (G) to 8-oxo-7,8-dihydro-2`-deoxyguanosine (OG) in G-rich potential G-quadruplex sequences (PQSs) in gene promoters impacts the level of gene expression up or down depending on the position of the PQS in the promoter. In the present report, bioinformatic analysis found that the 390 human DNA repair genes in the genome ontology initiative harbor 2,936 PQSs in their promoters and 5`-untranslated regions (5`-UTRs). The average density of PQSs in human DNA repair genes was found to be nearly twofold greater than the average density of PQSs in all coding and non-coding human genes (7.5 vs. 4.3 per gene). The distribution of the PQSs in the DNA repair genes on the non-transcribed (coding) vs. transcribed strands reflects that of PQSs in all human genes. Next, literature data were interrogated to select 30 PQSs to catalog their ability to adopt G-quadruplex (G4) folds in vitro using five different experimental tests. The G4 characterization experiments concluded that 26 of the 30 sequences could adopt G4 topologies in solution. Last, four PQSs were synthesized into the promoter of a luciferase plasmid and co-transfected with the G4-specific ligands pyridostatin, Phen-DC3, or BRACO-19 in human cells to determine whether the PQSs could adopt G4 folds. The cell studies identified changes in luciferase expression when the G4 ligands were present, and the magnitude of the expression changes dependent on the PQS and the coding vs. template strand on which the sequence resided. Our studies demonstrate PQSs exist at a high density in human DNA repair gene promoters and a subset of the identified sequences fold in vitro and in vivo.

  3. Cloning and characterization of the Drosophila homolog of the xeroderma pigmentosum complementation group B correcting gene, ERCC3.

    NARCIS (Netherlands)

    M.H.M. Koken (Marcel); C. Vreeken; S.A.M. Bol (Sandra); N.C. Cheng (Ngan Ching); I. Jaspers-Dekker (Iris); J.H.J. Hoeijmakers (Jan); J.C.J. Eeken; G. Weeda (Geert); A. Pastink (Albert)

    1992-01-01

    textabstractPreviously the human nucleotide excision repair gene ERCC3 was shown to be responsible for a rare combination of the autosomal recessive DNA repair disorders xeroderma pigmentosum (complementation group B) and Cockayne's syndrome (complementation group C). The human and mouse ERCC3

  4. Haplotype analyses of DNA repair gene polymorphisms and their role in ulcerative colitis.

    Directory of Open Access Journals (Sweden)

    Avinash Bardia

    Full Text Available Ulcerative colitis (UC is a major clinical form of inflammatory bowel disease. UC is characterized by mucosal inflammation limited to the colon, always involving the rectum and a variable extent of the more proximal colon in a continuous manner. Genetic variations in DNA repair genes may influence the extent of repair functions, DNA damage, and thus the manifestations of UC. This study thus evaluated the role of polymorphisms of the genes involved in DNA repair mechanisms. A total of 171 patients and 213 controls were included. Genotyping was carried out by ARMS PCR and PCR-RFLP analyses for RAD51, XRCC3 and hMSH2 gene polymorphisms. Allelic and genotypic frequencies were computed in both control & patient groups and data was analyzed using appropriate statistical tests. The frequency of 'A' allele of hMSH2 in the UC group caused statistically significant increased risk for UC compared to controls (OR 1.64, 95% CI 1.16-2.31, p = 0.004. Similarly, the CT genotype of XRCC3 gene was predominant in the UC group and increased the risk for UC by 1.75 fold compared to controls (OR 1.75, 95% CI 1.15-2.67, p = 0.03, further confirming the risk of 'T' allele in UC. The GC genotype frequency of RAD51 gene was significantly increased (p = 0.02 in the UC group (50.3% compared to controls (38%. The GC genotype significantly increased the risk for UC compared to GG genotype by 1.73 fold (OR 1.73, 95% CI 1.14-2.62, p = 0.02 confirming the strong association of 'C' allele with UC. Among the controls, the SNP loci combination of hMSH2:XRCC3 were in perfect linkage. The GTC and ACC haplotypes were found to be predominant in UC than controls with a 2.28 and 2.93 fold significant increase risk of UC.

  5. Role of APC and DNA mismatch repair genes in the development of colorectal cancers

    Directory of Open Access Journals (Sweden)

    Roy Deodutta

    2003-12-01

    Full Text Available Abstract Colorectal cancer is the third most common cause of cancer-related death in both men and women in the western hemisphere. According to the American Cancer Society, an estimated 105,500 new cases of colon cancer with 57,100 deaths will occur in the U.S. in 2003, accounting for about 10% of cancer deaths. Among the colon cancer patients, hereditary risk contributes approximately 20%. The main inherited colorectal cancers are the familial adenomatous polyposis (FAP and the hereditary nonpolyposis colorectal cancers (HNPCC. The FAP and HNPCC are caused due to mutations in the adenomatous polyposis coli (APC and DNA mismatch repair (MMR genes. The focus of this review is to summarize the functions of APC and MMR gene products in the development of colorectal cancers.

  6. DNA Repair Variants, Indoor Tanning and Risk of Melanoma

    Science.gov (United States)

    Torres, Salina M.; Luo, Li; Lilyquist, Jenna; Stidley, Christine A; Flores, Kristina; White, Kirsten A. M.; Erdei, Esther; Gonzales, Melissa; Paine, Susan; Vogel, Rachel Isaksson; Lazovich, DeAnn; Berwick, Marianne

    2013-01-01

    Summary Although ultraviolet radiation (UV) exposure from indoor tanning has been linked to an increased risk of melanoma, the role of DNA repair genes in this process is unknown. We evaluated the association of 92 single nucleotide polymorphisms (SNPs) in 20 DNA repair genes with the risk of melanoma and indoor tanning among 929 melanoma patients and 817 controls from the Minnesota Skin Health Study. Significant associations with melanoma risk were identified for SNPs in ERCC4, ERCC6, RFC1, XPC, MGMT, and FBRSL1 genes; with a cut-off of p<0.05. ERCC6 and FBRSL1 gene variants and haplotypes interacted with indoor tanning. However, none of the 92 SNPs tested met the correction criteria for multiple comparisons. This study, based on an a priori interest in investigating the role of DNA repair capacity using variants in base excision and nucleotide excision repair, identified several genes that may play a role in resolving UV-induced DNA damage. PMID:23659246

  7. BRD4 Promotes DNA Repair and Mediates the Formation of TMPRSS2-ERG Gene Rearrangements in Prostate Cancer

    Directory of Open Access Journals (Sweden)

    Xiangyi Li

    2018-01-01

    Full Text Available BRD4 belongs to the bromodomain and extraterminal (BET family of chromatin reader proteins that bind acetylated histones and regulate gene expression. Pharmacological inhibition of BRD4 by BET inhibitors (BETi has indicated antitumor activity against multiple cancer types. We show that BRD4 is essential for the repair of DNA double-strand breaks (DSBs and mediates the formation of oncogenic gene rearrangements by engaging the non-homologous end joining (NHEJ pathway. Mechanistically, genome-wide DNA breaks are associated with enhanced acetylation of histone H4, leading to BRD4 recruitment, and stable establishment of the DNA repair complex. In support of this, we also show that, in clinical tumor samples, BRD4 protein levels are negatively associated with outcome after prostate cancer (PCa radiation therapy. Thus, in addition to regulating gene expression, BRD4 is also a central player in the repair of DNA DSBs, with significant implications for cancer therapy.

  8. Tax Avoidance in Excise Tax

    OpenAIRE

    VARGOVÁ, Monika

    2007-01-01

    The taxes from consumption are an excise taxes and value adds tax. I will focus on an excise tax. The excise tax is one of the most important income to state budget. The excise taxes belong between indirect taxes and is divided into five selected products, such as cigarettes, bear, vine, alcohol and mineral oils.

  9. Toll pathway is required for wound-induced expression of barrier repair genes in the Drosophila epidermis

    Science.gov (United States)

    Capilla, Amalia; Karachentsev, Dmitry; Patterson, Rachel A.; Hermann, Anita; Juarez, Michelle T.; McGinnis, William

    2017-01-01

    The epidermis serves as a protective barrier in animals. After epidermal injury, barrier repair requires activation of many wound response genes in epidermal cells surrounding wound sites. Two such genes in Drosophila encode the enzymes dopa decarboxylase (Ddc) and tyrosine hydroxylase (ple). In this paper we explore the involvement of the Toll/NF-κB pathway in the localized activation of wound repair genes around epidermal breaks. Robust activation of wound-induced transcription from ple and Ddc requires Toll pathway components ranging from the extracellular ligand Spätzle to the Dif transcription factor. Epistasis experiments indicate a requirement for Spätzle ligand downstream of hydrogen peroxide and protease function, both of which are known activators of wound-induced transcription. The localized activation of Toll a few cell diameters from wound edges is reminiscent of local activation of Toll in early embryonic ventral hypoderm, consistent with the hypothesis that the dorsal–ventral patterning function of Toll arose from the evolutionary cooption of a morphogen-responsive function in wound repair. Furthermore, the combinatorial activity of Toll and other signaling pathways in activating epidermal barrier repair genes can help explain why developmental activation of the Toll, ERK, or JNK pathways alone fail to activate wound repair loci. PMID:28289197

  10. Cystic fibrosis gene repair: correction of ΔF508 using ZFN and CRISPR/Cas9 guide RNA gene editing tools

    OpenAIRE

    Hollywood, Jennifer

    2013-01-01

    Cystic Fibrosis (CF) is an autosomal recessive monogenic disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene with the ΔF508 mutation accounting for approximately 70% of all CF cases worldwide. This thesis investigates whether existing zinc finger nucleases designed in this lab and CRISPR/gRNAs designed in this thesis can mediate efficient homology-directed repair (HDR) with appropriate donor repair plasmids to correct CF-causing mutations in a C...

