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Sample records for base excision repair

  1. 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...

  2. Base excision repair in sugarcane

    Directory of Open Access Journals (Sweden)

    Agnez-Lima Lucymara F.

    2001-01-01

    Full Text Available DNA damage can be induced by a large number of physical and chemical agents from the environment as well as compounds produced by cellular metabolism. This type of damage can interfere with cellular processes such as replication and transcription, resulting in cell death and/or mutations. The low frequency of mutagenesis in cells is due to the presence of enzymatic pathways which repair damaged DNA. Several DNA repair genes (mainly from bacteria, yeasts and mammals have been cloned and their products characterized. The high conservation, especially in eukaryotes, of the majority of genes related to DNA repair argues for their importance in the maintenance of life on earth. In plants, our understanding of DNA repair pathways is still very poor, the first plant repair genes having only been cloned in 1997 and the mechanisms of their products have not yet been characterized. The objective of our data mining work was to identify genes related to the base excision repair (BER pathway, which are present in the database of the Sugarcane Expressed Sequence Tag (SUCEST Project. This search was performed by tblastn program. We identified sugarcane clusters homologous to the majority of BER proteins used in the analysis and a high degree of conservation was observed. The best results were obtained with BER proteins from Arabidopsis thaliana. For some sugarcane BER genes, the presence of more than one form of mRNA is possible, as shown by the occurrence of more than one homologous EST cluster.

  3. uv photobiology: excision repair

    International Nuclear Information System (INIS)

    The following topics are discussed: steps in nucleotide excision; damage to DNA by uv-endonuclease; use of complementation to study DNA repair in Escherichia coli and mammalian cells; role of BUDR photolysis in excision repair, relation between DNA repair defect and human disease; base excision repair; and excision repair by removal of damaged region of a base in DNA without excision

  4. Sequence Context Specific Mutagenesis and Base Excision Repair

    OpenAIRE

    Donigan, Katherine; Sweasy, Joann B.

    2009-01-01

    Base excision repair is critical for the maintenance of genome stability because it repairs at least 20,000 endogenously generated DNA lesions per cell per day. Several enzymes within the base excision repair pathway exhibit sequence context dependency during the excision and DNA synthesis steps of repair. New evidence is emerging that germ line and tumor-associated variants of enzymes in this repair pathway exhibit sequence context dependence that is different from their ancestral counterpar...

  5. Variant Base Excision Repair Proteins: Contributors to Genomic Instability

    Science.gov (United States)

    Nemec, Antonia A.; Wallace, Susan S.; Sweasy, Joann B.

    2012-01-01

    Cells sustain endogenous DNA damage at rates greater than 20,000 DNA lesions per cell per day. These damages occur largely as a result of the inherently unstable nature of DNA and the presence of reactive oxygen species within cells. The base excision repair system removes the majority of DNA lesions resulting from endogenous DNA damage. There are several enzymes that function during base excision repair. Importantly, there are over 100 germline single nucleotide polymorphisms in genes that function in base excision repair and that result in non-synonymous amino acid substitutions in the proteins they encode. Somatic variants of these enzymes are also found in human tumors. Variant repair enzymes catalyze aberrant base excision repair. Aberrant base excision repair combined with continuous endogenous DNA damage over time has the potential to lead to a mutator phenotype. Mutations that arise in key growth control genes, imbalances in chromosome number, chromosomal translocations, and loss of heterozygosity can result in the initiation of human cancer or its progression. PMID:20955798

  6. Base excision repair mechanisms and relevance to cancer susceptibility

    International Nuclear Information System (INIS)

    The base excision repair (BER) pathway is considered the predominant DNA repair system in mammalian cells for eliminating small DNA lesions generated at DNA bases either exogenously by environmental agents or endogenously by normal cellular metabolic processes (e.g. production of oxyradical species, alkylating agents, etc). The main goal of this project is the understanding of the involvement of BER in genome stability and in particular in sporadic cancer development associated with inflammation such as gastric cancer (GC). A major risk factor of GC is the infection by Helicobacter pylori, which causes oxidative stress. Oxidative DNA damage is mainly repaired by BER

  7. Ku80-Deleted Cells are Defective at Base Excision Repair

    OpenAIRE

    Li, Han; Marple, Teresa; Hasty, Paul

    2013-01-01

    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 repa...

  8. Nucleosomes determine their own patch size in base excision repair.

    Science.gov (United States)

    Meas, Rithy; Smerdon, Michael J

    2016-01-01

    Base excision repair (BER) processes non-helix distorting lesions (e.g., uracils and gaps) and is composed of two subpathways that differ in the number of nucleotides (nts) incorporated during the DNA synthesis step: short patch (SP) repair incorporates 1 nt and long patch (LP) repair incorporates 2-12 nts. This choice for either LP or SP repair has not been analyzed in the context of nucleosomes. Initial studies with uracil located in nucleosome core DNA showed a distinct DNA polymerase extension profile in cell-free extracts that specifically limits extension to 1 nt, suggesting a preference for SP BER. Therefore, we developed an assay to differentiate long and short repair patches in 'designed' nucleosomes containing a single-nucleotide gap at specific locations relative to the dyad center. Using cell-free extracts or purified enzymes, we found that DNA lesions in the nucleosome core are preferentially repaired by DNA polymerase β and there is a significant reduction in BER polymerase extension beyond 1 nt, creating a striking bias for incorporation of short patches into nucleosomal DNA. These results show that nucleosomes control the patch size used by BER. PMID:27265863

  9. Base Excision Repair, a Pathway Regulated by Posttranslational Modifications.

    Science.gov (United States)

    Carter, Rachel J; Parsons, Jason L

    2016-05-15

    Base excision repair (BER) is an essential DNA repair pathway involved in the maintenance of genome stability and thus in the prevention of human diseases, such as premature aging, neurodegenerative diseases, and cancer. Protein posttranslational modifications (PTMs), including acetylation, methylation, phosphorylation, SUMOylation, and ubiquitylation, have emerged as important contributors in controlling cellular BER protein levels, enzymatic activities, protein-protein interactions, and protein cellular localization. These PTMs therefore play key roles in regulating the BER pathway and are consequently crucial for coordinating an efficient cellular DNA damage response. In this review, we summarize the presently available data on characterized PTMs of key BER proteins, the functional consequences of these modifications at the protein level, and also the impact on BER in vitro and in vivo. PMID:26976642

  10. X-ray repair cross complementing protein 1 in base excision repair

    DEFF Research Database (Denmark)

    Hanssen-Bauer, Audun; Solvang-Garten, Karin; Akbari, Mansour;

    2012-01-01

    X-ray Repair Cross Complementing protein 1 (XRCC1) acts as a scaffolding protein in the converging base excision repair (BER) and single strand break repair (SSBR) pathways. XRCC1 also interacts with itself and rapidly accumulates at sites of DNA damage. XRCC1 can thus mediate the assembly of large...... multiprotein DNA repair complexes as well as facilitate the recruitment of DNA repair proteins to sites of DNA damage. Moreover, XRCC1 is present in constitutive DNA repair complexes, some of which associate with the replication machinery. Because of the critical role of XRCC1 in DNA repair, its common...... variants Arg194Trp, Arg280His and Arg399Gln have been extensively studied. However, the prevalence of these variants varies strongly in different populations, and their functional influence on DNA repair and disease remains elusive. Here we present the current knowledge about the role of XRCC1 and its...

  11. 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.

  12. Base excision repair deficiency in acute myeloid leukemia

    International Nuclear Information System (INIS)

    Acute myeloid leukemia (AML) is an aggressive malignancy of the hematopoietic system arising from a transformed myeloid progenitor cell. Genomic instability is the hallmark of AML and characterized by a variety of cytogenetic and molecular abnormalities. Whereas 10% to 20% of AML cases reflect long-term sequelae of cytotoxic therapies for a primary disorder, the etiology for the majority of AMLs remains unknown. The integrity of DNA is under continuous attack from a variety of exogenous and endogenous DNA damaging agents. The majority of DNA damage is caused by constantly generated reactive oxygen species (ROS) resulting from metabolic by-products. Base excision repair (BER) is the major DNA repair mechanism dealing with DNA base lesions that are induced by oxidative stress or alkylation. In this study we investigated the BER in AML. Primary AML patients samples as well as AML cell lines were treated with hydrogen peroxide (H2O2). DNA damage induction and repair was monitored by the alkaline comet assay. In 15/30 leukemic samples from patients with therapy-related AML, in 13/35 with de novo AML and 14/26 with AML following a myelodysplastic syndrome, significantly reduced single strand breaks (SSBs) representing BER intermediates were found. In contrast, normal SSB formation was seen in mononuclear cells of 30 healthy individuals and 30/31 purified hematopoietic stem- and progenitor cell preparations obtained from umbilical cord blood. Additionally, in 5/10 analyzed AML cell lines, no SSBs were formed upon H2O2 treatment, either. Differences in intracellular ROS concentrations or apoptosis could be excluded as reason for this phenomenon. A significantly diminished cleavage capacity for 7,8-dihydro-8-oxoguanine as well as for Furan was observed in cell lines that exhibited no SSB formation. These data demonstrate for the first time that initial steps of BER are impaired in a proportion of AML cell lines and leukemic cells from patients with different forms of AML

  13. Repair of 3-methyladenine and abasic sites by base excision repair mediates glioblastoma resistance to temozolomide

    Directory of Open Access Journals (Sweden)

    John R Silber

    2012-11-01

    Full Text Available Alkylating agents have long played a central role in the adjuvant therapy of glioblastoma multiforme (GBM. More recently, inclusion of temozolomide (TMZ, an orally administered methylating agent with low systemic toxicity, during radiotherapy and afterward has markedly improved survival. Extensive in vitro and in vivo evidence has shown that TMZ-induced O6-methylguanine (O6-meG mediates GBM cell killing. Moreover, low or absent expression of O6-methylguanine-DNA methyltransferase (MGMT, the sole human repair protein that removes O6-meG from DNA, is frequently associated with longer survival in GBMs treated with TMZ, promoting interest in developing inhibitors of MGMT to counter resistance. However, the clinical efficacy of TMZ is unlikely to be due solely to O6-meG, as the agent produces approximately a dozen additional DNA adducts, including cytotoxic N3-methyladenine (3-meA and abasic sites. Repair of 3-meA and abasic sites, both of which are produced in greater abundance than O6-meG, is mediated by the base excision repair (BER pathway, and occurs independently of removal of O6-meG. These observations indicate that BER activities are also potential targets for strategies to potentiate TMZ cytotoxicity. Here we review the evidence that 3-meA and abasic sites mediate killing of GBM cells. We also present in vitro and in vivo evidence that alkyladenine-DNA–glycosylase, the sole repair activity that excises 3-meA from DNA, and Ape1, the major human abasic site endonuclease, mediate TMZ resistance in GBMs and represent potential anti-resistance targets.

  14. Chk2-dependent phosphorylation of XRCC1 in the DNA damage response promotes base excision repair

    OpenAIRE

    Chou, Wen-Cheng; Wang, Hui-Chun; Wong, Fen-Hwa; Ding, Shian-ling; Wu, Pei-Ei; Shieh, Sheau-Yann; Shen, Chen-Yang

    2008-01-01

    The DNA damage response (DDR) has an essential function in maintaining genomic stability. Ataxia telangiectasia-mutated (ATM)-checkpoint kinase 2 (Chk2) and ATM- and Rad3-related (ATR)-Chk1, triggered, respectively, by DNA double-strand breaks and blocked replication forks, are two major DDRs processing structurally complicated DNA damage. In contrast, damage repaired by base excision repair (BER) is structurally simple, but whether, and how, the DDR is involved in repairing this damage is un...

  15. 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

    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 of p...... 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....

  16. 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

    Oxidative damage to DNA is thought to play a role in carcinogenesis by causing mutations, and indeed accumulation of oxidized DNA bases has been observed in samples obtained from tumors but not from surrounding tissue within the same patient. Base excision repair (BER) is the main pathway for the...

  17. 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 glyc

  18. 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

    mitochondria, whereas base excision repair (BER) has been comprehensively characterized in these organelles. The BER proteins are associated with the inner membrane in mitochondria and thus with the mitochondrial nucleoid, where TFAM is also situated. However, a function for TFAM in BER has not yet been...

  19. Vertebrate POLQ and POLβ Cooperate in Base Excision Repair of Oxidative DNA Damage

    OpenAIRE

    Yoshimura, Michio; Kohzaki, Masaoki; Nakamura, Jun; Asagoshi, Kenjiro; Sonoda, Eiichiro; Hou, Esther; Prasad, Rajendra; Wilson, Samuel H.; TANO, KEIZO; Yasui, Akira; Lan, Li; Seki, Mineaki; Wood, Richard D.; Arakawa, Hiroshi; Buerstedde, Jean-Marie

    2006-01-01

    Base excision repair (BER) plays an essential role in protecting cells from mutagenic base damage caused by oxidative stress, hydrolysis, and environmental factors. POLQ is a DNA polymerase, which appears to be involved in translesion DNA synthesis (TLS) past base damage. We disrupted POLQ, and its homologs HEL308 and POLN in chicken DT40 cells, and also created polq/hel308 and polq/poln double mutants. We found that POLQ-deficient mutants exhibit hypersensitivity to oxidative base damage ind...

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

    DEFF Research Database (Denmark)

    Knudsen, Nina Østergaard; Andersen, Sofie Dabros; Lützen, Anne; Nielsen, Finn Cilius; Rasmussen, Lene Juel

    2009-01-01

    DNA mutations are circumvented by dedicated specialized excision repair systems, such as the base excision repair (BER), nucleotide excision repair (NER), and mismatch repair (MMR) pathways. Although the individual repair pathways have distinct roles in suppressing changes in the nuclear DNA, it is...... co-import appears to be a mechanism employed by the composite repair systems NER and MMR to enhance and regulate nuclear accumulation of repair proteins thereby ensuring faithful DNA repair....

  1. XRCC1 and Base Excision Repair Balance in Response to Nitric Oxide

    OpenAIRE

    Mutamba, James T; Svilar, David; Prasongtanakij, Somsak; Wang, Xiao-hong; Lin, Ying-Chih; Dedon, Peter C; Sobol, Robert W.; Engelward, Bevin P.

    2011-01-01

    Inflammation associated reactive oxygen and nitrogen species (RONs), including peroxynitrite (ONOO−) and nitric oxide (NO· ), create base lesions that potentially play a role in the toxicity and large-scale genomic rearrangements associated with many malignancies. Nevertheless, little is known about the functional role of base excision repair (BER) deficiencies following exposure to RONs. Here, we explore the role of XRCC1 in modulating the levels of RONs-induced genotoxicity. XRCC1 is a scaf...

  2. Gut Microbiota Imbalance and Base Excision Repair Dynamics in Colon Cancer

    Science.gov (United States)

    Ray, Debolina; Kidane, Dawit

    2016-01-01

    Gut microbiota are required for host nutrition, energy balance, and regulating immune homeostasis, however, in some cases, this mutually beneficial relationship becomes twisted (dysbiosis), and the gut flora can incite pathological disorders including colon cancer. Microbial dysbiosis promotes the release of bacterial genotoxins, metabolites, and causes chronic inflammation, which promote oxidative DNA damage. Oxidized DNA base lesions are removed by base excision repair (BER), however, the role of this altered function of BER, as well as microbiota-mediated genomic instability and colon cancer development, is still poorly understood. In this review article, we will discuss how dysbiotic microbiota induce DNA damage, its impact on base excision repair capacity, the potential link of host BER gene polymorphism, and the risk of dysbiotic microbiota mediated genomic instability and colon cancer.

  3. Repair of DNA damage in mammalian cells after treatment with UV and dimethyl sulphate: discrimination between nucleotide and base excision repair by their temperature dependence.

    Science.gov (United States)

    Hjertvik, M; Erixon, K; Ahnström, G

    1998-03-01

    Alkylating agents have been reported to give rise to both short and long patches of repair. The reason for the different patch sizes is not known. One possibility is that alkylating agents can trigger both base and nucleotide excision repair. Another possibility is that base excision repair itself can result in different patch sizes. Recognition and incision at lesions is the rate limiting step in excision repair. In order to discriminate between base and nucleotide excision repair it would be desirable to be able to distinguish between different incision activities. In order to accurately measure incision rates, the rejoining of the strand-breaks formed must be inhibited. We have used two inhibitors, aphidicolin and 3-aminobenzamide. Aphidicolin, an inhibitor of DNA polymerases alpha/delta/epsilon. caused accumulation of single-strand breaks both after UV and dimethylsulphate. 3-Aminobenzamide, an inhibitor of poly(ADP-ribose)-polymerase caused accumulation of single-strand breaks only after alkylating agents and is thus specific for base excision repair. Enzymatic activities can be characterised by their activation energy. In order to discriminate between base and nucleotide excision repair the temperature dependence of incision activities was determined. When the temperature is decreased, the incision rate is reduced to a larger extent for UV than for DMS-induced repair. Incisions in UV-irradiated cells are practically cut off at temperatures of 15 degrees C and below, whereas DMS-exposed cells still are actively repairing at this temperature. In DMS treated cells the temperature dependence was the same whether aphidicolin or 3-aminobenzamide was used, speaking against an involvement of nucleotide excision repair. In addition, cell lines deficient in nucleotide excision repair responded in the same way to aphidicolin after DMS treatment as normal cells and were able to make incisions at 15 degrees C. This indicates that nucleotide excision repair is not to any

  4. Trypanosoma cruzi contains a single detectable uracil-DNA glycosylase and repairs uracil exclusively via short patch base excision repair

    DEFF Research Database (Denmark)

    Pena Diaz, Javier; Akbari, Mansour; Sundheim, Ottar; Farez-Vidal, M Esther; Andersen, Sonja; Sneve, Ragnhild; Gonzalez-Pacanowska, Dolores; Krokan, Hans E; Slupphaug, Geir

    2004-01-01

    Enzymes involved in genomic maintenance of human parasites are attractive targets for parasite-specific drugs. The parasitic protozoan Trypanosoma cruzi contains at least two enzymes involved in the protection against potentially mutagenic uracil, a deoxyuridine triphosphate nucleotidohydrolase (d......UTPase) and a uracil-DNA glycosylase belonging to the highly conserved UNG-family. Uracil-DNA glycosylase activities excise uracil from DNA and initiate a multistep base-excision repair (BER) pathway to restore the correct nucleotide sequence. Here we report the biochemical characterisation of T.cruzi UNG (Tc......UNG) and its contribution to the total uracil repair activity in T.cruzi. TcUNG is shown to be the major uracil-DNA glycosylase in T.cruzi. The purified recombinant TcUNG exhibits substrate preference for removal of uracil in the order ssU>U:G>U:A, and has no associated thymine-DNA glycosylase activity. T.cruzi...

  5. Initial steps of the base excision repair pathway within the nuclear architecture

    International Nuclear Information System (INIS)

    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)

  6. Mammalian Base Excision Repair: Functional Partnership between PARP-1 and APE1 in AP-Site Repair.

    Directory of Open Access Journals (Sweden)

    Rajendra Prasad

    Full Text Available The apurinic/apyrimidinic- (AP- site in genomic DNA arises through spontaneous base loss and base removal by DNA glycosylases and is considered an abundant DNA lesion in mammalian cells. The base excision repair (BER pathway repairs the AP-site lesion by excising and replacing the site with a normal nucleotide via template directed gap-filling DNA synthesis. The BER pathway is mediated by a specialized group of proteins, some of which can be found in multiprotein complexes in cultured mouse fibroblasts. Using a DNA polymerase (pol β immunoaffinity-capture technique to isolate such a complex, we identified five tightly associated and abundant BER factors in the complex: PARP-1, XRCC1, DNA ligase III, PNKP, and Tdp1. AP endonuclease 1 (APE1, however, was not present. Nevertheless, the complex was capable of BER activity, since repair was initiated by PARP-1's AP lyase strand incision activity. Addition of purified APE1 increased the BER activity of the pol β complex. Surprisingly, the pol β complex stimulated the strand incision activity of APE1. Our results suggested that PARP-1 was responsible for this effect, whereas other proteins in the complex had no effect on APE1 strand incision activity. Studies of purified PARP-1 and APE1 revealed that PARP-1 was able to stimulate APE1 strand incision activity. These results illustrate roles of PARP-1 in BER including a functional partnership with APE1.

  7. Distinct spatiotemporal patterns and PARP dependence of XRCC1 recruitment to single-strand break and base excision repair

    NARCIS (Netherlands)

    A. Campalans (Anna); R. Amouroux (Rachel); H. Menoni (Hervé); W. Vermeulen (Wim); J.P. Radicella (Pablo)

    2013-01-01

    textabstractSingle-strand break repair (SSBR) and base excision repair (BER) of modified bases and abasic sites share several players. Among them is XRCC1, an essential scaffold protein with no enzymatic activity, required for the coordination of both pathways. XRCC1 is recruited to SSBR by PARP-1,

  8. ALKBH1 is dispensable for abasic site cleavage during base excision repair and class switch recombination.

    Science.gov (United States)

    Müller, Tina A; Yu, Kefei; Hausinger, Robert P; Meek, Katheryn

    2013-01-01

    Potential roles of the abasic site lyase activity associated with AlkB homolog 1 (ALKBH1) were assessed by studies focusing on the two cellular processes that create abasic sites as intermediates: base excision repair and class switch recombination. Alkbh1(-/-) pups (lacking exon 3) were born at a lower than expected frequency from heterozygous parents, suggesting a reduced survival rate and non-Mendelian inheritance, and they exhibited a gender bias in favor of males (70% males and 30% females). To study ALKBH1's potential involvement in DNA repair, fibroblasts were isolated from Alkbh1(-/-) mice, spontaneously immortalized and tested for resistance to DNA damaging agents. Alkbh1(-/-) and isogenic cells expressing hALKBH1 showed no difference in survival to the DNA damaging agents methyl-methionine sulfate or H2O2. This result indicates that ALKBH1 does not play a major role in the base excision repair pathway. To assess ALKBH1's role in class switch recombination, splenic B cells were isolated from Alkbh1(-/-) and Alkbh1(+/+) mice and subjected to switching from IgM to IgG1. No differences were found in IgG1 switching, suggesting that Alkbh1 is not involved in class switch recombination of the immunoglobulin heavy chain during B lymphocyte activation. PMID:23825659

  9. ALKBH1 is dispensable for abasic site cleavage during base excision repair and class switch recombination.

    Directory of Open Access Journals (Sweden)

    Tina A Müller

    Full Text Available Potential roles of the abasic site lyase activity associated with AlkB homolog 1 (ALKBH1 were assessed by studies focusing on the two cellular processes that create abasic sites as intermediates: base excision repair and class switch recombination. Alkbh1(-/- pups (lacking exon 3 were born at a lower than expected frequency from heterozygous parents, suggesting a reduced survival rate and non-Mendelian inheritance, and they exhibited a gender bias in favor of males (70% males and 30% females. To study ALKBH1's potential involvement in DNA repair, fibroblasts were isolated from Alkbh1(-/- mice, spontaneously immortalized and tested for resistance to DNA damaging agents. Alkbh1(-/- and isogenic cells expressing hALKBH1 showed no difference in survival to the DNA damaging agents methyl-methionine sulfate or H2O2. This result indicates that ALKBH1 does not play a major role in the base excision repair pathway. To assess ALKBH1's role in class switch recombination, splenic B cells were isolated from Alkbh1(-/- and Alkbh1(+/+ mice and subjected to switching from IgM to IgG1. No differences were found in IgG1 switching, suggesting that Alkbh1 is not involved in class switch recombination of the immunoglobulin heavy chain during B lymphocyte activation.

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

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    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.

  11. Nucleotide excision repair in humans.

    Science.gov (United States)

    Spivak, Graciela

    2015-12-01

    The demonstration of DNA damage excision and repair replication by Setlow, Howard-Flanders, Hanawalt and their colleagues in the early 1960s, constituted the discovery of the ubiquitous pathway of nucleotide excision repair (NER). The serial steps in NER are similar in organisms from unicellular bacteria to complex mammals and plants, and involve recognition of lesions, adducts or structures that disrupt the DNA double helix, removal of a short oligonucleotide containing the offending lesion, synthesis of a repair patch copying the opposite undamaged strand, and ligation, to restore the DNA to its original form. The transcription-coupled repair (TCR) subpathway of NER, discovered nearly two decades later, is dedicated to the removal of lesions from the template DNA strands of actively transcribed genes. In this review I will outline the essential factors and complexes involved in NER in humans, and will comment on additional factors and metabolic processes that affect the efficiency of this important process. PMID:26388429

  12. On-bead fluorescent DNA nanoprobes to analyze base excision repair activities

    International Nuclear Information System (INIS)

    Graphical abstract: -- Highlights: •On magnetic beads fluorescent enzymatic assays. •Simple, easy, non-radioactive and electrophoresis-free functional assay. •Lesion-containing hairpin DNA probes are selective for repair enzymes. •The biosensing platform allows the measurement of DNA repair activities from purified enzymes or within cell free extracts. -- Abstract: DNA integrity is constantly threatened by endogenous and exogenous agents that can modify its physical and chemical structure. Changes in DNA sequence can cause mutations sparked by some genetic diseases or cancers. Organisms have developed efficient defense mechanisms able to specifically repair each kind of lesion (alkylation, oxidation, single or double strand break, mismatch, etc). Here we report the adjustment of an original assay to detect enzymes’ activity of base excision repair (BER), that supports a set of lesions including abasic sites, alkylation, oxidation or deamination products of bases. The biosensor is characterized by a set of fluorescent hairpin-shaped nucleic acid probes supported on magnetic beads, each containing a selective lesion targeting a specific BER enzyme. We have studied the DNA glycosylase alkyl-adenine glycosylase (AAG) and the human AP-endonuclease (APE1) by incorporating within the DNA probe a hypoxanthine lesion or an abasic site analog (tetrahydrofuran), respectively. Enzymatic repair activity induces the formation of a nick in the damaged strand, leading to probe's break, that is detected in the supernatant by fluorescence. The functional assay allows the measurement of DNA repair activities from purified enzymes or in cell-free extracts in a fast, specific, quantitative and sensitive way, using only 1 pmol of probe for a test. We recorded a detection limit of 1 μg mL−1 and 50 μg mL−1 of HeLa nuclear extracts for APE1 and AAG enzymes, respectively. Finally, the on-bead assay should be useful to screen inhibitors of DNA repair activities

  13. 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. PMID:27062014

  14. XRCC1 and base excision repair balance in response to nitric oxide.

    Science.gov (United States)

    Mutamba, James T; Svilar, David; Prasongtanakij, Somsak; Wang, Xiao-Hong; Lin, Ying-Chih; Dedon, Peter C; Sobol, Robert W; Engelward, Bevin P

    2011-12-10

    Inflammation associated reactive oxygen and nitrogen species (RONs), including peroxynitrite (ONOO(-)) and nitric oxide (NO), create base lesions that potentially play a role in the toxicity and large genomic rearrangements associated with many malignancies. Little is known about the role of base excision repair (BER) in removing these endogenous DNA lesions. Here, we explore the role of X-ray repair cross-complementing group 1 (XRCC1) in attenuating RONs-induced genotoxicity. XRCC1 is a scaffold protein critical for BER for which polymorphisms modulate the risk of cancer. We exploited CHO and human glioblastoma cell lines engineered to express varied levels of BER proteins to study XRCC1. Cytotoxicity and the levels of DNA repair intermediates (single-strand breaks; SSB) were evaluated following exposure of the cells to the ONOO(-) donor, SIN-1, and to gaseous NO. XRCC1 null cells were slightly more sensitive to SIN-1 than wild-type cells. We used small-scale bioreactors to expose cells to NO and found that XRCC1-deficient CHO cells were not sensitive. However, using a molecular beacon assay to test lesion removal in vitro, we found that XRCC1 facilitates AAG-initiated excision of two key NO-induced DNA lesions: 1,N(6)-ethenoadenine and hypoxanthine. Furthermore, overexpression of AAG rendered XRCC1-deficient cells sensitive to NO-induced DNA damage. These results show that AAG is a key glycosylase for BER of NO-induced DNA damage and that XRCC1's role in modulating sensitivity to RONs is dependent upon the cellular level of AAG. This demonstrates the importance of considering the expression of other components of the BER pathway when evaluating the impact of XRCC1 polymorphisms on cancer risk. PMID:22041025

  15. 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.

  16. 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.

  17. Pol β associated complex and base excision repair factors in mouse fibroblasts.

    Science.gov (United States)

    Prasad, Rajendra; Williams, Jason G; Hou, Esther W; Wilson, Samuel H

    2012-12-01

    During mammalian base excision repair (BER) of lesion-containing DNA, it is proposed that toxic strand-break intermediates generated throughout the pathway are sequestered and passed from one step to the next until repair is complete. This stepwise process is termed substrate channeling. A working model evaluated here is that a complex of BER factors may facilitate the BER process. FLAG-tagged DNA polymerase (pol) β was expressed in mouse fibroblasts carrying a deletion in the endogenous pol β gene, and the cell extract was subjected to an 'affinity-capture' procedure using anti-FLAG antibody. The pol β affinity-capture fraction (ACF) was found to contain several BER factors including polymerase-1, X-ray cross-complementing factor1-DNA ligase III and enzymes involved in processing 3'-blocked ends of BER intermediates, e.g. polynucleotide kinase and tyrosyl-DNA phosphodiesterase 1. In contrast, DNA glycosylases, apurinic/aprymidinic endonuclease 1 and flap endonuclease 1 and several other factors involved in BER were not present. Some of the BER factors in the pol β ACF were in a multi-protein complex as observed by sucrose gradient centrifugation. The pol β ACF was capable of substrate channeling for steps in vitro BER and was proficient in in vitro repair of substrates mimicking a 3'-blocked topoisomerase I covalent intermediate or an oxidative stress-induced 3'-blocked intermediate. PMID:23042675

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

    International Nuclear Information System (INIS)

    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

  19. 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.

  20. Nonhomologous end joining of complex DNA double-strand breaks with proximal thymine glycol and interplay with base excision repair.

    Science.gov (United States)

    Almohaini, Mohammed; Chalasani, Sri Lakshmi; Bafail, Duaa; Akopiants, Konstantin; Zhou, Tong; Yannone, Steven M; Ramsden, Dale A; Hartman, Matthew C T; Povirk, Lawrence F

    2016-05-01

    DNA double-strand breaks induced by ionizing radiation are often accompanied by ancillary oxidative base damage that may prevent or delay their repair. In order to better define the features that make some DSBs repair-resistant, XLF-dependent nonhomologous end joining of blunt-ended DSB substrates having the oxidatively modified nonplanar base thymine glycol at the first (Tg1), second (Tg2), third (Tg3) or fifth (Tg5) positions from one 3' terminus, was examined in human whole-cell extracts. Tg at the third position had little effect on end-joining even when present on both ends of the break. However, Tg as the terminal or penultimate base was a major barrier to end joining (>10-fold reduction in ligated products) and an absolute barrier when present at both ends. Dideoxy trapping of base excision repair intermediates indicated that Tg was excised from Tg1, Tg2 and Tg3 largely if not exclusively after DSB ligation. However, Tg was rapidly excised from the Tg5 substrate, resulting in a reduced level of DSB ligation, as well as slow concomitant resection of the opposite strand. Ligase reactions containing only purified Ku, XRCC4, ligase IV and XLF showed that ligation of Tg3 and Tg5 was efficient and only partially XLF-dependent, whereas ligation of Tg1 and Tg2 was inefficient and only detectable in the presence of XLF. Overall, the results suggest that promoting ligation of DSBs with proximal base damage may be an important function of XLF, but that Tg can still be a major impediment to repair, being relatively resistant to both trimming and ligation. Moreover, it appears that base excision repair of Tg can sometimes interfere with repair of DSBs that would otherwise be readily rejoined. PMID:27049455

  1. Measurement of DNA base and nucleotide excision repair activities in mammalian cells and tissues using the comet assay - A methodological overview

    Czech Academy of Sciences Publication Activity Database

    Azqueta, A.; Langie, S. A. S.; Slyšková, Jana; Collins, A. R.

    2013-01-01

    Roč. 12, č. 11 (2013), s. 1007-1010. ISSN 1568-7864 Grant ostatní: EU FP6(XE) LSHB-CT-2006-037575 Institutional support: RVO:68378041 Keywords : comet assay * base excision repair * nucleotide excision repair Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.362, year: 2013

  2. Nucleotide excision repair in the test tube.

    NARCIS (Netherlands)

    N.G.J. Jaspers (Nicolaas); J.H.J. Hoeijmakers (Jan)

    1995-01-01

    textabstractThe eukaryotic nucleotide excision-repair pathway has been reconstituted in vitro, an achievement that should hasten the full enzymological characterization of this highly complex DNA-repair pathway.

  3. Comparative assessment of plasmid and oligonucleotide DNA substrates in measurement of in vitro base excision repair activity

    OpenAIRE

    Hou, Esther W.; Prasad, Rajendra; Asagoshi, Kenjiro; Masaoka, Aya; Wilson, Samuel H.

    2007-01-01

    Mammalian base excision repair (BER) is mediated through at least two subpathways designated ‘single-nucleotide’ (SN) and ‘long-patch’ (LP) BER (2-nucleotides long/more repair patch). Two forms of DNA substrate are generally used for in vitro BER assays: oligonucleotide- and plasmid-based. For plasmid-based BER assays, the availability of large quantities of substrate DNA with a specific lesion remains the limiting factor. Using sequence-specific endonucleases that cleave only one strand of D...

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

    International Nuclear Information System (INIS)

    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

  5. 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.

  6. 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

    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...... 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...... 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...

  7. 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.

  8. Reprint of "Oxidant and environmental toxicant-induced effects compromise DNA ligation during base excision DNA repair".

    Science.gov (United States)

    Çağlayan, Melike; Wilson, Samuel H

    2015-12-01

    DNA lesions arise from many endogenous and environmental agents, and such lesions can promote deleterious events leading to genomic instability and cell death. Base excision repair (BER) is the main DNA repair pathway responsible for repairing single strand breaks, base lesions and abasic sites in mammalian cells. During BER, DNA substrates and repair intermediates are channeled from one step to the next in a sequential fashion so that release of toxic repair intermediates is minimized. This includes handoff of the product of gap-filling DNA synthesis to the DNA ligation step. The conformational differences in DNA polymerase β (pol β) associated with incorrect or oxidized nucleotide (8-oxodGMP) insertion could impact channeling of the repair intermediate to the final step of BER, i.e., DNA ligation by DNA ligase I or the DNA Ligase III/XRCC1 complex. Thus, modified DNA ligase substrates produced by faulty pol β gap-filling could impair coordination between pol β and DNA ligase. Ligation failure is associated with 5'-AMP addition to the repair intermediate and accumulation of strand breaks that could be more toxic than the initial DNA lesions. Here, we provide an overview of the consequences of ligation failure in the last step of BER. We also discuss DNA-end processing mechanisms that could play roles in reversal of impaired BER. PMID:26596511

  9. Differential modes of DNA binding by mismatch uracil DNA glycosylase from Escherichia coli: implications for abasic lesion processing and enzyme communication in the base excision repair pathway

    OpenAIRE

    Grippon, Seden; Zhao, Qiyuan; Robinson, Tom; Marshall, Jacqueline J. T.; O’Neill, Rory J.; Manning, Hugh; Kennedy, Gordon; Dunsby, Christopher; Neil, Mark; Halford, Stephen E.; French, Paul M. W.; Baldwin, Geoff S.

    2010-01-01

    Mismatch uracil DNA glycosylase (Mug) from Escherichia coli is an initiating enzyme in the base-excision repair pathway. As with other DNA glycosylases, the abasic product is potentially more harmful than the initial lesion. Since Mug is known to bind its product tightly, inhibiting enzyme turnover, understanding how Mug binds DNA is of significance when considering how Mug interacts with downstream enzymes in the base-excision repair pathway. We have demonstrated differential binding modes o...

  10. Mutants of the Base Excision Repair Glycosylase, Endonuclease III: DNA Charge Transport as a First Step in Lesion Detection

    OpenAIRE

    Romano, Christine A.; Sontz, Pamela A.; Barton, Jacqueline K.

    2011-01-01

    Endonuclease III (EndoIII) is a base excision repair glycosylase that targets damaged pyrimidines and contains a [4Fe-4S] cluster. We have proposed a model where BER proteins that contain redox-active [4Fe-4S] clusters utilize DNA charge transport (CT) as a first step in the detection of DNA lesions. Here, several mutants of EndoIII were prepared to probe their efficiency of DNA/protein charge transport. Cyclic voltammetry experiments on DNA-modified electrodes show that aromatic residues F30...

  11. Distinct spatio temporal patterns and PARP dependence of XRCC1 recruitment to single-strand break and base excision repair

    International Nuclear Information System (INIS)

    Single-strand break repair (SSBR) and base excision repair (BER) of modified bases and abasic sites share several players. Among them is XRCC1, an essential scaffold protein with no enzymatic activity, required for the coordination of both pathways. XRCC1 is recruited to SSBR by PARP-1, responsible for the initial recognition of the break. The recruitment of XRCC1 to BER is still poorly understood. Here we show by using both local and global induction of oxidative DNA base damage that XRCC1 participation in BER complexes can be distinguished from that in SSBR by several criteria. We show first that XRCC1 recruitment to BER is independent of PARP. Second, unlike SSBR complexes that are assembled within minutes after global damage induction, XRCC1 is detected later in BER patches, with kinetics consistent with the repair of oxidized bases. Third, while XRCC1-containing foci associated with SSBR are formed both in eu- and heterochromatin domains, BER complexes are assembled in patches that are essentially excluded from heterochromatin and where the oxidized bases are detected. (authors)

  12. Histone displacement during nucleotide excision repair

    DEFF Research Database (Denmark)

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

    2012-01-01

    Nucleotide excision repair (NER) is an important DNA repair mechanism required for cellular resistance against UV light and toxic chemicals such as those found in tobacco smoke. In living cells, NER efficiently detects and removes DNA lesions within the large nuclear macromolecular complex called......, thus allowing repair proteins to efficiently access DNA. On the other hand, after completion of the repair, the chromatin must be returned to its previous undamaged state. Chromatin remodeling can refer to three separate but interconnected processes, histone post-translational modifications, insertion...