  11. Two human homologs of Rad23 are functionally interchangeable in complex formation and stimulation of XPC repair activity.

    NARCIS (Netherlands)

    K. Sugasawa (Kaoru); J.M.Y. Ng (Jessica); C. Masutani (Chikahide); T. Maekawa; A. Uchida; P.J. van der Spek (Peter); A.P.M. Eker (André); S. Rademakers (Suzanne); C.E. Visser (Cécile); A. Aboussekhra; R.D. Wood (Richard); F. Hanaoka (Fumio); D. Bootsma (Dirk); J.H.J. Hoeijmakers (Jan)

    1997-01-01

    textabstractXPC-hHR23B protein complex is specifically involved in nucleotide excision repair (NER) of DNA lesions on transcriptionally inactive sequences as well as the nontranscribed strand of active genes. Here we demonstrate that not only highly purified recombinant hHR23B (rhHR23B) but also a

  12. Horizontal gene transfer regulation in bacteria as a "spandrel" of DNA repair mechanisms.

    Directory of Open Access Journals (Sweden)

    Saliou Fall

    Full Text Available Horizontal gene transfer (HGT is recognized as the major force for bacterial genome evolution. Yet, numerous questions remain about the transferred genes, their function, quantity and frequency. The extent to which genetic transformation by exogenous DNA has occurred over evolutionary time was initially addressed by an in silico approach using the complete genome sequence of the Ralstonia solanacearum GMI1000 strain. Methods based on phylogenetic reconstruction of prokaryote homologous genes families detected 151 genes (13.3% of foreign origin in the R. solanacearum genome and tentatively identified their bacterial origin. These putative transfers were analyzed in comparison to experimental transformation tests involving 18 different genomic DNA positions in the genome as sites for homologous or homeologous recombination. Significant transformation frequency differences were observed among these positions tested regardless of the overall genomic divergence of the R. solanacearum strains tested as recipients. The genomic positions containing the putative exogenous DNA were not systematically transformed at the highest frequencies. The two genomic "hot spots", which contain recA and mutS genes, exhibited transformation frequencies from 2 to more than 4 orders of magnitude higher than positions associated with other genes depending on the recipient strain. These results support the notion that the bacterial cell is equipped with active mechanisms to modulate acquisition of new DNA in different genomic positions. Bio-informatics study correlated recombination "hot-spots" to the presence of Chi-like signature sequences with which recombination might be preferentially initiated. The fundamental role of HGT is certainly not limited to the critical impact that the very rare foreign genes acquired mainly by chance can have on the bacterial adaptation potential. The frequency to which HGT with homologous and homeologous DNA happens in the environment

  13. DNA repair activity in fish and interest in ecotoxicology: a review.

    Science.gov (United States)

    Kienzler, Aude; Bony, Sylvie; Devaux, Alain

    2013-06-15

    The knowledge of DNA repair in a target species is of first importance as it is the primary line of defense against genotoxicants, and a better knowledge of DNA repair capacity in fish could help to interpret genotoxicity data and/or assist in the choice of target species, developmental stage and tissues to focus on, both for environmental biomonitoring studies and DNA repair testing. This review focuses in a first part on what is presently known on a mechanistic basis, about the various DNA repair systems in fish, in vivo and in established cell lines. Data on base excision repair (BER), direct reversal with O⁶-alkylguanine transferase and double strand breaks repair, although rather scarce, are being reviewed, as well as nucleotide excision repair (NER) and photoreactivation repair (PER), which are by far the most studied repair mechanisms in fish. Most of these repair mechanisms seem to be strongly species and tissue dependent; they also depend on the developmental stage of the organisms. BER is efficient in vivo, although no data has been found on in vitro models. NER activity is quite low or even inexistent depending on the studies; however this lack is partly compensated by a strong PER activity, especially in early developmental stage. In a second part, a survey of the ecotoxicological studies integrating DNA repair as a parameter responding to single or mixture of contaminant is realized. Three main approaches are being used: the measurement of DNA repair gene expression after exposure, although it has not yet been clearly established whether gene expression is indicative of repair capacity; the monitoring of DNA damage removal by following DNA repair kinetics; and the modulation of DNA repair activity following exposure in situ, in order to assess the impact of exposure history on DNA repair capacity. Since all DNA repair processes are possible targets for environmental pollutants, we can also wonder at which extent such a modulation of repair capacities

  14. Enzymatic repair of selected cross-linked homoduplex molecules enhances nuclear gene rescue from Pompeii and Herculaneum remains.

    Science.gov (United States)

    Di Bernardo, Giovanni; Del Gaudio, Stefania; Cammarota, Marcella; Galderisi, Umberto; Cascino, Antonino; Cipollaro, Marilena

    2002-02-15

    Ancient DNA (aDNA) samples extracted from the bone remains of six equids buried by the Vesuvius eruption in 79 AD were investigated to test pre-amplification and enzymatic repair procedures designed to enhance the rescue of nuclear genes. The extracts, which proved all positive for Equidae mtDNA amplification, proved positive only four times out of 18 when tested for single-copy Equidae nuclear genes (epsilon globin, p53 and gamma interferon). Pre-amplification did not change the number of retrieved aDNA sequences but 10 times out of 14 enzymatic repair restored the amplifiability of the genes analysed, proving that repair increases the rate of successful rescue from 22 to alpha(lambda)mu(omicron)sigma(tau) 80%. These findings support the hypothesis that some of these cross-linked aDNA molecules, which are not completely separated when DNA is extracted under denaturing conditions, become homoduplex substrates for Pol I and/or T4 ligase action upon renaturation. aDNA authenticity is proved by the homology of the nucleotide sequences of loci tested to the corresponding modern Equidae sequences. Data also indicate that cross-linked homoduplex molecules selected by denaturation of the extract are repaired without any chimera formation. The general features of aDNA amplification with and without denaturation and enzymatic repair are discussed.

  15. Transcription pattern of p53-targeted DNA repair genes in the hypoxia-tolerant subterranean mole rat Spalax.

    Science.gov (United States)

    Shams, Imad; Malik, Assaf; Manov, Irena; Joel, Alma; Band, Mark; Avivi, Aaron

    2013-04-12

    The tumor suppressor gene p53 induces growth arrest and/or apoptosis in response to DNA damage/hypoxia. Inactivation of p53 confers a selective advantage to tumor cells under a hypoxic microenvironment during tumor progression. The subterranean blind mole rat, Spalax, spends its life underground at low-oxygen tensions, hence developing a wide range of respiratory/molecular adaptations to hypoxic stress, including critical changes in p53 structure and signaling pathway. The highly conserved p53 Arg(R)-172 is substituted by lysine (K) in Spalax, identical with a tumor-associated mutation. Functionality assays revealed that Spalax p53 is unable to activate apoptotic target genes but is still capable of activating cell cycle arrest genes. Furthermore, we have shown that the transcription patterns of representative p53-induced genes (Apaf1 and Mdm2) in Spalax are influenced by hypoxia. Cell cycle arrest allows the cells to repair DNA damage via different DNA repair genes. We tested the transcription pattern of three p53-related DNA repair genes (p53R2, Mlh1, and Msh2) under normoxia and short-acute hypoxia in Spalax, C57BL/6 wild-type mice, and two strains of mutant C57BL/6 mice, each carrying a different mutation at the R172 position. Our results show that while wild-type/mutant mice exhibit strong hypoxia-induced reductions of repair gene transcript levels, no such inhibition is found in Spalax under hypoxia. Moreover, unlike mouse p53R2, Spalax p53R2 transcript levels are strongly elevated under hypoxia. These results suggest that critical repair functions, which are known to be inhibited under hypoxia in mice, remain active in Spalax, as part of its unique hypoxia tolerance mechanisms. Copyright © 2013 Elsevier Ltd. All rights reserved.

  16. Clustered Mutation Signatures Reveal that Error-Prone DNA Repair Targets Mutations to Active Genes.

    Science.gov (United States)

    Supek, Fran; Lehner, Ben

    2017-07-27

    Many processes can cause the same nucleotide change in a genome, making the identification of the mechanisms causing mutations a difficult challenge. Here, we show that clustered mutations provide a more precise fingerprint of mutagenic processes. Of nine clustered mutation signatures identified from >1,000 tumor genomes, three relate to variable APOBEC activity and three are associated with tobacco smoking. An additional signature matches the spectrum of translesion DNA polymerase eta (POLH). In lymphoid cells, these mutations target promoters, consistent with AID-initiated somatic hypermutation. In solid tumors, however, they are associated with UV exposure and alcohol consumption and target the H3K36me3 chromatin of active genes in a mismatch repair (MMR)-dependent manner. These regions normally have a low mutation rate because error-free MMR also targets H3K36me3 chromatin. Carcinogens and error-prone repair therefore redistribute mutations to the more important regions of the genome, contributing a substantial mutation load in many tumors, including driver mutations. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. A Cross-Cancer Genetic Association Analysis of the DNA Repair and DNA Damage Signaling Pathways for Lung, Ovary, Prostate, Breast, and Colorectal Cancer.

    Science.gov (United States)

    Scarbrough, Peter M; Weber, Rachel Palmieri; Iversen, Edwin S; Brhane, Yonathan; Amos, Christopher I; Kraft, Peter; Hung, Rayjean J; Sellers, Thomas A; Witte, John S; Pharoah, Paul; Henderson, Brian E; Gruber, Stephen B; Hunter, David J; Garber, Judy E; Joshi, Amit D; McDonnell, Kevin; Easton, Doug F; Eeles, Ros; Kote-Jarai, Zsofia; Muir, Kenneth; Doherty, Jennifer A; Schildkraut, Joellen M

    2016-01-01

    DNA damage is an established mediator of carcinogenesis, although genome-wide association studies (GWAS) have identified few significant loci. This cross-cancer site, pooled analysis was performed to increase the power to detect common variants of DNA repair genes associated with cancer susceptibility. We conducted a cross-cancer analysis of 60,297 single nucleotide polymorphisms, at 229 DNA repair gene regions, using data from the NCI Genetic Associations and Mechanisms in Oncology (GAME-ON) Network. Our analysis included data from 32 GWAS and 48,734 controls and 51,537 cases across five cancer sites (breast, colon, lung, ovary, and prostate). Because of the unavailability of individual data, data were analyzed at the aggregate level. Meta-analysis was performed using the Association analysis for SubSETs (ASSET) software. To test for genetic associations that might escape individual variant testing due to small effect sizes, pathway analysis of eight DNA repair pathways was performed using hierarchical modeling. We identified three susceptibility DNA repair genes, RAD51B (P associations with cancer risk in the base excision repair, nucleotide excision repair, mismatch repair, and homologous recombination pathways. Only three susceptibility loci were identified, which had all been previously reported. In contrast, hierarchical modeling identified several pleiotropic cancer risk associations in key DNA repair pathways. Results suggest that many common variants in DNA repair genes are likely associated with cancer susceptibility through small effect sizes that do not meet stringent significance testing criteria. ©2015 American Association for Cancer Research.

  18. ABCB5 is a limbal stem cell gene required for corneal development and repair

    Science.gov (United States)

    Ksander, Bruce R.; Kolovou, Paraskevi E.; Wilson, Brian J.; Saab, Karim R.; Guo, Qin; Ma, Jie; McGuire, Sean P.; Gregory, Meredith S.; Vincent, William J. B.; Perez, Victor L.; Cruz-Guilloty, Fernando; Kao, Winston W. Y.; Call, Mindy K.; Tucker, Budd A.; Zhan, Qian; Murphy, George F.; Lathrop, Kira L.; Alt, Clemens; Mortensen, Luke J.; Lin, Charles P.; Zieske, James D.; Frank, Markus H.; Frank, Natasha Y.

    2014-01-01

    Corneal epithelial homeostasis and regeneration are sustained by limbal stem cells (LSCs)1–3, and LSC deficiency is a major cause of blindness worldwide4. Transplantation is often the only therapeutic option available to patients with LSC deficiency. However, while transplant success depends foremost on LSC frequency within grafts5, a gene allowing for prospective LSC enrichment has not been identified so far5. Here we show that ATP-binding cassette, sub-family B, member 5 (ABCB5)6,7 marks LSCs and is required for LSC maintenance, corneal development and repair. Furthermore, we demonstrate that prospectively isolated human or murine ABCB5-positive LSCs possess the exclusive capacity to fully restore the cornea upon grafting to LSC-deficient mice in xenogeneic or syngeneic transplantation models. ABCB5 is preferentially expressed on label-retaining LSCs2 in mice and p63α-positive LSCs8 in humans. Consistent with these findings, ABCB5-positive LSC frequency is reduced in LSC-deficient patients. Abcb5 loss of function in Abcb5 knockout mice causes depletion of quiescent LSCs due to enhanced proliferation and apoptosis, and results in defective corneal differentiation and wound healing. Our results from gene knockout studies, LSC tracing and transplantation models, as well as phenotypic and functional analyses of human biopsy specimens, provide converging lines of evidence that ABCB5 identifies mammalian LSCs. Identification and prospective isolation of molecularly defined LSCs with essential functions in corneal development and repair has important implications for the treatment of corneal disease, particularly corneal blindness due to LSC deficiency. PMID:25030174

  19. [Lentiviral vector-mediated RNA interfere gene Nogo receptor to repair spinal cord injury].

    Science.gov (United States)

    Lü, Bi-tao; Yuan, Wen; Xu, Sheng-ming

    2010-10-15

    To evaluate the effects of lentiviral vector-mediated RNA interfere gene Nogo receptor (NgR) of rat cortical neurons in repairing spinal cord injury. The recombinant-lentiviral vector with small inferring RNA siNgR199 which had been constructed was transfected into rat cortical neuron cells in vitro in 3 multiplicity of infection (MOI). The infection rate was determined with fluorescent microscope, and the target gene was detected by PCR analysis. Then, the recombinant was injected into the cortical motor area of the rats with severe spinal cord injury, and the saline was also injected into other rats with severe spinal cord injury as a match control. The functional recovery of the rats' hindlimb was assessed using BBB score and the nerve fiber of the injured region was observed by nerve tracing. The rate of recombinant infecting rat cortical neuron in vitro exceeded 99%. PCR analysis confirmed that the effect of lentiviral vector-mediated RNA interfering gene NgR of rat cortical neurons in vitro was 61%. Although all rats with spinal cord injury were observed to have the hindlimb functional recovery, these rats injected with recombinant had better hindlimb functional recovery than others showing by more BBB score (P vector with siNgR199 which had been constructed is able to promote the growth of nerve fiber and the functional recovery of the rats' hindlimb.

  20. Allelic variants of XRCC1 and XRCC3 repair genes and susceptibility of oral cancer in Brazilian patients

    DEFF Research Database (Denmark)

    Dos Reis, Mariana Bisarro; Losi-Guembarovski, Roberta; de Souza Fonseca Ribeiro, Enilze Maria

    2013-01-01

    genes have been found to be associated with oral cancer. The aim of this study was to investigate the relationship between the presence of allelic variants Arg194Trp (rs:1799782) and Arg399Gln (rs: 25487) of XRCC1 gene and Thr241Met (rs: 861539) of XRCC3 gene and susceptibility to oral cancer. We also...... variants of the XRCC1 gene within codon 194 (OR 0.82, 95% CI: 0.44-1.51) and codon 399 (OR 0.94, 95% CI: 0.59-1.50) and within the XRCC3 gene (OR 0.72; 95% CI: 0.45-1.16) were not associated with an increased risk of oral cancer. A combinational analysis of SNPs in both genes indicated no association......BACKGROUND: The capacity for DNA repair is essential in maintaining cellular functions and homeostasis; however, this capacity can be altered based on DNA sequence variations in DNA repair genes, which may contribute to the onset of cancer. Many single-nucleotide polymorphisms (SNPs) in repair...

  1. Biallelic germline mutations of mismatch-repair genes: a possible cause for multiple pediatric malignancies.

    Science.gov (United States)

    Poley, Jan-Werner; Wagner, Anja; Hoogmans, Monique M C P; Menko, Fred H; Tops, Carli; Kros, Johan M; Reddingius, Roel E; Meijers-Heijboer, Hanne; Kuipers, Ernst J; Dinjens, Winand N M

    2007-06-01

    Heterozygous defects in mismatch-repair (MMR) genes cause hereditary nonpolyposis colorectal cancer (HNPCC). In this syndrome, tumors typically arise from age 25 years onward. Case reports have shown that homozygosity or compound heterozygosity for MMR gene mutations can cause multiple tumors in childhood, sometimes combined with neurofibromatosis type I (NF1)-like features. Therefore, the authors studied the role of homozygosity or compound heterozygosity (CZ) for MMR gene defects in children with multiple primary tumors. A database that contained all pediatric oncology patients who were seen between 1982 and 2003 at the author's institution was queried to identify patients aged Saccharomyces cerevisiae 2 [PMS2] expression) and a Wilms tumor (high MSI; no MLH1 or PMS2 expression). Apart from >6 cafe-au-lait spots, he had no other signs of NF1. The patient had CZ identified for a pathogenic MLH1 mutation (593delAG frameshift) and an unclassified MLH1 variant (Met35Asn). There was strong evidence that this unclassified variant was a pathogenic mutation. The second patient was diagnosed with a non-Hodgkin lymphoma (no tissue available) and an anaplastic oligodendroglioma (low MSI; no MSH6 expression) at age 4 years and 6 years, respectively. His brother had died of a medulloblastoma at age 6 years (low MSI, no MSH6 expression). Both boys had cafe-au-lait spots. Further genetic testing was not possible. Carriage of biallelic MMR gene defects can be associated with multiple malignancies in childhood that may differ from the standard spectrum of HNPCC tumor types. In 15 pediatric patients with multiple malignancies, the authors identified 1 clear case and 1 possible case of biallelic MMR gene defect. Recognition of the inherited nature of the tumors in these patients is important for counseling these patients and their families. (c) 2007 American Cancer Society.

  2. Genetic variations in the homologous recombination repair pathway genes modify risk of glioma.

    Science.gov (United States)

    Zhang, Haishi; Liu, Yanhong; Zhou, Keke; Zhou, Chengcheng; Zhou, Renke; Cheng, Chunxia; Wei, Qingyi; Lu, Daru; Zhou, Liangfu

    2016-01-01

    Accumulative epidemiological evidence suggests that single nucleotide polymorphisms (SNPs) in genes involved in homologous recombination (HR) DNA repair pathway play an important role in glioma susceptibility. However, the effects of such SNPs on glioma risk remain unclear. We used a used a candidate pathway-based approach to elucidate the relationship between glioma risk and 12 putative functional SNPs in genes involved in the HR pathway. Genotyping was conducted on 771 histologically-confirmed glioma patients and 752 cancer-free controls from the Chinese Han population. Odds ratios (OR) were calculated both for each SNP individually and for grouped analyses, examining the effects of the numbers of adverse alleles on glioma risk, and evaluated their potential gene-gene interactions using the multifactor dimensionality reduction (MDR). In the single-locus analysis, two variants, the NBS1 rs1805794 (OR 1.42, 95% CI 1.15-1.76, P = 0.001), and RAD54L rs1048771 (OR 1.61, 95% CI 1.17-2.22, P = 0.002) were significantly associated with glioma risk. When we examined the joint effects of the risk-conferring alleles of these three SNPs, we found a significant trend indicating that the risk increases as the number of adverse alleles increase (P = 0.005). Moreover, the MDR analysis suggested a significant three-locus interaction model involving NBS1 rs1805794, MRE11 rs10831234, and ATM rs227062. These results suggested that these variants of the genes involved in the HR pathway may contribute to glioma susceptibility.