  13. 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

  14. Nucleotide Excision Repair in Caenorhabditis elegans

    OpenAIRE

    Hannes Lans; Wim Vermeulen

    2011-01-01

    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 vi...

  15. 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.

  16. 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...... acetyltransferases is of key interest because of its potential importance in aging, DNA repair and transcription....

  17. 3CAPS – a structural AP–site analogue as a tool to investigate DNA base excision repair

    Science.gov (United States)

    Schuermann, David; Scheidegger, Simon P.; Weber, Alain R.; Bjørås, Magnar; Leumann, Christian J.; Schär, Primo

    2016-01-01

    Abasic sites (AP-sites) are frequent DNA lesions, arising by spontaneous base hydrolysis or as intermediates of base excision repair (BER). The hemiacetal at the anomeric centre renders them chemically reactive, which presents a challenge to biochemical and structural investigation. Chemically more stable AP-site analogues have been used to avoid spontaneous decay, but these do not fully recapitulate the features of natural AP–sites. With its 3′–phosphate replaced by methylene, the abasic site analogue 3CAPS was suggested to circumvent some of these limitations. Here, we evaluated the properties of 3CAPS in biochemical BER assays with mammalian proteins. 3CAPS-containing DNA substrates were processed by APE1, albeit with comparably poor efficiency. APE1-cleaved 3CAPS can be extended by DNA polymerase β but repaired only by strand displacement as the 5′–deoxyribophosphate (dRP) cannot be removed. DNA glycosylases physically and functionally interact with 3CAPS substrates, underlining its structural integrity and biochemical reactivity. The AP lyase activity of bifunctional DNA glycosylases (NTH1, NEIL1, FPG), however, was fully inhibited. Notably, 3CAPS-containing DNA also effectively inhibited the activity of bifunctional glycosylases on authentic substrates. Hence, the chemically stable 3CAPS with its preserved hemiacetal functionality is a potent tool for BER research and a potential inhibitor of bifunctional DNA glycosylases. PMID:26733580

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

    Czech Academy of Sciences Publication Activity Database

    Pardini, B.; Rosa, F.; Barone, E.; Di Gaetano, C.; Slyšková, Jana; Novotný, J.; Levý, M.; Garritano, S.; Vodičková, Ludmila; Buchler, T.; Gemignani, F.; Landi, S.; Vodička, Pavel; Naccarati, Alessio

    2013-01-01

    Roč. 19, č. 21 (2013), s. 6044-6056. ISSN 1078-0432 R&D Projects: GA ČR GAP304/10/1286; GA ČR(CZ) GAP304/12/1585 Institutional support: RVO:68378041 Keywords : colorectal cancer * base excision repair * survival Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 8.193, year: 2013

  19. 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...

  20. Functional, genetic and epigenetic aspects of base and nucleotide excision repair in colorectal carcinomas

    Czech Academy of Sciences Publication Activity Database

    Slyšková, Jana; Korenková, Vlasta; Collins, A. R.; Procházka, Pavel; Vodičková, Ludmila; Švec, Jiří; Lipská, L.; Levý, M.; Schneiderová, M.; Liška, V.; Holubec, L.; Kumar, R.; Souček, P.; Naccarati, Alessio; Vodička, Pavel

    2012-01-01

    Roč. 18, č. 21 (2012), s. 5878-5887. ISSN 1078-0432 R&D Projects: GA ČR GAP304/12/1585; GA ČR(CZ) GAP304/10/1286; GA MZd NT12025 Grant ostatní: UICC(XE) ICR/11/068/2011; EEA- research fund:(NO) B/CZ0046/40031 Institutional research plan: CEZ:AV0Z50390512 Institutional support: RVO:68378041 ; RVO:86652036 Keywords : DNA repair capacity * DNA repair gene expression * methylation Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 7.837, year: 2012

  1. DNA polymerase X from Deinococcus radiodurans implicated in bacterial tolerance to DNA damage is characterized as a short patch base excision repair polymerase.

    Science.gov (United States)

    Khairnar, Nivedita P; Misra, Hari S

    2009-09-01

    The Deinococcus radiodurans R1 genome encodes an X-family DNA repair polymerase homologous to eukaryotic DNA polymerase beta. The recombinant deinococcal polymerase X (PolX) purified from transgenic Escherichia coli showed deoxynucleotidyltransferase activity. Unlike the Klenow fragment of E. coli, this enzyme showed short patch DNA synthesis activity on heteropolymeric DNA substrate. The recombinant enzyme showed 5'-deoxyribose phosphate (5'-dRP) lyase activity and base excision repair function in vitro, with the help of externally supplied glycosylase and AP endonuclease functions. A polX disruption mutant of D. radiodurans expressing 5'-dRP lyase and a truncated polymerase domain was comparatively less sensitive to gamma-radiation than a polX deletion mutant. Both mutants showed higher sensitivity to hydrogen peroxide. Excision repair mutants of E. coli expressing this polymerase showed functional complementation of UV sensitivity. These results suggest the involvement of deinococcal polymerase X in DNA-damage tolerance of D. radiodurans, possibly by contributing to DNA double-strand break repair and base excision repair. PMID:19542005

  2. 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; Hart, Anne C

    2016-03-01

    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

  3. 碱基切除修复与抗肿瘤药物耐药%Base excision repair and antineoplastic drug resistance

    Institute of Scientific and Technical Information of China (English)

    况里杉; 王宇亮; 周向东

    2013-01-01

    Chemotherapy is one of the main methods to treat malignant tumors in clinical practice. Resistance to antineoplastic agents is one of the important reasons for treatment failure. The antineoplastic mechanism of various chemotherapeutic agents is to cause DNA damage, then result in apoptosis of tumor cells. It is suggested that the function of DNA repair is directly associated with the efficacy of antineoplastic agents. Current studies suggest that there are four major DNA repair pathways including BER (base excision repair), NER (nucleotide excision repair), MMR (mismatch repair) and DSBR (double strand break repair). Of these four pathways, BER is one of the main mechanisms of DNA repair and its malfunction is closely related to the resistance to antineoplastic agents. Recently, many kinds of agents and strategies targeting BER have been developed to reverse chemoresistance. This review summarizes the progress in research in this area and discusses the mechanism of resistance to antineoplastic agents and the potential preventive and therapeutic strategies.%化疗是目前临床上治疗肿瘤的主要方法之一,抗肿瘤药物耐药则是导致肿瘤治疗失败的重要原因之一.多种化疗药物抗肿瘤的主要机制是引起DNA损伤,进而导致肿瘤细胞凋亡;因此,DNA修复功能状态与抗肿瘤药物疗效有着直接的关系.目前,已知有4种主要的DNA修复途径:碱基切除修复(base excision repair,BER)、核苷酸切除修复(nucleotide excision repair,NER)、错配修复(mismatch repair,MMR)和双键断裂修复(double strand break repair,DSBR).其中,BER是主要的DNA修复机制之一,其修复功能异常与抗肿瘤药物耐药有着密切的联系.近年来,以BER为靶点开发了多种逆转耐药的药物或方法.本文将简要综述相关的研究进展,深入探讨抗肿瘤药物耐药的发生机制及防治措施.

  4. Regulation of nucleotide excision repair through ubiquitination

    Institute of Scientific and Technical Information of China (English)

    Jia Li; Audesh Bhat; Wei Xiao

    2011-01-01

    Nucleotide excision repair (NER) is the most versatile DNA-repair pathway in all organisms.While bacteria require only three proteins to complete the incision step of NER,eukaryotes employ about 30 proteins to complete the same step.Here we summarize recent studies demonstrating that ubiquitination,a post-translational modification,plays critical roles in regulating the NER activity either dependent on or independent of ubiquitin-proteolysis.Several NER components have been shown as targets of ubiquitination while others are actively involved in the ubiquitination process.We argue through this analysis that ubiquitination serves to coordinate various steps of NER and meanwhile connect NER with other related pathways to achieve the efficient global DNA-damage response.

  5. Recombinant methods for screening human DNA excision repair proficiency

    International Nuclear Information System (INIS)

    A method for measuring DNA excision repair in response to ultraviolet radiation (UV)-induced DNA damage has been developed, validated, and field-tested in cultured human lymphocytes. The methodology is amenable to population-based screening and should facilitate future epidemiologic studies seeking to investigate associations between excision repair proficiency and cancer susceptibility. The impetus for such endeavors derives from the belief that the high incidence of skin cancer in the genetic disorder xeroderma pigmentosum (XP) primarily is a result of the reduced capacity of patients cells to repair UV-induced DNA damage. For assay, UV-irradiated non-replicating recombinant plasmid DNA harboring a chloramphenicol acetyltransferase (CAT) indicator gene is introduced into lymphocytes using DEAE-dextran short-term transfection conditions. Exposure to UV induces transcriptionally-inactivating DNA photoproducts in the plasmid DNA which inactivate CAT gene expression. Excision repair of the damaged CAT gene is monitored indirectly as a function of reactivated CAT enzyme activity following a 40 hour repair/expression incubation period

  6. 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

  7. 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

  8. Poly(ADP-ribose) polymerase 1 regulates activity of DNA polymerase β in long patch base excision repair

    International Nuclear Information System (INIS)

    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 β (Pol β)-mediated long patch (LP) BER DNA synthesis that is accomplished through a cooperation between Pol β and apurinic/apyrimidinic endonuclease1 (APE1) or flap endonuclease 1 (FEN1) and gap-filling activity of Pol β. 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 β via strand-displacement mechanism. PARP1 also represses Pol β/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 β both via APE1-mediated strand-displacement and FEN1-mediated 'gap translation' mechanism. Thus PARP1 may act as a negative regulator of Pol β activity in LP BER pathway and poly(ADP-ribosyl)ation of PARP1 seems to play a critical role in enablement of Pol β-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 β and the overall efficiency of SP BER is practically unaffected by PARP1. Thus, PARP1 differentially influences DNA synthesis in SP- and

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

    International Nuclear Information System (INIS)

    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

  10. 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.

  11. Oxidative stress alters base excision repair pathway and increases apoptotic response in Apurinic/apyrimidinic endonuclease 1/Redox factor-1 haploinsufficient mice

    OpenAIRE

    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.

    2009-01-01

    Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is the redox regulator of multiple stress-inducible transcription factors, such as NF-κB, and the major 5’-endonuclease in base excision repair (BER). We utilized mice containing 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 re...

  12. Tissue-specific accelerated aging in nucleotide excision repair deficiency

    OpenAIRE

    Laura J. Niedernhofer

    2008-01-01

    Nucleotide excision repair (NER) is a multi-step DNA repair mechanism that removes helix-distorting modified nucleotides from the genome. NER is divided into two subpathways depending on the location of DNA damage in the genome and how it is first detected. Global genome NER identifies and repairs DNA lesions throughout the genome. This subpathway of NER primarily protects against the accumulation of mutations in the genome. Transcription-coupled (TC) NER rapidly repairs lesions in the transc...

  13. 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.

  14. 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. PMID:19268524

  15. Kin-cohort estimates for familial breast cancer risk in relation to variants in DNA base excision repair, BRCA1 interacting and growth factor genes

    International Nuclear Information System (INIS)

    Subtle functional deficiencies in highly conserved DNA repair or growth regulatory processes resulting from polymorphic variation may increase genetic susceptibility to breast cancer. Polymorphisms in DNA repair genes can impact protein function leading to genomic instability facilitated by growth stimulation and increased cancer risk. Thus, 19 single nucleotide polymorphisms (SNPs) in eight genes involved in base excision repair (XRCC1, APEX, POLD1), BRCA1 protein interaction (BRIP1, ZNF350, BRCA2), and growth regulation (TGFß1, IGFBP3) were evaluated. Genomic DNA samples were used in Taqman 5'-nuclease assays for most SNPs. Breast cancer risk to ages 50 and 70 were estimated using the kin-cohort method in which genotypes of relatives are inferred based on the known genotype of the index subject and Mendelian inheritance patterns. Family cancer history data was collected from a series of genotyped breast cancer cases (N = 748) identified within a cohort of female US radiologic technologists. Among 2,430 female first-degree relatives of cases, 190 breast cancers were reported. Genotypes associated with increased risk were: XRCC1 R194W (WW and RW vs. RR, cumulative risk up to age 70, risk ratio (RR) = 2.3; 95% CI 1.3–3.8); XRCC1 R399Q (QQ vs. RR, cumulative risk up to age 70, RR = 1.9; 1.1–3.9); and BRIP1 (or BACH1) P919S (SS vs. PP, cumulative risk up to age 50, RR = 6.9; 1.6–29.3). The risk for those heterozygous for BRCA2 N372H and APEX D148E were significantly lower than risks for homozygotes of either allele, and these were the only two results that remained significant after adjusting for multiple comparisons. No associations with breast cancer were observed for: APEX Q51H; XRCC1 R280H; IGFPB3 -202A>C; TGFß1 L10P, P25R, and T263I; BRCA2 N289H and T1915M; BRIP1 -64A>C; and ZNF350 (or ZBRK1) 1845C>T, L66P, R501S, and S472P. Some variants in genes within the base-excision repair pathway (XRCC1) and BRCA1 interacting proteins (BRIP1) may play a role

  16. POLYMORPHISMS IN THE DNA BASE EXCISION REPAIR GENES APEX1 AND XRCC1 AND LUNG CANCER RISK IN XUAN WEI, CHINA

    Science.gov (United States)

    The lung cancer mortality rate in Xuan Wei County is among the highest in China and has been attributed to exposure to indoor smoky coal emissions that contain very high levels of polycyclic aromatic hydrocarbons (PAHs). Nucleotide excision repair (NER) plays a key role in revers...

  17. Nucleotide excision repair by dual incisions in plants.

    Science.gov (United States)

    Canturk, Fazile; Karaman, Muhammet; Selby, Christopher P; Kemp, Michael G; Kulaksiz-Erkmen, Gulnihal; Hu, Jinchuan; Li, Wentao; Lindsey-Boltz, Laura A; Sancar, Aziz

    2016-04-26

    Plants use light for photosynthesis and for various signaling purposes. The UV wavelengths in sunlight also introduce DNA damage in the form of cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts [(6-4)PPs] that must be repaired for the survival of the plant. Genome sequencing has revealed the presence of genes for both CPD and (6-4)PP photolyases, as well as genes for nucleotide excision repair in plants, such as Arabidopsis and rice. Plant photolyases have been purified, characterized, and have been shown to play an important role in plant survival. In contrast, even though nucleotide excision repair gene homologs have been found in plants, the mechanism of nucleotide excision repair has not been investigated. Here we used the in vivo excision repair assay developed in our laboratory to demonstrate that Arabidopsis removes CPDs and (6-4)PPs by a dual-incision mechanism that is essentially identical to the mechanism of dual incisions in humans and other eukaryotes, in which oligonucleotides with a mean length of 26-27 nucleotides are removed by incising ∼20 phosphodiester bonds 5' and 5 phosphodiester bonds 3' to the photoproduct. PMID:27071131

  18. Polymorphisms in base excision DNA repair genes and association with melanoma risk in a pilot study on Central-South Italian population.

    Science.gov (United States)

    Santonocito, Concetta; Scapaticci, Margherita; Penitente, Romina; Paradisi, Andrea; Capizzi, Rodolfo; Lanza-Silveri, Sara; Ficarra, Silvana; Landi, Francesco; Zuppi, Cecilia; Capoluongo, Ettore

    2012-10-01

    Base excision repair plays a key role in the removing of DNA damage from exposure to endogenous and exogenous carcinogens. The BER pathway removes alterations of a single oxidized, reduced or methylated base. Recently some studies have explored the association between risk for cutaneous melanoma and non-synonymous single-nucleotide polymorphisms (nsSNPs) in DNA-repair genes, although with contradictory results. We hypothesized that common nsSNPs of BER genes, specifically ADPRT rs1136410, XRCC1 rs25487, rs25489, rs1799782, APEX1 rs1130409, OGG1 rs1052133, LIG3 rs3136025 and MUTYH rs3219466, may contribute to risk of melanoma. The aim of this study is to investigate whether or not a correlation between these nsSNPs and melanoma risk and/or aggressiveness is present. 167 melanoma patients and 186 healthy control subjects were analysed. By multivariate statistical analysis no association was found between nsSNP and melanoma aggressiveness, while only the two XRCC1 (rs25487 and rs25489) nsSNPs showed a strong correlation (p<0.001) with melanoma risk. To our knowledge this is the first study reporting an association between BER nsSNPs and melanoma risk in Central-South Italian individuals. Our findings, if confirmed in larger population studies, will allow the inclusion of these XRCC1 nsSNPs in a screening panel for those individuals at higher risk for melanoma. PMID:22687647

  19. 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

    International Nuclear Information System (INIS)

    Highlights: • We examine effect of volume of extraction buffer relative to volume of isolated nuclei on repair activity of nuclear extract. • Base excision repair activity of nuclear extracts prepared from the same batch and number of nuclei varies inversely with the volume of nuclear extraction buffer. • Effect of the volume of extraction buffer on BER activity of nuclear extracts can only be partially reversed after concentration of the more diluted extract by ultrafiltration. - Abstract: 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, candidate proteins in extracts can be inhibited or depleted in a controlled way, making defined extracts an important source for mechanistic studies. The major drawback is that there is no standardized method of preparing nuclear extract for BER studies, and it does not appear to be a topic given much 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 extraction buffer relative to nuclei volume, in spite of identical protein concentrations in the BER assay mixture. Surprisingly, the uracil–DNA glycosylase activity (mainly UNG2), but not amount of UNG2, also correlated negatively with the volume of extraction buffer. These studies demonstrate

  20. Both base excision repair and O6 -methylguanine-DNA methyltransferase protect against methylation-induced colon carcinogenesis

    OpenAIRE

    Wirtz, Stefan; Nagel, Georg; Eshkind, Leonid; Neurath, Markus F; Samson, Leona D.; Kaina, Bernd

    2010-01-01

    Methylating agents are widely distributed environmental carcinogens. Moreover, they are being used in cancer chemotherapy. The primary target of methylating agents is DNA, and therefore, DNA repair is the first-line barrier in defense against their toxic and carcinogenic effects. Methylating agents induce in the DNA O[superscript 6]-methylguanine (O[superscript 6]MeG) and methylations of the ring nitrogens of purines. The lesions are repaired by O[superscript 6]-methylguanine-DNA methyltransf...

  1. 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.

  2. 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

    Increasing evidence suggests that oxidative DNA damage accumulates in atherosclerosis. Recently, we showed that a genetic variant in the human DNA repair enzyme NEIL3 was associated with increased risk of myocardial infarction. Here, we explored the role of Neil3/NEIL3 in atherogenesis by both cl...

  3. 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

    cells both had capacity for single- and two-nucleotide insertion BER activity. However, patch size longer than two nucleotides was only detected in extracts from proliferating cells. Relative to extracts from proliferating cells, extracts from non-proliferating cells had approximately two-fold higher...... concentration of POLbeta, which contributed to most of two-nucleotide insertion BER. In contrast, two-nucleotide insertion in extracts from proliferating cells was not dependent on POLbeta. BER fidelity was two- to three-fold lower in extracts from the non-proliferating compared with extracts of proliferating...... cells. Furthermore, although one-nucleotide deletion was the predominant type of repair error in both extracts, the pattern of repair errors was somewhat different. These results establish two-nucleotide patch BER as a distinct POLbeta-dependent mechanism in non-proliferating cells and demonstrate that...

  4. 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)

    José Carlos P. De Mattos

    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.

  5. Correlation between base-excision repair gene polymorphisms and levels of in-vitro BPDE-induced DNA adducts in cultured peripheral blood lymphocytes.

    Directory of Open Access Journals (Sweden)

    Hongping Yu

    Full Text Available In vitro benzo[a]pyrene diol epoxide (BPDE-induced DNA adducts in cultured peripheral lymphocytes have been shown to be a phenotypic biomarker of individual's DNA repair phenotype that is associated with cancer risk. In this study, we explored associations between genotypes of base-excision repair genes (PARP1 Val762Ala, APEX1 Asp148Glu, and XRCC1 Arg399Gln and in vitro BPDE-induced DNA adducts in cultured peripheral blood lymphocytes in 706 cancer-free non-Hispanic white subjects. We found that levels of BPDE-induced DNA adducts were significantly higher in ever smokers than in never smokers and that individuals with the Glu variant genotypes (i.e., Asp/Glu and Glu/Glu exhibited lower levels of BPDE-induced DNA adducts than did individuals with the common Asp/Asp homozygous genotype (median RAL levels: 32.0 for Asp/Asp, 27.0 for Asp/Glu, and 17.0 for Glu/Glu, respectively; P(trend = 0.030. Further stratified analysis showed that compared with individuals with the common APEX1-148 homozygous Asp/Asp genotype, individuals with the APEX1-148Asp/Glu genotype or the Glu/Glu genotype had a lower risk of having higher-level adducts (adjusted OR = 0.60, 95% CI: 0.36-0.98 and adjusted OR = 0.47, 95% CI: 0.26-0.86, respectively; P(trend = 0.012 among smokers. Such an effect was not observed in non-smokers. However, there was no significant interaction between the APEX1 Asp148Glu polymorphism and smoking exposure in this study population (P = 0.512. Additional genotype-phenotype analysis found that the APEX1-148Glu allele had significantly increased expression of APEX1 mRNA in 270 Epstein-Barr virus-transformed lymphoblastoid cell lines, which is likely associated with more active repair activity. Our findings suggest that the functional APEX1-148Glu allele is associated with reduced risk of having high levels of BPDE-induced DNA adducts mediated with high levels of mRNA expression.

  6. Single-nucleotide polymorphisms in base excision repair, nucleotide excision repair, and double strand break genes as markers for response to radiotherapy in patients with Stage I to II head-and-neck cancer

    International Nuclear Information System (INIS)

    Purpose: Polymorphisms in DNA repair genes can influence response to radiotherapy. We analyzed single-nucleotide polymorphisms (SNP) in nine DNA repair genes in 108 patients with head-and-neck cancer (HNSCC) who had received radiotherapy only. Methods and Materials: From May 1993 to December 2004, patients with Stage I and II histopathologically confirmed HNSCC underwent radiotherapy. DNA was obtained from paraffin-embedded tissue, and SNP analysis was performed using a real-time polymerase chain reaction allelic discrimination TaqMan assay with minor modifications. Results: Patients were 101 men (93.5%) and 7 (6.5%) women, with a median age of 64 years (range, 40 to 89 years). Of the patients, 76 (70.4%) patients were Stage I and 32 (29.6%) were Stage II. The XPF/ERCC1 SNP at codon 259 and XPG/ERCC5 at codon 46 emerged as significant predictors of progression (p 0.00005 and 0.049, respectively) and survival (p = 0.0089 and 0.0066, respectively). Similarly, when variant alleles of XPF/ERCC1, XPG/ERCC5 and XPA were examined in combination, a greater number of variant alleles was associated with shorter time to progression (p = 0.0003) and survival (p 0.0002). Conclusions: Genetic polymorphisms in XPF/ERCC1, XPG/ERCC5, and XPA may significantly influence response to radiotherapy; large studies are warranted to confirm their role in HNSCC

  7. 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.

  8. Effect of the uvrD mutation on excision repair

    International Nuclear Information System (INIS)

    A pair of related Escherichia coli K-12 strains, one of which contains the uvrD101 mutation, were constructed and compared for ability to perform various steps in the excision repair of deoxyribonucleic acid damage inflicted by ultraviolet radiation. The results of this study indicated: (i) ultraviolet sensitivity in the uvrD101 mutant was greater than that of wild type but less than that measured in an incision-deficient uvrA mutant; (ii) host cell reactivation paralleled the survival data; (iii) postirradiation deoxyribonucleic acid degradation was virtually identical in the two strains; (iv) incision, presumably at the sites of pyrimidine dimers, proceeded normally in the uvrD101 strain; (v) excision of pyrimidine dimers was markedly reduced in both rate and extent in the uvrD101 mutant; (vi) the amount of repair resynthesis was the same in both strains, and there was no evidence of abnormally long repair patches in the uvrD mutant; and (vii) rejoining of incision breaks was slow and incomplete in the uvrD strain. These data suggest that the ultraviolet sensitivity conferred by the uvrD mutation arises from inefficient removal of pyrimidine dimers or from failure to close incision breaks. The data are compatible with the notion that the uvrD+ gene product affects the conformation of incised deoxyribonucleic acid molecules

  9. Effect of the uvrD mutation on excision repair

    Energy Technology Data Exchange (ETDEWEB)

    Kuemmerle, N.B.; Masker, W.E.

    1980-05-01

    A pair of related Escherichia coli K-12 strains, one of which contains the uvrD101 mutation, were constructed and compared for ability to perform various steps in the excision repair of deoxyribonucleic acid damage inflicted by ultraviolet radiation. The results of this study indicated: (i) ultraviolet sensitivity in the uvrD101 mutant was greater than that of wild type but less than that measured in an incision-deficient uvrA mutant; (ii) host cell reactivation paralleled the survival data; (iii) postirradiation deoxyribonucleic acid degradation was virtually identical in the two strains; (iv) incision, presumably at the sites of pyrimidine dimers, proceeded normally in the uvrD101 strain; (v) excision of pyrimidine dimers was markedly reduced in both rate and extent in the uvrD101 mutant; (vi) the amount of repair resynthesis was the same in both strains, and there was no evidence of abnormally long repair patches in the uvrD mutant; and (vii) rejoining of incision breaks was slow and incomplete in the uvrD strain. These data suggest that the ultraviolet sensitivity conferred by the uvrD mutation arises from inefficient removal of pyrimidine dimers or from failure to close incision breaks. The data are compatible with the notion that the uvrD+ gene product affects the conformation of incised deoxyribonucleic acid molecules.

  10. Evidence implying DNA polymerase beta function in excision repair.

    OpenAIRE

    Siedlecki, J A; Szyszko, J.; Pietrzykowska, I; Zmudzka, B

    1980-01-01

    Comparison was made of the ability of calf thymus DNA polymerases alpha and beta to replicate the following templates: native E. coli CR-34 DNA (T-DNA), calf thymus DNA activated by DNase I (act.DNA), BU-DNA (from E. coli CR-34 cells cultured on BUdR-containing medium) with damages resulting from incomplete excision repair, as well as thermally denatured act.DNA and BU-DNA (s.s.act.DNA and s.s.BU-DNA). 3H-TTP incorporation during extensive replication of act.DNA was similar for both enzymes, ...

  11. Effect of the uvrD mutation on excision repair.

    OpenAIRE

    Kuemmerle, N B; Masker, W E

    1980-01-01

    A pair of related Escherichia coli K-12 strains, one of which contains the uvrD101 mutation, were constructed and compared for ability to perform various steps in the excision repair of deoxyribonucleic acid damage inflicted by ultraviolet radiation. The results of this study indicated: (i) ultraviolet sensitivity in the uvrD101 mutant was greater than that of wild type but less than that measured in an incision-deficient uvrA mutant; (ii) host cell reactivation paralleled the survival data; ...

  12. Mechanisms of DNA Repair by Photolyase and Excision Nuclease (Nobel Lecture).

    Science.gov (United States)

    Sancar, Aziz

    2016-07-18

    Ultraviolet light damages DNA by converting two adjacent thymines into a thymine dimer which is potentially mutagenic, carcinogenic, or lethal to the organism. This damage is repaired by photolyase and the nucleotide excision repair system in E. coli by nucleotide excision repair in humans. The work leading to these results is presented by Aziz Sancar in his Nobel Lecture. PMID:27337655

  13. Eukaryotic nucleotide excision repair: from understanding mechanisms to influencing biology

    Institute of Scientific and Technical Information of China (English)

    Sarah C Shuck; Emily A Short; John J Turchi

    2008-01-01

    Repair of bulky DNA adducts by the nucleotide excision repair (NER) pathway is one of the more versatile DNA repair pathways for the removal of DNA lesions. There are two subsets of the NER pathway, global genomic-NER (GG-NER) and transcription-coupled NER (TC-NER), which differ only in the step involving recognition of the DNA lesion. Following recognition of the damage, the sub-pathways then converge for the incision/excision steps and subsequent gap filling and ligation steps. This review will focus on the GGR sub-pathway of NER while the TCR sub-pathway will be covered in another article in this issue. The ability of the NER pathway to repair a wide array of adducts stems, in part, from the mechanisms involved in the initial recognition step of the damaged DNA and results in NER impacting an equally wide array of human physiological responses and events. In this review, the impact of NER on carcinogenesis, neurological function, sensitivity to environmental factors and sensitivity to cancer therapeutics will be discussed. The knowledge generated in our understanding of the NER pathway over the past 40 years has resulted from advances in the fields of animal model systems, mammalian genetics and in vitro biochemistry, as well as from reconstitution studies and structural analyses of the proteins and enzymes that participate in this pathway. Each of these avenues of research has contributed significantly to our understanding of how the NER pathway works and how alterations in NER activity, both positive and negative, influence human biology.

  14. Low-dose formaldehyde delays DNA damage recognition and DNA excision repair in human cells.

    Directory of Open Access Journals (Sweden)

    Andreas Luch

    Full Text Available 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 (<100 μM 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.

  15. Human DNA mismatch repair: coupling of mismatch recognition to strand-specific excision

    OpenAIRE

    WANG Huixian; Hays, John B.

    2007-01-01

    Eukaryotic mismatch-repair (MMR) proteins MutSα and MutLα couple recognition of base mismatches to strand-specific excision, initiated in vivo at growing 3′ ends and 5′ Okazaki-fragment ends or, in human nuclear extracts, at nicks in exogenous circular substrates. We addressed five biochemical questions relevant to coupling models. Excision remained fully efficient at DNA:MutSα ratios of nearly 1 to 1 at various mismatch-nick distances, suggesting a requirement for only one MutSα molecule per...

  16. Global-genome Nucleotide Excision Repair Controlled by Ubiquitin/Sumo Modifiers

    Directory of Open Access Journals (Sweden)

    Peter eRuethemann

    2016-04-01

    Full Text Available Global-genome nucleotide excision repair (GG-NER prevents genome instability by excising a wide range of structurally unrelated DNA base adducts and crosslinks induced by chemical carcinogens, ultraviolet (UV radiation or intracellular metabolic by-products. As a versatile damage sensor, xeroderma pigmentosum group C (XPC protein initiates this generic defense reaction by locating the damage and recruiting the subunits of a large lesion demarcation complex that, in turn, triggers the excision of aberrant DNA by endonucleases. In the very special case of a DNA repair response to UV radiation, the function of this XPC initiator is tightly controlled by the dual action of cullin-type CRL4DDB2 and sumo-targeted RNF111 ubiquitin ligases. This twofold protein ubiquitination system promotes GG-NER reactions by spatially and temporally regulating the interaction of XPC protein with damaged DNA across the nucleosome landscape of chromatin. In the absence of either CRL4DDB2 or RNF111, the DNA excision repair of UV lesions is inefficient, indicating that these two ubiquitin ligases play a critical role in mitigating the adverse biological effects of UV light in the exposed skin.

  17. Global-genome Nucleotide Excision Repair Controlled by Ubiquitin/Sumo Modifiers.

    Science.gov (United States)

    Rüthemann, Peter; Balbo Pogliano, Chiara; Naegeli, Hanspeter

    2016-01-01

    Global-genome nucleotide excision repair (GG-NER) prevents genome instability by excising a wide range of different DNA base adducts and crosslinks induced by chemical carcinogens, ultraviolet (UV) light or intracellular side products of metabolism. As a versatile damage sensor, xeroderma pigmentosum group C (XPC) protein initiates this generic defense reaction by locating the damage and recruiting the subunits of a large lesion demarcation complex that, in turn, triggers the excision of aberrant DNA by endonucleases. In the very special case of a DNA repair response to UV radiation, the function of this XPC initiator is tightly controlled by the dual action of cullin-type CRL4(DDB2) and sumo-targeted RNF111 ubiquitin ligases. This twofold protein ubiquitination system promotes GG-NER reactions by spatially and temporally regulating the interaction of XPC protein with damaged DNA across the nucleosome landscape of chromatin. In the absence of either CRL4(DDB2) or RNF111, the DNA excision repair of UV lesions is inefficient, indicating that these two ubiquitin ligases play a critical role in mitigating the adverse biological effects of UV light in the exposed skin. PMID:27200078

  18. Cdt2-mediated XPG degradation promotes gap-filling DNA synthesis in nucleotide excision repair

    OpenAIRE

    Han, Chunhua; Wani, Gulzar; Zhao, Ran; Qian, Jiang; Sharma, Nidhi; He, Jinshan; Zhu, Qianzheng; Wang, Qi-En; Wani, Altaf A.

    2014-01-01

    Xeroderma pigmentosum group G (XPG) protein is a structure-specific repair endonuclease, which cleaves DNA strands on the 3′ side of the DNA damage during nucleotide excision repair (NER). XPG also plays a crucial role in initiating DNA repair synthesis through recruitment of PCNA to the repair sites. However, the fate of XPG protein subsequent to the excision of DNA damage has remained unresolved. Here, we show that XPG, following its action on bulky lesions resulting from exposures to UV ir...

  19. Nucleotide-excision repair of DNA in cell-free extracts of the yeast Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    A wide spectrum of DNA lesions are repaired by the nucleotide-excision repair (NER) pathway in both eukaryotic and prokaryotic cells. We have developed a cell-free system in Saccharomyces cerevisiae that supports NER. NER was monitored by measuring repair synthesis in DNA treated with cisplatin or with UV radiation. Repair synthesis in vitro was defective in extracts of rad1, rad2, and rad10 mutant cells, all of which have mutations in genes whose products are known to be required for NER in vivo. Additionally, repair synthesis was complemented by mixing different mutant extracts, or by adding purified Rad1 or Rad10 protein to rad1 or rad10 mutant extracts, respectively. The latter observation demonstrates that the Rad1 and Rad10 proteins directly participate in the biochemical pathway of NER. NER supported by nuclear extracts requires ATP and Mg2+ and is stimulated by polyethylene glycol and by small amounts of whole cell extract containing overexpressed Rad2 protein. The nuclear extracts also contain base-excision repair activity that is present at wild-type levels in rad mutant extracts. This cell-free system is expected to facilitate studies on the biochemical pathway of NER in S. cerevisiae

  20. Slow base excision by human alkyladenine DNA glycosylase limits the rate of formation of AP sites and AP endonuclease 1 does not stimulate base excision.

    Science.gov (United States)

    Maher, Robyn L; Vallur, Aarthy C; Feller, Joyce A; Bloom, Linda B

    2007-01-01

    The base excision repair pathway removes damaged DNA bases and resynthesizes DNA to replace the damage. Human alkyladenine DNA glycosylase (AAG) is one of several damage-specific DNA glycosylases that recognizes and excises damaged DNA bases. AAG removes primarily damaged adenine residues. Human AP endonuclease 1 (APE1) recognizes AP sites produced by DNA glycosylases and incises the phophodiester bond 5' to the damaged site. The repair process is completed by a DNA polymerase and DNA ligase. If not tightly coordinated, base excision repair could generate intermediates that are more deleterious to the cell than the initial DNA damage. The kinetics of AAG-catalyzed excision of two damaged bases, hypoxanthine and 1,N6-ethenoadenine, were measured in the presence and absence of APE1 to investigate the mechanism by which the base excision activity of AAG is coordinated with the AP incision activity of APE1. 1,N6-ethenoadenine is excised significantly slower than hypoxanthine and the rate of excision is not affected by APE1. The excision of hypoxanthine is inhibited to a small degree by accumulated product, and APE1 stimulates multiple turnovers by alleviating product inhibition. These results show that APE1 does not significantly affect the kinetics of base excision by AAG. It is likely that slow excision by AAG limits the rate of AP site formation in vivo such that AP sites are not created faster than can be processed by APE1. PMID:17018265

  1. Excision repair of 5,6-dihydroxydihydrothymine from the DNA of Micrococcus radiodurans

    International Nuclear Information System (INIS)

    One of the major ionizing radiation products, 5,6-dihydroxydihydrothymine (thymine glycol), was measured in the DNA of Micrococcus radiodurans following exposure of cells to 6.8-MeV electrons or 254-nm ultraviolet light. Removal of 5,6-dihydroxydihydrothymine was measured in both an ionizing radiation-sensitive strain (262) and a highly radioresistant strain (the wild type W+) of Micrococcus radiodurans. Within 30 min of incubation (330C) following exposure to ultraviolet light (2400 J/m2) approximately 60% of the thymine glycols were excised, whereas in the case of ionizing radiation (250 krad) only 35% were removed from the cellular DNA of the wild-type strain. In contrast less than 50% of the thymine glycols were excised from the sensitive strain. The amount of DNA degradation induced by radiation was less than 10% in both strains. The results suggest a possible correlation between reduced excision repair of base damage and increased radiation sensitivity

  2. Excision repair in MUT-mutants of Proteus mirabilis after UV-irradiation

    International Nuclear Information System (INIS)

    The behaviour of MUT-mutants of P.mirabilis to perform certain steps of excision repair after U.V.-irradiation is described. MUT-mutants introduce single-strand breaks in the DNA immediately after U.V.-irradiation, but their ability to excise pyrimidine dimers from the DNA is very diminished. Moreover, they are not able to accomplish the excision repair by rejoining of the single-strand breaks. The connection between the incomplete excision repair and the mutator phenotype of these mutants is discussed. (author)

  3. Repair of triplex-directed DNA alkylation by nucleotide excision repair

    OpenAIRE

    Ziemba, Amy; Derosier, L. Chris; Methvin, Russell; Song, Chun-Yan; Clary, Eric; Kahn, Wendy; Milesi, David; Gorn, Vladimir; Reed, Mike; Ebbinghaus, Scot

    2001-01-01

    Triplex-forming oligonucleotides (TFOs) are being investigated as highly specific DNA binding agents to inhibit the expression of clinically relevant genes. So far, they have been shown to inhibit transcription from the HER-2/neu gene in vitro, whereas their use in vivo has been studied to a limited extent. This study uses a TFO–chlorambucil (chl) conjugate capable of forming site-specific covalent guanine adducts within the HER-2/neu promoter. We demonstrate that nucleotide excision repair (...

  4. 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. A...

  5. Excision repair and patch size in UV-irradiated bacteriophage T4.

    OpenAIRE

    Yarosh, D B; Rosenstein, B S; Setlow, R B

    1981-01-01

    We determined the average size of excision repair patches in repair of UV lesions in bacteriophage T4 by measuring the photolysis of bromodeoxyuridine incorporated during repair. The average patch was small, approximately four nucleotides long. In control experiments with the denV1 excision-deficient mutant, we encountered an artifact, a protein(s) which remained bound to phenol-extracted DNA and prevented nicking by the UV-specific endonucleases of Micrococcus luteus and bacteriophage T4.

  6. Excision repair cross complementation group 1 is a chemotherapy-tolerating gene in cisplatin-based treatment for non-small cell lung cancer.

    Science.gov (United States)

    Wang, Shoufeng; Pan, Hong; Liu, Desen; Mao, Naiquan; Zuo, Chuantian; Li, Li; Xie, Tong; Huang, Dingming; Huang, Yaoyuan; Pan, Qi; Yang, Li; Wu, Junwei

    2015-02-01

    This study aimed to evaluate the biological functions of excision repair cross complementation goup 1 (ERCC1) in cell proliferation, cell cycle, invasion and cisplatin response of non-small cell lung cancer (NSCLC) cells. Firstly, ERCC1 gene was successfully transfected into H1299 cells by gene cloning and transfection techniques. Then, cell proliferation was determined with the cell growth curve and colony-forming assays. Flow cytometry (FCM) was employed to investigate the cell cycle distribution. The ability of cell invasion was estimated by means of Matrigel invasion assays. Response of NSCLC cells to cisplatin was detected utilizing MTT assays, and the intracellular drug concentrations were determined by the high performance liquid chromatography (HPLC) analysis. Expression of the two cell membrane proteins, P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP), was also evaluated utilizing FCM technique. By contrast, ERCC1 expression in the NSCLC A549 cells was silenced by small interfering RNA (siRNA) through RNAi technique. In addition, the cytotoxic effect of cisplatin on A549 cells was detected by MTT assays. In the present study, the results demonstrated that ERCC1 had no effect on cell proliferation, cell cycle and the ability of invasion, but showed significant impact on cisplatin response of the NSCLC H1299 cells. Furthermore, siRNA-induced suppression of ERCC1 evidently enhanced sensitivity to cisplatin of NSCLC A549 cells. Therefore, it is confirmed that ERCC1 is a chemotherapy-tolerating gene and a promising predictor in tailoring chemotherapy of NSCLC. PMID:25434755

  7. Reconstitution of DNA base excision-repair with purified human proteins: interaction between DNA polymerase beta and the XRCC1 protein.

    OpenAIRE

    Kubota, Y; Nash, R. A.; Klungland, A; Schär, P; Barnes, D E.; Lindahl, T

    1996-01-01

    Repair of a uracil-guanine base pair in DNA has been reconstituted with the recombinant human proteins uracil-DNA glycosylase, apurinic/apyrimidinic endonuclease, DNA polymerase beta and DNA ligase III. The XRCC1 protein, which is known to bind DNA ligase III, is not absolutely required for the reaction but suppresses strand displacement by DNA polymerase beta, allowing for more efficient ligation after filling of a single nucleotide patch. We show that XRCC1 interacts directly with DNA polym...

  8. Resistance to Nucleotide Excision Repair of Bulky Guanine Adducts Opposite Abasic Sites in DNA Duplexes and Relationships between Structure and Function.

    Directory of Open Access Journals (Sweden)

    Zhi Liu

    Full Text Available The nucleotide excision repair of certain bulky DNA lesions is abrogated in some specific non-canonical DNA base sequence contexts, while the removal of the same lesions by the nucleotide excision repair mechanism is efficient in duplexes in which all base pairs are complementary. Here we show that the nucleotide excision repair activity in human cell extracts is moderate-to-high in the case of two stereoisomeric DNA lesions derived from the pro-carcinogen benzo[a]pyrene (cis- and trans-B[a]P-N2-dG adducts in a normal DNA duplex. By contrast, the nucleotide excision repair activity is completely abrogated when the canonical cytosine base opposite the B[a]P-dG adducts is replaced by an abasic site in duplex DNA. However, base excision repair of the abasic site persists. In order to understand the structural origins of these striking phenomena, we used NMR and molecular spectroscopy techniques to evaluate the conformational features of 11mer DNA duplexes containing these B[a]P-dG lesions opposite abasic sites. Our results show that in these duplexes containing the clustered lesions, both B[a]P-dG adducts adopt base-displaced intercalated conformations, with the B[a]P aromatic rings intercalated into the DNA helix. To explain the persistence of base excision repair in the face of the opposed bulky B[a]P ring system, molecular modeling results suggest how the APE1 base excision repair endonuclease, that excises abasic lesions, can bind productively even with the trans-B[a]P-dG positioned opposite the abasic site. We hypothesize that the nucleotide excision repair resistance is fostered by local B[a]P residue-DNA base stacking interactions at the abasic sites, that are facilitated by the absence of the cytosine partner base in the complementary strand. More broadly, this study sets the stage for elucidating the interplay between base excision and nucleotide excision repair in processing different types of clustered DNA lesions that are substrates of

  9. 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.

  10. 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

    2009-07-01

    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 MutSbeta were sensitive to psoralen ICLs. To further investigate the role of MutSbeta 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 MutSbeta and NER proteins with Tdp-ICLs. We found that MutSbeta bound to Tdp-ICLs with high affinity and specificity in vitro and in vivo, and that MutSbeta 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

  11. Incomplete excision repair process after UV-irradiation in MUT-mutants of Proteus mirabillis

    International Nuclear Information System (INIS)

    MUT-mutants of P. mirabilis seem to be able to perform the incision step in the course of excision repair. In contrast to the corresponding wildtype strains with MUT-mutants the number of single-strand breaks formed after UV-irradiation is independent of the UV-dose up to about 720 erg/mm2. Incubation in minimal medium over a longer time does not result in completion of excision repair; about 3-6 single-strand breaks in the DNA of these mutants remain open. Likewise, the low molecular weight of the newly synthesized daughter DNA confirms an incompletely proceeding or delayed repair process. As a possible reason for the mutator phenotype an alteration of the DNA-polymerase playing a role in excision and resynthesis steps of excision repair is discussed. (author)

  12. 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. PMID:26939044

  13. Physical interaction between components of DNA mismatch repair and nucleotide excision repair

    International Nuclear Information System (INIS)

    Nucleotide excision repair (NER) and DNA mismatch repair are required for some common processes although the biochemical basis for this requirement is unknown. Saccharomyces cerevisiae RAD14 was identified in a two-hybrid screen using MSH2 as 'bait,' and pairwise interactions between MSH2 and RAD1, RAD2, RAD3, RAD10, RAD14, and RAD25 subsequently were demonstrated by two-hybrid analysis. MSH2 coimmunoprecipitated specifically with epitope-tagged versions of RAD2, RAD10, RAD14, and RAD25. MSH2 and RAD10 were found to interact in msh3 msh6 and mlh1 pms1 double mutants, suggesting a direct interaction with MSH2. Mutations in MSH2 increased the UV sensitivity of NER-deficient yeast strains, and msh2 mutations were epistatic to the mutator phenotype observed in NER-deficient strains. These data suggest that MSH2 and possibly other components of DNA mismatch repair exist in a complex with NER proteins, providing a biochemical and genetical basis for these proteins to function in common processes

  14. DDB2 (Damaged DNA binding protein 2) in nucleotide excision repair and DNA damage response

    OpenAIRE

    Stoyanova, Tanya; Roy, Nilotpal; Kopanja, Dragana; Raychaudhuri, Pradip; Bagchi, Srilata

    2009-01-01

    DDB2 was identified as a protein involved in the Nucleotide Excision Repair (NER), a major DNA repair mechanism that repairs UV damage to prevent accumulation of mutations and tumorigenesis. However, recent studies indicated additional functions of DDB2 in the DNA damage response pathway. Herein, we discuss the proposed mechanisms by which DDB2 activates NER and programmed cell death upon DNA damage through its E3 ligase activity.

  15. Repair of single-strand breaks induced in the DNA of Proteus mirabilis by excision repair after UV-irradiation

    International Nuclear Information System (INIS)

    Single-strand breaks have been produced in the DNA of P. mirabilis after UV-irradiation in dependence on the incident UV-doses. It has been found that there exists a discrepancy between the single-strand breaks estimated from sedimentation in alkaline sucrose gradients and the expected single-strand breaks approximated from measurements of dimer excision. The low number in incision breaks observed by sedimentation experiments is an indication that the cells are able to repair the excision-induced breaks as fast as they are formed. Toluenized cells have been used for investigation of the incision step independently of subsequent repair processes. In presence of NMN the appearance of more single-strand breaks in the DNA has been observed. Furthermore, the number of incision breaks in toluenized cells increased in presence of exogenous ATP. The completion of the excision repair process has been investigated by observing the rejoining of incision breaks. After irradiation with UV-doses higher than approximately 240 erg/mm2 the number of single-strand breaks remaining unrepaired in the DNA increased. Studies of the influence of nutrition conditions on the repair process have shown approximately the same capacity for repair of single-strand breaks in growth medium as well as in buffer. Progress in the excision repair was also followed by investigation of the DNA synthesized at the template-DNA containing the pyrimidine dimers. In comparison with E. coli, P. mirabilis showed a somewhat lower efficiency for the repair of single-strand breaks during the excision repair. (author)

  16. The ING1b tumor suppressor facilitates nucleotide excision repair by promoting chromatin accessibility to XPA

    International Nuclear Information System (INIS)

    ING1b is the most studied ING family protein and perhaps the most ubiquitously and abundantly expressed. This protein is involved in the regulation of various biological functions ranging from senescence, cell cycle arrest, apoptosis, to DNA repair. ING1b is upregulated by UV irradiation and enhances the removal of bulky nucleic acid photoproducts. In this study, we provide evidence that ING1b mediates nucleotide excision repair by facilitating the access to damaged nucleosomal DNA. We demonstrate that ING1b is not recruited to UV-induced DNA lesions but enhances nucleotide excision repair only in XPC-proficient cells, implying an essential role in early steps of the 'access, repair, restore' model. We also find that ING1b alters histone acetylation dynamics upon exposure to UV radiation and induces chromatin relaxation in microccocal nuclease digestion assay, revealing that ING1b may allow better access to nucleotide excision repair machinery. More importantly, ING1b associates with chromatin in a UV-inducible manner and facilitates DNA access to nucleotide excision repair factor XPA. Furthermore, depletion of the endogenous ING1b results to the sensitization of cells at S-phase to UV irradiation. Taken together, these observations establish a role of ING1b acting as a chromatin accessibility factor for DNA damage recognition proteins upon genotoxic injury

  17. Substrate overlap and functional competition between human nucleotide excision repair and Escherichia coli photolyase and (A)BC excision nuclease

    International Nuclear Information System (INIS)

    Human cell free extract prepared by the method of Manley et al. carries out repair synthesis on UV-irradiated DNA. Removal of pyrimidine dimers by photoreactivation with DNA photolyase reduces repair synthesis by about 50%. With excess enzyme in the reaction mixture photolyase reduced the repair signal by the same amount even in the absence of photoreactivating light, presumably by binding to pyrimidine dimers and interfering with the binding of human damage recognition protein. Similarly, the UvrB subunit of Escherichia coli (A)BC excinuclease when loaded onto UV-irradiated or psoralen-adducted DNA inhibited repair synthesis by cell-free extract by 75-80%. The opposite was true also as HeLa cell free extract specifically inhibited the photorepair of a thymine dimer by DNA photolyase and its removal by (A)BC excinuclease. Cell-free extracts from xeroderma pigmentosum (XP) complementation groups A and C were equally effective in blocking the E. coli repair proteins, while extracts from complementation groups D and E were ineffective in blocking the E. coli enzyme. These results suggest that XP-D and XP-E cells are defective in the damage recognition subunits(s) of human excision nuclease

  18. Surgical problems in the excision and repair of radiated tissue

    International Nuclear Information System (INIS)

    Surgical procedures for tissues damaged by radiation are fraught with the possibility of multiple and serious complications. A knowledge of the altered pathophysiology of such tissues (chiefly diminished blood supply, fibrosis, and direct cellular reparative potential) is necessary for the surgeon to make a proper judgment. Recognition of the pathognomic signs in the skin is important. The possibility of malignant change must be kept in mind. Surgical treatment must include the excision of radiated tissues back to good blood supply, and then proper and adequate coverage by the simplest and best means. If the surrounding local tissues are so altered that they are unsuitable for coverage, structures from a distance must be used. Prior planning of the coverage before the excision pays dividends by preventing disastrous complications. (U.S.)

  19. Differing levels of excision repair in human fetal dermis and brain cells

    Energy Technology Data Exchange (ETDEWEB)

    Gibson, R.E. (Ohio State Univ., Columbus (USA). Dept. of Radiology); D' Ambrosio, S.M. (Ohio State Univ., Columbus (USA). Dept. of Radiology; Ohio State Univ., Columbus (USA). Dept. of Pharmacology)

    1982-01-01

    The levels of DNA excision repair, as measured by unscheduled DNA synthesis (UDS) and the UV-endonuclease sensitive site assay, were compared in cells derived from human fetal brain and dermal tissues. The level of UDS induced following ultraviolet (UV) irradiation was found to be lower (approx. 60%) in the fetal brain cells than in fetal dermal cells. It was determined, using the UV-endonuclease sensitive site assay to confirm the UDS observation, that 50% of the dimers induced by UV in fetal dermal cells were repaired in 8 h. while only 15% were removed in the fetal brain cells during the same period of time. Even after 24 h. only 44% of the dimers induced by UV in the fetal brain cells were repaired, while 65% were removed in the dermal cells. These data suggest that cultured human fetal brain cells exhibit lower levels of excision repair compared to cultured human fetal dermal cells.

  20. 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; Krokan, H E

    2007-01-01

    proteins also remove the oxidized cytosine derivatives isodialuric acid, alloxan and 5-hydroxyuracil. UNG1 and UNG2 have identical catalytic domain, but different N-terminal regions required for subcellular sorting. We demonstrate that mRNA for UNG1, but not UNG2, is increased after hydrogen peroxide......, 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 of...... 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....

  1. Evidence that DNA excision-repair in xeroderma pigmentosum group A is limited but biologically significant

    International Nuclear Information System (INIS)

    The loss of pyrimidine dimers in nondividing populations of an excision-repair deficient xeroderma pigmentosum group. A strain (XP12BE) was measured throughout long periods (up to 5 months) following exposure to low doses of ultraviolet light (UV, 254 nm) using a UV endonuclease-alkaline sedimentation assay. Excision of about 90% of the dimers induced by 1 J/m2 occurred during the first 50 days. The rate curve has some similarities with that of normal excision-repair proficient cultures that may not be coincidental. Rate curves for both XP12BE and normal cultures are characterized by a fast and slow component, with both rate constants for the XP12BE cultures (0.15 day-1 and 0.025 day-1) a factor of 10 smaller than those observed for the respective components of normal cell cultures. The slow components for both XP12BE and normal cultures extrapolate to about 30% of the initial number of dimers. No further excision was detected throughout an additional 90-day period even though the cultures were capable of excision-repair of other newly-introduced pyrimidine dimers. We conclude that nondividing XP12BE cells in addition to having a slower repair rate, cannot repair some of the UV-induced DNA damage. The repair in XP12BE is shown to have biological significance as detected by a cell-survival assay and dose-fractionation techniques. Nondividing XP12BE cells are more resistant to UV when irradiated chronically than when irradiated acutely with the same total dose. (orig.)

  2. 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

  3. POLYMORPHISMS IN THE DNA NUCLEOTIDE EXCISION REPAIR GENES AND LUNG CANCER RISK IN XUAN WEI, CHINA

    Science.gov (United States)

    The lung cancer mortality rate in Xuan Wei County, China is among the highest in the country and has been etiologically attributed to exposure to indoor smoky coal emissions that contain very high levels of polycyclic aromatic hydrocarbons (PAHs). Nucleotide excision repair (NE...

  4. Rearrangement of nucleosome structure during excision repair in xeroderma pigmentosum (group A) human fibroblasts

    International Nuclear Information System (INIS)

    Rearrangements of chromatic structure during excision repair were examined in xeroderma pigmentosum (XP; complementation group A) human fibroblasts treated with the small-molecule alkylating agent methyl methanesulfonate (MMS). We observed normal levels of repair synthesis in these cells during the first 12 h after exposure to MMS, in contrast to the near zero incorporation of repair patches following exposure to u.v. light. Our results indicate that the relative nuclease sensitivity of newly repaired regions in MMS-treated XP (group A) cells is quantitatively similar to that of newly repaired regions in MMS-treated normal human fibroblasts. This enhanced sensitivity is accompanied by a marked under-representation of repair-incorporated nucleotides in isolated nucleosome core DNA. Pulse-chase experiments demonstrated that these regions rapidly undergo rearrangements in chromatin structure, and both the rate and extent of these rearrangements are similar to those observed in normal cells. (author)

  5. Wound repair and anti-inflammatory potential of Lonicera japonica in excision wound-induced rats

    Directory of Open Access Journals (Sweden)

    Chen Wei-Cheng

    2012-11-01

    Full Text Available Abstract Background Lonicera japonica Thunb. (Caprifoliaceae, a widely used traditional Chinese medicinal plant, is used to treat some infectious diseases and it may have uses as a healthy food and applications in cosmetics and as an ornamental groundcover. The ethanol extract of the flowering aerial parts of L. japonica (LJEE was investigated for its healing efficiency in a rat excision wound model. Methods Excision wounds were inflicted upon three groups of eight rats each. Healing was assessed by the rate of wound contraction in skin wound sites in rats treated with simple ointment base, 10% (w/w LJEE ointment, or the reference standard drug, 0.2% (w/w nitrofurazone ointment. The effects of LJEE on the contents of hydroxyproline and hexosamine during healing were estimated. The antimicrobial activity of LJEE against microorganisms was also assessed. The in vivo anti-inflammatory activity of LJEE was investigated to understand the mechanism of wound healing. Results LJEE exhibited significant antimicrobial activity against Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Candida albicans, and Candida tropicalis. The ointment formulation prepared with 10% (w/w LJEE exhibited potent wound healing capacity as evidenced by the wound contraction in the excision wound model. The contents of hydroxyproline and hexosamine also correlated with the observed healing pattern. These findings were supported by the histopathological characteristics of healed wound sections, as greater tissue regeneration, more fibroblasts, and angiogenesis were observed in the 10% (w/w LJEE ointment-treated group. The results also indicated that LJEE possesses potent anti-inflammatory activity, as it enhanced the production of anti-inflammatory cytokines that suppress proinflammatory cytokine production. Conclusions The results suggest that the antimicrobial and anti-inflammatory activities of LJEE act synergistically to accelerate wound repair.

  6. DNA damage and nucleotide excision repair capacity in healthy individuals

    Czech Academy of Sciences Publication Activity Database

    Slyšková, Jana; Naccarati, Alessio; Poláková, Veronika; Pardini, Barbara; Vodičková, Ludmila; Štětina, R.; Schmuczerová, Jana; Šmerhovský, Z.; Lipská, L.; Vodička, Pavel

    2011-01-01

    Roč. 25, č. 7 (2011), s. 511-517. ISSN 0893-6692 R&D Projects: GA ČR GAP304/10/1286; GA MŠk 7F10069 Grant ostatní: GA MŠk(CZ) GAUK124710 Institutional research plan: CEZ:AV0Z50390512 Keywords : BPDE-induced DNA repair capacity * comet assay * interindividual variability Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.709, year: 2011

  7. Tfb5 Is Partially Dispensable for Rad26 Mediated Transcription Coupled Nucleotide Excision Repair in Yeast

    OpenAIRE

    Ding, Baojin; Ruggiero, Christine; Chen, Xuefeng; Li, Shisheng

    2007-01-01

    Nucleotide excision repair (NER) is a conserved DNA repair mechanism capable of removing a variety of helix-distorting DNA lesions. A specialized NER pathway, called transcription coupled NER (TC-NER), refers to preferential repair in the transcribed strand of an actively transcribed gene. To be distinguished from TCR-NER, the genome-wide NER process is termed as global genomic NER (GG-NER). In Saccharomyces cerevisiae, GG-NER is dependent on Rad7, whereas TC-NER is mediated by Rad26, the hom...

  8. Differentiation-dependent p53 regulation of nucleotide excision repair in keratinocytes.

    OpenAIRE

    Li, G.; Ho, V. C.; D. L. Mitchell; Trotter, M. J.; Tron, V A

    1997-01-01

    The role of the tumor suppressor p53 in repair of ultraviolet light (UV)-induced DNA damage was evaluated using a host-cell reactivation (HCR) assay. HCR determines a cell's ability to repair UV-damaged DNA through reactivation of a transfected CAT reported plasmid. Most UV damage is removed through nucleotide excision repair (NER). Primary murine keratinocytes isolated from p53-deficient and wild-type p53 mice were used in the HCR assay. The NER was reduced in p53-/- keratinocytes as compare...

  9. Genetic Characterization of the Nucleotide Excision Repair System of Neisseria gonorrhoeae▿

    OpenAIRE

    LeCuyer, Brian E.; Criss, Alison K.; Seifert, H. Steven

    2009-01-01

    Nucleotide excision repair (NER) is universally used to recognize and remove many types of DNA damage. In eubacteria, the NER system typically consists of UvrA, UvrB, UvrC, the UvrD helicase, DNA polymerase I, and ligase. In addition, when DNA damage blocks transcription, transcription-repair coupling factor (TRCF), the product of the mfd gene, recruits the Uvr complex to repair the damage. Previous work using selected mutants and assays have indicated that pathogenic Neisseria spp. carry a f...

  10. Modeling of induced mutation process in bacterial cells with defects in excision repair system

    International Nuclear Information System (INIS)

    A mathematical model of the UV-induced mutation process in Escherichia coli bacteria cells with defects in uvrA and polA genes has been developed. The model describes in detail the reaction kinetics for excision repair system. The number of mismatches as results from translesion-synthesis is calculated for both wild-type and mutant cells. An effect of temporal modulation for amount of single-stranded DNA during post-replication repair is predicted. A comparison of repair system efficiency is conducted

  11. EXCISED DAMAGED BASE DETERMINES THE TURNOVER OF HUMAN N-METHYLPURINE-DNA GLYCOSYLASE

    OpenAIRE

    Adhikari, Sanjay; Üren, Aykut; Roy, Rabindra

    2009-01-01

    N-Methylpurine-DNA glycosylase (MPG) initiates base excision repair in DNA by removing a wide variety of alkylated, deaminated, and lipid peroxidation-induced purine adducts. In this study, we tested the role of excised base on MPG’s enzymatic activity. After the reaction, MPG produced two products: free damaged base and AP-site containing DNA. Our results showed that MPG excises 1, N6-ethenoadenine (εA) from εA-containing oligonucleotide (εA-DNA) at a similar or slightly increased efficiency...

  12. Molecular mechanisms of DNA damage recognition for mammalian nucleotide excision repair.

    Science.gov (United States)

    Sugasawa, Kaoru

    2016-08-01

    For faithful DNA repair, it is crucial for cells to locate lesions precisely within the vast genome. In the mammalian global genomic nucleotide excision repair (NER) pathway, this difficult task is accomplished through multiple steps, in which the xeroderma pigmentosum group C (XPC) protein complex plays a central role. XPC senses the presence of oscillating 'normal' bases in the DNA duplex, and its binding properties contribute to the extremely broad substrate specificity of NER. Unlike XPC, which acts as a versatile sensor of DNA helical distortion, the UV-damaged DNA-binding protein (UV-DDB) is more specialized, recognizing UV-induced photolesions and facilitating recruitment of XPC. Recent single-molecule analyses and structural studies have advanced our understanding of how UV-DDB finds its targets, particularly in the context of chromatin. After XPC binds DNA, it is necessary to verify the presence of damage in order to avoid potentially deleterious incisions at damage-free sites. Accumulating evidence suggests that XPA and the helicase activity of transcription factor IIH (TFIIH) cooperate to verify abnormalities in DNA chemistry. This chapter reviews recent findings about the mechanisms underlying the efficiency, versatility, and accuracy of NER. PMID:27264556

  13. Nucleotide Excision Repair Pathway Polymorphisms and Pancreatic Cancer Risk: Evidence for role of MMS19L

    Science.gov (United States)

    McWilliams, Robert R.; Bamlet, William R.; de Andrade, Mariza; Rider, David N.; Cunningham, Julie M.; Petersen, Gloria M.

    2009-01-01

    Background Nucleotide excision repair (NER) is a vital response to DNA damage, including damage from tobacco exposure. Single nucleotide polymorphisms (SNPs) in the NER pathway may encode alterations that affect DNA repair function and therefore influence risk for pancreatic cancer development. Methods A clinic based case-control study in non-Hispanic white persons compared 1,143 patients with pancreatic adenocarcinoma with 1,097 healthy controls. Twenty-seven genes directly and indirectly involved in the NER pathway were identified and 236 tag-SNPs were selected from 26 of these (one had no SNPs identified). Association studies were performed at the gene level by principal components analysis, while recursive partitioning analysis was utilized to identify potential gene-gene and gene-environment interactions within the pathway. At the individual SNP level, adjusted additive, dominant, and recessive models were investigated, and gene-environment interactions were also assessed. Results Gene level analyses showed an association of MMS19L genotype (chromosome 10q24.1) with altered pancreatic cancer risk (p=0.023). Haplotype analysis of MMS19L also showed a significant association (p=0.0132). Analyses of 7 individual SNPs in this gene showed both protective and risk associations for minor alleles, broadly distributed across patient subgroups defined by smoking status, sex, and age. Conclusion In a candidate pathway SNP association study analysis, common variation in a NER gene, MMS19L, was associated with risk for pancreatic cancer. PMID:19318433

  14. Excision repair of ultraviolet-irradiated deoxyribonucleic acid in plasmolyzed cells of Escherichia coli

    International Nuclear Information System (INIS)

    A system of cells made permeable by treatment with high concentrations of sucrose (plasmolysis) has been exploited to study the excision repair of ultraviolet-irradiated deoxyribonucleic acid in Escherichia coli. It is demonstrated that adenosine 5'-triphosphate is required for incision breaks to be made in the bacterial chromosome as well as in covalently closed bacteriophage lambda deoxyribonucleic acid. After plasmolysis, uvrC mutant strains appear as defective in the incision step as the uvrA-mutated strains. This is in contrast to the situation in intact cells where uvrC mutants accumulate single-strand breaks during postirradiation incubation. These observations have led to the proposal of a model for excision repair, in which the ultraviolet-specific endonuclease, coded for by the uvrA and uvrB genes, exists in a complex with the uvrC gene product. The complex is responsible for the incision and possibly also the excision steps of repair. The dark-repair inhibitors acriflavine and caffeine are both shown to interfere with the action of the adenosine 5'-triphosphate-dependent enzyme

  15. Molecular cloning and biological characterization of the human excision repair gene ERCC-3

    International Nuclear Information System (INIS)

    In this report we present the cloning, partial characterization, and preliminary studies of the biological activity of a human gene, designated ERCC-3, involved in early steps of the nucleotide excision repair pathway. The gene was cloned after genomic DNA transfection of human (HeLa) chromosomal DNA together with dominant marker pSV3gptH to the UV-sensitive, incision-defective Chinese hamster ovary (CHO) mutant 27-1. This mutant belongs to complementation group 3 of repair-deficient rodent mutants. After selection of UV-resistant primary and secondary 27-1 transformants, human sequences associated with the induced UV resistance were rescued in cosmids from the DNA of a secondary transformant by using a linked dominant marker copy and human repetitive DNA as probes. From coinheritance analysis of the ERCC-3 region in independent transformants, we deduce that the gene has a size of 35 to 45 kilobases, of which one essential segment has so far been refractory to cloning. Conserved unique human sequences hybridizing to a 3.0-kilobase mRNA were used to isolate apparently full-length cDNA clones. Upon transfection to 27-1 cells, the ERCC-3 cDNA, inserted in a mammalian expression vector, induced specific and (virtually) complete correction of the UV sensitivity and unscheduled DNA synthesis of mutants of complementation group 3 with very high efficiency. Mutant 27-1 is, unlike other mutants of complementation group 3, also very sensitive toward small alkylating agents. This unique property of the mutant is not corrected by introduction of the ERCC-3 cDNA, indicating that it may be caused by an independent second mutation in another repair function. By hybridization to DNA of a human x rodent hybrid cell panel, the ERCC-3 gene was assigned to chromosome 2, in agreement with data based on cell fusion

  16. SUMO and ubiquitin-dependent XPC exchange drives nucleotide excision repair

    DEFF Research Database (Denmark)

    Van Cuijk, Loes; Van Belle, Gijsbert J.; Turkyilmaz, Yasemin;

    2015-01-01

    XPC recognizes UV-induced DNA lesions and initiates their removal by nucleotide excision repair (NER). Damage recognition in NER is tightly controlled by ubiquitin and SUMO modifications. Recent studies have shown that the SUMO-targeted ubiquitin ligase RNF111 promotes K63-linked ubiquitylation of...... SUMOylated XPC after DNA damage. However, the exact regulatory function of these modifications in vivo remains elusive. Here we show that RNF111 is required for efficient repair of ultraviolet-induced DNA lesions. RNF111-mediated ubiquitylation promotes the release of XPC from damaged DNA after NER...

  17. Molecular cloning and biological characterization of the human excision repair gene ERCC-3.

    OpenAIRE

    Weeda, G; van Ham, R C; Masurel, R; Westerveld, A; Odijk, H; Wit, J.; Bootsma, D; van der Eb, A J; Hoeijmakers, J. H.

    1990-01-01

    In this report we present the cloning, partial characterization, and preliminary studies of the biological activity of a human gene, designated ERCC-3, involved in early steps of the nucleotide excision repair pathway. The gene was cloned after genomic DNA transfection of human (HeLa) chromosomal DNA together with dominant marker pSV3gptH to the UV-sensitive, incision-defective Chinese hamster ovary (CHO) mutant 27-1. This mutant belongs to complementation group 3 of repair-deficient rodent m...

  18. The yeast TFB1 and SSL1 genes, which encode subunits of transcription factor IIH, are required for nucleotide excision repair and RNA polymerase II transcription.

    OpenAIRE

    Z. Wang; Buratowski, S; Svejstrup, J Q; Feaver, W J; Wu, X; Kornberg, R D; Donahue, T F; Friedberg, E C

    1995-01-01

    The essential TFB1 and SSL1 genes of the yeast Saccharomyces cerevisiae encode two subunits of the RNA polymerase II transcription factor TFIIH (factor b). Here we show that extracts of temperature-sensitive mutants carrying mutations in both genes (tfb1-101 and ssl1-1) are defective in nucleotide excision repair (NER) and RNA polymerase II transcription but are proficient for base excision repair. RNA polymerase II-dependent transcription at the CYC1 promoter was normal at permissive tempera...

  19. Mutagenic specificity of solar UV light in nucleotide excision repair-deficient rodent cells.

    OpenAIRE

    Sage, E.; Lamolet, B; Brulay, E; Moustacchi, E; Chteauneuf, A; Drobetsky, E A

    1996-01-01

    To investigate the role of nucleotide excision repair (NER) in the cellular processing of carcinogenic DNA photoproducts induced by defined, environmentally relevant portions of the solar wavelength spectrum, we have determined the mutagenic specificity of simulated sunlight (310-1100 nm), UVA (350-400 nm), and UVB (290-320 nm), as well as of the "nonsolar" model mutagen 254-nm UVC, at the adenine phosphoribosyltransferase (aprt) locus in NER-deficient (ERCC1) Chinese hamster ovary (CHO) cell...

  20. Genetic polymorphisms in the nucleotide excision repair pathway and lung cancer risk: A meta-analysis

    Directory of Open Access Journals (Sweden)

    Chikako Kiyohara, Kouichi Yoshimasu

    2007-01-01

    Full Text Available Various DNA alterations can be caused by exposure to environmental and endogenous carcinogens. Most of these alterations, if not repaired, can result in genetic instability, mutagenesis and cell death. DNA repair mechanisms are important for maintaining DNA integrity and preventing carcinogenesis. Recent lung cancer studies have focused on identifying the effects of single nucleotide polymorphisms (SNPs in candidate genes, among which DNA repair genes are increasingly being studied. Genetic variations in DNA repair genes are thought to modulate DNA repair capacity and are suggested to be related to lung cancer risk. We identified a sufficient number of epidemiologic studies on lung cancer to conduct a meta-analysis for genetic polymorphisms in nucleotide excision repair pathway genes, focusing on xeroderma pigmentosum group A (XPA, excision repair cross complementing group 1 (ERCC1, ERCC2/XPD, ERCC4/XPF and ERCC5/XPG. We found an increased risk of lung cancer among subjects carrying the ERCC2 751Gln/Gln genotype (odds ratio (OR = 1.30, 95% confidence interval (CI = 1.14 - 1.49. We found a protective effect of the XPA 23G/G genotype (OR = 0.75, 95% CI = 0.59 - 0.95. Considering the data available, it can be conjectured that if there is any risk association between a single SNP and lung cancer, the risk fluctuation will probably be minimal. Advances in the identification of new polymorphisms and in high-throughput genotyping techniques will facilitate the analysis of multiple genes in multiple DNA repair pathways. Therefore, it is likely that the defining feature of future epidemiologic studies will be the simultaneous analysis of large samples.

  1. Excision-repair of γ-ray damaged thymine in bacterial and mammalian systems

    International Nuclear Information System (INIS)

    The selective excision of products of the 5,6-dihydroxy-dihydrothymine type (t') for γ-irradiated or OsO4-oxidized DNA or synthetic poly [d(A-T)] was observed with crude extracts of Escherichia coli and isolated nuclei from human carcinoma HeLa S-3 cells and Chinese hamster ovary cells. The results with E. coli extracts allow the following conclusion: The uvrA-gene product is not required for t' excision; radiation-induced strand breakage is not required for product excision; experiments with extracts of E. coli polAexl showed that the 5' → 3' exonuclease associated with polymerase I is responsible for the removal of t'; experiments with extracts of E. coli endo I lig 4 and the ligase inhibitor nicotinamide mononucleotide showed that polynucleotide ligase accomplishes the last strand resealing step in the excision-repair of t'. Isolated nuclei from HeLa and Chinese hamster ovary cells possess the necessary enzymes for the selective excision of t' from γ-irradiated or osmium tetroxide oxidized DNA. Approximately 25 to 35 percent of the products were removed from DNA within 60 min. Unspecific DNA degradation was very low. Radiation-induced strand breakage is not required for product removal

  2. The molecular genetics of the incision step in the DNA excision repair process

    International Nuclear Information System (INIS)

    In this historical review one particular aspect of excision repair, DNA incision, and how it is controlled at the genetic level in bacteriophage, bacteria, S. cerevisae, D. melanogaster, rodent cells and humans is examined. In phage T4, DNA is incised by a DNA glycosylase-AP endonuclease coded for by the denV gene. In E. coli, products of three genes, uvrA, uvrB and uvrC, are required to form UVRABC excinuclease that cleaves DNA and releases a fragment 12-13 nucleotides long containing the damage site. In S. cerevisiae, genes complementing five mutants of the RAD3 epistasis group, rad1, rad2, rad3, rad4 and rad10 have been cloned and analyzed. Rodent cells sensitive to a variety of mutagenic agents and deficient in excision repair are being used in molecular studies to identify and clone human repair genes (e.g. ERCC1) capable of complementing mammalian repair defects. Most studies of the human system, have been done with cells isolated from patients xeroderma pigmentosum, and these are now beginning to be characterized at the molecular level. (author)

  3. Repair of UV-irradiated plasmid DNA in excision repair deficient mutants of Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    The repair of UV-irradiated DNA of plasmid YEp13 was studied in the incision defective strains by measurement of cell transformation frequency. In Saccharomyces cerevisiae, rad1,2,3 and 4 mutants could repair UV-damaged plasmid DNA. In Escherichia coli, uvrA mutant was unable to repair UV-damaged plasmid DNA; however, pretreatment of the plasmid with Micrococcus luteus endonuclease increased repair. It was concluded that all the mutations of yeast were probably limited only to the nuclear DNA. (author)

  4. 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.

  5. 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.