  3. Sequence conservation of the rad21 Schizosaccharomyces pombe DNA double-strand break repair gene in human and mouse.

    NARCIS (Netherlands)

    M.J. McKay (Michael); C. Troelstra (Christine); P.J. van der Spek (Peter); R. Kanaar (Roland); B. Smit (Bep); A. Hagemeijer (Anne); D. Bootsma (Dirk); J.H.J. Hoeijmakers (Jan)

    1996-01-01

    textabstractThe rad21 gene of Schizosaccharomyces pombe is involved in the repair of ionizing radiation-induced DNA double-strand breaks. The isolation of mouse and human putative homologs of rad21 is reported here. Alignment of the predicted amino acid sequence of Rad21 with the mammalian proteins

  4. Pharmacogenetic Study in Rectal Cancer Patients Treated With Preoperative Chemoradiotherapy: Polymorphisms in Thymidylate Synthase, Epidermal Growth Factor Receptor, GSTP1, and DNA Repair Genes

    Energy Technology Data Exchange (ETDEWEB)

    Paez, David, E-mail: dpaez@santpau.cat [Department of Medical Oncology, Hospital de la Santa Creu i Sant Pau, Universitat Autonoma de Barcelona, Barcelona (Spain); Salazar, Juliana; Pare, Laia [Centre for Biomedical Network Research on Rare Diseases, Barcelona (Spain); Department of Genetics, Hospital de la Santa Creu i Sant Pau, Universitat Autonoma de Barcelona, Barcelona (Spain); Pertriz, Lourdes [Department of Radiotherapy, Hospital de la Santa Creu i Sant Pau, Universitat Autonoma de Barcelona, Barcelona (Spain); Targarona, Eduardo [Department of Surgery, Hospital de la Santa Creu i Sant Pau, Universitat Autonoma de Barcelona, Barcelona (Spain); Rio, Elisabeth del [Centre for Biomedical Network Research on Rare Diseases, Barcelona (Spain); Department of Genetics, Hospital de la Santa Creu i Sant Pau, Universitat Autonoma de Barcelona, Barcelona (Spain); Barnadas, Agusti; Marcuello, Eugenio [Department of Medical Oncology, Hospital de la Santa Creu i Sant Pau, Universitat Autonoma de Barcelona, Barcelona (Spain); Baiget, Montserrat [Centre for Biomedical Network Research on Rare Diseases, Barcelona (Spain); Department of Genetics, Hospital de la Santa Creu i Sant Pau, Universitat Autonoma de Barcelona, Barcelona (Spain)

    2011-12-01

    Purpose: Several studies have been performed to evaluate the usefulness of neoadjuvant treatment using oxaliplatin and fluoropyrimidines for locally advanced rectal cancer. However, preoperative biomarkers of outcome are lacking. We studied the polymorphisms in thymidylate synthase, epidermal growth factor receptor, glutathione S-transferase pi 1 (GSTP1), and several DNA repair genes to evaluate their usefulness as pharmacogenetic markers in a cohort of 128 rectal cancer patients treated with preoperative chemoradiotherapy. Methods and Materials: Blood samples were obtained from 128 patients with Stage II-III rectal cancer. DNA was extracted from the peripheral blood nucleated cells, and the genotypes were analyzed by polymerase chain reaction amplification and automated sequencing techniques or using a 48.48 dynamic array on the BioMark system. The germline polymorphisms studied were thymidylate synthase, (VNTR/5 Prime UTR, 2R G>C single nucleotide polymorphism [SNP], 3R G>C SNP), epidermal growth factor receptor (Arg497Lys), GSTP1 (Ile105val), excision repair cross-complementing 1 (Asn118Asn, 8092C>A, 19716G>C), X-ray repair cross-complementing group 1 (XRCC1) (Arg194Trp, Arg280His, Arg399Gln), and xeroderma pigmentosum group D (Lys751Gln). The pathologic response, pathologic regression, progression-free survival, and overall survival were evaluated according to each genotype. Results: The Asterisk-Operator 3/ Asterisk-Operator 3 thymidylate synthase genotype was associated with a greater response rate (pathologic complete remission and microfoci residual tumor, 59% in Asterisk-Operator 3/ Asterisk-Operator 3 vs. 35% in Asterisk-Operator 2/ Asterisk-Operator 2 and Asterisk-Operator 2/ Asterisk-Operator 3; p = .013). For the thymidylate synthase genotype, the median progression-free survival was 103 months for the Asterisk-Operator 3/ Asterisk-Operator 3 patients and 84 months for the Asterisk-Operator 2/ Asterisk-Operator 2 and Asterisk-Operator 2/ Asterisk

  5. Pharmacogenetic study in rectal cancer patients treated with preoperative chemoradiotherapy: polymorphisms in thymidylate synthase, epidermal growth factor receptor, GSTP1, and DNA repair genes.

    Science.gov (United States)

    Páez, David; Salazar, Juliana; Paré, Laia; Pertriz, Lourdes; Targarona, Eduardo; del Rio, Elisabeth; Barnadas, Agusti; Marcuello, Eugenio; Baiget, Montserrat

    2011-12-01

    Several studies have been performed to evaluate the usefulness of neoadjuvant treatment using oxaliplatin and fluoropyrimidines for locally advanced rectal cancer. However, preoperative biomarkers of outcome are lacking. We studied the polymorphisms in thymidylate synthase, epidermal growth factor receptor, glutathione S-transferase pi 1 (GSTP1), and several DNA repair genes to evaluate their usefulness as pharmacogenetic markers in a cohort of 128 rectal cancer patients treated with preoperative chemoradiotherapy. Blood samples were obtained from 128 patients with Stage II-III rectal cancer. DNA was extracted from the peripheral blood nucleated cells, and the genotypes were analyzed by polymerase chain reaction amplification and automated sequencing techniques or using a 48.48 dynamic array on the BioMark system. The germline polymorphisms studied were thymidylate synthase, (VNTR/5'UTR, 2R G>C single nucleotide polymorphism [SNP], 3R G>C SNP), epidermal growth factor receptor (Arg497Lys), GSTP1 (Ile105val), excision repair cross-complementing 1 (Asn118Asn, 8092C>A, 19716G>C), X-ray repair cross-complementing group 1 (XRCC1) (Arg194Trp, Arg280His, Arg399Gln), and xeroderma pigmentosum group D (Lys751Gln). The pathologic response, pathologic regression, progression-free survival, and overall survival were evaluated according to each genotype. The ∗3/∗3 thymidylate synthase genotype was associated with a greater response rate (pathologic complete remission and microfoci residual tumor, 59% in ∗3/∗3 vs. 35% in ∗2/∗2 and ∗2/∗3; p=.013). For the thymidylate synthase genotype, the median progression-free survival was 103 months for the ∗3/∗3 patients and 84 months for the ∗2/∗2 and ∗2/∗3 patients (p=.039). For XRCC1 Arg399Gln SNP, the median progression-free survival was 101 months for the G/G, 78 months for the G/A, and 31 months for the A/A patients (p=.048). The thymidylate synthase genotype and XRCC1 Arg399Gln polymorphism might help to

  6. Multidirectional Vector Excision Leads to Better Outcomes than Traditional Elliptical Excision of Facial Congenital Melanocytic Nevus

    Directory of Open Access Journals (Sweden)

    Seung Il Oh

    2013-09-01

    Full Text Available Background The elliptical excision is the standard method of removing benign skin lesions,such as congenital melanocytic nevi. This technique allows for primary closure, with little to nodog-ear deformity, but may sacrifice normal tissue adjacent to the lesion, resulting in scarswhich are unnecessarily long. This study was designed to compare the predicted results ofelliptical excision with those resulting from our excision technique.Methods Eighty-two patients with congenital melanocytic nevus on the face were prospectivelystudied. Each lesion was examined and an optimal ellipse was designed and marked onthe skin. After an incision on one side of the nevus margin, subcutaneous undermining wasperformed in the appropriate direction. The skin flap was pulled up and approximated alongseveral vectors to minimize the occurrence of dog-ear deformity.Results Overall, the final wound length was 21.1% shorter than that achieved by ellipticalexcision. Only 8.5% of the patients required dog-ear repair. There was no significant distortionof critical facial structures. All of the scars were deemed aesthetically acceptable based ontheir Patient and Observer Scar Assessment Scale scores.Conclusions When compared to elliptical excision, our technique appears to minimize dogeardeformity and decrease the final wound length. This technique should be considered analternative method for excision of facial nevi.

  7. Structure of the DNA Repair Helicase XPD

    OpenAIRE

    Liu, Huanting; Rudolf, Jana; Johnson, Kenneth A.; McMahon, Stephen A.; Oke, Muse; Carter, Lester; McRobbie, Anne-Marie; Brown, Sara E.; Naismith, James H.; White, Malcolm F.

    2008-01-01

    The XPD helicase (Rad3 in Saccharomyces cerevisiae) is a component of transcription factor IIH (TFIIH), which functions in transcription initiation and Nucleotide Excision Repair in eukaryotes, catalysing DNA duplex opening localised to the transcription start site or site of DNA damage, respectively. XPD has a 5′ to 3′ polarity and the helicase activity is dependent on an iron-sulfur cluster binding domain, a feature that is conserved in related helicases such as FancJ. The xpd gene is the t...