  6. Ku protein complex is involved in nucleotide excision repair of DNA

    International Nuclear Information System (INIS)

    The repair of ultraviolet light (UV-C, 254 nm) DNA lesions by nucleotide excision repair (NER) has been studied in the rodent cell line xrs6 belonging to complementation group 5 of ionising radiation sensitive (IRs) mutants. xrs6 cell line shows e defect in he DNA-end binding protein complex Ku which is involved in the repair of double-strand breaks (DSB) due to IR. In agreement with IR sensitivity, a bleomycin sensitive phenotype of xrs6 cell line was found as compared to the parental CHO-Kl line (factor> 8 fold). xrs6 exhibited also a slight (factor 2) but reproducible sensitivity to UV-C-light, while a revertant cell line for Ku DNA-end binding activity, xrs6rev, showed a restoration of both IR and UV-C sensitivities to the parental level. The NER activity of these cell lines was measured in vitro in nuclear protein extracts in the presence of plasmid DNA repair substrate damaged with UV-C lesions repaired by NER: xrs6 cell extracts exhibited only 55 % of NER activity as compared to the control CHO-Kl and xrs6rev cell extracts. These indicate that the Ku DSB repair protein in involved also in the NER process. (authors). 31 refs., 1 fig., 1 tab

  7. 1-β-D-arabinofuranosylcytosine is cytotoxic in quiescent normal lymphocytes undergoing DNA excision repair

    International Nuclear Information System (INIS)

    We have sought to clarify the potential activity of the S-phase-specific antileukemic agent 1-β-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 rejoining by ligation, were evaluated using the single cell gel electrophoresis (Comet) assay and the thymidine incorporation assay. When normal lymphocytes were treated with ultraviolet C or with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), the processes of DNA excision repair were promptly initiated and rapidly completed. When the cells were incubated with ara-C prior to irradiation or BCNU treatment, the steps of DNA synthesis and rejoining in the repair processes were both inhibited. The ara-C-mediated inhibition of the repair processes was concentration-dependent, with the effect peaking at 10μM. The combination of ara-C with these DNA repair initiators exerted subsequent cytotoxicity, which was proportional to the extent of the repair inhibition in the presence of ara-C. In conclusion, ara-C was cytotoxic in quiescent cells undergoing DNA repair. This might be attributed to unrepaired DNA damage that remained in the cells, thereby inducing lethal cytotoxicity. Alternatively, ara-C might exert its own cytotoxicity by inhibiting DNA synthesis in the repair processes. Such a strategy may be effective against a dormant subpopulation in acute leukemia that survives chemotherapy. (author)

  8. 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.

  9. 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.

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

    OpenAIRE

    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 ...

  11. Cdt2-mediated XPG degradation promotes gap-filling DNA synthesis in nucleotide excision repair.

    Science.gov (United States)

    Han, Chunhua; Wani, Gulzar; Zhao, Ran; Qian, Jiang; Sharma, Nidhi; He, Jinshan; Zhu, Qianzheng; Wang, Qi-En; Wani, Altaf A

    2015-01-01

    Xeroderma pigmentosum group G (XPG) protein is a structure-specific repair endonuclease, which cleaves DNA strands on the 3' side of the DNA damage during nucleotide excision repair (NER). XPG also plays a crucial role in initiating DNA repair synthesis through recruitment of PCNA to the repair sites. However, the fate of XPG protein subsequent to the excision of DNA damage has remained unresolved. Here, we show that XPG, following its action on bulky lesions resulting from exposures to UV irradiation and cisplatin, is subjected to proteasome-mediated proteolytic degradation. Productive NER processing is required for XPG degradation as both UV and cisplatin treatment-induced XPG degradation is compromised in NER-deficient XP-A, XP-B, XP-C, and XP-F cells. In addition, the NER-related XPG degradation requires Cdt2, a component of an E3 ubiquitin ligase, CRL4(Cdt2). Micropore local UV irradiation and in situ Proximity Ligation assays demonstrated that Cdt2 is recruited to the UV-damage sites and interacts with XPG in the presence of PCNA. Importantly, Cdt2-mediated XPG degradation is crucial to the subsequent recruitment of DNA polymerase δ and DNA repair synthesis. Collectively, our data support the idea of PCNA recruitment to damage sites which occurs in conjunction with XPG, recognition of the PCNA-bound XPG by CRL4(Cdt2) for specific ubiquitylation and finally the protein degradation. In essence, XPG elimination from DNA damage sites clears the chromatin space needed for the subsequent recruitment of DNA polymerase δ to the damage site and completion of gap-filling DNA synthesis during the final stage of NER. PMID:25483071

  12. A Case of Microangiopathic Hemolytic Anemia after Myxoma Excision and Mitral Valve Repair Presenting as Hemolytic Uremic Syndrome

    Science.gov (United States)

    Park, Young Joo; Kim, Sang Pil; Shin, Ho-Jin

    2016-01-01

    Microangiopathic hemolytic anemia occurs in a diverse group of disorders, including thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, and prosthetic cardiac valves. Hemolytic anemia also occurs as a rare complication after mitral valve repair. In this report, we describe a case of microangiopathic hemolytic anemia following myxoma excision and mitral valve repair, which was presented as hemolytic uremic syndrome. PMID:27081450

  13. Induced excision repair is required for repair of lesions in the vicinity of DNA replication forks

    International Nuclear Information System (INIS)

    A technique for resolving DNA fragments containing replication forks from linear DNA fragments by two-dimensional agarose gel electrophoresis is described. The technique is based on the altered mobility in agarose of branched structures relative to linear double-stranded molecules as a function of gel concentration and voltage. When pulse-labeled DNA is isolated, purified, fragmented by digestion with restriction nucleases, and run in the two-dimensional gel system, the bulk of the DNA migrates in a single arc visible by staining with ethidium bromide. However, when autoradiograms are prepared from the gels, it can be seen that the nascent DNA, represented by the radioactive pulse label, is contained in a second distinct arc. We have shown by a variety of criteria that the nascent DNA migrating in this minor arc has properties consistent with replication fork structures. We have now applied this technique to testing the hypothesis that long patch repair occurs at lesions in the vicinity of DNA replication forks. 2 figs

  14. UV-induced DNA excision repair in rat fibroblasts during immortalization and terminal differentiation in vitro

    International Nuclear Information System (INIS)

    UV-induced DNA excision repair was studied as DNA repair synthesis and dimer removal in rat fibroblast cultures, initiated from either dense or sparse inocula of primary cells grown from skin biopsies. During passaging in vitro an initial increase in DNA repair synthesis, determined both autoradiographically as unscheduled DNA synthesis (UDS) and by means of the BrdU photolysis assay as the number and average size of repair patches, was found to be associated with a morphological shift from small spindle-shaped to large pleiomorphic cells observed over the first twenty generations. In cell populations in growth crisis, a situation exclusively associated with thin-inoculum cultures in which the population predominantly consisted of large pleiomorphic cells, UDS was found to occur at a low level. After development of secondary cultures into immortal cell lines, both repair synthesis and morphology appeared to be the same as in the original primary spindle-shaped cells. At all passages the capacity to remove UV-induced pyrimidine dimers was found to be low, as indicated by the persistence of Micrococcus luteus UV endonuclease-sensitive sites. These results are discussed in the context of terminal differentiation and immortalization of rat fibroblasts upon establishment in vitro

  15. Tfb5 interacts with Tfb2 and facilitates nucleotide excision repair in yeast

    OpenAIRE

    Ying ZHOU; Kou, Haiping; Wang, Zhigang

    2007-01-01

    TFIIH is indispensable for nucleotide excision repair (NER) and RNA polymerase II transcription. Its tenth subunit was recently discovered in yeast as Tfb5. Unlike other TFIIH subunits, Tfb5 is not essential for cell survival. We have analyzed the role of Tfb5 in NER. NER was deficient in the tfb5 deletion mutant cell extracts, and was specifically complemented by purified Tfb5 protein. In contrast to the extreme ultraviolet (UV) sensitivity of rad14 mutant cells that lack any NER activity, t...

  16. Role of the Escherichia coli Nucleotide Excision Repair Proteins in DNA Replication

    OpenAIRE

    Moolenaar, Geri F.; Moorman, Celine; Goosen, Nora

    2000-01-01

    DNA polymerase I (PolI) functions both in nucleotide excision repair (NER) and in the processing of Okazaki fragments that are generated on the lagging strand during DNA replication. Escherichia coli cells completely lacking the PolI enzyme are viable as long as they are grown on minimal medium. Here we show that viability is fully dependent on the presence of functional UvrA, UvrB, and UvrD (helicase II) proteins but does not require UvrC. In contrast, ΔpolA cells grow even better when the u...

  17. 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

  18. Recovery of DNA synthesis after ultraviolet irradiation of xeroderma pigmentosum cells depends on excision repair and is blocked by caffeine

    International Nuclear Information System (INIS)

    Normal human and xeroderma pigmentosum (XP, excision-defective group A) cells (both SV40-transformed) pulse-labeled with [3H] thymidine at various times after irradiation with ultraviolet light showed a decline and recovery of both the molecular weights of newly synthesized DNA and the rated of synthesis per cell. At the same ultraviolet dose, both molecular weights and rates of synthesis were inhibited more in XP than in normal cells. This indicates that excision repair plays a role in minimizing the inhibition of chain growth, possibly by excision of dimers ahead of the growing point. The ability to synthesize normal-sized DNA recovered more rapidly than rates of synthesis in normal cells, but both parameters recovered in phase in XP cells. During recovery in normal cells there are therefore fewer actively replicating clusters of replicons because the single-strand breaks involved in the excision of dimers inhibit replicon initiation. XP cells have few excision repair events and therefore fewer breaks to interfere with initiation, but chain growth is blocked by unexcised dimers. In both cell types recovery of the ability to synthesize normal-sized DNA was prevented by growing cells in caffeine after irradiation, possibly because of competition between the DNA binding properties of caffeine and replication proteins. These observations imply that excision repair and semiconservative replication interact strongly in irradiated cells to produce a complex spectrum of changes in DNA replication which may be confused with parts of alternative systems such as post-replication repair. (author)

  19. 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.

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

    International Nuclear Information System (INIS)

    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

  1. TGF-β signaling links E-cadherin loss to suppression of nucleotide excision repair.

    Science.gov (United States)

    Qiang, L; Shah, P; Barcellos-Hoff, M H; He, Y Y

    2016-06-23

    E-cadherin is a cell adhesion molecule best known for its function in suppressing tumor progression and metastasis. Here we show that E-cadherin promotes nucleotide excision repair through positively regulating the expression of xeroderma pigmentosum complementation group C (XPC) and DNA damage-binding protein 1 (DDB1). Loss of E-cadherin activates the E2F4 and p130/107 transcription repressor complexes to suppress the transcription of both XPC and DDB1 through activating the transforming growth factor-β (TGF-β) pathway. Adding XPC or DDB1, or inhibiting the TGF-β pathway, increases the repair of ultraviolet (UV)-induced DNA damage in E-cadherin-inhibited cells. In the mouse skin and skin tumors, UVB radiation downregulates E-cadherin. In sun-associated premalignant and malignant skin neoplasia, E-cadherin is downregulated in association with reduced XPC and DDB1 levels. These findings demonstrate a crucial role of E-cadherin in efficient DNA repair of UV-induced DNA damage, identify a new link between epithelial adhesion and DNA repair and suggest a mechanistic link of early E-cadherin loss in tumor initiation. PMID:26477308

  2. Alar base reduction: the boomerang-shaped excision.

    Science.gov (United States)

    Foda, Hossam M T

    2011-04-01

    A boomerang-shaped alar base excision is described to narrow the nasal base and correct the excessive alar flare. The boomerang excision combined the external alar wedge resection with an internal vestibular floor excision. The internal excision was inclined 30 to 45 degrees laterally to form the inner limb of the boomerang. The study included 46 patients presenting with wide nasal base and excessive alar flaring. All cases were followed for a mean period of 18 months (range, 8 to 36 months). The laterally oriented vestibular floor excision allowed for maximum preservation of the natural curvature of the alar rim where it meets the nostril floor and upon its closure resulted in a considerable medialization of alar lobule, which significantly reduced the amount of alar flare and the amount of external alar excision needed. This external alar excision measured, on average, 3.8 mm (range, 2 to 8 mm), which is significantly less than that needed when a standard vertical internal excision was used ( P < 0.0001). Such conservative external excisions eliminated the risk of obliterating the natural alar-facial crease, which did not occur in any of our cases. No cases of postoperative bleeding, infection, or vestibular stenosis were encountered. Keloid or hypertrophic scar formation was not encountered; however, dermabrasion of the scars was needed in three (6.5%) cases to eliminate apparent suture track marks. The boomerang alar base excision proved to be a safe and effective technique for narrowing the nasal base and elimination of the excessive flaring and resulted in a natural, well-proportioned nasal base with no obvious scarring. PMID:21404164

  3. Genetic characterization of the nucleotide excision repair system of Neisseria gonorrhoeae.

    Science.gov (United States)

    LeCuyer, Brian E; Criss, Alison K; Seifert, H Steven

    2010-02-01

    Nucleotide excision repair (NER) is universally used to recognize and remove many types of DNA damage. In eubacteria, the NER system typically consists of UvrA, UvrB, UvrC, the UvrD helicase, DNA polymerase I, and ligase. In addition, when DNA damage blocks transcription, transcription-repair coupling factor (TRCF), the product of the mfd gene, recruits the Uvr complex to repair the damage. Previous work using selected mutants and assays have indicated that pathogenic Neisseria spp. carry a functional NER system. In order to comprehensively examine the role of NER in Neisseria gonorrhoeae DNA recombination and repair processes, the predicted NER genes (uvrA, uvrB, uvrC, uvrD, and mfd) were each disrupted by a transposon insertion, and the uvrB and uvrD mutants were complemented with a copy of each gene in an ectopic locus. Each uvr mutant strain was highly sensitive to UV irradiation and also showed sensitivity to hydrogen peroxide killing, confirming that all of the NER genes in N. gonorrhoeae are functional. The effect of RecA expression on UV survival was minor in uvr mutants but much larger in the mfd mutant. All of the NER mutants demonstrated wild-type levels of pilin antigenic variation and DNA transformation. However, the uvrD mutant exhibited higher frequencies of PilC-mediated pilus phase variation and spontaneous mutation, a finding consistent with a role for UvrD in mismatch repair. We conclude that NER functions are conserved in N. gonorrhoeae and are important for the DNA repair capabilities of this strict human pathogen. PMID:19933360

  4. mei-9/sup a/ mutant of Drosophila melanogaster increases mutagen sensitivity and decreases excision repair

    International Nuclear Information System (INIS)

    The mei-9/sup a/ mutant of Drosophila melanogaster, which reduces meiotic recombination in females, is deficient in the excision of uv-induced pyrimidine dimers in both sexes. Assays were performed in primary cultures and established cell lines derived from embryos. An endonuclease preparation from M. luteus, which is specific for pyrimidine dimers, was employed to monitor uv-induced dimers in cellular DNA. The rate of disappearance of endonuclease-sensitive sites from DNA of control cells is 10-20 times faster than that from mei-9/sup a/ cells. The mutant mei-218, which is also deficient in meiotic recombination, removes nuclease-sensitive sites at control rates. The mei-9/sup a/ cells exhibit control levels of photorepair, postreplication repair and repair of single strand breaks. In mei-9 cells DNA synthesis and possibly postreplication repair are weakly sensitive to caffeine. Larvae which are hemizygous for either of the two mutants that define the mei-9 locus are hypersensitive to killing by the mutagens methyl methanesulfonate, nitrogen mustard and 2-acetylaminofluorene. Larvae hemizygous for the mei-218 mutant are insensitive to each of these reagents. These data demonstrate that the mei-9 locus is active in DNA repair of somatic cells. Thus functions involved in meiotic recombination are also active in DNA repair in this higher eukaryote. The results are consistent with the earlier suggestions that the mei-9 locus functions in the exchange events of meiosis. The mei-218 mutation behaves differently in genetic tests and our data suggest its function may be restricted to meiosis. These studies demonstrate that currently recognized modes of DNA repair can be efficiently detected in primary cell cultures derived from Drosophila embryos

  5. Genotoxicity of soluble and particulate cadmium compounds: impact on oxidative DNA damage and nucleotide excision repair.

    Science.gov (United States)

    Schwerdtle, Tanja; Ebert, Franziska; Thuy, Christina; Richter, Constanze; Mullenders, Leon H F; Hartwig, Andrea

    2010-02-15

    Water-soluble and particulate cadmium compounds are carcinogenic to humans. While direct interactions with DNA are unlikely to account for carcinogenicity, induction of oxidative DNA damage and interference with DNA repair processes might be more relevant underlying modes of action (recently summarized, for example, in Joseph , P. (2009) Tox. Appl. Pharmacol. 238 , 271 - 279). The present study aimed to compare genotoxic effects of particulate CdO and soluble CdCl(2) in cultured human cells (A549, VH10hTert). Both cadmium compounds increased the baseline level of oxidative DNA damage. Even more pronounced, both cadmium compounds inhibited the nucleotide excision repair (NER) of BPDE-induced bulky DNA adducts and UVC-induced photolesions in a dose-dependent manner at noncytotoxic concentrations. Thereby, the uptake of cadmium in the nuclei strongly correlated with the repair inhibition of bulky DNA adducts, indicating that independent of the cadmium compound applied Cd(2+) is the common species responsible for the observed repair inhibition. Regarding the underlying molecular mechanisms in human cells, CdCl(2) (as shown before by Meplan, C., Mann, K. and Hainaut, P. (1999) J. Biol. Chem. 274 , 31663 - 31670 ) and CdO altered the conformation of the zinc binding domain of the tumor suppressor protein p53. In further studies applying only CdCl(2), cadmium decreased the total nuclear protein level of XPC, which is believed to be the principle initiator of global genome NER. This led to diminished association of XPC to sites of local UVC damage, resulting in decreased recruitment of further NER proteins. Additionally, CdCl(2) strongly disturbed the disassembly of XPC and XPA. In summary, our data indicate a general nucleotide excision repair inhibition by cadmium compounds, which is most likely caused by a diminished assembly and disassembly of the NER machinery. These data reveal new insights into the mechanisms involved in cadmium carcinogenesis and provide further

  6. Hydrogen peroxide induced genomic instability in nucleotide excision repair-deficient lymphoblastoid cells

    Directory of Open Access Journals (Sweden)

    Gopalakrishnan Kalpana

    2010-12-01

    Full Text Available Abstract Background The Nucleotide Excision Repair (NER pathway specialises in UV-induced DNA damage repair. Inherited defects in the NER can predispose individuals to Xeroderma Pigmentosum (XP. UV-induced DNA damage cannot account for the manifestation of XP in organ systems not directly exposed to sunlight. While the NER has recently been implicated in the repair of oxidative DNA lesions, it is not well characterised. Therefore we sought to investigate the role of NER factors Xeroderma Pigmentosum A (XPA, XPB and XPD in oxidative DNA damage-repair by subjecting lymphoblastoid cells from patients suffering from XP-A, XP-D and XP-B with Cockayne Syndrome to hydrogen peroxide (H2O2. Results Loss of functional XPB or XPD but not XPA led to enhanced sensitivity towards H2O2-induced cell death. XP-deficient lymphoblastoid cells exhibited increased susceptibility to H2O2-induced DNA damage with XPD showing the highest susceptibility and lowest repair capacity. Furthermore, XPB- and XPD-deficient lymphoblastoid cells displayed enhanced DNA damage at the telomeres. XPA- and XPB-deficient lymphoblastoid cells also showed differential regulation of XPD following H2O2 treatment. Conclusions Taken together, our data implicate a role for the NER in H2O2-induced oxidative stress management and further corroborates that oxidative stress is a significant contributing factor in XP symptoms. Resistance of XPA-deficient lymphoblastoid cells to H2O2-induced cell death while harbouring DNA damage poses a potential cancer risk factor for XPA patients. Our data implicate XPB and XPD in the protection against oxidative stress-induced DNA damage and telomere shortening, and thus premature senescence.

  7. Transcription-coupled nucleotide excision repair in mammalian cells: molecular mechanisms and biological effects

    Institute of Scientific and Technical Information of China (English)

    Mafia Fousteri; Leon HF Mullenders

    2008-01-01

    The encounter of elongating RNA polymerase Ⅱ (RNAPIIo) with DNA lesions has severe consequences for the cell as this event provides a strong signal for P53-dependent apoptosis and cell cycle arrest. To counteract prolonged blockage of transcription, the cell removes the RNAPllo-hlocking DNA lesions by transcription-coupled repair (TC-NER), a specialized subpathway of nucleotide excision repair (NER). Exposure of mice to UVB light or chemicals has elucidated that TC-NER is a critical survival pathway protecting against acute toxic and long-term effects (cancer) of genotoxic exposure. Deficiency in TC-NER is associated with mutations in the CSA and CSB genes giving rise to the rare hu-man disorder Cockayne syndrome (CS). Recent data suggest that CSA and CSB play differential roles in mammalian TC-NER: CSB as a repair coupling factor to attract NER proteins, chromatin remodellers and the CSA- E3-ubiquitin iigase complex to the stalled RNAPI io. CSA is dispensable for attraction of NER proteins, yet in cooperation with CSB is required to recruit XAB2, the nucleosomal binding protein HMGNl and TFIIS. The emerging picture of TC-NER is complex: repair of transcription-blocking lesions occurs without displacement of the DNA damage-stalled RNAPIIo, and requires at least two essential assembly factors (CSA and CSB), the core NER factors (except for XPC-RAD23B), and TC-NER specific factors. These and yet unidentified proteins will accomplish not only efficient repair of transcrip-tion-blocking lesions, but are also likely to contribute to DNA damage signalling events.

  8. 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

  9. 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;

    2008-01-01

    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......Polymorphisms in nucleotide excision repair genes have been associated with risk for lung cancer. We examined gene-environment interactions in relation to lung cancer in 430 cases and 790 comparison persons identified within a prospective cohort of 57,053 persons. We included polymorphisms in the...

  10. Host DNA replication or excision repair requirement for ultraviolet induction of bacteriophage lambda lysogens

    International Nuclear Information System (INIS)

    It is stated that the mechanism for prophage induction by radiation, or chemical agents, is not known, although a variety of hypothesis have been advanced during recent years. Biochemical data have been described that seem to favour the suggestion that DNA intermediates in the repair of DNA damage compete with prophage operators for repressor binding. When sufficient repressor is bound none remains for prophage repression and induction occurs. If this is so the prediction may be made that induction should not occur in the absence of normal repair processes. Some experimental work is described with a view to testing and verifying this prediction. A bacteriophage lambda lysogen was used in the work. Irradiation was at 420C, and samples were removed at intervals and assayed for free plaque-forming units, little induction was observed over a wide range of UV doses in non-replicating non-excising lysogens, in contradiction with some earlier results. Competition between prophage operators and repair intermediates for lambda repressor appears to be the simplest way to account for the observed results, although other possibilities are discussed. (U.K.)

  11. The Role of Altered Nucleotide Excision Repair and UVB-Induced DNA Damage in Melanomagenesis

    Directory of Open Access Journals (Sweden)

    Timothy Budden

    2013-01-01

    Full Text Available UVB radiation is the most mutagenic component of the UV spectrum that reaches the earth’s surface and causes the development of DNA damage in the form of cyclobutane pyrimidine dimers and 6-4 photoproducts. UV radiation usually results in cellular death, but if left unchecked, it can affect DNA integrity, cell and tissue homeostasis and cause mutations in oncogenes and tumour-suppressor genes. These mutations, if unrepaired, can lead to abnormal cell growth, increasing the risk of cancer development. Epidemiological data strongly associates UV exposure as a major factor in melanoma development, but the exact biological mechanisms involved in this process are yet to be fully elucidated. The nucleotide excision repair (NER pathway is responsible for the repair of UV-induced lesions. Patients with the genetic disorder Xeroderma Pigmentosum have a mutation in one of eight NER genes associated with the XP complementation groups XP-A to XP-G and XP variant (XP-V. XP is characterized by diminished repair capacity, as well as a 1000-fold increase in the incidence of skin cancers, including melanoma. This has suggested a significant role for NER in melanoma development as a result of UVB exposure. This review discusses the current research surrounding UVB radiation and NER capacity and how further investigation of NER could elucidate the role of NER in avoiding UV-induced cellular death resulting in melanomagenesis.

  12. XPC is essential for nucleotide excision repair of zidovudine-induced DNA damage in human hepatoma cells

    International Nuclear Information System (INIS)

    Zidovudine (3'-azido-3'-dexoythymidine, AZT), a nucleoside reverse transcriptase inhibitor, can be incorporated into DNA and cause DNA damage. The mechanisms underlying the repair of AZT-induced DNA damage are unknown. To investigate the pathways involved in the recognition and repair of AZT-induced DNA damage, human hepatoma HepG2 cells were incubated with AZT for 2 weeks and the expression of DNA damage signaling pathways was determined using a pathway-based real-time PCR array. Compared to control cultures, damaged DNA binding and nucleotide excision repair (NER) pathways showed significantly increased gene expression. Further analysis indicated that AZT treatment increased the expression of genes associated with NER, including XPC, XPA, RPA1, GTF2H1, and ERCC1. Western blot analysis demonstrated that the protein levels of XPC and GTF2H1 were also significantly up-regulated. To explore further the function of XPC in the repair of AZT-induced DNA damage, XPC expression was stably knocked down by 71% using short hairpin RNA interference. In the XPC knocked-down cells, 100 μM AZT treatment significantly increased [3H]AZT incorporation into DNA, decreased the total number of viable cells, increased the release of lactate dehydrogenase, induced apoptosis, and caused a more extensive G2/M cell cycle arrest when compared to non-transfected HepG2 cells or HepG2 cells transfected with a scrambled short hairpin RNA sequence. Overall, these data indicate that XPC plays an essential role in the NER repair of AZT-induced DNA damage.

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

    Directory of Open Access Journals (Sweden)

    A.S. Fonseca

    2015-01-01

    Full Text Available 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 T4 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 T4 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 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.

  14. 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)

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

    Science.gov (United States)

    Pawlowska, Elzbieta; Wysokinski, Daniel; Blasiak, Janusz

    2016-01-01

    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. PMID:27058533

  16. Nucleotide Excision Repair and Vitamin D—Relevance for Skin Cancer Therapy

    Directory of Open Access Journals (Sweden)

    Elzbieta Pawlowska

    2016-04-01

    Full Text Available 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.

  17. Nucleotide Excision Repair and Vitamin D—Relevance for Skin Cancer Therapy

    Science.gov (United States)

    Pawlowska, Elzbieta; Wysokinski, Daniel; Blasiak, Janusz

    2016-01-01

    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. PMID:27058533

  18. Structural Basis for Bulky-Adduct DNA-Lesion Recognition by the Nucleotide Excision Repair Protein Rad14.

    Science.gov (United States)

    Simon, Nina; Ebert, Charlotte; Schneider, Sabine

    2016-07-25

    Heterocyclic aromatic amines react with purine bases and result in bulky DNA adducts that cause mutations. Such structurally diverse lesions are substrates for the nucleotide excision repair (NER). It is thought that the NER machinery recognises and verifies distorted DNA conformations, also involving the xeroderma pigmentosum group A and C proteins (XPA, XPC) that act as a scaffold between the DNA substrate and several other NER proteins. Here we present the synthesis of DNA molecules containing the polycyclic, aromatic amine C8-guanine lesions acetylaminophenyl, acetylaminonaphthyl, acetylaminoanthryl, and acetylaminopyrenyl, as well as their crystal structures in complex with the yeast XPA homologue Rad14. This work further substantiates the indirect lesion-detection mechanism employed by the NER system that recognises destabilised and deformable DNA structures. PMID:27223336

  19. Oxidative damage to RPA limits the nucleotide excision repair capacity of human cells

    Science.gov (United States)

    Guven, Melisa; Brem, Reto; Macpherson, Peter; Peacock, Matthew; Karran, Peter

    2015-01-01

    Nucleotide excision repair (NER) protects against sunlight-induced skin cancer. Defective NER is associated with photosensitivity and a high skin cancer incidence. Some clinical treatments that cause photosensitivity can also increase skin cancer risk. Among these, the immunosuppressant azathioprine and the fluoroquinolone antibiotics ciprofloxacin and ofloxacin, interact with UVA radiation to generate reactive oxygen species (ROS) that diminish NER capacity by causing protein damage. The RPA DNA binding protein plays a pivotal role in DNA metabolism and is an essential component of NER. The relationship between protein oxidation and NER inhibition was investigated in cultured human cells expressing different levels of RPA. We show here that RPA is limiting for NER and that oxidative damage to RPA compromises NER capability. Our findings reveal that cellular RPA is surprisingly vulnerable to oxidation and we identify oxidized forms of RPA that are associated with impaired NER. The vulnerability of NER to inhibition by oxidation provides a connection between cutaneous photosensitivity, protein damage and increased skin cancer risk. Our findings emphasize that damage to DNA repair proteins, as well as to DNA itself is likely to be an important contributor to skin cancer risk. PMID:26134950

  20. Excision repair of UVR-induced pyrimidine dimers in corneal DNR

    International Nuclear Information System (INIS)

    We measured excision repair of ultraviolet radiation (UVR)-induced pyrimidine dimers in DNA of the corneal epithelium of the marsupial, Monodelphis domestica, using damage-specific nucleases from Micrococcus luteus in conjunction with agarose gel electrophoresis. We observed that 100 J m-2 of UVR from a FS-40 sunlamp (280-400 nm) induced an average of 2.2 +- 0.2x 10-2 endonuclease-sensitive sites per kilobase (ESS/kb) (pyrimidine dimers) and that ∼ 50% of the dimers were repaired within 12 h after exposure. We also determined that an exposure of 400 J m-2 was needed to induce comparable numbers of pyrimidine dimers (2.5 x 10-2) in the DNA of skin of M. domestica in vivo. In addition, we found that 50% of the dimers were also removed from the epidermal cells of M. domestica within 12 h after exposure. A dose of 100 J m-2 was necessary to induce similar levels of pyrimidine dimers (2.0 +- 0.2 x 10-2) in the DNA of the cultured marsupial cell line Pt K2 (Potorous tridactylus). (author)

  1. Excision repair of UVR-induced pyrimidine dimers in corneal DNR

    Energy Technology Data Exchange (ETDEWEB)

    Freeman, S.E.; Applegate, L.A.; Ley, R.D.

    1988-01-01

    We measured excision repair of ultraviolet radiation (UVR)-induced pyrimidine dimers in DNA of the corneal epithelium of the marsupial, Monodelphis domestica, using damage-specific nucleases from Micrococcus luteus in conjunction with agarose gel electrophoresis. We observed that 100 J m/sup -2/ of UVR from a FS-40 sunlamp (280-400 nm) induced an average of 2.2 +- 0.2x 10/sup -2/ endonuclease-sensitive sites per kilobase (ESS/kb) (pyrimidine dimers) and that approx. 50% of the dimers were repaired within 12 h after exposure. We also determined that an exposure of 400 J m/sup -2/ was needed to induce comparable numbers of pyrimidine dimers (2.5 x 10/sup -2/) in the DNA of skin of M. domestica in vivo. In addition, we found that 50% of the dimers were also removed from the epidermal cells of M. domestica within 12 h after exposure. A dose of 100 J m/sup -2/ was necessary to induce similar levels of pyrimidine dimers (2.0 +- 0.2 x 10/sup -2/) in the DNA of the cultured marsupial cell line Pt K2 (Potorous tridactylus).

  2. Oxidative Damage to RPA Limits the Nucleotide Excision Repair Capacity of Human Cells.

    Science.gov (United States)

    Guven, Melisa; Brem, Reto; Macpherson, Peter; Peacock, Matthew; Karran, Peter

    2015-11-01

    Nucleotide excision repair (NER) protects against sunlight-induced skin cancer. Defective NER is associated with photosensitivity and a high skin cancer incidence. Some clinical treatments that cause photosensitivity can also increase skin cancer risk. Among these, the immunosuppressant azathioprine and the fluoroquinolone antibiotics ciprofloxacin and ofloxacin interact with UVA radiation to generate reactive oxygen species that diminish NER capacity by causing protein damage. The replication protein A (RPA) DNA-binding protein has a pivotal role in DNA metabolism and is an essential component of NER. The relationship between protein oxidation and NER inhibition was investigated in cultured human cells expressing different levels of RPA. We show here that RPA is limiting for NER and that oxidative damage to RPA compromises NER capability. Our findings reveal that cellular RPA is surprisingly vulnerable to oxidation, and we identify oxidized forms of RPA that are associated with impaired NER. The vulnerability of NER to inhibition by oxidation provides a connection between cutaneous photosensitivity, protein damage, and increased skin cancer risk. Our findings emphasize that damage to DNA repair proteins, as well as to DNA itself, is likely to be an important contributor to skin cancer risk. PMID:26134950

  3. DNA with Damage in Both Strands as Affinity Probes and Nucleotide Excision Repair Substrates.

    Science.gov (United States)

    Lukyanchikova, N V; Petruseva, I O; Evdokimov, A N; Silnikov, V N; Lavrik, O I

    2016-03-01

    Nucleotide excision repair (NER) is a multistep process of recognition and elimination of a wide spectrum of damages that cause significant distortions in DNA structure, such as UV-induced damage and bulky chemical adducts. A series of model DNAs containing new bulky fluoro-azidobenzoyl photoactive lesion dC(FAB) and well-recognized nonnucleoside lesions nFlu and nAnt have been designed and their interaction with repair proteins investigated. We demonstrate that modified DNA duplexes dC(FAB)/dG (probe I), dC(FAB)/nFlu+4 (probe II), and dC(FAB)/nFlu-3 (probe III) have increased (as compared to unmodified DNA, umDNA) structure-dependent affinity for XPC-HR23B (Kdum > KdI > KdII ≈ KdIII) and differentially crosslink to XPC and proteins of NER-competent extracts. The presence of dC(FAB) results in (i) decreased melting temperature (ΔTm = -3°C) and (ii) 12° DNA bending. The extended dC(FAB)/dG-DNA (137 bp) was demonstrated to be an effective NER substrate. Lack of correlation between the affinity to XPC-HR23B and substrate properties of the model DNA suggests a high impact of the verification stage on the overall NER process. In addition, DNAs containing closely positioned, well-recognized lesions in the complementary strands represent hardly repairable (dC(FAB)/nFlu+4, dC(FAB)/nFlu-3) or irreparable (nFlu/nFlu+4, nFlu/nFlu-3, nAnt/nFlu+4, nAnt/nFlu-3) structures. Our data provide evidence that the NER system of higher eukaryotes recognizes and eliminates damaged DNA fragments on a multi-criterion basis. PMID:27262196

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

    International Nuclear Information System (INIS)

    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 M1dG adducts. No differences in M1dG 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 (γH2AX). This reduction in γH2AX 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.

  5. 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.

  6. Impaired nucleotide excision repair pathway as a possible factor in pathogenesis of head and neck cancer

    Energy Technology Data Exchange (ETDEWEB)

    Sliwinski, T. [Department of Molecular Genetics, University of Lodz, Lodz (Poland); Markiewicz, L. [Department of Clinical Chemistry and Biochemistry, Medical University of Lodz, Lodz (Poland); Rusin, P. [Department of Molecular Genetics, University of Lodz, Lodz (Poland); Department of Clinical Chemistry and Biochemistry, Medical University of Lodz, Lodz (Poland); Kabzinski, J. [Department of Clinical Chemistry and Biochemistry, Medical University of Lodz, Lodz (Poland); Dziki, L. [Department of General and Colorectal Surgery, Medical University of Lodz, Lodz (Poland); Milonski, J.; Olszewski, J. [Department of Otolaryngology and Oncology, Medical University of Lodz, Lodz (Poland); Blaszczyk, J. [Department of Human Physiology, Medical University of Lodz, Lodz (Poland); Szemraj, J. [Department of Medical Biochemistry, Medical University of Lodz, Lodz (Poland); Majsterek, I., E-mail: ireneusz.majsterek@umed.lodz.pl [Department of Clinical Chemistry and Biochemistry, Medical University of Lodz, Lodz (Poland)

    2011-11-01

    Tobacco smoking is one of the major risk factors in pathogenesis of head and neck squamous cell carcinomas (HNSCC). Many of the chemical compounds present in tobacco are well-known carcinogens which form adducts with DNA. Cells remove these adducts mainly by the nucleotide excision repair pathway (NER). NER also eliminates a broad spectrum of pyrimidine dimers (CPD) and photo-products (6-4PP) induced by UV-radiation or DNA cross-links after cisplatin anti-cancer treatment. In this study DNA damage and repair was examined in peripheral blood lymphocytes obtained from 20 HNSCC patients and 20 healthy controls as well as HTB-43 larynx and SSC-25 tongue cancer cell lines. DNA repair kinetics in the examined cells after cisplatin or UV-radiation treatment were investigated using alkaline comet assay during 240 min of post-treatment incubation. MTT assay was used to analyse cell viability and the Annexin V-FITC kit specific for kinase-3 was employed to determine apoptosis after treating the cells with UV-radiation at dose range from 0.5 to 60 J/m{sup 2}. NER capability was assessed in vitro with cell extracts by the use of a bacterial plasmid irradiated with UV-light as a substrate for the repair. The results show that lymphocytes from HNSCC patients and HTB-43 or SSC-25 cancer cells were more sensitive to genotoxic treatment with UV-radiation and displayed impaired DNA repair. Also evidenced was a higher rate of apoptosis induction after UV-radiation treatment of lymphocytes from the HNSCC patients and the HTB-43 cancer cells than after treatment of those from healthy donors. Finally, our results showed that there was a significant decrease in NER capacity in HTB-43 or SSC-25 cancer cells as well as in peripheral blood lymphocytes of HNSCC patients compared to controls. In conclusion, we suggest that the impaired NER pathway might be a critical factor in pathogenesis of head and neck cancer.

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

    International Nuclear Information System (INIS)

    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

  8. Replicon size and excision repair as factors in the inhibition and recovery of DNA synthesis from ultraviolet damage

    International Nuclear Information System (INIS)

    Initiation of DNA replication and chain growth, analyzed by alkaline sucrose gradient sedimentation, was interrupted to different extents in different cell types by irradiation with ultraviolet light. Within the first hour of irradiation DNA replication was reduced in a manner that depended on the average number of lesions per replicating unit (replicon). At low numbers of lesions per replicon, inhibition of replicon initiation was the predominant response; at higher numbers of lesions per replicon, blockage of chain growth was also observed. After irradiation with a dose that initially blocked chain growth, the rate at which cells recovered their ability to synthesize increasingly more and larger size DNA was a function both of replicon size and of excision repair capacity. Cells with small replicons recovered more rapidly than cells with large replicons, and excision repair-deficient cells recovered less rapidly than excision-competent cells. These observations indicate that excision repair capacity and replicon size play major roles in the response of DNA replication to ultraviolet damage. (Auth.)

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

    OpenAIRE

    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 sh...

  10. Molecular analysis of plasmid DNA repair within ultraviolet-irradiated Escherichia coli. II. UvrABC-initiated excision repair and photolyase-catalyzed dimer monomerization

    International Nuclear Information System (INIS)

    In this study, a novel approach to the analysis of DNA repair in Escherichia coli was employed which allowed the first direct determination of the mechanisms by which endogenous DNA repair enzymes encounter target sites in vivo. An in vivo plasmid DNA repair analysis was employed to discriminate between two possible mechanisms of target site location: a processive DNA scanning mechanism or a distributive random diffusion mechanism. The results demonstrate that photolyase acts by a distributive mechanism within E. coli. In contrast, UvrABC-initiated excision repair occurs by a limited processive DNA scanning mechanism. A majority of the dimer sites on a given plasmid molecule were repaired prior to the dissociation of the UvrABC complex. Furthermore, plasmid DNA repair catalyzed by the UvrABC complex occurs without a detectable accumulation of nicked plasmid intermediates despite the fact that the UvrABC complex generates dual incisions in the DNA at the site of a pyrimidine dimer. Therefore, the binding or assembly of the UvrABC complex on DNA at the site of a pyrimidine dimer represents the rate-limiting step in the overall process of UvrABC-initiated excision repair in vivo

  11. The DNA glycosylase AlkD uses a non-base-flipping mechanism to excise bulky lesions

    Science.gov (United States)

    Mullins, Elwood A.; Shi, Rongxin; Parsons, Zachary D.; Yuen, Philip K.; David, Sheila S.; Igarashi, Yasuhiro; Eichman, Brandt F.

    2015-11-01

    Threats to genomic integrity arising from DNA damage are mitigated by DNA glycosylases, which initiate the base excision repair pathway by locating and excising aberrant nucleobases. How these enzymes find small modifications within the genome is a current area of intensive research. A hallmark of these and other DNA repair enzymes is their use of base flipping to sequester modified nucleotides from the DNA helix and into an active site pocket. Consequently, base flipping is generally regarded as an essential aspect of lesion recognition and a necessary precursor to base excision. Here we present the first, to our knowledge, DNA glycosylase mechanism that does not require base flipping for either binding or catalysis. Using the DNA glycosylase AlkD from Bacillus cereus, we crystallographically monitored excision of an alkylpurine substrate as a function of time, and reconstructed the steps along the reaction coordinate through structures representing substrate, intermediate and product complexes. Instead of directly interacting with the damaged nucleobase, AlkD recognizes aberrant base pairs through interactions with the phosphoribose backbone, while the lesion remains stacked in the DNA duplex. Quantum mechanical calculations revealed that these contacts include catalytic charge-dipole and CH-π interactions that preferentially stabilize the transition state. We show in vitro and in vivo how this unique means of recognition and catalysis enables AlkD to repair large adducts formed by yatakemycin, a member of the duocarmycin family of antimicrobial natural products exploited in bacterial warfare and chemotherapeutic trials. Bulky adducts of this or any type are not excised by DNA glycosylases that use a traditional base-flipping mechanism. Hence, these findings represent a new model for DNA repair and provide insights into catalysis of base excision.

  12. 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.

  13. Studies on the molecular mechanism of nucleotide excision repair in human cells

    International Nuclear Information System (INIS)

    Studies in this laboratory have focused on attempts to define the mechanism of nucleotide excision repair of DNA in human cells, with a view to understanding the molecular pathogenesis of the disease XP. With the advent of recombinant DNA technology, they directed their efforts to the molecular cloning of human genes defective in XP, with a view to using the cloned genes to overexpress proteins of interest for biochemical investigations. Initial studies exploited the selectable phenotype of marked sensitivity to killing of XP group A cells by UV radiation and by other DNA damaging agents. However, except for a single report in 1982 there has been no reproducible demonstration of complementation of the UV sensitivity of XP cells by DNA-mediated transfection. The apparent difficulties associated with transfection of XP cells have been the subject of several recent studies. In view of the multiple problems associated with stable transfection of XP cells using total genomic DNA, they have embarked on an alternative strategy designed to facilitate the cloning of human XP genes. This strategy involves the transfer of single human chromosomes into XP cells and screening for this relatively high frequency event. The idea is to identify chromosomes on which particular XP genes reside and then to isolate non-complementing derivatives of these chromosomes so that highly enriched DNA pools containing genes of interest can be generated by employing one or more subtractive strategies

  14. 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. PMID:23577191

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

    International Nuclear Information System (INIS)

    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 IC50 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 (IC50 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 options in NER

  16. Expression of Excision Repair Cross-Complementation Group 1 as Predictive Marker for Nasopharyngeal Cancer Treated With Concurrent Chemoradiotherapy

    International Nuclear Information System (INIS)

    Purpose: Cisplatin-based concurrent chemoradiotherapy is the standard treatment of nasopharyngeal cancer. The expression of excision repair cross-complementation group 1 (ERCC1) has been reported to be associated with resistance to platinum-based chemotherapy. We evaluated whether ERCC1 expression could predict the treatment response and survival outcome of patients with locally advanced nasopharyngeal cancer who were treated with cisplatin-based concurrent chemoradiotherapy. Methods and Materials: Immunohistochemistry was used to examine the expression of ERCC1 in nasopharyngeal tumor tissue. Patients were categorized into either a resistant or sensitive group depending on their treatment response outcome. A total of 77 patients were assessed in the present study. Results: The resistant and sensitive groups included 25 and 52 patients, respectively. ERCC1 expression was positive in the tumor tissue for 39 of the 77 patients (51%). Significantly more ERCC1-negative tumors were in the sensitive group than in the resistant group (p = .035). In terms of survival outcome, univariate analysis determined that patients with ERCC1-negative tumors had longer disease-free survival (p = .076) and overall survival (p = .013) than patients with ERCC1-positive tumors. Multivariate analysis determined that negative ERCC expression in tumors was an independent predictor for prolonged overall survival (hazard ratio, 0.14; 95% confidence interval, 0.03-0.71). Conclusion: These results suggest that ERCC1 expression might be a useful predictive marker in patients with locally advanced nasopharyngeal cancer who are under consideration for cisplatin-based concurrent chemoradiotherapy.

  17. ATP-dependent chromatin remodeling facilitates nucleotide excision repair of UV-induced DNA lesions in synthetic dinucleosomes

    OpenAIRE

    Ura, Kiyoe; Araki, Marito; Saeki, Hideaki; Masutani, Chikahide; Ito, Takashi; Iwai, Shigenori; Mizukoshi, Toshimi; Kaneda, Yasufumi; Hanaoka, Fumio

    2001-01-01

    To investigate the relationship between chromatin dynamics and nucleotide excision repair (NER), we have examined the effect of chromatin structure on the formation of two major classes of UV-induced DNA lesions in reconstituted dinucleosomes. Furthermore, we have developed a model chromatin-NER system consisting of purified human NER factors and dinucleosome substrates that contain pyrimidine (6-4) pyrimidone photoproducts (6-4PPs) either at the center of the nucleosome or in the linker DNA....

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

    International Nuclear Information System (INIS)

    Inhibition of the gap-filling, polymerizing step of excision repair by 1-β-D-arabinofuranosylcytosine (ara-C) after irradiation with ultraviolet light in human diploid fibroblasts resulted in the formation of persistent DNA strand breaks in G1, G2, 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 μM 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 3H-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 3H-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 3H-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

  19. Evidence for an involvement of thymidine kinase in the excision repair of ultraviolet-irradiated herpes simplex virus in human cells

    International Nuclear Information System (INIS)

    A wild-type strain of herpes simplex virus type 1 (HSV-1:KOS) encoding a functional thymidine kinase (tk+) and a tk- mutant strain (HSV-1:PTK3B) were used to study the role of the viral tk in the repair of UV-irradiated HSV-1 in human cells. UV survival of HSV-1:PTK3B was substantially reduced compared with that of HSV-1:KOS when infecting normal human cells. In contrast, the UV survival of HSV-1:PTK3B was similar to that of HSV-1:KOS when infecting excision repair-deficient cells from a xeroderma pigmentosum patient from complementation group A. These results suggest that the repair of UV-irradiated HSV-1 in human cells depends, in part at least, on expression of the viral tk and that the repair process influenced by tk activity is excision repair or a process dependent on excision repair

  20. Twist-open mechanism of DNA damage recognition by the Rad4/XPC nucleotide excision repair complex.

    Science.gov (United States)

    Velmurugu, Yogambigai; Chen, Xuejing; Slogoff Sevilla, Phillip; Min, Jung-Hyun; Ansari, Anjum

    2016-04-19

    DNA damage repair starts with the recognition of damaged sites from predominantly normal DNA. In eukaryotes, diverse DNA lesions from environmental sources are recognized by the xeroderma pigmentosum C (XPC) nucleotide excision repair complex. Studies of Rad4 (radiation-sensitive 4; yeast XPC ortholog) showed that Rad4 "opens" up damaged DNA by inserting a β-hairpin into the duplex and flipping out two damage-containing nucleotide pairs. However, this DNA lesion "opening" is slow (˜5-10 ms) compared with typical submillisecond residence times per base pair site reported for various DNA-binding proteins during 1D diffusion on DNA. To address the mystery as to how Rad4 pauses to recognize lesions during diffusional search, we examine conformational dynamics along the lesion recognition trajectory using temperature-jump spectroscopy. Besides identifying the ˜10-ms step as the rate-limiting bottleneck towards opening specific DNA site, we uncover an earlier ˜100- to 500-μs step that we assign to nonspecific deformation (unwinding/"twisting") of DNA by Rad4. The β-hairpin is not required to unwind or to overcome the bottleneck but is essential for full nucleotide-flipping. We propose that Rad4 recognizes lesions in a step-wise "twist-open" mechanism, in which preliminary twisting represents Rad4 interconverting between search and interrogation modes. Through such conformational switches compatible with rapid diffusion on DNA, Rad4 may stall preferentially at a lesion site, offering time to open DNA. This study represents the first direct observation, to our knowledge, of dynamical DNA distortions during search/interrogation beyond base pair breathing. Submillisecond interrogation with preferential stalling at cognate sites may be common to various DNA-binding proteins. PMID:27035942

  1. Base excision by thymine DNA glycosylase mediates DNA-directed cytotoxicity of 5-fluorouracil.

    Directory of Open Access Journals (Sweden)

    Christophe Kunz

    2009-04-01

    Full Text Available 5-Fluorouracil (5-FU, a chemotherapeutic drug commonly used in cancer treatment, imbalances nucleotide pools, thereby favoring misincorporation of uracil and 5-FU into genomic DNA. The processing of these bases by DNA repair activities was proposed to cause DNA-directed cytotoxicity, but the underlying mechanisms have not been resolved. In this study, we investigated a possible role of thymine DNA glycosylase (TDG, one of four mammalian uracil DNA glycosylases (UDGs, in the cellular response to 5-FU. Using genetic and biochemical tools, we found that inactivation of TDG significantly increases resistance of both mouse and human cancer cells towards 5-FU. We show that excision of DNA-incorporated 5-FU by TDG generates persistent DNA strand breaks, delays S-phase progression, and activates DNA damage signaling, and that the repair of 5-FU-induced DNA strand breaks is more efficient in the absence of TDG. Hence, excision of 5-FU by TDG, but not by other UDGs (UNG2 and SMUG1, prevents efficient downstream processing of the repair intermediate, thereby mediating DNA-directed cytotoxicity. The status of TDG expression in a cancer is therefore likely to determine its response to 5-FU-based chemotherapy.

  2. Nucleotide excision repair DNA synthesis by excess DNA polymerase beta: a potential source of genetic instability in cancer cells.

    Science.gov (United States)

    Canitrot, Y; Hoffmann, J S; Calsou, P; Hayakawa, H; Salles, B; Cazaux, C

    2000-09-01

    The nucleotide excision repair pathway contributes to genetic stability by removing a wide range of DNA damage through an error-free reaction. When the lesion is located, the altered strand is incised on both sides of the lesion and a damaged oligonucleotide excised. A repair patch is then synthesized and the repaired strand is ligated. It is assumed that only DNA polymerases delta and/or epsilon participate to the repair DNA synthesis step. Using UV and cisplatin-modified DNA templates, we measured in vitro that extracts from cells overexpressing the error-prone DNA polymerase beta exhibited a five- to sixfold increase of the ultimate DNA synthesis activity compared with control extracts and demonstrated the specific involvement of Pol beta in this step. By using a 28 nt gapped, double-stranded DNA substrate mimicking the product of the incision step, we showed that Pol beta is able to catalyze strand displacement downstream of the gap. We discuss these data within the scope of a hypothesis previously presented proposing that excess error-prone Pol beta in cancer cells could perturb the well-defined specific functions of DNA polymerases during error-free DNA transactions. PMID:10973926

  3. 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.

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

    International Nuclear Information System (INIS)

    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

  5. Correlation among the rates of dimer excision, DNA repair replication, and recovery of human cells from potentially lethal damage induced by ultraviolet radiation

    International Nuclear Information System (INIS)

    The kinetics of excision repair in confluent cultures of diploid human fibroblasts after ultraviolet irradiation at varying doses was measured by three different methods: (1) removal of thymine-containing dimers, (2) DNA excision repair synthesis, and (3) biological recovery of cells from the potentially lethal effects of the irradiation. Each method gave similar results and indicated that the excision rate was dependent upon the number of thymine-containing dimers induced (substrate concentration). For example, at a dose of 40 J/m2 (0.2% dimerization), the repair rate was 1.6 J/m2 per h as determined by a modified method to measure the number of thymine-containing dimers remaining in DNA and 1.65 J/m2 as measured by excision repair synthesis. At a dose of 7.5 J/m2, the repair rate was 0.5 J/m2 per h as measured by biological recovery, and at a dose of 7 J/m2, the repair rate was 0.46 J/m2 per h as measured by excision repair synthesis

  6. The nucleotide excision repair system of Borrelia burgdorferi is the sole pathway involved in repair of DNA damage by UV light.

    Science.gov (United States)

    Hardy, Pierre-Olivier; Chaconas, George

    2013-05-01

    To survive and avoid accumulation of mutations caused by DNA damage, the genomes of prokaryotes encode a variety of DNA repair pathways most well characterized in Escherichia coli. Some of these are required for the infectivity of various pathogens. In this study, the importance of 25 DNA repair/recombination genes for Borrelia burgdorferi survival to UV-induced DNA damage was assessed. In contrast to E. coli, where 15 of these genes have an effect on survival of UV irradiation, disruption of recombinational repair, transcription-coupled repair, methyl-directed mismatch correction, and repair of arrested replication fork pathways did not decrease survival of B. burgdorferi exposed to UV light. However, the disruption of the B. burgdorferi nucleotide excision repair (NER) pathway (uvrA, uvrB, uvrC, and uvrD) resulted in a 10- to 1,000-fold increase in sensitivity to UV light. A functional NER pathway was also shown to be required for B. burgdorferi resistance to nitrosative damage. Finally, disruption of uvrA, uvrC, and uvrD had only a minor effect upon murine infection by increasing the time required for dissemination. PMID:23475971

  7. Silibinin enhances the repair of ultraviolet B-induced DNA damage by activating p53-dependent nucleotide excision repair mechanism in human dermal fibroblasts.

    Science.gov (United States)

    Guillermo-Lagae, Ruth; Deep, Gagan; Ting, Harold; Agarwal, Chapla; Agarwal, Rajesh

    2015-11-24

    Ultraviolet radiation B (UVB) is the main cause of DNA damage in epidermal cells; and if not repaired, this DNA damage leads to skin cancer. In earlier studies, we have reported that natural flavonolignan silibinin exerts strong chemopreventive efficacy against UVB-induced skin damage and carcinogenesis; however mechanistic studies are still being actively pursued. Here, we investigated the role of nucleotide excision repair (NER) pathway in silibinin's efficacy to repair UVB-induced DNA damage. Normal human dermal fibroblasts (NHDFs) were exposed to UVB (1 mJ/cm2) with pre- or post- silibinin (100 μM) treatment, and cyclobutane pyrimidine dimers (CPDs) formation/repair was measured. Results showed that post-UVB silibinin treatment accelerates DNA repair via activating the NER pathway including the expression of XPA (xeroderma pigmentosum complementation group A), XPB, XPC, and XPG. In UVB exposed fibroblasts, silibinin treatment also increased p53 and GADD45α expression; the key regulators of the NER pathway and DNA repair. Consistently, post-UVB silibinin treatment increased the mRNA transcripts of XPA and GADD45α. Importantly, silibinin showed no effect on UVB-induced DNA damage repair in XPA- and XPB-deficient human dermal fibroblasts suggesting their key role in silibinin-mediated DNA damage repair. Moreover, in the presence of pifithrin-α, an inhibitor of p53, the DNA repair efficacy of silibinin was compromised associated with a reduction in XPA and GADD45α transcripts. Together, these findings suggest that silibinin's efficacy against UVB-induced photodamage is primarily by inhibiting NER and p53; and these findings further support silibinin's usage as a potential inexpensive, effective, and non-toxic agent for skin cancer chemoprevention. PMID:26447614

  8. Mms19 protein functions in nucleotide excision repair by sustaining an adequate cellular concentration of the TFIIH component Rad3

    OpenAIRE

    Kou, Haiping; Ying ZHOU; Gorospe, R.M. Charlotte; Wang, Zhigang

    2008-01-01

    Nucleotide excision repair (NER) is a major cellular defense mechanism against DNA damage. We have investigated the role of Mms19 in NER in the yeast Saccharomyces cerevisiae. NER was deficient in the mms19 deletion mutant cell extracts, which was complemented by the NER/transcription factor TFIIH, but not by purified Mms19 protein. In mms19 mutant cells, protein levels of the core TFIIH component Rad3 (XPD homologue) and Ssl2 (XPB homologue) were significantly reduced by up to 3.5- and 2.2-f...

  9. HCMV-infected cells maintain efficient nucleotide excision repair of the viral genome while abrogating repair of the host genome.

    Directory of Open Access Journals (Sweden)

    John M O'Dowd

    Full Text Available Many viruses subvert the host cell's ability to mount and complete various DNA damage responses (DDRs after infection. HCMV infection of permissive fibroblasts activates host DDRs at the time of viral deposition and during replication, but the DDRs remain uncompleted without arrest or apoptosis. We believe this was in part due to partitioning of the damage response and double strand break repair components. After extraction of soluble proteins, the localization of these components fell into three groups: specifically associated with the viral replication centers (RCs, diffused throughout the nucleoplasm and excluded from the RCs. Others have shown that cells are incapable of processing exogenously introduced damage after infection. We hypothesized that the inability of the cells to process damage might be due to the differential association of repair components within the RCs and, in turn, potentially preferential repair of the viral genome and compromised repair of the host genome. To test this hypothesis we used multiple strategies to examine repair of UV-induced DNA damage in mock and virus-infected fibroblasts. Comet assays indicated that repair was initiated, but was not completed in infected cells. Quantitative analysis of immunofluorescent localization of cyclobutane pyrimidine dimers (CPDs revealed that after 24 h of repair, CPDs were significantly reduced in viral DNA, but not significantly changed in the infected host DNA. To further quantitate CPD repair, we developed a novel dual-color Southern protocol allowing visualization of host and viral DNA simultaneously. Combining this Southern methodology with a CPD-specific T4 endonuclease V alkaline agarose assay to quantitate repair of adducts, we found efficient repair of CPDs from the viral DNA but not host cellular DNA. Our data confirm that NER functions in HCMV-infected cells and almost exclusively repairs the viral genome to the detriment of the host's genome.

  10. Metal binding mediated conformational change of XPA protein:a potential cytotoxic mechanism of nickel in the nucleotide excision repair.

    Science.gov (United States)

    Hu, Jianping; Hu, Ziheng; Zhang, Yan; Gou, Xiaojun; Mu, Ying; Wang, Lirong; Xie, Xiang-Qun

    2016-07-01

    Nucleotide excision repair (NER) is a pivotal life process for repairing DNA nucleotide mismatch caused by chemicals, metal ions, radiation, and other factors. As the initiation step of NER, the xeroderma pigmentosum complementation group A protein (XPA) recognizes damaged DNA molecules, and recruits the replication protein A (RPA), another important player in the NER process. The stability of the Zn(2+)-chelated Zn-finger domain of XPA center core portion (i.e., XPA98-210) is the foundation of its biological functionality, while the displacement of the Zn(2+) by toxic metal ions (such as Ni(2+), a known human carcinogen and allergen) may impair the effectiveness of NER and hence elevate the chance of carcinogenesis. In this study, we first calculated the force field parameters for the bonded model in the metal center of the XPA98-210 system, showing that the calculated results, including charges, bonds, angles etc., are congruent with previously reported results measured by spectrometry experiments and quantum chemistry computation. Then, comparative molecular dynamics simulations using these parameters revealed the changes in the conformation and motion mode of XPA98-210 Zn-finger after the substitution of Zn(2+) by Ni(2+). The results showed that Ni(2+) dramatically disrupted the relative positions of the four Cys residues in the Zn-finger structure, forcing them to collapse from a tetrahedron into an almost planar structure. Finally, we acquired the binding mode of XPA98-210 with its ligands RPA70N and DNA based on molecular docking and structural alignment. We found that XPA98-210's Zn-finger domain primarily binds to a V-shaped cleft in RPA70N, while the cationic band in its C-terminal subdomain participates in the recognition of damaged DNA. In addition, this article sheds light on the multi-component interaction pattern among XPA, DNA, and other NER-related proteins (i.e., RPA70N, RPA70A, RPA70B, RPA70C, RPA32, and RPA14) based on previously reported

  11. Inhibition of nucleotide excision repair by fludarabine in normal lymphocytes in vitro, measured by the alkaline single cell gel electrophoresis (comet) assay

    International Nuclear Information System (INIS)

    Alkylating agents or platinum analogues initiate several excision repair mechanisms, which involve incision of the DNA strand, excision of the damaged nucleotide, gap filling by DNA resynthesis, and rejoining by ligation. The previous study described that nucleotide excision repair permitted incorporation of fludarabine nucleoside (F-area-A) into the repair patch, thereby inhibiting the DNA resynthesis. In the present study, to clarify the repair kinetics in view of the inhibition by F-ara-A, normal lymphocytes were stimulated to undergo nucleotide excision repair by ultraviolet C (UV) irradiation in the presence or absence of F-ara-A. The repair kinetics were determined as DNA single strand breaks resulting from the incision and the rejoining using the alkaline single cell gel electrophoresis (comet) assay. DNA resynthesis was evaluated in terms of the uptake of tritiated thymidine into DNA. The lymphocytes initiated the incision step maximally at 1 h, and completed the rejoining process within 4 h after UV exposure. UV also initiated thymidine uptake, which increased time-dependently and reached a plateau at 4 h. A 2-h pre-incubation with F-ara-A inhibited the repair in a concentration-dependent manner, with the maximal inhibition by 5 μM. This inhibitory effect was demonstrated by the reduction of the thymidine uptake and by the inhibition of the rejoining. A DNA polymerase inhibitor, aphidicolin, and a ribonucleotide reductase inhibitor, hydroxyurea, were not so inhibitory to the repair process as F-ara-A at equimolar concentrations. The present findings suggest that inhibition of nucleotide excision repair may represent a novel therapeutic strategy against cancer, especially in the context of resistant cells with an increased repair capacity. (author)

  12. Resveratrol mediated cell death in cigarette smoke transformed breast epithelial cells is through induction of p21Waf1/Cip1 and inhibition of long patch base excision repair pathway

    International Nuclear Information System (INIS)

    Cigarette smoking is a key factor for the development and progression of different cancers including mammary tumor in women. Resveratrol (Res) is a promising natural chemotherapeutic agent that regulates many cellular targets including p21, a cip/kip family of cyclin kinase inhibitors involved in DNA damage-induced cell cycle arrest and blocking of DNA replication and repair. We have recently shown that cigarette smoke condensate (CSC) prepared from commercially available Indian cigarette can cause neoplastic transformation of normal breast epithelial MCF-10A cell. Here we studied the mechanism of Res mediated apoptosis in CSC transformed (MCF-10A-Tr) cells in vitro and in vivo. Res mediated apoptosis in MCF-10A-Tr cells was a p21 dependent event. It increased the p21 protein expression in MCF-10A-Tr cells and MCF-10A-Tr cells-mediated tumors in xenograft mice. Res treatment reduced the tumor size(s) and expression of anti-apoptotic proteins (e.g. PI3K, AKT, NFκB) in solid tumor. The expressions of cell cycle regulatory (Cyclins, CDC-2, CDC-6, etc.), BER associated (Pol-β, Pol-δ, Pol-ε, Pol-η, RPA, Fen-1, DNA-Ligase-I, etc.) proteins and LP-BER activity decreased in MCF-10A-Tr cells but remain significantly unaltered in isogenic p21 null MCF-10A-Tr cells after Res treatment. Interestingly, no significant changes were noted in SP-BER activity in both the cell lines after Res exposure. Finally, it was observed that increased p21 blocks the LP-BER in MCF-10A-Tr cells by increasing its interaction with PCNA via competing with Fen-1 after Res treatment. Thus, Res caused apoptosis in CSC-induced cancer cells by reduction of LP-BER activity and this phenomenon largely depends on p21. - Highlights: • Resveratrol (Res) caused reduction of MCF-10A-Tr cell growth by inducing apoptosis. • Res caused cell cycle arrest and DNA damage in p21 dependent manner. • Res mediated LP-BER reduction in MCF-10A-Tr cells was a p21 dependent phenomenon. • Res inhibits BER and PI

  13. Resveratrol mediated cell death in cigarette smoke transformed breast epithelial cells is through induction of p21Waf1/Cip1 and inhibition of long patch base excision repair pathway

    Energy Technology Data Exchange (ETDEWEB)

    Mohapatra, Purusottam; Satapathy, Shakti Ranjan; Das, Dipon; Siddharth, Sumit [Cancer Biology Division, KIIT School of Biotechnology, KIIT University, Campus-11, Patia, Bhubaneswar, Orissa 751024 (India); Choudhuri, Tathagata [Institute of Life Sciences, Nalco Square, Bhubaneswar, Orissa 751023 (India); Department of Biotechnology, Visva Bharati University, Santiniketan, West Bengal (India); Kundu, Chanakya Nath, E-mail: cnkundu@gmail.com [Cancer Biology Division, KIIT School of Biotechnology, KIIT University, Campus-11, Patia, Bhubaneswar, Orissa 751024 (India)

    2014-03-15

    Cigarette smoking is a key factor for the development and progression of different cancers including mammary tumor in women. Resveratrol (Res) is a promising natural chemotherapeutic agent that regulates many cellular targets including p21, a cip/kip family of cyclin kinase inhibitors involved in DNA damage-induced cell cycle arrest and blocking of DNA replication and repair. We have recently shown that cigarette smoke condensate (CSC) prepared from commercially available Indian cigarette can cause neoplastic transformation of normal breast epithelial MCF-10A cell. Here we studied the mechanism of Res mediated apoptosis in CSC transformed (MCF-10A-Tr) cells in vitro and in vivo. Res mediated apoptosis in MCF-10A-Tr cells was a p21 dependent event. It increased the p21 protein expression in MCF-10A-Tr cells and MCF-10A-Tr cells-mediated tumors in xenograft mice. Res treatment reduced the tumor size(s) and expression of anti-apoptotic proteins (e.g. PI3K, AKT, NFκB) in solid tumor. The expressions of cell cycle regulatory (Cyclins, CDC-2, CDC-6, etc.), BER associated (Pol-β, Pol-δ, Pol-ε, Pol-η, RPA, Fen-1, DNA-Ligase-I, etc.) proteins and LP-BER activity decreased in MCF-10A-Tr cells but remain significantly unaltered in isogenic p21 null MCF-10A-Tr cells after Res treatment. Interestingly, no significant changes were noted in SP-BER activity in both the cell lines after Res exposure. Finally, it was observed that increased p21 blocks the LP-BER in MCF-10A-Tr cells by increasing its interaction with PCNA via competing with Fen-1 after Res treatment. Thus, Res caused apoptosis in CSC-induced cancer cells by reduction of LP-BER activity and this phenomenon largely depends on p21. - Highlights: • Resveratrol (Res) caused reduction of MCF-10A-Tr cell growth by inducing apoptosis. • Res caused cell cycle arrest and DNA damage in p21 dependent manner. • Res mediated LP-BER reduction in MCF-10A-Tr cells was a p21 dependent phenomenon. • Res inhibits BER and PI

  14. Ultraviolet-induced DNA excision repair in human B and T lymphocytes. 3. Repair in lymphocyte from chronic lymphocytic leukaemia

    International Nuclear Information System (INIS)

    This study examined the capacity of lymphocytes from individuals with chronic lymphocytic leukaemia (CLL) to undertake ultraviolet (u.v.)-induced DNA repair in comparison to control and age-matched purified B and T lymphocytes. The technique was independent of incorporation of radioactive precursor, i.e. by the recovery of normal sedimentation behaviour of nucleoid bodies obtained from these cells by lysis in high salt and non-ionic detergent. Recovery of normal sedimentation was associated with restoration of DNA supercoiling. CLL cells were found to be as sensitive to u.v. and to repair at similar rates as age-matched B controls. They were considerably more sensitive than young B cells and repaired less efficiently. Reasons for previous reported discrepancies in CLL repair were discussed. (author)

  15. Conserved XPB Core Structure and Motifs for DNA Unwinding:Implications for Pathway Selection of Transcription or ExcisionRepair

    Energy Technology Data Exchange (ETDEWEB)

    Fan, Li; Arval, Andrew S.; Cooper, Priscilla K.; Iwai, Shigenori; Hanaoka, Fumio; Tainer, John A.

    2005-04-01

    The human xeroderma pigmentosum group B (XPB) helicase is essential for transcription, nucleotide excision repair, and TFIIH functional assembly. Here, we determined crystal structures of an Archaeoglobus fulgidus XPB homolog (AfXPB) that characterize two RecA-like XPB helicase domains and discover a DNA damage recognition domain (DRD), a unique RED motif, a flexible thumb motif (ThM), and implied conformational changes within a conserved functional core. RED motif mutations dramatically reduce helicase activity, and the DRD and ThM, which flank the RED motif, appear structurally as well as functionally analogous to the MutS mismatch recognition and DNA polymerase thumb domains. Substrate specificity is altered by DNA damage, such that AfXPB unwinds dsDNA with 3' extensions, but not blunt-ended dsDNA, unless it contains a lesion, as shown for CPD or (6-4) photoproducts. Together, these results provide an unexpected mechanism of DNA unwinding with Implications for XPB damage verification in nucleotide excision repair.

  16. Disruption and characterization of the excision repair pathway in the extremely radioresistant bacterium Deinococcus SP. BR501

    International Nuclear Information System (INIS)

    Deinococcus sp. BR501, an extremely radioresistant bacterium may contain two nucleotide excision repair pathways: the UV damage endonuclease β (UvsE)-dependent excision repair pathway and the UvrABC-dependent pathway. And the UvsE (coded by dr1819) and UvrABC(Unit A coded by dr1771) are their key enzymes respectively. PCR primers were designed and homologous genes were cloned and disrupted in vitro according to the completely nucleotide sequence of Deinococcus radiodurans R1 genome. Then PCR production was transformed to BR501, and the disrupted mutants (triangle open dr1771, triangle open dr1819 and triangle open dr1771dr1819) were checked and confirmed by homologous recombination. These mutants and the wild type were irradiated by UV light and exposed to the DNA-damaging agents MMC and H2O2. The results showed that these pathways were existed in BR501 and only the two pathway losses could result in increased sensitivity to UV and MMC. (authors)

  17. The influence of some prostaglandins on DNA synthesis and DNA excision repair in mouse spleen cells ''in vitro''

    International Nuclear Information System (INIS)

    ''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 Fsub(2α) were tested in concentrations of 10 pg, 5 ng and 2,5μg per ml cell suspension. DNA synthesis was significantly increased by PgFsub(2α) 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 μg/ml but increased by PgFsub(2α) in the two lower concentrations. The rejoining of DNA-strand breaks after gamma-irradiation was slightly reduced by PgE1, PgE2 and PgF2 at 2,5 μg/ml. (author)

  18. Inhibition of DNA excision repair by methotrexate in Chinese hamster ovary cells following exposure to ultraviolet irradiation or ethylmethanesulfonate

    International Nuclear Information System (INIS)

    Previous results have suggested that methotrexate (MTX) could interfere with the repair of spontaneous DNA damage. To determine its effects on induced DNA damage, MTX was compared to hydroxyurea and arabinofuranosylcytosine (H/A), a drug combination known to block the DNA polymerase step of excision repair, for its ability to cause the accumulation of single-strand breaks (SSB) following exposure to either UV light or the alkylating agent ethylmethanesulfonate in Chinese hamster ovary cells. SSB were measured by alkaline elution 1, 2, and 6 h after exposure to either 1.8 mg/ml of ethylmethanesulfonate or 10 J/m2 of UV in cells pretreated with MTX or H/A. Following exposure to ethylmethanesulfonate, significant accumulation of SSB occurred in cells pretreated with either H/A or MTX. Coadministration of hypoxanthine and thymidine in MTX-treated cells prevented SSB accumulation, indicating that nucleotide depletion by MTX had inhibited repair synthesis. After UV irradiation, SSB accumulation was much less in MTX- than in H/A-treated cells. MTX was found to have no effect on the incision of UV damage. These results indicate that nucleotide depletion by MTX can affect the repair of DNA damage by exogenous agents, and that the extent of inhibition is dependent on the type of damage induced

  19. Excision repair in ataxia telangiectasia, Fanconi's anemia, Cockayne syndrome, and Bloom's syndrome after treatment with ultraviolet radiation and N-acetoxy-2-acetylaminofluorene

    International Nuclear Information System (INIS)

    Excision repair of damage due to ultraviolet radiation, N-acetoxy-2-acetylaminofluorene and a combination of both agents was studied in normal human fibroblasts and various cells from cancer prone patients (ataxia telangiectasia, Fanconi's anemia, Cockayne syndrome and Bloom's syndrome). Three methods giving similar results were used: unscheduled DNA synthesis by radioautography, photolysis of bromodeoxyuridine incorporated into parental DNA during repair, and loss of sites sensitive to an ultraviolet endonuclease. All cell lines were proficient in repair of ultraviolet and acetoxy acetylaminofluorene damage and at saturation doses of both agents repair was additive. We interpret these data as indicating that the rate limiting step in excision repair of ultraviolet and acetoxy acetylaminofluorene is different and that there are different enzyme(s) working on incision of both types of damages. (Auth.)

  20. Affinity of yeast nucleotide excision repair factor 2, consisting of the Rad4 and Rad23 proteins, for ultraviolet damaged DNA

    International Nuclear Information System (INIS)

    Saccharomyces cerevisiae Rad4 and Rad23 proteins are required for the nucleotide excision repair of UV light-damaged DNA. Previous studies have indicated that these two DNA repair proteins are associated in a tight complex, which we refer to as nucleotide excision repair factor 2 (NEF2). In a reconstituted nucleotide excision repair reaction, incision of UV-damaged DNA is dependent on NEF2, indicating a role of NEF2 in an early step of the repair process. NEF2 does not, however, possess an enzymatic activity, and its function in the damage-specific incision reaction has not yet been defined. Here we use a DNA mobility shift assay to demonstrate that NEF2 binds specifically to UV-damaged DNA. Elimination of cyclobutane pyrimidine dimers from the UV-damaged DNA by enzymatic photoreactivation has little effect on the affinity of NEF2 for the DNA, suggesting that NEF2 recognizes the 6-(1, 2)-dihydro-2-oxo-4-pyrimidinyl)-5-methyl-2,4-(1H,3H)-pyrimidinedione photoproducts in the damaged DNA. These results highlight the intricacy of the DNA damage-demarcation reaction during nucleotide excision repair in eukaryotes. (author)

  1. Active transcriptomic and proteomic reprogramming in the C. elegans nucleotide excision repair mutant xpa-1.

    Science.gov (United States)

    Kassahun, Henok; Nilsen, Hilde

    2013-10-01

    Oxidative stress promotes human aging and contributes to common neurodegenerative diseases. Endogenous DNA damage induced by oxidative stress is believed to be an important promoter of neurodegenerative diseases. Although a large amount of evidence correlates a reduced DNA repair capacity with aging and neurodegenerative disease, there is little direct evidence of causality. Moreover, the contribution of oxidative DNA damage to the aging process is poorly understood. We have used the nematode Caenorhabditis elegans to study the contribution of oxidative DNA damage and repair to aging. C. elegans is particularly well suited to tackle this problem because it has a minimum complexity DNA repair system, which enables us to circumvent the important limitation presented by the extensive redundancy of DNA repair enzymes in mammals. PMID:24744987

  2. Effect of cordycepin(3'-deoxyadenosine) on excision repair of 5,6-dihydroxy-dihydrothymine-type products from the DNA of Micrococcus radiodurans

    International Nuclear Information System (INIS)

    Cordycepin(3'-deoxyadenosine), a nucleoside analog, has been shown to enhance radiation-induced cell killing. In an effort to elucidate the possible mechanism for enhancement of cell killing, the effect of cordycepin on the excision repair of radiation-induced 5,6-dihydroxy-dihydrothymine-type (t') products from the DNA of wild type Micrococcus radiodurans was investigated. The capacity of M. radiodurans to excise nondimeric (t') products from its DNA was significantly impaired after cordycepin treatment. The results suggest that the increased radiation sensitivity of cordycepin-treated cells could be due to alterations in cellular processes that repair DNA damage

  3. HHR23A, a human homolog of Saccharomyces cerevisiae Rad23, regulates xeroderma pigmentosum C protein and is required for nucleotide excision repair

    International Nuclear Information System (INIS)

    HHR23A and hHR23B are the human homologs of Saccharomyces cerevisiae Rad23. hHR23B is associated with the nucleotide excision repair (NER) factor xeroderma pigmentosum C (XPC) protein and is required for global genome repair. The function of hHR23A is not yet clear. In this study, the potential function of the hHR23A protein was investigated using RNA interference techniques. The hHR23A knock-down (KD) construct diminished the RNA level of hHR23A protein by approximately 60%, and it did not interfere with expression of the hHR23B gene. Based on Southwestern immunoblot and host-cell reactivation assays, hHR23AKD cells were found to be deficient in DNA repair activity against the DNA damage caused by UVC irradiation. In these hHR23AKD cells, the XPC gene was not normally induced by UVC irradiation, indicating that the hHR23A protein is involved in NER through regulation of the DNA damage recognition protein XPC. Co-immunoprecipitation experiments revealed that hHR23A was associated with a small portion of hHR23B and the majority of p53 protein, indicating that hHR23A regulates the function of XPC by its association with the NER activator p53

  4. Deoxyribonucleic acid excision repair in chromatin after ultraviolet irradiation of human fibroblasts in culture

    International Nuclear Information System (INIS)

    We have exposed confluent normal human fibroblasts to ultraviolet (UV) fluences of 5, 14, or 40 J/m2 and monitored the specific activity of post-uv repair synthesis in chromatin with [3H]thymidine pulses. We have shown that under conditions where no semiconservative deoxyribonucleic acid (DNA) synthesis is detectable, the specific activity of repair label one-fifth that in bulk DNA at all three uv fluences. On the other hand, the distribution of thymine-containing pyrimidine dimers in bulk and nuclease-resistant regions measured either immediately after irradiation or at later times showed no significant differences; preferential labeling of linker (nuclease-sensitive) DNA during repair synthesis is thus apparently not due to a predominance of uv-induced photoproducts in linker relative to core particle DNA in the nucleosoome. Pulse and pulse-chase experiments at 14 or 40 J/m2 with normal human or repair-deficient xeroderma pigmentosum (XP) cells showed that at most 30% of repair label in all these cells shifts from nuclease-sensitive (linker) DNA to nuclease-resistant (core particle) DNA

  5. Variation in PAH-related DNA adduct levels among non-smokers: the role of multiple genetic polymorphisms and nucleotide excision repair phenotype

    OpenAIRE

    Etemadi, Arash; Islami, Farhad; Phillips, David H.; Godschalk, Roger; Golozar, Asieh; Kamangar, Farin; Malekshah, Akbar Fazel-Tabar; Pourshams, Akram; Elahi, Seerat; Ghojaghi, Farhad; Strickland, Paul T.; Taylor, Philip R.; Boffetta, Paolo; Abnet, Christian C.; Dawsey, Sanford M.