  8. Spectrum of mismatch repair gene mutations and clinical presentation of Hispanic individuals with Lynch syndrome.

    Science.gov (United States)

    Sunga, Annette Y; Ricker, Charité; Espenschied, Carin R; Castillo, Danielle; Melas, Marilena; Herzog, Josef; Bannon, Sarah; Cruz-Correa, Marcia; Lynch, Patrick; Solomon, Ilana; Gruber, Stephen B; Weitzel, Jeffrey N

    2017-04-01

    Lynch syndrome (LS), the most common hereditary colorectal cancer syndrome, is caused by mismatch repair (MMR) gene mutations. However, data about MMR mutations in Hispanics are limited. This study aims to describe the spectrum of MMR mutations in Hispanics with LS and explore ancestral origins. This case series involved an IRB-approved retrospective chart review of self-identified Hispanic patients (n = 397) seen for genetic cancer risk assessment at four collaborating academic institutions in California, Texas, and Puerto Rico who were evaluated by MMR genotyping and/or tumor analysis. A literature review was conducted for all mutations identified. Of those who underwent clinical genetic testing (n = 176), 71 had MMR gene mutations. Nine mutations were observed more than once. One third (3/9) of recurrent mutations and two additional mutations (seen only once) were previously reported in Spain, confirming the influence of Spanish ancestry on MMR mutations in Hispanic populations. The recurrent mutations identified (n = 9) included both previously reported mutations as well as unique mutations not in the literature. This is the largest report of Hispanic MMR mutations in North America; however, a larger sample and haplotype analyses are needed to better understand recurrent MMR mutations in Hispanic populations. Copyright © 2017. Published by Elsevier Inc.

  9. DNA damage repair and response proteins as targets for cancer therapy.

    Science.gov (United States)

    Lieberman, Howard B

    2008-01-01

    The cellular response to DNA damage is critical for determining whether carcinogenesis, cell death or other deleterious biological effects will ensue. Numerous cellular enzymatic mechanisms can directly repair damaged DNA, or allow tolerance of DNA lesions, and thus reduce potential harmful effects. These processes include base excision repair, nucleotide excision repair, nonhomologous end joining, homologous recombinational repair and mismatch repair, as well as translesion synthesis. Furthermore, DNA damage-inducible cell cycle checkpoint systems transiently delay cell cycle progression. Presumably, this allows extra time for repair before entry of cells into critical phases of the cell cycle, an event that could be lethal if pursued with damaged DNA. When damage is excessive apoptotic cellular suicide mechanisms can be induced. Many of the survival-promoting pathways maintain genomic integrity even in the absence of exogenous agents, thus likely processing spontaneous damage caused by the byproducts of normal cellular metabolism. DNA damage can initiate cancer, and radiological as well as chemical agents used to treat cancer patients often cause DNA damage. Many genes are involved in each of the DNA damage processing mechanisms, and the encoded proteins could ultimately serve as targets for therapy, with the goal of neutralizing their ability to repair damage in cancer cells. Therefore, modulation of DNA damage responses coupled with more conventional radiotherapy and chemotherapy approaches could sensitize cancer cells to treatment. Alteration of DNA damage response genes and proteins should thus be considered an important though as of yet not fully exploited avenue to enhance cancer therapy.

  10. Identification of Rad23-4 gene required for pollen development in ...

    African Journals Online (AJOL)

    雨林木风

    2012-05-31

    May 31, 2012 ... Key words: Nucleotide excision repair (NER), rad23-4 gene, anthocyanin, pollen grains, seeds abortion. INTRODUCTION. Plants are ... Rad23 gene in Arabidopsis were purchased from the ABRC. (Arabidopsis Biological ... exposed to enhanced UV-B for 0-6 h daily for one week. After UV-B treatment, the ...

  11. Xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy: do the genes explain the diseases?

    Science.gov (United States)

    Chu, G; Mayne, L

    1996-05-01

    Xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy are three distinct human syndromes associated with sensitivity to ultraviolet radiation. We review evidence that these syndromes overlap with each other and arise from mutations in genes involved in nucleotide-excision repair and RNA transcription. Attempts have been made to explain the syndromes in terms of defects in repair and transcription. These two biochemical pathways do not easily account for all the features of the syndromes. Therefore, we propose a third pathway, in which the syndromes are due, in part, to defects in a demethylation mechanism involving the excision of methylated cytosine. Perturbation of demethylation could affect the developmentally regulated expression of some genes.

  12. Nucleotide fluctuation of RecA repair gene in Siberian permafrost Psychrobacter cryohalolentis K5

    Science.gov (United States)

    Tremberger, George, Jr.; Holden, T.; Cheung, E.; Subramaniam, R.; Sullivan, R.; Schneider, P.; Flamholz, A.; Marchese, P.; Lieberman, D.; Cheung, T.

    2008-08-01

    A nucleotide sequence can be expressed as a numerical sequence when each nucleotide is assigned its proton number. A resulting gene numerical sequence can be investigated for its fractal dimension in terms of evolution and chemical properties for comparative studies. We have investigated such nucleotide fluctuation in the RecA repair gene of Psychrobacter cryohalolentis K5, Psychrobacter arcticus 273-4, and Psychrobacter sp. PRwf-1. The fractal dimension was found to correlate with the gene's operating temperature with the highest fractal dimension associated with P. cryohalolentis K5 living at the low temperatures found in Siberian permafrost. The CpG dinucleotide content was found to be about 5% for the three species of Psychrobacters, which is substantially lower than that of Deinococcus radiodurans at about 12%. The average nucleotide pair-wise free energy was found to be lowest for Psychrobacter sp. PRwf-1, the species with the lowest fractal dimension of the three, consistent with the recent finding that Psychrobacter sp. PRw-f1 has a temperature growth maximum of 15-20°C higher than P. arcticus 273-4 and P. cryohaloentis K5. The results suggest that microbial vitality in extreme environments is associated with fractal dimension as well as high CpG dinucleotide content, while the average nucleotide pair-wise free energy is related to the operating environment. Evidence that extreme temperature operation would impose constraints measurable by Shannon entropy is also discussed. A quantitative estimate of an entropy-based measure having the characteristics of a mechanical pressure shows that the Psychrobacter RecA sequence experiences lower pressure than that of the human HAR1 sequence.

  13. Association of common variants in mismatch repair genes and breast cancer susceptibility: a multigene study

    Directory of Open Access Journals (Sweden)

    Pina Julieta

    2009-09-01

    Full Text Available Abstract Background MMR is responsible for the repair of base-base mismatches and insertion/deletion loops. Besides this, MMR is also associated with an anti-recombination function, suppressing homologous recombination. Losses of heterozygosity and/or microsatellite instability have been detected in a large number of skin samples from breast cancer patients, suggesting a potential role of MMR in breast cancer susceptibility. Methods We carried out a hospital-based case-control study in a Caucasian Portuguese population (287 cases and 547 controls to estimate the susceptibility to non-familial breast cancer associated with some polymorphisms in mismatch repair genes (MSH3, MSH4, MSH6, MLH1, MLH3, PMS1 and MUTYH. Results Using unconditional logistic regression we found that MLH3 (L844P, G>A polymorphism GA (Leu/Pro and AA (Pro/Pro genotypes were associated with a decreased risk: OR = 0.65 (0.45-0.95 (p = 0.03 and OR = 0.62 (0.41-0.94 (p = 0.03, respectively. Analysis of two-way SNP interaction effects on breast cancer revealed two potential associations to breast cancer susceptibility: MSH3 Ala1045Thr/MSH6 Gly39Glu - AA/TC [OR = 0.43 (0.21-0.83, p = 0.01] associated with a decreased risk; and MSH4 Ala97Thr/MLH3 Leu844Pro - AG/AA [OR = 2.35 (1.23-4.49, p = 0.01], GG/AA [OR = 2.11 (1.12-3,98, p = 0.02], and GG/AG [adjusted OR = 1.88 (1.12-3.15, p = 0.02] all associated with an increased risk for breast cancer. Conclusion It is possible that some of these common variants in MMR genes contribute significantly to breast cancer susceptibility. However, further studies with a large sample size will be needed to support our results.

  14. Combined effects of scaffold stiffening and mechanical preconditioning cycles on construct biomechanics, gene expression, and tendon repair biomechanics.

    Science.gov (United States)

    Nirmalanandhan, Victor Sanjit; Juncosa-Melvin, Natalia; Shearn, Jason T; Boivin, Gregory P; Galloway, Marc T; Gooch, Cynthia; Bradica, Gino; Butler, David L

    2009-08-01

    Our group has previously reported that in vitro mechanical stimulation of tissue-engineered tendon constructs significantly increases both construct stiffness and the biomechanical properties of the repair tissue after surgery. When optimized using response surface methodology, our results indicate that a mechanical stimulus with three components (2.4% strain, 3000 cycles/day, and one cycle repetition) produced the highest in vitro linear stiffness. Such positive correlations between construct and repair stiffness after surgery suggest that enhancing structural stiffness before surgery could not only accelerate repair stiffness but also prevent premature failures in culture due to poor mechanical integrity. In this study, we examined the combined effects of scaffold crosslinking and subsequent mechanical stimulation on construct mechanics and biology. Autologous tissue-engineered constructs were created by seeding mesenchymal stem cells (MSCs) from 15 New Zealand white rabbits on type I collagen sponges that had undergone additional dehydrothermal crosslinking (termed ADHT in this manuscript). Both constructs from each rabbit were mechanically stimulated for 8h/day for 12 consecutive days with half receiving 100 cycles/day and the other half receiving 3000 cycles/day. These paired MSC-collagen autologous constructs were then implanted in bilateral full-thickness, full-length defects in the central third of rabbit patellar tendons. Increasing the number of in vitro cycles/day delivered to the ADHT constructs in culture produced no differences in stiffness or gene expression and no changes in biomechanical properties or histology 12 weeks after surgery. Compared to MSC-based repairs from a previous study that received no additional treatment in culture, ADHT crosslinking of the scaffolds actually lowered the 12-week repair stiffness. Thus, while ADHT crosslinking may initially stiffen a construct in culture, this specific treatment also appears to mask any benefits