    2012-01-01

    Polycyclic aromatic hydrocarbons (PAHs) likely play a role in many cancers even in never-smokers. We tried to find a model to explain the relationship between variation in PAH-related DNA adduct levels among people with similar exposures, multiple genetic polymorphisms in genes related to metabolic and repair pathways, and nucleotide excision repair (NER) capacity. In 111 randomly selected female never-smokers from the Golestan Cohort Study in Iran, we evaluated 21 SNPs in 14 genes related to...

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

    OpenAIRE

    Newton, Kelley N.; Courcelle, Charmain T; Justin Courcelle

    2012-01-01

    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...

  7. Excision repair of gamma-ray-induced alkali-stable DNA lesions with the help of γ-endonuclease from Micrococcus luteus

    International Nuclear Information System (INIS)

    γ-endonuclease Y, an enzyme that hydrolyses phosphodiester bonds at alkali-stable lesions in γ-irradiated (N2, tris buffer) DNA, has been partially purified from Micrococcus luteus. The enzyme has a molecular weight of about 19 000, induces single-strand breaks with 3'OH-5'PO4 termini and contains endonuclease activity towards DNA treated with 7-bromomethylbenz(a)anthracene. γ-endonuclease Y induces breaks in OsO4-treated poly(dA-dT) and apparently is specific towards γ-ray-induced base lesions of the t' type. The complete excision repair of γ-endonuclease Y substrate sites has been performed in vitro by γ-endonuclease Y, DNA polymerase and ligase. (author)

  8. In vitro Repair of Oxidative DNA Damage by Human Nucleotide Excision Repair System: Possible Explanation for Neurodegeneration in Xeroderma Pigmentosum Patients

    Science.gov (United States)

    Reardon, Joyce T.; Bessho, Tadayoshi; Kung, Hsiang Chuan; Bolton, Philip H.; Sancar, Aziz

    1997-08-01

    Xeroderma pigmentosum (XP) patients fail to remove pyrimidine dimers caused by sunlight and, as a consequence, develop multiple cancers in areas exposed to light. The second most common sign, present in 20-30% of XP patients, is a set of neurological abnormalities caused by neuronal death in the central and peripheral nervous systems. Neural tissue is shielded from sunlight-induced DNA damage, so the cause of neurodegeneration in XP patients remains unexplained. In this study, we show that two major oxidative DNA lesions, 8-oxoguanine and thymine glycol, are excised from DNA in vitro by the same enzyme system responsible for removing pyrimidine dimers and other bulky DNA adducts. Our results suggest that XP neurological disease may be caused by defective repair of lesions that are produced in nerve cells by reactive oxygen species generated as by-products of an active oxidative metabolism.

  9. 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.

  10. 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.

  11. Calcium-Binding Capacity of Centrin2 Is Required for Linear POC5 Assembly but Not for Nucleotide Excision Repair

    Science.gov (United States)

    Dantas, Tiago J.; Daly, Owen M.; Conroy, Pauline C.; Tomas, Martin; Wang, Yifan; Lalor, Pierce; Dockery, Peter; Ferrando-May, Elisa; Morrison, Ciaran G.

    2013-01-01

    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. PMID:23844208

  12. Polymorphisms in miRNA binding sites of nucleotide excision repair genes and colorectal cancer risk

    Czech Academy of Sciences Publication Activity Database

    Naccarati, Alessio; Pardini, Barbara; Landi, S.; Landi, D.; Slyšková, Jana; Novotný, J.; Levý, M.; Poláková, Veronika; Lipská, L.; Vodička, Pavel

    2012-01-01

    Roč. 33, č. 7 (2012), s. 1346-1351. ISSN 0143-3334 R&D Projects: GA ČR GAP304/10/1286; GA ČR GP305/09/P194 Institutional research plan: CEZ:AV0Z50390703 Keywords : DNA repair * polymorphisms * miRNA binding sites Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.635, year: 2012

  13. Differences in nucleotide excision repair capacity between newly diagnosed colorectal cancer patients and healthy controls

    Czech Academy of Sciences Publication Activity Database

    Slyšková, Jana; Naccarati, Alessio; Pardini, Barbara; Poláková, Veronika; Vodičková, Ludmila; Šmerhovský, Z.; Levý, M.; Lipská, L.; Liška, V.; Vodička, Pavel (ed.)

    2012-01-01

    Roč. 27, č. 2 (2012), s. 225-232. ISSN 0267-8357 R&D Projects: GA ČR GAP304/10/1286; GA MZd NS10230 Grant ostatní: EEA-research fund:(NO) A/CZ0046/2/0012 Institutional research plan: CEZ:AV0Z50390512 Keywords : biomarkers * DNA damage * DNA repair capacity Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.500, year: 2012

  14. Active transcriptomic and proteomic reprogramming in the C. elegans nucleotide excision repair mutant xpa-1

    OpenAIRE

    Kassahun, Henok; Nilsen, Hilde

    2013-01-01

    Oxidative stress promotes human aging and contributes to common neurodegenerative diseases. Endogenous DNA damage induced by oxidative stress is believed to be an important promoter of neurodegenerative diseases. Although a large amount of evidence correlates a reduced DNA repair capacity with aging and neurodegenerative disease, there is little direct evidence of causality. Moreover, the contribution of oxidative DNA damage to the aging process is poorly understood. We have used the nematode...

  15. Sealing of chromosomal DNA nicks during nucleotide excision repair requires XRCC1 and DNA ligase III alpha in a cell-cycle-specific manner

    OpenAIRE

    Moser, Jill; Kool, Hanneke; Giakzidis, Ioannis; Caldecott, Keith; Mullenders, Leon H. F.; Fousteri, Maria I.

    2007-01-01

    Impaired gap filling and sealing of chromosomal DNA in nucleotide excision repair (NER) leads to genome instability. XRCC1-DNA ligase IIIa (XRCC1-Lig3) plays a central role in the repair of DNA single-strand breaks but has never been implicated in NER. Here we show that XRCC1-Lig3 is indispensable for ligation of NER-induced breaks and repair of UV lesions in quiescent cells. Furthermore, our results demonstrate that two distinct complexes differentially carry out gap filling in NER. XRCC1-Li...

  16. Combination of Aβ Secretion and Oxidative Stress in an Alzheimer-Like Cell Line Leads to the Over-Expression of the Nucleotide Excision Repair Proteins DDB2 and XPC

    Directory of Open Access Journals (Sweden)

    Anne Forestier

    2015-07-01

    Full Text Available Repair of oxidative DNA damage, particularly Base Excision Repair (BER, impairment is often associated with Alzheimer’s disease pathology. Here, we aimed at investigating the complete Nucleotide Excision Repair (NER, a DNA repair pathway involved in the removal of bulky DNA adducts, status in an Alzheimer-like cell line. The level of DNA damage was quantified using mass spectrometry, NER gene expression was assessed by qPCR, and the NER protein activity was analysed through a modified version of the COMET assay. Interestingly, we found that in the presence of the Amyloid β peptide (Aβ, NER factors were upregulated at the mRNA level and that NER capacities were also specifically increased following oxidative stress. Surprisingly, NER capacities were not differentially improved following a typical NER-triggering of ultraviolet C (UVC stress. Oxidative stress generates a differential and specific DNA damage response in the presence of Aβ. We hypothesized that the release of NER components such as DNA damage binding protein 2 (DDB2 and Xeroderma Pigmentosum complementation group C protein (XPC following oxidative stress might putatively involve their apoptotic role rather than DNA repair function.

  17. Yeast rad7-rad16 complex, specific for the nucleotide excision repair of the nontranscribed DNA strand, is an ATP-dependent DNA damage sensor

    International Nuclear Information System (INIS)

    In eukaryotes, nucleotide excision repair of ultraviolet light-damaged DNA is a highly intricate process that requires a large number of evolutionary conserved protein factors. Genetic studies in the yeast Saccharomyces cerevisiae have indicated a specific role of the RAD7 and RAD16 genes in the repair of transcriptionally inactive DNA. Here we show that the RAD7- and RAD16-encoded products exist as a complex of 1:1 stoichiometry, exhibiting an apparent dissociation constant (Kd) of <4 x 10(-10) M. The Rad7-Rad16 complex has been purified to near homogeneity in this study and is shown to bind, in an ATP-dependent manner and with high specificity, to DNA damaged by ultraviolet light. Importantly, inclusion of the Rad7-Rad16 complex in the in vitro nucleotide excision repair system that consists entirely of purified components results in a marked stimulation of damage specific incision. Thus, Rad7-Rad16 complex is the ATP-dependent DNA damage sensor that specifically functions with the ensemble of nucleotide excision repair factor (NEF) 1, NEF2, NEF3, and replication protein A in the repair of transcriptionally inactive DNA. We name this novel complex of Rad7 and Rad16 proteins NEF4. (author)

  18. 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.

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

    Science.gov (United States)

    Newton, Kelley N; Courcelle, Charmain T; Courcelle, Justin

    2012-01-01

    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. PMID:23056919

  20. Post-UV survival and mutagenesis in DNA repair-proficient and -deficient strains of Escherichia coli K-12 grown in 5-azacytidine to inhibit DNA cytosine methylation: evidence for mutagenic excision repair.

    Science.gov (United States)

    Radnedge, L; Pinney, R J

    1993-03-01

    Inhibition of cytosine methylation by growth in 5-azacytidine (5-azaC), did not affect the sensitivities to DNA damage induced by exposure to ultraviolet light (UV) of Escherichia coli K-12 strains AB1157 dcm+, which is fully DNA repair-proficient, LR68 (a dcm derivative of AB1157), JC3890 dcm+ uvrB, deficient in error-free excision repair, TK702 dcm+ umuC, deficient in error-prone repair, or TK501 dcm+ uvrB umuC, which lacks both excision repair and error-prone repair. However, growth in 5-azaC increased the post-UV survival of strains AB2463 recA(Def), AB2470 recB and AB2494 lexA(Ind-), which are deficient in the induction or expression of recombination repair or error-prone repair of DNA. Spontaneous mutation frequencies were increased in strains LR68, AB2463, AB2470 and AB2494 by growth in 5-azaC, but remained unaltered in strains AB1157, JC3890, TK702 or TK501. Growth in 5-azaC significantly increased UV-induced mutation frequencies in strains AB2463 and AB2470, significantly reduced UV-induced mutation in strain JC3890, but had little effect on UV-induced mutation in the other strains. The results suggest that 5-azaC may induce a normally error-free DNA repair pathway to become error-prone and therefore genotoxic. PMID:7683337

  1. Post-UV survival and mutagenesis in DNA repair-proficient and -deficient strains of Escherichia coli K-12 grown in 5-azacytidine to inhibit DNA cytosine methylation: evidence for mutagenic excision repair

    International Nuclear Information System (INIS)

    Inhibition of cytosine methylation by growth in 5-azacytidine (5-azaC), did not affect the sensitivities to DNA damage induced by exposure to ultraviolet light (UV) of Escherichia coli K-12 strains AB1157 dcm+, which is fully DNA repair-proficient, LR68 (a dcm derivative of AB1157), JC3890 dcm+ uvr B, deficient in error-free excision repair, TK702 dcm+ umuC, deficient in error-prone repair, or TK501 dcm+ uvrB umuC, which lacks both excision repair and error-prone repair. However, growth in 5-azaC increased the post-UV survival of strains AB2463 recA(Def), AB2470 recB and AB2494 lexA(Ind-), which are deficient in the induction or expression of recombination repair or error-prone repair of DNA. Spontaneous mutation frequencies were increased in strains LR68, AB2463, AB2470 and AB2494 by growth in 5-azaC, but remained unaltered in strains AB1157, JC3890, TK702 or TK501. Growth in 5-azaC significantly increased UV-induced mutation frequencies in strains AB2463 and AB2470, significantly reduced UV-induced mutation in strain JC3890, but had little effect on UV-induced mutation in the other strains. The results suggest that 5-azaC may induce a normally error-free DNA repair pathway to become error-prone and therefore genotoxic. (Author)

  2. 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

  3. Up-regulation of nucleotide excision repair in mouse lung and liver following chronic exposure to aflatoxin B1 and its dependence on p53 genotype

    International Nuclear Information System (INIS)

    Aflatoxin B1 (AFB1) 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-Trp53tm1BrdN5, Taconic) and wild-type mice were exposed to 0, 0.2 or 1.0 ppm AFB1 for 26 weeks. NER activity was assessed with an in vitro assay, using AFB1-epoxide adducted plasmid DNA as a substrate. For wild-type mice, repair of AFB1–N7-Gua adducts was 124% and 96% greater in lung extracts from mice exposed to 0.2 ppm and 1.0 ppm AFB1 respectively, and 224% greater in liver extracts from mice exposed to 0.2 ppm AFB1 (p < 0.05). In heterozygous p53 knockout mice, repair of AFB1–N7-Gua was only 45% greater in lung extracts from mice exposed to 0.2 ppm AFB1 (p < 0.05), and no effect was observed in lung extracts from mice treated with 1.0 ppm AFB1 or in liver extracts from mice treated with either AFB1 concentration. p53 genotype did not affect basal levels of repair. AFB1 exposure did not alter repair of AFB1-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 AFB1 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 B1 (AFB1). • The effects of AFB1 and p53 status on nucleotide excision repair are investigated. • AFB1 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 nucleotide excision repair after AFB1 exposure

  4. Catalytic sites for 3' and 5' incision of Escherichia coli nucleotide excision repair are both located in UvrC.

    Science.gov (United States)

    Verhoeven, E E; van Kesteren, M; Moolenaar, G F; Visse, R; Goosen, N

    2000-02-18

    Nucleotide excision repair in Escherichia coli is a multistep process in which DNA damage is removed by incision of the DNA on both sides of the damage, followed by removal of the oligonucleotide containing the lesion. The two incision reactions take place in a complex of damaged DNA with UvrB and UvrC. It has been shown (Lin, J. -J., and Sancar, A. (1992) J. Biol. Chem. 267, 17688-17692) that the catalytic site for incision on the 5' side of the damage is located in the UvrC protein. Here we show that the catalytic site for incision on the 3' side is in this protein as well, because substitution R42A abolishes 3' incision, whereas formation of the UvrBC-DNA complex and the 5' incision reaction are unaffected. Arg(42) is part of a region that is homologous to the catalytic domain of the homing endonuclease I-TevI. We propose that the UvrC protein consists of two functional parts, with the N-terminal half for the 3' incision reaction and the C-terminal half containing all the determinants for the 5' incision reaction. PMID:10671556

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

    Directory of Open Access Journals (Sweden)

    Moccia Claudia

    2012-05-01

    Full Text Available Abstract Background 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. Results 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. Conclusions 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.

  6. Complementary Roles of Yeast Rad4p and Rad34p in Nucleotide Excision Repair of Active and Inactive rRNA Gene Chromatin▿

    OpenAIRE

    Tremblay, Maxime; Teng, Yumin; Paquette, Michel; Waters, Raymond; Conconi, Antonio

    2008-01-01

    Nucleotide excision repair (NER) removes a plethora of DNA lesions. It is performed by a large multisubunit protein complex that finds and repairs damaged DNA in different chromatin contexts and nuclear domains. The nucleolus is the most transcriptionally active domain, and in yeast, transcription-coupled NER occurs in RNA polymerase I-transcribed genes (rDNA). Here we have analyzed the roles of two members of the xeroderma pigmentosum group C family of proteins, Rad4p and Rad34p, during NER ...

  7. The unstructured C-terminal extension of UvrD interacts with UvrB, but is dispensable for nucleotide excision repair

    OpenAIRE

    Manelyte, Laura; Guy, Colin P.; Smith, Rachel M.; Dillingham, Mark S.; McGlynn, Peter; Savery, Nigel J

    2009-01-01

    During nucleotide excision repair (NER) in bacteria the UvrC nuclease and the short oligonucleotide that contains the DNA lesion are removed from the post-incision complex by UvrD, a superfamily 1A helicase. Helicases are frequently regulated by interactions with partner proteins, and immunoprecipitation experiments have previously indicated that UvrD interacts with UvrB, a component of the post-incision complex. We examined this interaction using 2-hybrid analysis and surface plasmon resonan...

  8. 碱基切除修复基因HOGG1特异性锤头状核酶表达载体的构建及其功能的初步研究%Constructing the Eukaryotic Expression Vector to Study Preliminarily the Functions of Hammerhead Ribozyme Targeting Base Excision Repair Gene HOGG1

    Institute of Scientific and Technical Information of China (English)

    张遵真; 张勤; 吴媚

    2006-01-01

    Objective Adriamycin is widely used as an effective anti-tumor drug clinically treating a number of human cancers, but the effect of adriamycin is limited by drug resistance. The various kinds of investigations indicated that the anti-tumor activity of adriamycin resulted from drug-induced free radical formation. The free radicals could lead to oxidative DNA damage, and the lesion would be repaired by base excision repair (BER) pathway. Human 8-oxoguanine DNA glycosylase 1 (HOGG1) is a key enzyme on BER pathway. To study the influence and biological mechanism of the HOGG1 to adriamycin drug-sensitivity, the eukaryotic expression vector with gene of hammerhead ribozyme targeting HOGG1 mRNA would be constructed and identified, and then the change of drug-sensitivity in lung cancer A549 cells would be investigated. Methods According to computer design, two specific restriction site BamHⅠ and EcoRⅠ were added to both ends of the ribozyme gene, then the modified ribozyme gene was synthesized and cloned into the eukaryotic expression vector pcDNA3.1(+). The positive recombinants were screened by ampicillin resistance, and plasmids were extracted from the positive recombinants and digested by BamH Ⅰ and EcoR Ⅰ, and then were analyzed by agarose gel electrophoresis and DNA sequencing. The recombinants were transiently transfected into A549 cells. The positive recombinants were identified by reverse transcription-polymerase chain reaction (RT-PCR) targeting to NEO gene, which was a neomycin resistance gene for selection of stable cell lines and only existed in vectors. The changes of HOGG1 mRNA in A549 cells were detected by RT-PCR. Then the cellular sensitivity to adriamycin was tested by comparison between untransfected cells and transfected cells by MTT assay. The adriamycin-induced DNA damage was investigated by comet assay or single cell gel electrophoresis (SCGE) between untransfected and transfected cells. Results The recombinants containing the ribozyme gene

  9. Excision Repair Cross-complementation Group 1 is a Prognostic Biomarker in Patients with Colorectal Cancer Receiving Chemotherapy

    Institute of Scientific and Technical Information of China (English)

    Mu-Xing Li; Xin-Yu Bi; Hong Zhao; Zhen Huang; Yue Han; Dong-Bin Zhao; Jian-Jun Zhao

    2016-01-01

    Background:Conflicting results about the association between expression level of excision repair cross-complementation group 1 (ERCC1) and clinical outcome in patients with colorectal cancer (CRC) receiving chemotherapy have been reported.Thus,we searched the available articles and performed the meta-analysis to elucidate the prognostic role of ERCC1 expression in patients with CRC.Methods:A thorough literature search using PubMed (Medline),Embase,Cochrane Library,Web of Science databases,and Chinese Science Citation Database was conducted to obtain the relevant studies.Pooled hazard ratios (HRs) or odds ratios (ORs) with 95% confidence intervals (CIs) were calculated to estimate the results.Results:A total of 11 studies were finally enrolled in this meta-analysis.Compared with patients with lower ERCC1 expression,patients with higher ERCC1 expression tended to have unfavorable overall survival (OS) (HR =2.325,95% CI:1.720-3.143,P < 0.001),progression-free survival (PFS) (HR =1.917,95% CI:1.366-2.691,P < 0.001) and poor response to chemotherapy (OR =0.491,95% CI:0.243-0.990,P =0.047).Subgroup analyses by treatment setting,ethnicity,HR extraction,detection methods,survival analysis,and study design demonstrated that our results were robust.Conclusions:ERCC1 expression may be taken as an effective prognostic factor predicting the response to chemotherapy,OS,and PFS.Further studies with better study design and longer follow-up are warranted in order to gain a deeper understanding of ERCC 1's prognostic value.

  10. DNA excision repair in human cells treated with ultraviolet radiation and 7,12-dimethylbenz[a]anthracene 5,6-oxide

    International Nuclear Information System (INIS)

    Excision repair was measured in normal human and xeroderma pigmentosum group C cells treated with 7,12-dimethylbenz[a]anthracene 5,6-oxide and with ultraviolet radiation by the techniques of unscheduled DNA synthesis, repair replication, a modification of bromodeoxyuridine photolysis employing the dye Hoechst 33258 and 365 nm radiation, and endonuclease-sensitive sites assay. Radioautography and repair replication showed that in normal cells the magnitude of repair after a saturation dose of the epoxide (approx. 10 μM) to be 0.1-0.2 that after a saturating ultraviolet dose (20 J/m2 at 254), though survival data showed that both doses gave nearly similar killings. Repair was of the long-patch type and repair kinetics after the epoxide treatment were similar to ultraviolet. After a combined treatment with both agents, unscheduled synthesis in normal cells was more than additive, although, considering the experimental errors, these data and those of repair replication are consistent with additivity. The epoxide did not inhibit loss of sites sensitive to the ultraviolet endonuclease. However, after a combined treatment to xeroderma pigmentosum cells there was appreciably less unscheduled synthesis than for the sum of both treatments and the epoxide inhibited the loss of nuclease-sensitive sites. (Auth.)

  11. Post-UV survival and mutagenesis in DNA repair-proficient and -deficient strains of Escherichia coli K-12 grown in 5-azacytidine to inhibit DNA cytosine methylation: evidence for mutagenic excision repair

    Energy Technology Data Exchange (ETDEWEB)

    Radnedge, L.; Pinney, R.J. (London Univ. (United Kingdom). Dept. of Pharmaceutics)

    1993-03-01

    Inhibition of cytosine methylation by growth in 5-azacytidine (5-azaC), did not affect the sensitivities to DNA damage induced by exposure to ultraviolet light (UV) of Escherichia coli K-12 strains AB1157 dcm[sup +], which is fully DNA repair-proficient, LR68 (a dcm derivative of AB1157), JC3890 dcm[sup +] uvr B, deficient in error-free excision repair, TK702 dcm[sup +] umuC, deficient in error-prone repair, or TK501 dcm[sup +] uvrB umuC, which lacks both excision repair and error-prone repair. However, growth in 5-azaC increased the post-UV survival of strains AB2463 recA(Def), AB2470 recB and AB2494 lexA(Ind[sup -]), which are deficient in the induction or expression of recombination repair or error-prone repair of DNA. Spontaneous mutation frequencies were increased in strains LR68, AB2463, AB2470 and AB2494 by growth in 5-azaC, but remained unaltered in strains AB1157, JC3890, TK702 or TK501. Growth in 5-azaC significantly increased UV-induced mutation frequencies in strains AB2463 and AB2470, significantly reduced UV-induced mutation in strain JC3890, but had little effect on UV-induced mutation in the other strains. The results suggest that 5-azaC may induce a normally error-free DNA repair pathway to become error-prone and therefore genotoxic. (Author).

  12. [A Nobel Prize for DNA repair].

    Science.gov (United States)

    Jordan, Bertrand

    2016-01-01

    This year's Nobel Prize for chemistry recognizes the seminal contributions of three researchers who discovered the existence and the basic mechanisms of DNA repair: base excision repair, mismatch repair, and nucleotide excision repair. They have since been joined by many scientists elucidating diverse aspects of these complex mechanisms that now constitute a thriving research field with many applications, notably for understanding oncogenesis and devising more effective therapies. PMID:26850617

  13. Xeroderma pigmentosum, DNA repair and carcinogenesis

    International Nuclear Information System (INIS)

    The following topics are reviewed: Symptoms of xeroderma pigmentosum; xeroderma pigmentosum as a defect in the biochemistry of repair of radiation damage; major classes of DNA damage and repair mechanisms; excision repair in relation to biochemical steps and the XP defect; sensitivity of xeroderma pigmentosum cells; host-cell reactivation of UV-damaged viruses; excision of pyrimidine dimers from human cells; formation and sealing of single strand breaks during dimer excision; insertion of new bases to repair DNA; and DNA repair, carcinogens, and carcinogenesis

  14. Protective Effect of Diphlorethohydroxycarmalol against Ultraviolet B Radiation-Induced DNA Damage by Inducing the Nucleotide Excision Repair System in HaCaT Human Keratinocytes

    Directory of Open Access Journals (Sweden)

    Mei Jing Piao

    2015-09-01

    Full Text Available We investigated the protective properties of diphlorethohydroxycarmalol (DPHC, a phlorotannin, against ultraviolet B (UVB radiation-induced cyclobutane pyrimidine dimers (CPDs in HaCaT human keratinocytes. The nucleotide excision repair (NER system is the pathway by which cells identify and repair bulky, helix-distorting DNA lesions such as ultraviolet (UV radiation-induced CPDs and 6-4 photoproducts. CPDs levels were elevated in UVB-exposed cells; however, this increase was reduced by DPHC. Expression levels of xeroderma pigmentosum complementation group C (XPC and excision repair cross-complementing 1 (ERCC1, which are essential components of the NER pathway, were induced in DPHC-treated cells. Expression of XPC and ERCC1 were reduced following UVB exposure, whereas DPHC treatment partially restored the levels of both proteins. DPHC also increased expression of transcription factor specificity protein 1 (SP1 and sirtuin 1, an up-regulator of XPC, in UVB-exposed cells. DPHC restored binding of the SP1 to the XPC promoter, which is reduced in UVB-exposed cells. These results indicate that DPHC can protect cells against UVB-induced DNA damage by inducing the NER system.

  15. Effect of strand-specific excision repair on the spectra of mutations induced by benzo[a]pyrene-diol epoxide and ultraviolet radiation in diploid human cells

    International Nuclear Information System (INIS)

    To study the effect of excision repair on the spectra of mutations induced in diploid human cells by UV and ±-7β, 8α-dihydroxy-9α,10α-epoxy- 7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE), the author synchronized repair-proficient cells, treated them at the beginning of S phase or in G1 phase several hours prior to the onset of S phase, selected for thioguanine resistant cells, and determined the spectra of mutations in the coding region of the hyproxanthine(guanine)phosphoribosyl-transferase (HPRT) gene in the mutants. As a control, the spectra of mutations similarly induced in repair-deficient xeroderma pigmentosum (XP) cells were compared. There was no difference in the kinds of mutations observed in mutants derived from either cell strain treated with a particular mutagen either in S or in G1. With BPDE, the majority were G.C→T.A transversions; with UV, they were mainly G.C.→A.T transitions. The strand distribution of premutagenic lesions in mutants from repair-proficient cells treated in S or G1 differed significantly. The results strongly support the hypothesis that human cells preferentially repair UV- and BPDE-induced lesions from the transcribed strand of the HPRT gene. To test this, the rate of repair of BPDE adducts from individual strands of the HPRT gene was measured, using the UvrABC exinuclease and Southern hybridizations with strand-specific probes to detect lesions remaining. BPDE lesions were removed from the transcribed strand at a significantly faster rate than from the nontranscribed strand, consistent with my hypothesis. It was found that BPDE adducts were removed faster from either strand of the HPRT gene than from a transcriptionally inactive locus, indicating preferential repair of active genes. The results of these studies provide biochemical and biological evidence of strand-specific DNA repair of BPDE adducts in human cells

  16. Molecular cloning of eucaryotic genes required for excision repair of UV-irradiated DNA: isolation and partial characterization of the RAD3 gene of Saccharomyces cerevisiae.

    OpenAIRE

    Naumovski, L; Friedberg, E C

    1982-01-01

    We describe the molecular cloning of a 6-kilobase (kb) fragment of yeast chromosomal DNA containing the RAD3 gene of Saccharomyces cerevisiae. When present in the autonomously replicating yeast cloning vector YEp24, this fragment transformed two different UV-sensitive, excision repair-defective rad3 mutants of S. cerevisiae to UV resistance. The same result was obtained with a variety of other plasmids containing a 4.5-kb subclone of the 6-kb fragment. The UV sensitivity of mutants defective ...

  17. Important Role for Mycobacterium tuberculosis UvrD1 in Pathogenesis and Persistence apart from Its Function in Nucleotide Excision Repair

    OpenAIRE

    Houghton, Joanna; Townsend, Carolin; Williams, Alan R.; Rodgers, Angela; Rand, Lucinda; Walker, K. Barry; Böttger, Erik C; Springer, Burkhard; Davis, Elaine O.

    2012-01-01

    Mycobacterium tuberculosis survives and replicates in macrophages, where it is exposed to reactive oxygen and nitrogen species that damage DNA. In this study, we investigated the roles of UvrA and UvrD1, thought to be parts of the nucleotide excision repair pathway of M. tuberculosis. Strains in which uvrD1 was inactivated either alone or in conjunction with uvrA were constructed. Inactivation of uvrD1 resulted in a small colony phenotype, although growth in liquid culture was not significant...

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

    International Nuclear Information System (INIS)

    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. (paper)

  19. Differential expression of SOS genes in an E. coli mutant producing unstable lexA protein enhances excision repair but inhibits mutagenesis

    International Nuclear Information System (INIS)

    The SOS response is displayed following treatments which damage DNA or inhibit DNA replication. Two associated activities include enhanced capacity for DNA repair resulting from derepression of the recA, uvrA, uvrB and uvrD genes and increased mutagenesis due to derepression of recA, umuC and umuD. These changes are the consequence of the derepression of at least seventeen unlinked operons negatively regulated by LexA repressor. Following treatments that induce the SOS response, a signal molecule interacts with RecA protein, converting it to an activated form. Activated RecA protein facilitates the proteolytic cleavage of LexA repressor, which results in derepression of the regulon. The cell then enters a new physiological state during which time DNA repair processes are augmented. The lexA41 mutant of E. coli is a uv-resistant derivative of another mutant, lexA3, which produces a repressor that is not cleaved following inducing treatments. The resultant protein is unstable. Lac operon fusions to most of the genes in the SOS regulon were used to show that the various damage-inducible genes were derepressed to different extents. uvrA, B, and D were almost fully derepressed. Consistent with this finding, the rate of removal of T4 endonuclease V-sensitive sites was more rapid in the uv-irradiated lexA41 mutant than in normal cells, suggesting a more active excision repair system. We propose that the instability of the LexA41 protein reduces the intracellular concentration of repressor to a level that allows a high level of excision repair. The additional observation that SOS mutagenesis was only weakly induced in a lexA41 uvrA- mutant implies that the mutant protein partially represses one or more genes whose products promote SOS mutagenesis. 17 refs., 4 figs., 1 tab

  20. Low-intensity red and infrared lasers affect mRNA expression of DNA nucleotide excision repair in skin and muscle tissue.

    Science.gov (United States)

    Sergio, Luiz Philippe S; Campos, Vera Maria A; Vicentini, Solange C; Mencalha, Andre Luiz; de Paoli, Flavia; Fonseca, Adenilson S

    2016-04-01

    Lasers emit light beams with specific characteristics, in which wavelength, frequency, power, fluence, and emission mode properties determine the photophysical, photochemical, and photobiological responses. Low-intensity lasers could induce free radical generation in biological tissues and cause alterations in macromolecules, such as DNA. Thus, the aim of this work was to evaluate excision repair cross-complementing group 1 (ERCC1) and excision repair cross-complementing group 2 (ERCC2) messenger RNA (mRNA) expression in biological tissues exposed to low-intensity lasers. Wistar rat (n = 28, 4 for each group) skin and muscle were exposed to low-intensity red (660 nm) and near-infrared (880 nm) lasers at different fluences (25, 50, and 100 J/cm(2)), and samples of these tissues were withdrawn for RNA extraction, cDNA synthesis, and gene expression evaluation by quantitative polymerase chain reaction. Laser exposure was in continuous wave and power of 100 mW. Data show that ERCC1 and ERCC2 mRNA expressions decrease in skin (p tissue (p  0.05), but ERCC2 mRNA expression decreases in skin (p tissue (p tissue exposed to low-intensity lasers depending on wavelengths and fluences used in therapeutic protocols. PMID:26796702

  1. Molecular cloning and characterization of a mammalian excision repair gene that partially restores UV resistance to xeroderma pigmentosum complementation group D cells

    International Nuclear Information System (INIS)

    A hamster DNA repair gene has been isolated by cosmid rescue after two rounds of transfection of an immortalized xeroderma pigmentosum (XP) complementation group D cell line with neomycin-resistance gene (neo)-tagged normal hamster DNA and selection with G418 and ultraviolet irradiation. The functional length of the sequence has been defined as 11.5 kilobase pairs by measurement of the region of overlap between two hamster DNA-containing cosmids, cloned by selection for the integrated neo gene, that are able to confer an increase in resistance to ultraviolet irradiation on two XP-D cell line but not on an XP-A line. Detailed molecular characterization of the hamster repair gene has revealed no obvious similarities to two human excision repair genes (ERCC1 and ERCC2) that correct repair-defective hamster cells but have no effect on XP cells. Hybridization analyses of normal human and XP cell genomic DNAs and mRNAs, using a cosmid-clone probe from which repeated sequences have been removed, show that homologues are present and expressed in all cases

  2. Sealing of chromosomal DNA nicks during nucleotide excision repair requires XRCC1 and DNA ligase III alpha in a cell-cycle-specific manner.

    Science.gov (United States)

    Moser, Jill; Kool, Hanneke; Giakzidis, Ioannis; Caldecott, Keith; Mullenders, Leon H F; Fousteri, Maria I

    2007-07-20

    Impaired gap filling and sealing of chromosomal DNA in nucleotide excision repair (NER) leads to genome instability. XRCC1-DNA ligase IIIalpha (XRCC1-Lig3) plays a central role in the repair of DNA single-strand breaks but has never been implicated in NER. Here we show that XRCC1-Lig3 is indispensable for ligation of NER-induced breaks and repair of UV lesions in quiescent cells. Furthermore, our results demonstrate that two distinct complexes differentially carry out gap filling in NER. XRCC1-Lig3 and DNA polymerase delta colocalize and interact with NER components in a UV- and incision-dependent manner throughout the cell cycle. In contrast, DNA ligase I and DNA polymerase epsilon are recruited to UV-damage sites only in proliferating cells. This study reveals an unexpected and key role for XRCC1-Lig3 in maintenance of genomic integrity by NER in both dividing and nondividing cells and provides evidence for cell-cycle regulation of NER-mediated repair synthesis in vivo. PMID:17643379

  3. The unstructured C-terminal extension of UvrD interacts with UvrB, but is dispensable for nucleotide excision repair.

    Science.gov (United States)

    Manelyte, Laura; Guy, Colin P; Smith, Rachel M; Dillingham, Mark S; McGlynn, Peter; Savery, Nigel J

    2009-11-01

    During nucleotide excision repair (NER) in bacteria the UvrC nuclease and the short oligonucleotide that contains the DNA lesion are removed from the post-incision complex by UvrD, a superfamily 1A helicase. Helicases are frequently regulated by interactions with partner proteins, and immunoprecipitation experiments have previously indicated that UvrD interacts with UvrB, a component of the post-incision complex. We examined this interaction using 2-hybrid analysis and surface plasmon resonance spectroscopy, and found that the N-terminal domain and the unstructured region at the C-terminus of UvrD interact with UvrB. We analysed the properties of a truncated UvrD protein that lacked the unstructured C-terminal region and found that it showed a diminished affinity for single-stranded DNA, but retained the ability to displace both UvrC and the lesion-containing oligonucleotide from a post-incision nucleotide excision repair complex. The interaction of the C-terminal region of UvrD with UvrB is therefore not an essential feature of the mechanism by which UvrD disassembles the post-incision complex during NER. In further experiments we showed that PcrA helicase from Bacillus stearothermophilus can also displace UvrC and the excised oligonucleotide from a post-incision NER complex, which supports the idea that PcrA performs a UvrD-like function during NER in gram-positive organisms. PMID:19762288

  4. Polymorphisms in DNA Repair Genes, Smoking, and Pancreatic Adenocarcinoma Risk

    OpenAIRE

    Robert R McWilliams; William R Bamlet; Cunningham, Julie M.; Goode, Ellen L.; de ANDRADE, MARIZA; Lisa A Boardman; Petersen, Gloria M.