  15. Use of the comet-FISH assay to compare DNA damage and repair in p53 and hTERT genes following ionizing radiation.

    Directory of Open Access Journals (Sweden)

    Declan J McKenna

    Full Text Available The alkaline single cell gel electrophoresis (comet assay can be combined with fluorescent in situ hybridisation (FISH methodology in order to investigate the localisation of specific gene domains within an individual cell. The number and position of the fluorescent signal(s provides information about the relative damage and subsequent repair that is occurring in the targeted gene domain(s. In this study, we have optimised the comet-FISH assay to detect and compare DNA damage and repair in the p53 and hTERT gene regions of bladder cancer cell-lines RT4 and RT112, normal fibroblasts and Cockayne Syndrome (CS fibroblasts following γ-radiation. Cells were exposed to 5Gy γ-radiation and repair followed for up to 60 minutes. At each repair time-point, the number and location of p53 and hTERT hybridisation spots was recorded in addition to standard comet measurements. In bladder cancer cell-lines and normal fibroblasts, the p53 gene region was found to be rapidly repaired relative to the hTERT gene region and the overall genome, a phenomenon that appeared to be independent of hTERT transcriptional activity. However, in the CS fibroblasts, which are defective in transcription coupled repair (TCR, this rapid repair of the p53 gene region was not observed when compared to both the hTERT gene region and the overall genome, proving the assay can detect variations in DNA repair in the same gene. In conclusion, we propose that the comet-FISH assay is a sensitive and rapid method for detecting differences in DNA damage and repair between different gene regions in individual cells in response to radiation. We suggest this increases its potential for measuring radiosensitivity in cells and may therefore have value in a clinical setting.

  16. Use of the Comet-FISH Assay to Compare DNA Damage and Repair in p53 and hTERT Genes following Ionizing Radiation

    Science.gov (United States)

    McKenna, Declan J.; Doherty, Bernadette A.; Downes, C. Stephen; McKeown, Stephanie R.; McKelvey-Martin, Valerie J.

    2012-01-01

    The alkaline single cell gel electrophoresis (comet) assay can be combined with fluorescent in situ hybridisation (FISH) methodology in order to investigate the localisation of specific gene domains within an individual cell. The number and position of the fluorescent signal(s) provides information about the relative damage and subsequent repair that is occurring in the targeted gene domain(s). In this study, we have optimised the comet-FISH assay to detect and compare DNA damage and repair in the p53 and hTERT gene regions of bladder cancer cell-lines RT4 and RT112, normal fibroblasts and Cockayne Syndrome (CS) fibroblasts following γ-radiation. Cells were exposed to 5Gy γ-radiation and repair followed for up to 60 minutes. At each repair time-point, the number and location of p53 and hTERT hybridisation spots was recorded in addition to standard comet measurements. In bladder cancer cell-lines and normal fibroblasts, the p53 gene region was found to be rapidly repaired relative to the hTERT gene region and the overall genome, a phenomenon that appeared to be independent of hTERT transcriptional activity. However, in the CS fibroblasts, which are defective in transcription coupled repair (TCR), this rapid repair of the p53 gene region was not observed when compared to both the hTERT gene region and the overall genome, proving the assay can detect variations in DNA repair in the same gene. In conclusion, we propose that the comet-FISH assay is a sensitive and rapid method for detecting differences in DNA damage and repair between different gene regions in individual cells in response to radiation. We suggest this increases its potential for measuring radiosensitivity in cells and may therefore have value in a clinical setting. PMID:23145163

  17. RNA polymerase II is released from the DNA template during transcription-coupled repair in mammalian cells.

    Science.gov (United States)

    Chiou, Yi-Ying; Hu, Jinchuan; Sancar, Aziz; Selby, Christopher P

    2018-02-16

    In mammalian cells, bulky DNA adducts located in the template but not the coding strand of genes block elongation by RNA polymerase II (RNAPII). The blocked RNAPII targets these transcription-blocking adducts to undergo more rapid excision repair than adducts located elsewhere in the genome. In excision repair, coupled incisions are made in the damaged DNA strand on both sides of the adduct. The fate of RNAPII in the course of this transcription-coupled repair (TCR) pathway is unclear. To address the fate of RNAPII, we used methods that control transcription to initiate a discrete "wave" of elongation complexes. Analyzing genome-wide transcription and repair by next-generation sequencing, we identified locations of elongation complexes and transcription-repair coupling events in genes throughout the genome. Using UV-exposed human skin fibroblasts, we found that, at the dose used, a single wave of elongation complexes was blocked within the first 25 kb of genes. TCR occurred where the elongation complexes were blocked, and repair was associated with the dissociation of these complexes. These results indicate that individual elongation complexes do not engage in multiple rounds of TCR with successive lesions. Our results are consistent with a model in which RNAPII is dissociated after the dual incision of the transcription-blocking lesion, perhaps by Cockayne syndrome group B translocase, or during the synthesis of a repair patch. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Exome sequencing identifies rare deleterious mutations in DNA repair genes FANCC and BLM as potential breast cancer susceptibility alleles.

    Directory of Open Access Journals (Sweden)

    Ella R Thompson

    2012-09-01

    Full Text Available Despite intensive efforts using linkage and candidate gene approaches, the genetic etiology for the majority of families with a multi-generational breast cancer predisposition is unknown. In this study, we used whole-exome sequencing of thirty-three individuals from 15 breast cancer families to identify potential predisposing genes. Our analysis identified families with heterozygous, deleterious mutations in the DNA repair genes FANCC and BLM, which are responsible for the autosomal recessive disorders Fanconi Anemia and Bloom syndrome. In total, screening of all exons in these genes in 438 breast cancer families identified three with truncating mutations in FANCC and two with truncating mutations in BLM. Additional screening of FANCC mutation hotspot exons identified one pathogenic mutation among an additional 957 breast cancer families. Importantly, none of the deleterious mutations were identified among 464 healthy controls and are not reported in the 1,000 Genomes data. Given the rarity of Fanconi Anemia and Bloom syndrome disorders among Caucasian populations, the finding of multiple deleterious mutations in these critical DNA repair genes among high-risk breast cancer families is intriguing and suggestive of a predisposing role. Our data demonstrate the utility of intra-family exome-sequencing approaches to uncover cancer predisposition genes, but highlight the major challenge of definitively validating candidates where the incidence of sporadic disease is high, germline mutations are not fully penetrant, and individual predisposition genes may only account for a tiny proportion of breast cancer families.

  19. Contribution of DNA double-strand break repair gene XRCC3 genotypes to oral cancer susceptibility in Taiwan.

    Science.gov (United States)

    Tsai, Chia-Wen; Chang, Wen-Shin; Liu, Juhn-Cherng; Tsai, Ming-Hsui; Lin, Cheng-Chieh; Bau, Da-Tian

    2014-06-01

    The DNA repair gene X-ray repair cross complementing protein 3 (XRCC3) is thought to play a major role in double-strand break repair and in maintaining genomic stability. Very possibly, defective double-strand break repair of cells can lead to carcinogenesis. Therefore, a case-control study was performed to reveal the contribution of XRCC3 genotypes to individual oral cancer susceptibility. In this hospital-based research, the association of XRCC3 rs1799794, rs45603942, rs861530, rs3212057, rs1799796, rs861539, rs28903081 genotypes with oral cancer risk in a Taiwanese population was investigated. In total, 788 patients with oral cancer and 956 age- and gender-matched healthy controls were genotyped. The results showed that there was significant differential distribution among oral cancer and controls in the genotypic (p=0.001428) and allelic (p=0.0013) frequencies of XRCC3 rs861539. As for the other polymorphisms, there was no difference between case and control groups. In gene-lifestyle interaction analysis, we have provided the first evidence showing that there is an obvious joint effect of XRCC3 rs861539 genotype with individual areca chewing habits on oral cancer risk. In conclusion, the T allele of XRCC3 rs861539, which has an interaction with areca chewing habit in oral carcinogenesis, may be an early marker for oral cancer in Taiwanese. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  20. Constitutional Mismatch Repair Deficiency in Israel: High Proportion of Founder Mutations in MMR Genes and Consanguinity.

    Science.gov (United States)

    Baris, Hagit N; Barnes-Kedar, Inbal; Toledano, Helen; Halpern, Marisa; Hershkovitz, Dov; Lossos, Alexander; Lerer, Israela; Peretz, Tamar; Kariv, Revital; Cohen, Shlomi; Half, Elizabeth E; Magal, Nurit; Drasinover, Valerie; Wimmer, Katharina; Goldberg, Yael; Bercovich, Dani; Levi, Zohar

    2016-03-01

    Heterozygous germline mutations in any of the mismatch repair (MMR) genes, MLH1, MSH2, MSH6, and PMS2, cause Lynch syndrome (LS), an autosomal dominant cancer predisposition syndrome conferring a high risk of colorectal, endometrial, and other cancers in adulthood. Offspring of couples where both spouses have LS have a 1:4 risk of inheriting biallelic MMR gene mutations. These cause constitutional MMR deficiency (CMMRD) syndrome, a severe recessively inherited cancer syndrome with a broad tumor spectrum including mainly hematological malignancies, brain tumors, and colon cancer in childhood and adolescence. Many CMMRD children also present with café au lait spots and axillary freckling mimicking neurofibromatosis type 1. We describe our experience in seven CMMRD families demonstrating the role and importance of founder mutations and consanguinity on its prevalence. Clinical presentations included brain tumors, colon cancer, lymphoma, and small bowel cancer. In children from two nonconsanguineous Ashkenazi Jewish (AJ) families, the common Ashkenazi founder mutations were detected; these were homozygous in one family and compound heterozygous in the other. In four consanguineous families of various ancestries, different homozygous mutations were identified. In a nonconsanguineous Caucasus/AJ family, lack of PMS2 was demonstrated in tumor and normal tissues; however, mutations were not identified. CMMRD is rare, but, especially in areas where founder mutations for LS and consanguinity are common, pediatricians should be aware of it since they are the first to encounter these children. Early diagnosis will enable tailored cancer surveillance in the entire family and a discussion regarding prenatal genetic diagnosis. © 2015 Wiley Periodicals, Inc.