    2008-01-01

    Base excision repair and nucleotide excision repair are vital responses to multiple types of DNA damage, including damage from tobacco exposure. Single-nucleotide polymorphisms (SNP) in these pathways may affect DNA repair capacity and therefore influence risk for cancer development. We performed a clinic-based, case-control study comprising 481 consecutive patients with confirmed pancreatic adenocarcinoma and 625 healthy controls. Allele and genotype frequencies for 16 SNPs in DNA repair gen...

  5. Stem cell-based bone repair

    OpenAIRE

    Fei, Yurong; Xu, Ren-He; Hurley, Marja M.

    2012-01-01

    To accelerate bone repair, one strategy is to deliver the cells that make bone. The current review focuses on stem cell-based bone repair. Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) can self-renew unlimitedly and differentiate into the bone forming cells – osteoblasts. Scientists have been actively investigating culture conditions to stably and efficiently induce differentiation of these stem cells into osteoblasts. However, ESCs have the issues of ethnics, immune ...

  6. 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.

  7. Interaction of DNA replication with excision repair in ultraviolet light-irradiated human normal and xeroderma pigmentosum cells

    International Nuclear Information System (INIS)

    The molecular weights and rates of DNA synthesis per cell in human normal (E11 and GM 637) and xeroderma pigmentosum (XPICH and XP 12RO) cells irradiated with UV light and pulse labeled with 3H-thymidine undergo transient reduction and recovery during the hours immediately following irradiation. Recovery of the rate of DNA synthesis lags the recovery in molecular weight in cells capable of excision (normal and XP-varient) but dose not lag in excision defective XP group A (XP 12RO) cells. This effect is interpreted as being due to excision breaks which relex DNA coils and thereby prevent the initiation of clusters of replicons. The transient changes in molecular weights appear to be due to DNA chain growth being blocked by DNA damage soon after irradiation, but cells thereafter increasing in their ability to replicate normal sized molecules. The recovery fits an exponential curve with a similar rate constant in normal cells and a slower rate is observed in normal transformed, XP group A and variant cells. (author)

  8. Variation in PAH-related DNA adduct levels among non-smokers: the role of multiple genetic polymorphisms and nucleotide excision repair phenotype.

    Science.gov (United States)

    Etemadi, Arash; Islami, Farhad; Phillips, David H; Godschalk, Roger; Golozar, Asieh; Kamangar, Farin; Malekshah, Akbar Fazel-Tabar; Pourshams, Akram; Elahi, Seerat; Ghojaghi, Farhad; Strickland, Paul T; Taylor, Philip R; Boffetta, Paolo; Abnet, Christian C; Dawsey, Sanford M; Malekzadeh, Reza; van Schooten, Frederik J

    2013-06-15

    Polycyclic aromatic hydrocarbons (PAHs) likely play a role in many cancers even in never-smokers. We tried to find a model to explain the relationship between variation in PAH-related DNA adduct levels among people with similar exposures, multiple genetic polymorphisms in genes related to metabolic and repair pathways, and nucleotide excision repair (NER) capacity. In 111 randomly selected female never-smokers from the Golestan Cohort Study in Iran, we evaluated 21 SNPs in 14 genes related to xenobiotic metabolism and 12 SNPs in eight DNA repair genes. NER capacity was evaluated by a modified comet assay, and aromatic DNA adduct levels were measured in blood by32P-postlabeling. Multivariable regression models were compared by Akaike's information criterion (AIC). Aromatic DNA adduct levels ranged between 1.7 and 18.6 per 10(8) nucleotides (mean: 5.8 ± 3.1). DNA adduct level was significantly lower in homozygotes for NAT2 slow alleles and ERCC5 non-risk-allele genotype, and was higher in the MPO homozygote risk-allele genotype. The sum of risk alleles in these genes significantly correlated with the log-adduct level (r = 0.4, p studies, with large inter-individual variation which could best be explained by a combination of Phase I genes and NER capacity. PMID:23175176

  9. H. pylori-Induced DNA Strand Breaks Are Introduced by Nucleotide Excision Repair Endonucleases and Promote NF-κB Target Gene Expression

    Directory of Open Access Journals (Sweden)

    Mara L. Hartung

    2015-10-01

    Full Text Available The human bacterial pathogen Helicobacter pylori exhibits genotoxic properties that promote gastric carcinogenesis. H. pylori introduces DNA double strand breaks (DSBs in epithelial cells that trigger host cell DNA repair efforts. Here, we show that H. pylori-induced DSBs are repaired via error-prone, potentially mutagenic non-homologous end-joining. A genome-wide screen for factors contributing to DSB induction revealed a critical role for the H. pylori type IV secretion system (T4SS. Inhibition of transcription, as well as NF-κB/RelA-specific RNAi, abrogates DSB formation. DSB induction further requires β1-integrin signaling. DSBs are introduced by the nucleotide excision repair endonucleases XPF and XPG, which, together with RelA, are recruited to chromatin in a highly coordinated, T4SS-dependent manner. Interestingly, XPF/XPG-mediated DNA DSBs promote NF-κB target gene transactivation and host cell survival. In summary, H. pylori induces XPF/XPG-mediated DNA damage through activation of the T4SS/β1-integrin signaling axis, which promotes NF-κB target gene expression and host cell survival.

  10. The absence of caffeine inhibition of post-replication repair in excision deficient strains of Escherichia coli B and K12

    International Nuclear Information System (INIS)

    The effect of caffeine on postreplication repair, as seen in alkaline sucrose gradients, conjugation, and ultraviolet light (UV) survival, was studied in excision deficient strains of Escherichia coli K12 and B. A caffeine concentration of 2 mg/ml was chosen for the study which did not inhibit colony formation. Both E. coli K12 AB2500 and E. coli B WWP2 were more sensitive to UV when plated on caffeine plates. Conjugation was not inhibited in the E. coli K12 strain; however, the same procedure confirmed caffeine inhibition in the E. coli B strain. Caffeine did not inhibit postreplication repair in either strain, as determined by sedimentation profile studies of DNA on alkaline sucrose gradients. No strand breakage or degradation was observed in parental or post-UV replicated DNA for as long as 50 min incubation in caffeine. Thus caffeine concentrations that inhibited two recA gene product related phenomena did not cause immediate changes in size of DNA or inhibit the rate of a DNA gap generating postreplication type of DNA repair

  11. Molecular analysis of plasmid DNA repair within ultraviolet-irradiated Escherichia coli. I. T4 endonuclease V-initiated excision repair

    International Nuclear Information System (INIS)

    The process by which DNA-interactive proteins locate specific sequences or target sites on cellular DNA within Escherichia coli is a poorly understood phenomenon. In this study, we present the first direct in vivo analysis of the interaction of a DNA repair enzyme, T4 endonuclease V, and its substrate, pyrimidine dimer-containing plasmid DNA, within UV-irradiated E. coli. A pyrimidine dimer represents a small target site within large domains of DNA. There are two possible paradigms by which endonuclease V could locate these small target sites: a processive mechanism in which the enzyme scans DNA for dimer sites or a distributive process in which dimers are located by random three-dimensional diffusion. In order to discriminate between these two possibilities in E. coli, an in vivo DNA repair assay was developed to study the kinetics of plasmid DNA repair and the dimer frequency (i.e. the number of dimer sites on a given plasmid molecule) in plasmid DNA as a function of time during repair. Our results demonstrate that the overall process of plasmid DNA repair initiated by T4 endonuclease V (expressed from a recombinant plasmid within repair-deficient E. coli) occurs by a processive mechanism. Furthermore, by reducing the temperature of the repair incubation, the endonuclease V-catalyzed incision step has been effectively decoupled from the subsequent steps including repair patch synthesis, ligation, and supercoiling. By this manipulation, it was determined that the overall processive mechanism is composed of two phases: a rapid processive endonuclease V-catalyzed incision reaction, followed by a slower processive mechanism, the ultimate product of which is the dimer-free supercoiled plasmid molecule

  12. The Nucleotide Excision Repair System of Borrelia burgdorferi Is the Sole Pathway Involved in Repair of DNA Damage by UV Light

    OpenAIRE

    Hardy, Pierre-Olivier; Chaconas, George

    2013-01-01

    To survive and avoid accumulation of mutations caused by DNA damage, the genomes of prokaryotes encode a variety of DNA repair pathways most well characterized in Escherichia coli. Some of these are required for the infectivity of various pathogens. In this study, the importance of 25 DNA repair/recombination genes for Borrelia burgdorferi survival to UV-induced DNA damage was assessed. In contrast to E. coli, where 15 of these genes have an effect on survival of UV irradiation, disruption of...

  13. Unplanned Excision of Extremity Soft Tissue Sarcoma in Korea: A Nationwide Study Based on a Claims Registry.

    Directory of Open Access Journals (Sweden)

    Seungcheol Kang

    Full Text Available Unplanned excision of extremity soft tissue sarcoma (STS is common and has detrimental effects not only on patients' oncologic outcomes but also on functional and economic issues. However, no study has analyzed a nationwide population-based database. To estimate the incidence and treatment pattern of unplanned excision in extremity STS in the Korean population, a nationwide epidemiologic study was performed using the Korean Health Insurance Review and Assessment Service database, a centralized nationwide healthcare claims registry of Korea that covers the entire Korean population. Among 1,517 patients with extremity STS in the 4-year study period, 553 (36.5% underwent unplanned excision (unplanned group. About 80% of unplanned excisions were performed in tertiary or general hospitals. Of the unplanned group, 240 (43.4% underwent re-excision with or without radiation therapy and/or chemotherapy, and 51 (9.2% did not undergo re-excision but were treated with radiation therapy and/or chemotherapy; whereas, 262 (47.4% did not undergo any further treatment following unplanned excision. This study is the first nationwide population-based study on the unplanned excision of extremity STS. The results may have implications in establishing preventive or therapeutic measures to reduce the burden of unplanned excision of extremity STS.

  14. Polymorphisms in DNA Repair Genes, Recreational Physical Activity and Breast Cancer Risk

    OpenAIRE

    McCullough, Lauren E.; Santella, Regina M.; Cleveland, Rebecca J.; Millikan, Robert C.; Olshan, Andrew F.; North, Kari E; Bradshaw, Patrick T.; Eng, Sybil M.; Terry, Mary Beth; Shen, Jing; Crew, Katherine D.; Rossner, Pavel; Teitelbaum, Susan L.; Neugut, Alfred I.; Gammon, Marilie D.

    2013-01-01

    The mechanisms driving the inverse association between recreational physical activity (RPA) and breast cancer risk are complex. While exercise is associated with increased reactive oxygen species production it may also improve damage repair systems, particularly those that operate on single-strand breaks including base excision repair (BER), nucleotide excision repair (NER) and mismatch repair (MMR). Of these repair pathways, the role of MMR in breast carcinogenesis is least investigated. Pol...

  15. DNA repair deficiency in neurodegeneration

    DEFF Research Database (Denmark)

    Jeppesen, Dennis Kjølhede; Bohr, Vilhelm A; Stevnsner, Tinna V.

    2011-01-01

    Deficiency in repair of nuclear and mitochondrial DNA damage has been linked to several neurodegenerative disorders. Many recent experimental results indicate that the post-mitotic neurons are particularly prone to accumulation of unrepaired DNA lesions potentially leading to progressive...... neurodegeneration. Nucleotide excision repair is the cellular pathway responsible for removing helix-distorting DNA damage and deficiency in such repair is found in a number of diseases with neurodegenerative phenotypes, including Xeroderma Pigmentosum and Cockayne syndrome. The main pathway for repairing oxidative...... base lesions is base excision repair, and such repair is crucial for neurons given their high rates of oxygen metabolism. Mismatch repair corrects base mispairs generated during replication and evidence indicates that oxidative DNA damage can cause this pathway to expand trinucleotide repeats, thereby...

  16. 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. PMID:26805762

  17. Inter-individual variation in nucleotide excision repair pathway is modulated by non-synonymous polymorphisms in ERCC4 and MBD4 genes

    International Nuclear Information System (INIS)

    Highlights: • We reported a large inter-individual variability of NER capacity. • ERCC4 rs1800124 and MBD4 rs10342 nsSNP variants were associated with DNA repair capacity. • DNA–protein interaction analyses showed alteration of binding for ERCC4 and MBD4 variants. • A new possible cross-talk between NER and BER pathways has been reported. - Abstract: Inter-individual differences in DNA repair capacity (DRC) may lead to genome instability and, consequently, modulate individual cancer risk. Among the different DNA repair pathways, nucleotide excision repair (NER) is one of the most versatile, as it can eliminate a wide range of helix-distorting DNA lesions caused by ultraviolet light irradiation and chemical mutagens. We performed a genotype–phenotype correlation study in 122 healthy subjects in order to assess if any associations exist between phenotypic profiles of NER and DNA repair gene single nucleotide polymorphisms (SNPs). Individuals were genotyped for 768 SNPs with a custom Illumina Golden Gate Assay, and peripheral blood mononuclear cells (PBMCs) of the same subjects were tested for a NER comet assay to measure DRC after challenging cells by benzo(a)pyrene diolepoxide (BPDE). We observed a large inter-individual variability of NER capacity, with women showing a statistically significant lower DRC (mean ± SD: 6.68 ± 4.76; p = 0.004) than men (mean ± SD: 8.89 ± 5.20). Moreover, DRC was significantly lower in individuals carrying a variant allele for the ERCC4 rs1800124 non-synonymous SNP (nsSNP) (p = 0.006) and significantly higher in subjects with the variant allele of MBD4 rs2005618 SNP (p = 0.008), in linkage disequilibrium (r2 = 0.908) with rs10342 nsSNP. Traditional in silico docking approaches on protein–DNA and protein–protein interaction showed that Gly875 variant in ERCC4 (rs1800124) decreases the DNA–protein interaction and that Ser273 and Thr273 variants in MBD4 (rs10342) indicate complete loss of protein–DNA interactions

  18. Inter-individual variation in nucleotide excision repair pathway is modulated by non-synonymous polymorphisms in ERCC4 and MBD4 genes

    Energy Technology Data Exchange (ETDEWEB)

    Allione, Alessandra, E-mail: alessandra.allione@hugef-torino.org [Human Genetics Foundation (HuGeF), Via Nizza 52, 10126 Turin (Italy); Guarrera, Simonetta; Russo, Alessia [Human Genetics Foundation (HuGeF), Via Nizza 52, 10126 Turin (Italy); Ricceri, Fulvio [Human Genetics Foundation (HuGeF), Via Nizza 52, 10126 Turin (Italy); Department of Medical Sciences, University of Turin, Via Santena 19, 10126 Turin (Italy); Purohit, Rituraj [Human Genetics Foundation (HuGeF), Via Nizza 52, 10126 Turin (Italy); Bioinformatics Division, School of Bio Sciences and Technology, Vellore Institute of Technology University, Vellore 632014, Tamil Nadu (India); Pagnani, Andrea; Rosa, Fabio; Polidoro, Silvia; Voglino, Floriana [Human Genetics Foundation (HuGeF), Via Nizza 52, 10126 Turin (Italy); Matullo, Giuseppe [Human Genetics Foundation (HuGeF), Via Nizza 52, 10126 Turin (Italy); Department of Medical Sciences, University of Turin, Via Santena 19, 10126 Turin (Italy)

    2013-11-15

    Highlights: • We reported a large inter-individual variability of NER capacity. • ERCC4 rs1800124 and MBD4 rs10342 nsSNP variants were associated with DNA repair capacity. • DNA–protein interaction analyses showed alteration of binding for ERCC4 and MBD4 variants. • A new possible cross-talk between NER and BER pathways has been reported. - Abstract: Inter-individual differences in DNA repair capacity (DRC) may lead to genome instability and, consequently, modulate individual cancer risk. Among the different DNA repair pathways, nucleotide excision repair (NER) is one of the most versatile, as it can eliminate a wide range of helix-distorting DNA lesions caused by ultraviolet light irradiation and chemical mutagens. We performed a genotype–phenotype correlation study in 122 healthy subjects in order to assess if any associations exist between phenotypic profiles of NER and DNA repair gene single nucleotide polymorphisms (SNPs). Individuals were genotyped for 768 SNPs with a custom Illumina Golden Gate Assay, and peripheral blood mononuclear cells (PBMCs) of the same subjects were tested for a NER comet assay to measure DRC after challenging cells by benzo(a)pyrene diolepoxide (BPDE). We observed a large inter-individual variability of NER capacity, with women showing a statistically significant lower DRC (mean ± SD: 6.68 ± 4.76; p = 0.004) than men (mean ± SD: 8.89 ± 5.20). Moreover, DRC was significantly lower in individuals carrying a variant allele for the ERCC4 rs1800124 non-synonymous SNP (nsSNP) (p = 0.006) and significantly higher in subjects with the variant allele of MBD4 rs2005618 SNP (p = 0.008), in linkage disequilibrium (r{sup 2} = 0.908) with rs10342 nsSNP. Traditional in silico docking approaches on protein–DNA and protein–protein interaction showed that Gly875 variant in ERCC4 (rs1800124) decreases the DNA–protein interaction and that Ser273 and Thr273 variants in MBD4 (rs10342) indicate complete loss of protein

  19. Nucleotide excision repair deficiency increases levels of acrolein-derived cyclic DNA adduct and sensitizes cells to apoptosis induced by docosahexaenoic acid and acrolein.

    Science.gov (United States)

    Pan, Jishen; Sinclair, Elizabeth; Xuan, Zhuoli; Dyba, Marcin; Fu, Ying; Sen, Supti; Berry, Deborah; Creswell, Karen; Hu, Jiaxi; Roy, Rabindra; Chung, Fung-Lung

    2016-07-01

    The acrolein derived cyclic 1,N(2)-propanodeoxyguanosine adduct (Acr-dG), formed primarily from ω-3 polyunsaturated fatty acids such as docosahexaenoic acid (DHA) under oxidative conditions, while proven to be mutagenic, is potentially involved in DHA-induced apoptosis. The latter may contribute to the chemopreventive effects of DHA. Previous studies have shown that the levels of Acr-dG are correlated with apoptosis induction in HT29 cells treated with DHA. Because Acr-dG is shown to be repaired by the nucleotide excision repair (NER) pathway, to further investigate the role of Acr-dG in apoptosis, in this study, NER-deficient XPA and its isogenic NER-proficient XAN1 cells were treated with DHA. The Acr-dG levels and apoptosis were sharply increased in XPA cells, but not in XAN1 cells when treated with 125μM of DHA. Because DHA can induce formation of various DNA damage, to specifically investigate the role of Acr-dG in apoptosis induction, we treated XPA knockdown HCT116+ch3 cells with acrolein. The levels of both Acr-dG and apoptosis induction increased significantly in the XPA knockdown cells. These results clearly demonstrate that NER deficiency induces higher levels of Acr-dG in cells treated with DHA or acrolein and sensitizes cells to undergo apoptosis in a correlative manner. Collectively, these results support that Acr-dG, a ubiquitously formed mutagenic oxidative DNA adduct, plays a role in DHA-induced apoptosis and suggest that it could serve as a biomarker for the cancer preventive effects of DHA. PMID:27036235

  20. Adaptive resynthesis of O/sup 6/-methylguanine-accepting protein can explain the differences between mammalian cells proficient and deficient in methyl excision repair

    Energy Technology Data Exchange (ETDEWEB)

    Waldstein, E.A.; Cao, E.H.; Setlow, R.B.

    1982-09-01

    Mammalian cells have been classified as proficient (Mer/sup +/) or deficient (Mer/sup -/) in methyl excision repair in terms of their cytotoxic reactions to agents that form O/sup 6/-alkylguanine and their abilities to reactivate alkylated adenoviruses. O/sup 6/-Methylguanine (O/sup 6/MeGua) is considered to be a lethal, mutagenic, and carciogenic lesion. We measured the abilities of cell extracts to transfer the methyl group from an exogenous DNA containing O/sup 6/MeGua to acceptor protein. The constitutive level of acceptor activity was independent of the Mer phenotype and was approx. = 100,000 acceptor sites per cell. Treatment of cells with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) results in a dose-dependent decrease in the acceptor activity in extracts because the rapid reaction between endogenous O/sup 6/MeGua and acceptor protein makes the latter unavailable for further reaction. Treatment of cells with multiple low doses of MNNG results in the enhanced production of O/sup 6/MeGua-accepting protein in levels 2.5-fold above the constitutive values for Mer/sup +/ tumor cells and to approx. = 1.5-fold for Mer/sup +/ fibroblasts or Mer/sup -/ simian virus 40-transformed cells. Such treatments reduce the activities in Mer/sup -/ tumor cells and Chinese hamster cells. We concluded; (i) estimates of O/sup 6/MeGua in cellular DNA shortly after treatment may be seriously in error because of the rapid repair of this lesion, and (ii) the adaptive resynthesis of acceptor protein, not its constitutive level, is the important correlate of cell resistance to methylating agents.

  1. Affinity purification and partial characterization of a yeast multiprotein complex for nucleotide excision repair using histidine-tagged Rad14 protein

    International Nuclear Information System (INIS)

    The nucleotide excision repair (NER) pathway of eukaryotes involves approximately 30 polypeptides. Reconstitution of this pathway with purified components is consistent with the sequential assembly of NER proteins at the DNA lesion. However, recent studies have suggested that NER proteins may be pre-assembled in a high molecular weight complex in the absence of DNA damage. To examine this model further, we have constructed a histidine-tagged version of the yeast DNA damage recognition protein Rad14. Affinity purification of this protein from yeast nuclear extracts resulted in the co-purification of Rad1, Rad7, Rad10, Rad16, Rad23, RPA, RPB1, and TFIIH proteins, whereas none of these proteins bound to the affinity resin in the absence of recombinant Rad14. Furthermore, many of the co-purifying proteins were present in approximately equimolar amounts. Co-elution of these proteins was also observed when the nuclear extract was fractionated by gel filtration, indicating that the NER proteins were associated in a complex with a molecular mass of >1000 kDa prior to affinity chromatography. The affinity purified NER complex catalyzed the incision of UV-irradiated DNA in an ATP-dependent reaction. We conclude that active high molecular weight complexes of NER proteins exist in undamaged yeast cells

  2. Effects of environmental exposure: Interplay between helix-distorting and oxidative DNA lesions and their repair

    OpenAIRE

    Lakså, Solveig Margrethe B.

    2012-01-01

    DNA lesions are introduced in all living organisms every day, both via endogenous processes and by exposure to an array of DNA damaging agents. DNA lesions require repair for the sustenance of life. Base excision repair (BER) and nucleotide excision repair (NER) are DNA repair pathways involved in removal of oxidative DNA lesions and helix-distorting DNA lesions, respectively. Several studies suggest interactions or crosstalk between these pathways, involving overlapping activities for remova...

  3. 核苷酸切除修复通路基因多态性与肺癌易感性%Gene polymorphisms in the nucleotide excision repair pathway and lung cancer susceptibility

    Institute of Scientific and Technical Information of China (English)

    钱莹莹; 束永前

    2013-01-01

    Nucleotide excision repair (NER) pathway is one of the principal ways of the repair of DNA damage.The single nucleotide polymorphisms (SNP) of its key genes such as xeroderma pigmentosum group A (XPA) gene,excision repair cross complementingl (ERCC1) gene and xeroderma pigmentosum group D (XPD) gene may be associated with differences in the DNA repair capacity and may influence an individual's risk of lung cancer,because the variant genotype in those polymorphisms might destroy or alter repair function.%核苷酸切除修复(NER)通路是修复DNA损伤的主要途径之一,其中着色性于皮病A型(XPA)基因、核苷酸切除修复交叉互补1(ERCC1)基因和着色性干皮病D型(XPD)基因的单核苷酸多态性(SNP)改变了NER通路中重要酶与蛋白的修复功能,引起DNA损伤修复能力的差异,最终导致个体肺癌易感性不同.

  4. A Novel Variable Index and Excision CFAR Based Ship Detection Method on SAR Imagery

    Directory of Open Access Journals (Sweden)

    Kefeng Ji

    2015-01-01

    Full Text Available When applying the constant false alarm rate (CFAR detector to ship detection on synthetic aperture radar (SAR imagery, multiple interferers such as upwelling, breaking waves, ambiguities, and neighboring ships in a dense traffic area will degrade the probability of detection. In this paper, we propose a novel variable index and excision CFAR (VIE-CFAR based ship detection method to alleviate the masking effect of multiple interferers. Firstly, we improve the variable index (VI CFAR with an excision procedure, which censors the multiple interferers from the reference cells. And then, the paper integrates the novel CFAR concept into a ship detection scheme on SAR imagery, which adopts the VIE-CFAR to screen reference cells and the distribution to derive detection threshold. Finally, we analyze the performances of the VIE-CFAR under different environments and validate the proposed method on both ENVISAT and TerraSAR-X SAR data. The results demonstrate that the proposed method outperforms other existing detectors, especially in the presence of multiple interferers.

  5. Triple-Helix Formation Induces Recombination in Mammalian Cells via a Nucleotide Excision Repair-Dependent Pathway

    OpenAIRE

    Faruqi, A. Fawad; Datta, Hirock J.; Carroll, Dana; Seidman, Michael M.; Glazer, Peter M.

    2000-01-01

    The ability to stimulate recombination in a site-specific manner in mammalian cells may provide a useful tool for gene knockout and a valuable strategy for gene therapy. We previously demonstrated that psoralen adducts targeted by triple-helix-forming oligonucleotides (TFOs) could induce recombination between tandem repeats of a supF reporter gene in a simian virus 40 vector in monkey COS cells. Based on work showing that triple helices, even in the absence of associated psoralen adducts, are...

  6. Functional evaluation of DNA repair in human biopsies and their relation to other cellular biomarkers

    Czech Academy of Sciences Publication Activity Database

    Slyšková, Jana; Langie, S. A. S.; Collins, A. R.; Vodička, Pavel

    2014-01-01

    Roč. 116, č. 5 (2014). ISSN 1664-8021 R&D Projects: GA ČR(CZ) GAP304/12/1585 Institutional support: RVO:68378041 Keywords : base excision repair * nucleotide excision repair * human solid tissue Subject RIV: EB - Genetics ; Molecular Biology

  7. Adaptive resynthesis of O6-methylguanine-accepting protein can explain the differences between mammalian cells proficient and deficient in methyl excision repair.

    Science.gov (United States)

    Waldstein, E A; Cao, E H; Setlow, R B

    1982-09-01

    Mammalian cells have been classified as proficient (Mer(+)) or deficient (Mer(-)) in methyl excision repair in terms of their cytotoxic reactions to agents that form O(6)-alkylguanine and their abilities to reactivate alkylated adenoviruses. O(6)-Methylguanine (O(6)MeGua) is considered to be a lethal, mutagenic, and carcinogenic lesion. We measured the abilities of cell extracts to transfer the methyl group from an exogenous DNA containing O(6)MeGua to acceptor protein. The constitutive level of acceptor activity was independent of the Mer phenotype and was approximately 100,000 acceptor sites per cell. Treatment of cells with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) results in a dose-dependent decrease in the acceptor activity in extracts because the rapid reaction between endogenous O(6)MeGua and acceptor protein makes the latter unavailable for further reaction. Treatment of cells with 1 muM MNNG for 15 min or 2 muM for approximately 2 min uses up >95% of the constitutive activity. However, Mer(+) cells, which are resistant to MNNG, rapidly resynthesize new acceptor protein, and the activity returns to the basal level in approximately 90 min. In Mer(-) tumor cells and Chinese hamster cells, which are sensitive to MNNG, resynthesis is not detectable in 90 min. Mer(-) simian virus 40-transformed fibroblasts, known to have an intermediate sensitivity to MNNG, have an intermediate resynthesis rate. Treatment of cells with multiple low doses of MNNG results in the enhanced production of O(6)MeGua-accepting protein in levels 2.5-fold above the constitutive values for Mer(+) tumor cells and to approximately 1.5-fold for Mer(+) fibroblasts or Mer(-) simian virus 40-transformed cells. Such treatments reduce the activities in Mer(-) tumor cells and Chinese hamster cells. We conclude: (i) estimates of O(6)MeGua in cellular DNA shortly after treatment may be seriously in error because of the rapid repair of this lesion, and (ii) the adaptive resynthesis of acceptor

  8. Use of metabolic inhibitors to investigate the excision repair of pyrimidine dimers and non-dimer DNA damages induced in human and ICR 2A frog cells by solar ultraviolet radiation

    International Nuclear Information System (INIS)

    ICR 2A frog and normal human skin fibroblasts were exposed to either 5J/m2 of 254 nm UV or 50 kJ/m2 of the Mylar-filtered solar UV wavelengths produced by a fluorescent sunlamp. Following these approximately equitoxic treatments, cells were incubated in medium containing the DNA synthesis inhibitors hydroxyurea (HU) and 1-β-D-arabinofuranosyl cytosine (ara C0 for 0-20 min (human fibroblasts) or 0-4 h (frog cells) to accumulate DNA breaks resulting from enzymatic incision during excision repair. It was found that breaks were formed in human cells at about a 200-fold-higher rate compared with the ICR 2A cells indicating a relatively low capacity for excision repair in the frog cells. In addition, the rate of DNA break formation in solar UV-irradiated cells was only one-third of the level detected in 254 nm-irradiated cells. This result is consistent with the conclusion that the pathway(s) involved in the repair of solar UV-induced DNA damages differs from the repair of lesions produced in cells exposed to 254 nm UV. (author)

  9. DNA repair initiated in chronic lymphocytic leukemia lymphocytes by 4-hydroperoxycyclophosphamide is inhibited by fludarabine and clofarabine.

    OpenAIRE

    Yamauchi, Takahiro; Nowak, Billie J.; Michael J Keating; Plunkett, William

    2001-01-01

    PURPOSE: Chronic lymphocytic leukemia (CLL) lymphocytes respond to DNA alkylation by excision repair, with the extent of repair increasing as the cells acquire resistance to alkylating agents. Because incorporation of nucleotide analogues into the repair patches elicits death signals in quiescent cells, the increased capacity for excision repair in alkylator-resistant cells could facilitate incorporation of nucleotide analogues. We hypothesized that the mechanism-based interaction of nucleosi...

  10. DNA glycosylases: in DNA repair and beyond

    OpenAIRE

    Jacobs, Angelika L.; Schär, Primo

    2011-01-01

    The base excision repair machinery protects DNA in cells from the damaging effects of oxidation, alkylation, and deamination; it is specialized to fix single-base damage in the form of small chemical modifications. Base modifications can be mutagenic and/or cytotoxic, depending on how they interfere with the template function of the DNA during replication and transcription. DNA glycosylases play a key role in the elimination of such DNA lesions; they recognize and excise damaged bases, thereb...

  11. Role of Xeroderma Pigmentosum D and Excision Cross Repair Cross Complementing Gene 1 Polymorphism in the Occurrence of Side Effects for Advanced Colorectal Cancer Patients with Treatment of Platinum Based Chemotherapy%XPD和ERCC1基因多态性与进展期结直肠癌患者铂剂为基础化疗方案治疗的毒副作用

    Institute of Scientific and Technical Information of China (English)

    张正华; 侯凯生; 谢芳; 金重华; 洪洁艳; 卫海民; 吴学勇

    2015-01-01

    目的:探讨着色性干皮病基因D(Xeroderma Pigmentosum D,XPD)和剪切修复交叉互补基因l(Excision Repair CrossComplementing Gene 1,ERCC1)多态性基因型与以铂类为基础的化疗方案治疗结直肠癌的毒副作用的关系.方法:采用聚合酶链反应-限制性片段长度多态性(Polymemse Chain Reaction-Restriction Fragment Length Polymorphism,PCR-RFLP)分析方法,对我院2010年12月至2013年12月应用含奥沙利铂方案治疗的42例汉族进展期结直肠癌患者的XPD和XRCC1的多态性基因型进行分析,比较不同基因型与临床病理因素及化疗不良反应的关系.结果:XPD、ERCC1的单核苷酸多态性(Single Nucleotide Polymorphism,SNP)分布与年龄、性别、淋巴转移、肿瘤的部位、化疗史、分化程度、器官转移个数差异无统计学意义(P>0.05); XPD基因型中,其中AA基因型以骨髓抑制、恶心呕吐为主,AG基因型以腹泻及肝肾损伤为主,GG基因型以神经毒性及口腔黏膜炎为主,差异有统计学意义(P<0.05);ERCC1基因型中,LG基因型以骨髓抑制、恶心呕吐及腹泻等症状为主,LL基因型以肝肾损伤、神经毒性及口腔黏膜炎为主,差异有统计学意义(P<0.05).结论:XPD和ERCC1的基因型可能与结直肠癌铂类药物化疗的不良反应有关.

  12. Human longevity and variation in DNA damage response and repair

    DEFF Research Database (Denmark)

    Debrabant, Birgit; Soerensen, 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...

  13. 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...... mainly seen in men, where those with ERCC1-negative tumors had a significantly increased survival compared to men with ERCC1-positive tumors (median survival, 11.8 months v 7.9 months; P = .005). Conversely, women who were ERCC1 negative did not have a survival advantage over ERCC1-positive women...

  14. 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.

  15. Regulation of DNA repair in serum-stimulated xeroderma pigmentosum cells

    OpenAIRE

    1984-01-01

    The regulation of DNA repair during serum stimulation of quiescent cells was examined in normal human cells, in fibroblasts from three xeroderma pigmentosum complementation groups (A, C, and D), in xeroderma pigmentosum variant cells, and in ataxia telangiectasia cells. The regulation of nucleotide excision repair was examined by exposing cells to ultraviolet irradiation at discrete intervals after cell stimulation. Similarly, base excision repair was quantitated after exposure to methylmetha...

  16. Base excision repair of oxidative DNA damage and association with cancer and aging

    DEFF Research Database (Denmark)

    Maynard, Scott; Schurman, Shepherd H; Harboe, Charlotte;

    2009-01-01

    Aging has been associated with damage accumulation in the genome and with increased cancer incidence. Reactive oxygen species (ROS) are produced from endogenous sources, most notably the oxidative metabolism in the mitochondria, and from exogenous sources, such as ionizing radiation. ROS attack D...

  17. 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

    carotid plaques, 8 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...... response of BER 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....

  18. Kinetic Mechanism for the Flipping and Excision of 1,N 6-Ethenoadenine by AlkA

    OpenAIRE

    Taylor, Erin L.; O’Brien, Patrick J.

    2014-01-01

    Escherichia coli 3-methyladenine DNA glycosylase II (AlkA), an adaptive response glycosylase with a broad substrate range, initiates base excision repair by flipping a lesion out of the DNA duplex and hydrolyzing the N-glycosidic bond. We used transient and steady state kinetics to determine the minimal mechanism for recognition and excision of 1,N 6-ethenoadenine (εA) by AlkA. The natural fluorescence of this endogenously produced lesion allowed us to directly monitor the nucleotide flipping...

  19. Applications of repaired endonucleases

    International Nuclear Information System (INIS)

    The possibilities of using antimutagenously various endonucleases are discussed. Since mutageniety of excision repair is considerably lower than the repair in the replication moment or after thereof the intensification of the excision repair of premutation disorders can suppress mutagenesis. Experimental aproaches of using repair endonucleases for sounding premutation changes are described. The optimal object has been chosen for endonuclear sounding of premutation DNA disorders following an ionizing radiation action

  20. Expressions of Thymidylate Synthase, Thymidine Phosphorylase, Class Ⅲ β-tubulin, and Excision Repair Cross-complementing Group 1 Predict Response in Advanced Gastric Cancer Patients Receiving Capecitabine Plus Paclitaxel or Cisplatin

    Institute of Scientific and Technical Information of China (English)

    Ming Lu; Jing Gao; Xi-cheng Wang; Lin Shen

    2011-01-01

    Objective:To evaluate the role of class Ⅲ β-tubulin (TUBB3),thymidylate synthase (TS),thymidine phosphorylase (TP),and excision repair cross-complementing group 1 (ERCC1) in clinical outcome of advanced gastric cancer patients receiving capecitabine plus paclitaxel or cisplatin.Methods:The clinical data and tumor specimens from 57 advanced gastric cancer patients receiving first-line capecitabine plus paclitaxel (cohort 1,n=36) and capecitabine plus cisplatin (cohort 2,n=21) were retrospectively collected,and TUBB3,TS,TP,and ERCC1 expressions were detected by real-time quantitative PCR.The associations between expressions of biomarkers and response or survival were analyzed statistically.Results:The median age of 57 patients was 57 years (range:27-75 years) with 38 males and 19 females.Of all patients,the response rates of patients with high TP,low TP and high TS,low TS expressions were 57.1%,27.6% (P=0.024),and 55.2%,28.6% (P=0.042),respectively.Among cohort 1,the response rates and median overall survivals of patients with low and high TUBB3 expressions were 61.1% vs.33.3% (P=0.095) and 13.8 months vs.6.6 months (P=0.019),respectively; the response rate (87.5%) of patients with low TUBB3 and high TP expressions was higher than that (14.3%) of patients with high TUBB3 and low TP expressions (P=0.01).Among cohort 2,the response rates of patients with low ERCC1 and high ERCC1 expressions were 45.5% and 20.0% respectively (P=0.361).Conclusion:TUBB3,TS and TP expressions could predict the response of advanced gastric cancer patients receiving capecitabine-based and paclitaxel-based chemotherapy.These results will be further confirmed in future large samples.