  1. Characterization of mammalian RAD51 double strand break repair using non-lethal dominant-negative forms

    OpenAIRE

    Lambert, Sarah; Lopez, Bernard S.

    2000-01-01

    In contrast to yeast RAD51, mammalian mRAD51 is an essential gene. Its role in double strand break (DSB) repair and its consequences on cell viability remain to be characterized precisely. Here, we used a hamster cell line carrying tandem repeat sequences with an I-SceI cleavage site. We characterized conservative recombination after I-SceI cleavage as gene conversion or intrachromatid crossing over associated with random reintegration of the excised reciprocal product. We identified two domi...

  2. Molecular Mechanisms of the Whole DNA Repair System: A Comparison of Bacterial and Eukaryotic Systems

    Directory of Open Access Journals (Sweden)

    Rihito Morita

    2010-01-01

    Full Text Available DNA is subjected to many endogenous and exogenous damages. All organisms have developed a complex network of DNA repair mechanisms. A variety of different DNA repair pathways have been reported: direct reversal, base excision repair, nucleotide excision repair, mismatch repair, and recombination repair pathways. Recent studies of the fundamental mechanisms for DNA repair processes have revealed a complexity beyond that initially expected, with inter- and intrapathway complementation as well as functional interactions between proteins involved in repair pathways. In this paper we give a broad overview of the whole DNA repair system and focus on the molecular basis of the repair machineries, particularly in Thermus thermophilus HB8.

  3. Association between DNA damage response and repair genes and risk of invasive serous ovarian cancer.

    Directory of Open Access Journals (Sweden)

    Joellen M Schildkraut

    Full Text Available BACKGROUND: We analyzed the association between 53 genes related to DNA repair and p53-mediated damage response and serous ovarian cancer risk using case-control data from the North Carolina Ovarian Cancer Study (NCOCS, a population-based, case-control study. METHODS/PRINCIPAL FINDINGS: The analysis was restricted to 364 invasive serous ovarian cancer cases and 761 controls of white, non-Hispanic race. Statistical analysis was two staged: a screen using marginal Bayes factors (BFs for 484 SNPs and a modeling stage in which we calculated multivariate adjusted posterior probabilities of association for 77 SNPs that passed the screen. These probabilities were conditional on subject age at diagnosis/interview, batch, a DNA quality metric and genotypes of other SNPs and allowed for uncertainty in the genetic parameterizations of the SNPs and number of associated SNPs. Six SNPs had Bayes factors greater than 10 in favor of an association with invasive serous ovarian cancer. These included rs5762746 (median OR(odds ratio(per allele = 0.66; 95% credible interval (CI = 0.44-1.00 and rs6005835 (median OR(per allele = 0.69; 95% CI = 0.53-0.91 in CHEK2, rs2078486 (median OR(per allele = 1.65; 95% CI = 1.21-2.25 and rs12951053 (median OR(per allele = 1.65; 95% CI = 1.20-2.26 in TP53, rs411697 (median OR (rare homozygote = 0.53; 95% CI = 0.35 - 0.79 in BACH1 and rs10131 (median OR( rare homozygote = not estimable in LIG4. The six most highly associated SNPs are either predicted to be functionally significant or are in LD with such a variant. The variants in TP53 were confirmed to be associated in a large follow-up study. CONCLUSIONS/SIGNIFICANCE: Based on our findings, further follow-up of the DNA repair and response pathways in a larger dataset is warranted to confirm these results.

  4. Repair genes expression profile of MLH1, MSH2 and ATM in the normal oral mucosa of chronic smokers.

    Science.gov (United States)

    Alves, Mônica Ghislaine Oliveira; Carta, Celina Faig Lima; de Barros, Patrícia Pimentel; Issa, Jaqueline Scholz; Nunes, Fábio Daumas; Almeida, Janete Dias

    2017-01-01

    The aim of this study was to evaluate the effect of chronic smoking on the expression profile of the repair genes MLH1, MSH2 and ATM in the normal oral mucosa of chronic smokers and never smokers. The sample consisted of thirty exfoliative cytology smears per group obtained from Smokers and Never Smokers. Total RNA was extracted and expression of the MLH1, MSH2 and ATM genes were evaluated by quantitative real-time and immunocytochemistry. The gene and protein expression data were correlated to the clinical data. Gene expression was analyzed statistically using the Student t-test and Pearson's correlation coefficient, with pATM genes were downregulated in the smoking group compared to the control with significant values for MLH1 (p=0.006), MSH2 (p=0.0001) and ATM (p=0.0001). Immunocytochemical staining for anti-MLH1, anti-MSH2 and anti-ATM was negative in Never Smokers; in Smokers it was rarely positive. No significant correlation was observed among the expression of MLH1, MSH2, ATM and age, number of cigarettes consumed per day, time of smoking during life, smoking history or levels of CO in expired air. The expression of genes and proteins related to DNA repair mechanism MLH1, MSH2 and ATM in the normal oral mucosa of chronic smokers was reduced. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Knockout targeting of the Drosophila nap1 gene and examination of DNA repair tracts in the recombination products.

    Science.gov (United States)

    Lankenau, Susanne; Barnickel, Thorsten; Marhold, Joachim; Lyko, Frank; Mechler, Bernard M; Lankenau, Dirk-Henner

    2003-02-01

    We used ends-in gene targeting to generate knockout mutations of the nucleosome assembly protein 1 (Nap1) gene in Drosophila melanogaster. Three independent targeted null-knockout mutations were produced. No wild-type NAP1 protein could be detected in protein extracts. Homozygous Nap1(KO) knockout flies were either embryonic lethal or poorly viable adult escapers. Three additional targeted recombination products were viable. To gain insight into the underlying molecular processes we examined conversion tracts in the recombination products. In nearly all cases the I-SceI endonuclease site of the donor vector was replaced by the wild-type Nap1 sequence. This indicated exonuclease processing at the site of the double-strand break (DSB), followed by replicative repair at donor-target junctions. The targeting products are best interpreted either by the classical DSB repair model or by the break-induced recombination (BIR) model. Synthesis-dependent strand annealing (SDSA), which is another important recombinational repair pathway in the germline, does not explain ends-in targeting products. We conclude that this example of gene targeting at the Nap1 locus provides added support for the efficiency of this method and its usefulness in targeting any arbitrary locus in the Drosophila genome.

  6. Polymorphisms in DNA repair genes, smoking, and bladder cancer risk: findings from the International Consortium of Bladder Cancer

    Science.gov (United States)

    Stern, Mariana C.; Lin, Jie; Figueroa, Jonine D.; Kelsey, Karl T.; Kiltie, Anne E.; Yuan, Jian-Min; Matullo, Giuseppe; Fletcher, Tony; Benhamou, Simone; Taylor, Jack A.; Placidi, Donatella; Zhang, Zuo-Feng; Steineck, Gunnar; Rothman, Nathaniel; Kogevinas, Manolis; Silverman, Debra; Malats, Nuria; Chanock, Stephen; Wu, Xifeng; Karagas, Margaret R.; Andrew, Angeline S.; Nelson, Heather H.; Bishop, D. Timothy; Sak, Sei Chung; Choudhury, Ananya; Barrett, Jennifer H; Elliot, Faye; Corral, Román; Joshi, Amit D.; Gago-Dominguez, Manuela; Cortessis, Victoria K.; Xiang, Yong-Bing; Vineis, Paolo; Sacerdote, Carlotta; Guarrera, Simonetta; Polidoro, Silvia; Allione, Alessandra; Gurzau, Eugen; Koppova, Kvetoslava; Kumar, Rajiv; Rudnai, Peter; Porru, Stefano; Carta, Angela; Campagna, Marcello; Arici, Cecilia; Park, SungShim Lani; Garcia-Closas, Montserrat

    2009-01-01

    Tobacco smoking is the most important and well-established bladder cancer risk factor, and a rich source of chemical carcinogens and reactive oxygen species that can induce damage to DNA in urothelial cells. Therefore, common variation in DNA repair genes might modify bladder cancer risk. In this study we present results from meta- and pooled analyses conducted as part of the International Consortium of Bladder Cancer. We included data on 10 single nucleotide polymorphisms corresponding to 7 DNA repair genes from 13 studies. Pooled- and meta-analyses included 5,282 cases and 5,954 controls of non-Latino white origin. We found evidence for weak but consistent associations with ERCC2 D312N (rs1799793) (per allele OR = 1.10; 95% CI = 1.01–1.19; p = 0.021), NBN E185Q (rs1805794) (per allele OR = 1.09; 95% CI = 1.01–1.18; p = 0.028), and XPC A499V (rs2228000) (per allele OR = 1.10; 95% CI = 1.00–1.21, p = 0.044). The association with NBN E185Q was limited to ever smokers (interaction p = 0.002), and was strongest for the highest levels of smoking dose and smoking duration. Overall, our study provides the strongest evidence to date for a role of common variants in DNA repair genes in bladder carcinogenesis. PMID:19706757

  7. Partial Complementation of the UV Sensitivity of Deinococcus Radiodurans Excision Repair Mutants by the Cloned denv Gene of Bacteriophage T4

    Science.gov (United States)

    1990-07-19

    photolyase (Figure 3). DNA photolyase activity has been detected in a large number of plant and animal cell extracts as well as in prokaryotic cells...containing pyrimidine dimers. FASEB Journal 2: 2696 -2701. Grunstein, M. and D. Hogness (1975), Colony hibridization : a method for the isolation of

  8. Genotoxic stress and DNA repair in plants: emerging functions and tools for improving crop productivity.

    Science.gov (United States)

    Balestrazzi, Alma; Confalonieri, Massimo; Macovei, Anca; Donà, Mattia; Carbonera, Daniela

    2011-03-01

    Crop productivity is strictly related to genome stability, an essential requisite for optimal plant growth/development. Genotoxic agents (e.g., chemical agents, radiations) can cause both chemical and structural damage to DNA. In some cases, they severely affect the integrity of plant genome by inducing base oxidation, which interferes with the basal processes of replication and transcription, eventually leading to cell death. The cell response to oxidative stress includes several DNA repair pathways, which are activated to remove the damaged bases and other lesions. Information concerning DNA repair in plants is still limited, although results from gene profiling and mutant analysis suggest possible differences in repair mechanisms between plants and other eukaryotes. The present review focuses on the base- and nucleotide excision repair (BER, NER) pathways, which operate according to the most common DNA repair rule (excision of damaged bases and replacement by the correct nucleotide), highlighting the most recent findings in plants. An update on DNA repair in organelles, chloroplasts and mitochondria is also provided. Finally, it is generally acknowledged that DNA repair plays a critical role during seed imbibition, preserving seed vigor. Despite this, only a limited number of studies, described here, dedicated to seeds are currently available.

  9. Alcohol Consumption and the Risk of Colorectal Cancer for Mismatch Repair Gene Mutation Carriers.

    Science.gov (United States)

    Dashti, S Ghazaleh; Buchanan, Daniel D; Jayasekara, Harindra; Ait Ouakrim, Driss; Clendenning, Mark; Rosty, Christophe; Winship, Ingrid M; Macrae, Finlay A; Giles, Graham G; Parry, Susan; Casey, Graham; Haile, Robert W; Gallinger, Steven; Le Marchand, Loïc; Thibodeau, Stephen N; Lindor, Noralane M; Newcomb, Polly A; Potter, John D; Baron, John A; Hopper, John L; Jenkins, Mark A; Win, Aung Ko

    2017-03-01

    Background: People with germline mutation in one of the DNA mismatch repair (MMR) genes have increased colorectal cancer risk. For these high-risk people, study findings of the relationship between alcohol consumption and colorectal cancer risk have been inconclusive. Methods: 1,925 MMR gene mutations carriers recruited into the Colon Cancer Family Registry who had completed a questionnaire on lifestyle factors were included. Weighted Cox proportional hazard regression models were used to estimate hazard ratios (HR) and 95% confidence intervals (CI) for the association between alcohol consumption and colorectal cancer. Results: Colorectal cancer was diagnosed in 769 carriers (40%) at a mean (SD) age of 42.6 (10.3) years. Compared with abstention, ethanol consumption from any alcoholic beverage up to 14 g/day and >28 g/day was associated with increased colorectal cancer risk (HR, 1.50; 95% CI, 1.09-2.07 and 1.69; 95% CI, 1.07-2.65, respectively; P trend = 0.05), and colon cancer risk (HR, 1.78; 95% CI, 1.27-2.49 and 1.94; 95% CI, 1.19-3.18, respectively; P trend = 0.02). However, there was no clear evidence for an association with rectal cancer risk. Also, there was no evidence for associations between consumption of individual alcoholic beverage types (beer, wine, spirits) and colorectal, colon, or rectal cancer risk. Conclusions: Our data suggest that alcohol consumption, particularly more than 28 g/day of ethanol (∼2 standard drinks of alcohol in the United States), is associated with increased colorectal cancer risk for MMR gene mutation carriers. Impact: Although these data suggested that alcohol consumption in MMR carriers was associated with increased colorectal cancer risk, there was no evidence of a dose-response, and not all types of alcohol consumption were associated with increased risk. Cancer Epidemiol Biomarkers Prev; 26(3); 366-75. ©2016 AACR . ©2016 American Association for Cancer Research.

  10. Transplantation of erythropoietin gene-modified neural stem cells improves the repair of injured spinal cord

    Directory of Open Access Journals (Sweden)

    Min-fei Wu

    2015-01-01

    Full Text Available The protective effects of erythropoietin on spinal cord injury have not been well described. Here, the eukaryotic expression plasmid pcDNA3.1 human erythropoietin was transfected into rat neural stem cells cultured in vitro. A rat model of spinal cord injury was established using a free falling object. In the human erythropoietin-neural stem cells group, transfected neural stem cells were injected into the rat subarachnoid cavity, while the neural stem cells group was injected with non-transfected neural stem cells. Dulbecco′s modified Eagle′s medium/F12 medium was injected into the rats in the spinal cord injury group as a control. At 1-4 weeks post injury, the motor function in the rat lower limbs was best in the human erythropoietin-neural stem cells group, followed by the neural stem cells group, and lastly the spinal cord injury group. At 72 hours, compared with the spinal cord injury group, the apoptotic index and Caspase-3 gene and protein expressions were apparently decreased, and the bcl-2 gene and protein expressions were noticeably increased, in the tissues surrounding the injured region in the human erythropoietin-neural stem cells group. At 4 weeks, the cavities were clearly smaller and the motor and somatosensory evoked potential latencies were remarkably shorter in the human erythropoietin-neural stem cells group and neural stem cells group than those in the spinal cord injury group. These differences were particularly obvious in the human erythropoietin-neural stem cells group. More CM-Dil-positive cells and horseradish peroxidase-positive nerve fibers and larger amplitude motor and somatosensory evoked potentials were found in the human erythropoietin-neural stem cells group and neural stem cells group than in the spinal cord injury group. Again, these differences were particularly obvious in the human erythropoietin-neural stem cells group. These data indicate that transplantation of erythropoietin gene-modified neural stem

  11. Transplantation of erythropoietin gene-modified neural stem cells improves the repair of injured spinal cord.

    Science.gov (United States)

    Wu, Min-Fei; Zhang, Shu-Quan; Gu, Rui; Liu, Jia-Bei; Li, Ye; Zhu, Qing-San

    2015-09-01

    The protective effects of erythropoietin on spinal cord injury have not been well described. Here, the eukaryotic expression plasmid pcDNA3.1 human erythropoietin was transfected into rat neural stem cells cultured in vitro. A rat model of spinal cord injury was established using a free falling object. In the human erythropoietin-neural stem cells group, transfected neural stem cells were injected into the rat subarachnoid cavity, while the neural stem cells group was injected with non-transfected neural stem cells. Dulbecco's modified Eagle's medium/F12 medium was injected into the rats in the spinal cord injury group as a control. At 1-4 weeks post injury, the motor function in the rat lower limbs was best in the human erythropoietin-neural stem cells group, followed by the neural stem cells group, and lastly the spinal cord injury group. At 72 hours, compared with the spinal cord injury group, the apoptotic index and Caspase-3 gene and protein expressions were apparently decreased, and the bcl-2 gene and protein expressions were noticeably increased, in the tissues surrounding the injured region in the human erythropoietin-neural stem cells group. At 4 weeks, the cavities were clearly smaller and the motor and somatosensory evoked potential latencies were remarkably shorter in the human erythropoietin-neural stem cells group and neural stem cells group than those in the spinal cord injury group. These differences were particularly obvious in the human erythropoietin-neural stem cells group. More CM-Dil-positive cells and horseradish peroxidase-positive nerve fibers and larger amplitude motor and somatosensory evoked potentials were found in the human erythropoietin-neural stem cells group and neural stem cells group than in the spinal cord injury group. Again, these differences were particularly obvious in the human erythropoietin-neural stem cells group. These data indicate that transplantation of erythropoietin gene-modified neural stem cells into the

  12. Germ line mutations of mismatch repair genes in hereditary nonpolyposis colorectal cancer patients with small bowel cancer: International Society for Gastrointestinal Hereditary Tumours Collaborative Study

    DEFF Research Database (Denmark)

    Park, Jae-Gahb; Kim, Duck-Woo; Hong, Chang Won

    2006-01-01

    PURPOSE: The aim of study was to determine the clinical characteristics and mutational profiles of the mismatch repair genes in hereditary nonpolyposis colorectal cancer (HNPCC) patients with small bowel cancer (SBC). EXPERIMENTAL DESIGN: A questionnaire was mailed to 55 members...... of the International Society for Gastrointestinal Hereditary Tumours, requesting information regarding patients with HNPCC-associated SBC and germ line mismatch repair gene mutations. RESULTS: The study population consisted of 85 HNPCC patients with identified mismatch repair gene mutations and SBCs. SBC was the first...... HNPCC-associated malignancy in 14 of 41 (34.1%) patients for whom a personal history of HNPCC-associated cancers was available. The study population harbored 69 different germ line mismatch repair gene mutations, including 31 mutations in MLH1, 34 in MSH2, 3 in MSH6, and 1 in PMS2. We compared...

  13. Influence of XRCC1 Genetic Polymorphisms on Ionizing Radiation-Induced DNA Damage and Repair

    Directory of Open Access Journals (Sweden)

    Silvia Sterpone

    2010-01-01

    Full Text Available It is well known that ionizing radiation (IR can damage DNA through a direct action, producing single- and double-strand breaks on DNA double helix, as well as an indirect effect by generating oxygen reactive species in the cells. Mammals have evolved several and distinct DNA repair pathways in order to maintain genomic stability and avoid tumour cell transformation. This review reports important data showing a huge interindividual variability on sensitivity to IR and in susceptibility to developing cancer; this variability is principally represented by genetic polymorphisms, that is, DNA repair gene polymorphisms. In particular we have focussed on single nucleotide polymorphisms (SNPs of XRCC1, a gene that encodes for a scaffold protein involved basically in Base Excision Repair (BER. In this paper we have reported and presented recent studies that show an influence of XRCC1 variants on DNA repair capacity and susceptibility to breast cance