  1. DNA Mismatch Repair and Oxidative DNA Damage: Implications for Cancer Biology and Treatment

    International Nuclear Information System (INIS)

    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

  2. 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.

  3. Functional evaluation of DNA repair in human biopsies and their relation to other cellular biomarkers

    OpenAIRE

    Slyskova, Jana; Langie, Sabine A. S.; Collins, Andrew R.; Vodicka, Pavel

    2014-01-01

    Thousands of DNA lesions are estimated to occur in each cell every day and almost all are recognized and repaired. DNA repair is an essential system that prevents accumulation of mutations which can lead to serious cellular malfunctions. Phenotypic evaluation of DNA repair activity of individuals is a relatively new approach. Methods to assess base and nucleotide excision repair pathways (BER and NER) in peripheral blood cells based on modified comet assay protocols have been widely applied i...

  4. Repair of oxidative DNA base damage in the host genome influences the HIV integration site sequence preference.

    Directory of Open Access Journals (Sweden)

    Geoffrey R Bennett

    Full Text Available Host base excision repair (BER proteins that repair oxidative damage enhance HIV infection. These proteins include the oxidative DNA damage glycosylases 8-oxo-guanine DNA glycosylase (OGG1 and mutY homolog (MYH as well as DNA polymerase beta (Polβ. While deletion of oxidative BER genes leads to decreased HIV infection and integration efficiency, the mechanism remains unknown. One hypothesis is that BER proteins repair the DNA gapped integration intermediate. An alternative hypothesis considers that the most common oxidative DNA base damages occur on guanines. The subtle consensus sequence preference at HIV integration sites includes multiple G:C base pairs surrounding the points of joining. These observations suggest a role for oxidative BER during integration targeting at the nucleotide level. We examined the hypothesis that BER repairs a gapped integration intermediate by measuring HIV infection efficiency in Polβ null cell lines complemented with active site point mutants of Polβ. A DNA synthesis defective mutant, but not a 5'dRP lyase mutant, rescued HIV infection efficiency to wild type levels; this suggested Polβ DNA synthesis activity is not necessary while 5'dRP lyase activity is required for efficient HIV infection. An alternate hypothesis that BER events in the host genome influence HIV integration site selection was examined by sequencing integration sites in OGG1 and MYH null cells. In the absence of these 8-oxo-guanine specific glycosylases the chromatin elements of HIV integration site selection remain the same as in wild type cells. However, the HIV integration site sequence preference at G:C base pairs is altered at several positions in OGG1 and MYH null cells. Inefficient HIV infection in the absence of oxidative BER proteins does not appear related to repair of the gapped integration intermediate; instead oxidative damage repair may participate in HIV integration site preference at the sequence level.

  5. A-T推进皮瓣修复眼周基底细胞癌皮肤缺损%A-T advanced flap for the repair of periorbital skin defect after basal cell carcinoma excision

    Institute of Scientific and Technical Information of China (English)

    乔星; 程定; 马少林; 马娟

    2011-01-01

    Objective To explore the clinical effect of A-T advanced skin flap for the treatment of periorbital skin and soft tissue defect after basal cell carcinoma cxcision. Methods The basal cell carcinoma at the periorbital region was excised and prepared as a triangular wound surface. A-T flap was designed and harvested for the repair of the defect with lateral skin flaps advanced and covered the wound defect. Totally 36 patients were received in this study from Septemher 2002 to September 2010. Results All flaps survived in all 36 cases without any obvious complications. After 1 to 2 years follow-up, satisfactory results were received with slight scars and non recurrence of the tumor. Conclusion A-T advanced flap is one of optimal methods in repairing periorbital defect after basal cell carcinoma excision, which could not only excise the tumor completely , but also could receive excellent appearance and function.%目的 探讨A-T推进皮瓣修复眼周基底细胞癌切除后缺损的临床效果.方法 将基底细胞癌病灶行椭圆形切除,并修建成三角形创面,在缺损邻近处设计A-T皮瓣,将缺损两侧皮肤向中间推进修复缺损.自2002年9月至2010年9月,共修复眼周基底细胞癌切除后缺损36例.结果 本组36例患者,术后皮瓣全部成活,无明显并发症发生,术后随访1~2年,瘢痕轻微,肿瘤无复发,效果满意.结论 A-T推进皮瓣是修复眼周基底细胞癌切除后缺损较理想的方式之一,不仅可以彻底根除病灶,也可兼顾术后功能和外形的完善统一.

  6. Relationship between genetic polymorphisms in nucleotide excision repair gene excision repair cross complement group 6, xeroderma pigmentosum group A and coal-burning-borne-arsenism%核苷酸切除修复基因ERCC6、XPA多态性与燃煤污染型地方性砷中毒的关系研究

    Institute of Scientific and Technical Information of China (English)

    徐晓静; 张爱华; 梁冰; 韩雪; 董学新; 黄晓欣

    2014-01-01

    Objective To investigate the relationship between genetic polymorphisms in nucleotide excision repair gene excision repair cross complementing group 6(ERCC6),xeroderma pigmentosum group A(XPA) and coal-burning-borne-arsenism in Guizhou Province.Method ERCC6 A3368G,ERCC6 C-6530G and XPA A23G gene polymorphisms were analyzed by polymerase chain reaction restriction fragment length polymorphism technique(PCR-RFLP) of 205 cases which were chosen as patients with arsenism and 187 residents as control group.Results The distributions of ERCC6 A3368G,ERCC6 C-6530G and XPA A23G in the case group were not statistically significant compared with those of the control group(x2 =3.209,2.963,3.335,all P > 0.05); individuals carrying G allelomorphic gene(AG + GG) had a lower risk than individuals carring A allelomorphic gene(ORadj =0.282,95%CI:0.126-0.628,P =0.002); relationship was not found between single genetic polymorphisms of ERCC6 C-6530G,XPA A23G and coal-burning-borne-arsenism; the risk of arsenism was decreased for individuals carrying the following five genotypes combination:ERCC6 A3368G(AG + GG) genotype and ERCC6 C-6530G CC genotype(ORadj =0.287,95%CI:0.087-0.946,P=0.040); ERCC6 A3368G(AG + GG) genotype and ERCC6 C-6530G(CG + GG) genotype (ORadj =0.226,95%CI:0.077-0.661,P =0.007); ERCC6 A3368G(AG + GG) genotype and XPA A23G AA genotype (ORadj =0.150,95%CI:0.038-0.596,P =0.007); ERCC6 A3368G (AG + GG) genotype and XPA A23G(AG + GG) genotype(ORadj =0.325,95%CI:0.118-0.897,P =0.030) ; ERCC6 C6530G (CG + GG) genotype and XPA A23G AA genotype (ORadj =0.397,95%CI:0.162-0.975,P=0.036).Conclusions Individuals carring ERCC6 A3368G (AG + GG) genotype have a low risk of arsenism.There are five genotypes combination of three gene polymorphisms in two genes,ERCC6 and XPA,which may reduce the risk of coal-burning-borne-arsenism.%目的 探讨核苷酸切除修复基因切除交叉互补基因6(ERCC6)、着色性干皮病基因(XPA)的多态性与贵州省燃煤污染

  7. DNA repair genotype and lung cancer risk in the beta-carotene and retinol efficacy trial

    OpenAIRE

    Doherty, Jennifer A; Sakoda, Lori C.; Loomis, Melissa M; Barnett, Matt J.; Julianto, Liberto; Thornquist, Mark D; Neuhouser, Marian L; Weiss, Noel S.; Goodman, Gary E.; Chen, Chu

    2013-01-01

    Many carcinogens in tobacco smoke cause DNA damage, and some of that damage can be mitigated by the actions of DNA repair enzymes. In a case-control study nested within the Beta-Carotene and Retinol Efficacy Trial, a randomized chemoprevention trial in current and former heavy smokers, we examined whether lung cancer risk was associated with variation in 26 base excision repair, mismatch repair, and homologous recombination repair genes. Analyses were limited to Caucasians (744 cases, 1477 co...

  8. 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

  9. Repair processes in diverse systems: overview

    International Nuclear Information System (INIS)

    Studies on DNA repair in several bacterial systems as well as in eukaryotes are reviewed. Some topics discussed are: excision repair of uv damage in Mycoplasma; repair replication in uv-irradiated Tetrahymena; repair of double-strand breaks in DNA of Micrococcus radiodurans; DNA repair in Neurospora; lack of dimer excision in x-irradiated Vicia faba; and DNA repair in Nicotiana, Haplopappus, and Chlamydomonas

  10. Faulty DNA-polymerase δ/ε-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

    International Nuclear Information System (INIS)

    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)

  11. Reduced nucleotide excision repair and GSTM1-null genotypes influence anti-B(a)PDE-DNA adduct levels in mononuclear white blood cells of highly PAH-exposed coke oven workers

    Energy Technology Data Exchange (ETDEWEB)

    Sofia Pavanello; Alessandra Pulliero; Ewa Siwinska; Danuta Mielzynska; Erminio Clonfero [University of Padova, Padova (Italy). Occupational Health Section, Department of Environmental Medicine and Public Health

    2005-07-01

    It is important to identify the potential genetic-susceptible factors that are able to modulate individual responses to exposure to carcinogenic polycyclic aromatic hydrocarbons (PAHs). In the present study we evaluated the influence of four polymorphisms of nucleotide excision repair (NER) genes and that of glutathione S-transferase {mu}1 (GSTM1-active or -null) on benzo(a)pyrene diol epoxide (B(a)PDE)-DNA adduct levels from the lympho-monocyte fraction (LMF) of highly PAH benzo(a)pyrene -exposed Polish coke oven workers with individual urinary post-shift excretion of 1-pyrenol exceeding the proposed biological exposure index. The bulky {+-}-r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene (anti-B(a)PDE)-DNA adduct levels were detected by high-performance liquid chromatography fluorescence analysis and genotypes by polymerase chain reaction. We found that workers with the low DNA repair capacity of XPC-PAT+/+ and XPA-A23A genotypes had increased anti-B(a)PDE-DNA adduct levels, DNA adducts were also raised in workers without GSTM1 activity. Workers with unfavourable XPC-PAT+/+ and XPA-A23A NER genotypes, alone or combined with GSTM1-null genotype were in the tertile with the highest adduct level. The increase in anti-B(a)PDE-DNA adduct levels was related in a multiple linear regression analysis to PAH exposure lack of GSTM1 activity and to low DNA repair capacity of the XPC-PAT+/+ genotype. The influence of the XPA-A23A genotype was not evident in this statistical analysis, and no associations with XPD polymorphisms, dietary habits or tobacco smoking were found. The modulation of anti-B(a)PDE-DNA adducts in the LMF by GSTM1-null and some low-activity NER genotypes may be considered as a potential genetic susceptibility factor capable of modulating individual responses to PAH genotoxic exposure and the consequent risk of cancer in coke oven workers.

  12. Charge-transport-mediated recruitment of DNA repair enzymes

    OpenAIRE

    Fok, Pak-Wing; Guo, Chin-Lin; Chou, Tom

    2008-01-01

    Damaged or mismatched bases in DNA can be repaired by base excision repair enzymes (BER) that replace the defective base. Although the detailed molecular structures of many BER enzymes are known, how they colocalize to lesions remains unclear. One hypothesis involves charge transport (CT) along DNA [Yavin et al., Proc. Natl. Acad. Sci. U.S.A. 102, 3546 (2005)]. In this CT mechanism, electrons are released by recently adsorbed BER enzymes and travel along the DNA. The electrons can scatter (by...

  13. Lack of SOS repair in Streptococcus pneumoniae

    International Nuclear Information System (INIS)

    Wild-type strains of Streptococcus pneumoniae were non-mutable by UV radiation and thymidine starvation. Moreover, UV-irradiated pneumococcal ω2 phages were not reactivated in an irradiated host. This suggests that, in pneumococcus, there is no efficient inducible repair process similar to the SOS repair described in detail for E. coli. We also report that mutations cannot be induced by a process thought to be linked to competence during transformation with isogenic wild-type DNA either on wild-type strains or in strains in which the hex function of excision and repair of mismatched bases is inactive. (orig.)

  14. Formation of isodialuric acid lesion within DNA oligomers via one-electron oxidation of 5-hydroxyuracil: characterization, stability and excision repair

    OpenAIRE

    Simon, Philippe; Gasparutto, Didier; Gambarelli, Serge; Saint-Pierre, Christine; Favier, Alain; Cadet, Jean

    2006-01-01

    5-Hydroxyuracil is a major oxidized nucleobase that can be generated by the action of •OH radical and one-electron oxidants. The latter modified base that exhibits a low ionization potential is highly susceptible to further degradation upon exposure to various oxidants. Emphasis was placed in thiswork on the formation and characterization of one-electron oxidation products of 5-hydroxyuracil within DNA fragments of defined sequence. For this purpose, 5-hydroxyuracil containing single- and dou...

  15. Correlation analysis and prognostic impact of 18F-FDG PET and excision repair cross-complementation group 1 (ERCC-1) expression in non-small cell lung cancer

    International Nuclear Information System (INIS)

    The aim of this study was to determine the relationship between [18]-2-fluoro-2-deoxy-D-glucose (FDG) uptake and excision repair cross-complementation group 1 (ERCC-1) expression and to evaluate the prognostic effect of these two factors in resectable non-small cell lung cancer (NSCLC) patients. We retrospectively reviewed 212 patients with resectable NSCLC who underwent FDG positron emission tomography/computed tomography (PET/CT) scan for cancer staging and ERCC-1 expression analysis between January 2008 to December 2011. All patients were then followed-up for survival analysis. Semiquantitative evaluation of ERCC-1 was performed with the H-scoring system and was correlated with maximum standardized uptake value (SUVmax) of NSCLC. Univariate and multivariate analyses were performed to evaluate for FDG uptake and ERCC-1 expression predicting overall survival. In 212 patients (139 male, median age 68 ± 9.11), 112 patients had ERCC-positive tumors and 100 patients had ERCC-negative tumors. There was no significant difference in SUVmax between ERCC-1-positive tumors (8.02 ±5.40) and ERCC-1-negative tumors (7.57 ± 6.56, p = 0.584). All patients were followed-up for a median of 40.5 months (95 % confidence interval [CI], 38.5–42.2 months). Univariate analysis and multivariate analysis for all patients showed that both ERCC-1 expression (hazard ratio [HR], 2.78; 95 % CI, 1.20–6.47) and FDG uptake (HR, 4.50; 95 % CI, 2.07–9.77) independently predicted overall survival. We have found no statistical correlation between FDG uptake and ERCC-1 expression in NSCLC. However, both higher FDG uptake and positive ERCC-1 expression are independent predictive markers of prognosis, suggesting that both should be obtained during patient workup

  16. Correlation analysis and prognostic impact of {sup 18}F-FDG PET and excision repair cross-complementation group 1 (ERCC-1) expression in non-small cell lung cancer

    Energy Technology Data Exchange (ETDEWEB)

    Jeong, Yong Hyu; Lee, Choong Kun; Jo, Kwan Hyeong; Hwang, Sang Hyun; Cha, Jong Tae; Lee, Jeong Won; Yun, Mi Jin; Cho, Arthur [Yonsei University College of Medicine, Seoul (Korea, Republic of)

    2015-06-15

    The aim of this study was to determine the relationship between [{sup 18}]-2-fluoro-2-deoxy-D-glucose (FDG) uptake and excision repair cross-complementation group 1 (ERCC-1) expression and to evaluate the prognostic effect of these two factors in resectable non-small cell lung cancer (NSCLC) patients. We retrospectively reviewed 212 patients with resectable NSCLC who underwent FDG positron emission tomography/computed tomography (PET/CT) scan for cancer staging and ERCC-1 expression analysis between January 2008 to December 2011. All patients were then followed-up for survival analysis. Semiquantitative evaluation of ERCC-1 was performed with the H-scoring system and was correlated with maximum standardized uptake value (SUV{sub max}) of NSCLC. Univariate and multivariate analyses were performed to evaluate for FDG uptake and ERCC-1 expression predicting overall survival. In 212 patients (139 male, median age 68 ± 9.11), 112 patients had ERCC-positive tumors and 100 patients had ERCC-negative tumors. There was no significant difference in SUV{sub max} between ERCC-1-positive tumors (8.02 ±5.40) and ERCC-1-negative tumors (7.57 ± 6.56, p = 0.584). All patients were followed-up for a median of 40.5 months (95 % confidence interval [CI], 38.5–42.2 months). Univariate analysis and multivariate analysis for all patients showed that both ERCC-1 expression (hazard ratio [HR], 2.78; 95 % CI, 1.20–6.47) and FDG uptake (HR, 4.50; 95 % CI, 2.07–9.77) independently predicted overall survival. We have found no statistical correlation between FDG uptake and ERCC-1 expression in NSCLC. However, both higher FDG uptake and positive ERCC-1 expression are independent predictive markers of prognosis, suggesting that both should be obtained during patient workup.

  17. A Corpus-based Study on Self-repairs in Chinese EFL Learners' Spoken English

    Institute of Scientific and Technical Information of China (English)

    张付花

    2009-01-01

    Self-repair mentioned in this paper actually refers to self-initiated same-turn self-repair, which comprises a particular set of repair strategies in which trouble source and repairing segments occur in the same turn and the repair is beth initiated and performed by the speaker of trouble source. This study aims to study the self-repair behaviour of Chinese EFL learners based on the Spoken English Corpus of Chinese Learners as the observing corpus and the reference corpus SBNC. This study is mainly concerned with use frequency, distributions and structures of the self-repair on the basis of a clear classification of the self-repalr, aiming to find out whether there are significant differences in these three aspects of the self-repair between the Chinese EFL learners and the native speakers. Some implications and applications of the findings are also available for English teaching and learning in China.

  18. The Escherichia coli Methyl-Directed Mismatch Repair System Repairs Base Pairs Containing Oxidative Lesions

    OpenAIRE

    Wyrzykowski, Jennifer; Volkert, Michael R.

    2003-01-01

    A major role of the methyl-directed mismatch repair (MMR) system of Escherichia coli is to repair postreplicative errors. In this report, we provide evidence that MMR also acts on oxidized DNA, preventing mutagenesis. When cells deficient in MMR are grown anaerobically, spontaneous mutation frequencies are reduced compared with those of the same cells grown aerobically. In addition, we show that a dam mutant has an increased sensitivity to hydrogen peroxide treatment that can be suppressed by...

  19. In vivo repair of alkylating and oxidative DNA damage in the mitochondrial and nuclear genomes of wild-type and glycosylase-deficient Caenorhabditis elegans

    OpenAIRE

    Hunter, Senyene E.; Gustafson, Margaret A; Margillo, Kathleen M; Lee, Sean A; Ryde, Ian T.; Meyer, Joel N.

    2012-01-01

    Base excision repair (BER) is an evolutionarily conserved DNA repair pathway that is critical for repair of many of the most common types of DNA damage generated both by endogenous metabolic pathways and exposure to exogenous stressors such as pollutants. C. elegans is an increasingly important model organism for the study of DNA damage-related processes including DNA repair, genotoxicity, and apoptosis, but BER is not well understood in this organism, and has not previously been measured in ...

  20. Nucleotide excision repair in aging & cancer

    NARCIS (Netherlands)

    Melis, Joost

    2012-01-01

    DNA damage, mutations and genomic instability are established driving forces of cancer and other age-related diseases. Mutations in tumor suppressor genes and oncogenes are very frequently found in tumors and genomic instability is the most common enabling characteristic of cancer. Aging is also bel

  1. A stem cell-based approach to cartilage repair.

    Science.gov (United States)

    Johnson, Kristen; Zhu, Shoutian; Tremblay, Matthew S; Payette, Joshua N; Wang, Jianing; Bouchez, Laure C; Meeusen, Shelly; Althage, Alana; Cho, Charles Y; Wu, Xu; Schultz, Peter G

    2012-05-11

    Osteoarthritis (OA) is a degenerative joint disease that involves the destruction of articular cartilage and eventually leads to disability. Molecules that promote the selective differentiation of multipotent mesenchymal stem cells (MSCs) into chondrocytes may stimulate the repair of damaged cartilage. Using an image-based high-throughput screen, we identified the small molecule kartogenin, which promotes chondrocyte differentiation (median effective concentration = 100 nM), shows chondroprotective effects in vitro, and is efficacious in two OA animal models. Kartogenin binds filamin A, disrupts its interaction with the transcription factor core-binding factor β subunit (CBFβ), and induces chondrogenesis by regulating the CBFβ-RUNX1 transcriptional program. This work provides new insights into the control of chondrogenesis that may ultimately lead to a stem cell-based therapy for osteoarthritis. PMID:22491093

  2. TTT-based tests for trend in repairable systems data

    International Nuclear Information System (INIS)

    A major aspect of analysis of failure data for repairable systems is the testing for a possible trend in interfailure times. This paper reviews some important and popular graphical methods and tests for the nonhomogeneous Poisson process model. In particular, the total time on test (TTT) plot is considered, and trend tests based on the TTT-statistic are motivated and derived. In particular, a test based on the Anderson-Darling statistic is suggested. The tests are evaluated and compared in a simulation study, both with respect to the achievement of correct significance level and rejection power. The considered alternatives to 'no trend' are the log-linear, power law and a class of bathtub-shaped intensity functions. The simulation study involves single systems, as well as the case where several independent systems of the same kind are observed

  3. 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

    synaptosomal fraction, which was associated with a decrease in the level of BER proteins. However, we did not observe changes between the synaptosomal BER activities of presymptomatic and symptomatic AD mice harboring mutated amyolid precursor protein (APP), Tau, and presinilin-1 (PS1) (3xTgAD). Our findings...

  4. Catalytic mechanism of the hOGG1 base-excision repair enzyme; the theoretical modeling of reaction pathway

    Czech Academy of Sciences Publication Activity Database

    Šebera, Jakub; Nencka, Radim; Trantírek, L.; Tanaka, Y.; Sychrovský, Vladimír

    Praha : MATFYZPRESS, 2015 - (Burda, J.). s. 73 ISBN 978-80-7378-303-7. [Modeling Interactions in Biomolecules /7./. 14.09.2015-18.09.2015, Praha] R&D Projects: GA ČR GA13-27676S Institutional support: RVO:61388963 Keywords : hOGG1 * DFT * enzyme Subject RIV: CF - Physical ; Theoretical Chemistry

  5. 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)

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

    International Nuclear Information System (INIS)

    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

  7. Formulation and evaluation of exotic fat based cosmeceuticals for skin repair

    Directory of Open Access Journals (Sweden)

    Mandawgade S

    2008-01-01

    Full Text Available Mango butter was explored as a functional, natural supplement and active skin ingredient in skin care formulations. A foot care cream was developed with mango butter to evaluate its medicinal value and protective function in skin repair. Qualitative comparison and clinical case studies of the product were carried out. Wound healing potential of foot care cream was investigated on the rat excision and incision wound models. Results of the clinical studies demonstrated complete repair of worn and cracked skin in all the human volunteers. Furthermore, foot care cream exhibited significant healing response in both the wound models. The project work could be concluded as establishment of high potential for mango butter to yield excellent emolliency for better skin protection. Improving the product features and medicinal functionality further validate mango butter as a specialty excipient in development of cosmeceuticals and has an immense value for its commercialization.

  8. Excised radicle tips as a source of genomic DNA for PCR-based genotyping and melting curve analysis in cotton

    Indian Academy of Sciences (India)

    P Srinivasa Rao; P Sateesh Kumar; Ramesh V Sonti

    2013-03-01

    Genomic DNA isolation in cotton is complicated because of the presence of secondary metabolites that are inhibitory to PCR amplification. We report here that radicle tips, but not other parts of cotton seedlings, yield high-quality DNA that is readily amenable for PCR. The radicle-tip-excised seedlings retain viability because of the formation of adventitious roots. We demonstrate the utility of this method in distinguishing homozygotes from heterozygotes in a cotton breeding population and in hybrid seed purity testing.

  9. Role of polynucleotide kinase/phosphatase in mitochondrial DNA repair

    OpenAIRE

    Tahbaz, Nasser; Subedi, Sudip; Weinfeld, Michael

    2011-01-01

    Mutations in mitochondrial DNA (mtDNA) are implicated in a broad range of human diseases and in aging. Compared to nuclear DNA, mtDNA is more highly exposed to oxidative damage due to its proximity to the respiratory chain and the lack of protection afforded by chromatin-associated proteins. While repair of oxidative damage to the bases in mtDNA through the base excision repair pathway has been well studied, the repair of oxidatively induced strand breaks in mtDNA has been less thoroughly exa...

  10. Repair or replacement of defective restorations by dentists in The Dental Practice-Based Research Network

    DEFF Research Database (Denmark)

    Gordan, Valeria V; Riley, Joseph L; Geraldeli, Saulo;

    2012-01-01

    The authors aimed to determine whether dentists in practices belonging to The Dental Practice-Based Research Network (DPBRN) were more likely to repair or to replace a restoration that they diagnosed as defective; to quantify dentists' specific reasons for repairing or replacing restorations......; and to test the hypothesis that certain dentist-, patient- and restoration-related variables are associated with the decision between repairing and replacing restorations....

  11. Stem cell-based therapy in neural repair.

    Science.gov (United States)

    Hsu, Yi-Chao; Chen, Su-Liang; Wang, Dan-Yen; Chiu, Ing-Ming

    2013-01-01

    Cell-based therapy could aid in alleviating symptoms or even reversing the progression of neurodegenerative diseases and nerve injuries. Fibroblast growth factor 1 (FGF1) has been shown to maintain the survival of neurons and induce neurite outgrowth. Accumulating evidence suggests that combination of FGF1 and cell-based therapy is promising for future therapeutic application. Neural stem cells (NSCs), with the characteristics of self-renewal and multipotency, can be isolated from embryonic stem cells, embryonic ectoderm, and developing or adult brain tissues. For NSC clinical application, several critical problems remain to be resolved: (1) the source of NSCs should be personalized; (2) the isolation methods and protocols of human NSCs should be standardized; (3) the clinical efficacy of NSC transplants must be evaluated in more adequate animal models; and (4) the mechanism of intrinsic brain repair needs to be better characterized. In addition, the ideal imaging technique for tracking NSCs would be safe and yield high temporal and spatial resolution, good sensitivity and specificity. Here, we discuss recent progress and future development of cell-based therapy, such as NSCs, induced pluripotent stem cells, and induced neurons, in neurodegenerative diseases and peripheral nerve injuries. PMID:23806879

  12. Mitochondrial DNA repair and association with aging--an update

    DEFF Research Database (Denmark)

    Diaz, Ricardo Gredilla; Bohr, Vilhelm A; Stevnsner, Tinna V.

    2010-01-01

    the aging process and to be particularly deleterious in post-mitotic cells. Thus, DNA repair is an important mechanism for maintenance of genomic integrity. Despite the importance of mitochondria in the aging process, it was thought for many years that mitochondria lacked an enzymatic DNA repair...... system comparable to that in the nuclear compartment. However, it is now well established that DNA repair actively takes place in mitochondria. Oxidative DNA damage processing, base excision repair mechanisms were the first to be described in these organelles, and consequently the best understood....... However, new proteins and novel DNA repair pathways, thought to be exclusively present in the nucleus, have recently been described also to be present in mitochondria. Here we review the main mitochondrial DNA repair pathways and their association with the aging process....

  13. The Reinforcement Effect of Nano-Zirconia on the Transverse Strength of Repaired Acrylic Denture Base

    Science.gov (United States)

    ArRejaie, Aws S.; Abdel-Halim, Mohamed Saber; Rahoma, Ahmed

    2016-01-01

    Objective. The aim of this study was to evaluate the effect of incorporation of glass fiber, zirconia, and nano-zirconia on the transverse strength of repaired denture base. Materials and Methods. Eighty specimens of heat polymerized acrylic resin were prepared and randomly divided into eight groups (n = 10): one intact group (control) and seven repaired groups. One group was repaired with autopolymerized resin while the other six groups were repaired using autopolymerized resin reinforced with 2 wt% or 5 wt% glass fiber, zirconia, or nano-zirconia particles. A three-point bending test was used to measure the transverse strength. The results were analyzed using SPSS and repeated measure ANOVA and post hoc least significance (LSD) test (P ≤ 0.05). Results. Among repaired groups it was found that autopolymerized resin reinforced with 2 or 5 wt% nano-zirconia showed the highest transverse strength (P ≤ 0.05). Repairs with autopolymerized acrylic resin reinforced with 5 wt% zirconia showed the lowest transverse strength value. There was no significant difference between the groups repaired with repair resin without reinforcement, 2 wt% zirconia, and glass fiber reinforced resin. Conclusion. Reinforcing of repair material with nano-zirconia may significantly improve the transverse strength of some fractured denture base polymers. PMID:27366150

  14. Stem cell-based biological tooth repair and regeneration

    OpenAIRE

    Volponi, Ana Angelova; Pang, Yvonne; Sharpe, Paul T.

    2010-01-01

    Teeth exhibit limited repair in response to damage, and dental pulp stem cells probably provide a source of cells to replace those damaged and to facilitate repair. Stem cells in other parts of the tooth, such as the periodontal ligament and growing roots, play more dynamic roles in tooth function and development. Dental stem cells can be obtained with ease, making them an attractive source of autologous stem cells for use in restoring vital pulp tissue removed because of infection, in regene...

  15. The 2015 Nobel Prize in Chemistry The Discovery of Essential Mechanisms that Repair DNA Damage.

    Science.gov (United States)

    Lindahl, Tomas; Modrich, Paul; Sancar, Aziz

    2016-01-01

    The Royal Swedish Academy awarded the Nobel Prize in Chemistry for 2015 to Tomas Lindahl, Paul Modrich and Aziz Sancar for their discoveries in fundamental mechanisms of DNA repair. This pioneering research described three different essential pathways that correct DNA damage, safeguard the integrity of the genetic code to ensure its accurate replication through generations, and allow proper cell division. Working independently of each other, Tomas Lindahl, Paul Modrich and Aziz Sancar delineated the mechanisms of base excision repair, mismatch repair and nucleotide excision repair, respectively. These breakthroughs challenged and dismissed the early view that the DNA molecule was very stable, paving the way for the discovery of human hereditary diseases associated with distinct DNA repair deficiencies and a susceptibility to cancer. It also brought a deeper understanding of cancer as well as neurodegenerative or neurological diseases, and let to novel strategies to treat cancer. PMID:27183258

  16. Cell-based and biomaterial approaches to connective tissue repair

    Science.gov (United States)

    Stalling, Simone Suzette

    vitro as well as in a subcutaneous mouse model. Stable MA-MC hydrogels, of varying weight percentages, demonstrated tunable swelling and mechanical properties in the absence of cytotoxic degradation products. In vivo, 6wt% MA-MC hydrogels maintained their shape and mechanical integrity while eliciting a minimal inflammatory response; highly desirable properties for soft tissue reconstruction. These cellulose-based photopolymerizable hydrogels can be further optimized for drug delivery and tissue engineering applications to enhance wound repair.

  17. Human DNA repair and recombination genes

    International Nuclear Information System (INIS)

    Several genes involved in mammalian DNA repair pathways were identified by complementation analysis and chromosomal mapping based on hybrid cells. Eight complementation groups of rodent mutants defective in the repair of uv radiation damage are now identified. At least seven of these genes are probably essential for repair and at least six of them control the incision step. The many genes required for repair of DNA cross-linking damage show overlap with those involved in the repair of uv damage, but some of these genes appear to be unique for cross-link repair. Two genes residing on human chromosome 19 were cloned from genomic transformants using a cosmid vector, and near full-length cDNA clones of each gene were isolated and sequenced. Gene ERCC2 efficiently corrects the defect in CHO UV5, a nucleotide excision repair mutant. Gene XRCC1 normalizes repair of strand breaks and the excessive sister chromatid exchange in CHO mutant EM9. ERCC2 shows a remarkable /approximately/52% overall homology at both the amino acid and nucleotide levels with the yeast RAD3 gene. Evidence based on mutation induction frequencies suggests that ERCC2, like RAD3, might also be an essential gene for viability. 100 refs., 4 tabs

  18. Characterization of DNA repair phenotypes of Xeroderma pigmentosum cell lines by a paralleled in vitro test

    International Nuclear Information System (INIS)

    DNA is constantly damaged modifying the genetic information for which it encodes. Several cellular mechanisms as the Base Excision Repair (BER) and the Nucleotide Excision Repair (NER) allow recovering the right DNA sequence. The Xeroderma pigmentosum is a disease characterised by a deficiency in the NER pathway. The aim of this study was to propose an efficient and fast test for the diagnosis of this disease as an alternative to the currently available UDS test. DNA repair activities of XP cell lines were quantified using in vitro miniaturized and paralleled tests in order to establish DNA repair phenotypes of XPA and XPC deficient cells. The main advantage of the tests used in this study is the simultaneous measurement of excision or excision synthesis (ES) of several lesions by only one cellular extract. We showed on one hand that the relative ES of the different lesions depend strongly on the protein concentration of the nuclear extract tested. Working at high protein concentration allowed discriminating the XP phenotype versus the control one, whereas it was impossible under a certain concentration's threshold. On the other hand, while the UVB irradiation of control cells stimulated their repair activities, this effect was not observed in XP cells. This study brings new information on the XPA and XPC protein roles during BER and NER and underlines the complexity of the regulations of DNA repair processes. (author)

  19. Fault self-repair strategy based on evolvable hardware and reparation balance technology

    Institute of Scientific and Technical Information of China (English)

    Zhang Junbin; Cai Jinyan; Meng Yafeng; Meng Tianzhen

    2014-01-01

    In the face of harsh natural environment applications such as earth-orbiting and deep space satellites, underwater sea vehicles, strong electromagnetic interference and temperature stress, the circuits faults appear easily. Circuit faults will inevitably lead to serious losses of availability or impeded mission success without self-repair over the mission duration. Traditional fault-repair methods based on redundant fault-tolerant technique are straightforward to implement, yet their area, power and weight cost can be excessive. Moreover they utilize all plug-in or component level circuits to realize redundant backup, such that their applicability is limited. Hence, a novel self-repair technology based on evolvable hardware (EHW) and reparation balance technology (RBT) is proposed. Its cost is low, and fault self-repair of various circuits and devices can be realized through dynamic configuration. Making full use of the fault signals, correcting circuit can be found through EHW technique to realize the balance and compensation of the fault out-put-signals. In this paper, the self-repair model was analyzed which based on EHW and RBT tech-nique, the specific self-repair strategy was studied, the corresponding self-repair circuit fault system was designed, and the typical faults were simulated and analyzed which combined with the actual electronic devices. Simulation results demonstrated that the proposed fault self-repair strategy was feasible. Compared to traditional techniques, fault self-repair based on EHW consumes fewer hardware resources, and the scope of fault self-repair was expanded significantly.

  20. Inspection-Repair based Availability Optimization of Distribution Systems using Teaching Learning based Optimization

    Science.gov (United States)

    Tiwary, Aditya; Arya, L. D.; Arya, Rajesh; Choube, S. C.

    2015-03-01

    This paper describes a technique for optimizing inspection and repair based availability of distribution systems. Optimum duration between two inspections has been obtained for each feeder section with respect to cost function and subject to satisfaction of availability at each load point. Teaching learning based optimization has been used for availability optimization. The developed algorithm has been implemented on radial and meshed distribution systems. The result obtained has been compared with those obtained with differential evolution.

  1. ATP–stimulated DNA–mediated Redox Signaling by XPD, a DNA Repair and Transcription Helicase

    OpenAIRE

    Mui, Timothy P.; Fuss, Jill O.; Ishida, Justin P.; Tainer, John A.; Barton, Jacqueline K.

    2011-01-01

    Using DNA-modified electrodes, we show DNA-mediated signaling by XPD, a helicase that contains a [4Fe-4S] cluster and is critical for nucleotide excision repair and transcription. The DNA-mediated redox signal resembles that of base excision repair proteins, with a DNA-bound redox potential of ~80 mV versus NHE. Significantly, this signal increases with ATP hydrolysis. Moreover, the redox signal is substrate-dependent, reports on the DNA conformational changes associated with enzymatic functi...

  2. Rad9 plays an important role in DNA mismatch repair through physical interaction with MLH1

    OpenAIRE

    He, Wei; Zhao, Yun; Zhang, Chunbo; An, Lili; Hu, Zhishang; Liu, Yuheng; Han, Lu; Bi, Lijun; Xie, Zhensheng; Xue, Peng; Yang, Fuquan; Hang, Haiying

    2008-01-01

    Rad9 is conserved from yeast to humans and plays roles in DNA repair (homologous recombination repair, and base-pair excision repair) and cell cycle checkpoint controls. It has not previously been reported whether Rad9 is involved in DNA mismatch repair (MMR). In this study, we have demonstrated that both human and mouse Rad9 interacts physically with the MMR protein MLH1. Disruption of the interaction by a single-point mutation in Rad9 leads to significantly reduced MMR activity. This disrup...

  3. Potential of bacteria-based repair solution as healing agent for porous network concrete

    NARCIS (Netherlands)

    Wiktor, V.A.C.; Sangadji, S.; Jonkers, H.M.; Schlangen, H.E.J.G.

    2013-01-01

    Bacterially induced calcium carbonate precipitation has received considerable attention for its potential application in enforcing or repairing construction material. The mechanism of bacterially mediated calcite precipitation in those studies is primarily based on the enzymatic hydrolysis of urea.

  4. Aero Repair and Overhaul Services at Rolls Royce Group Plc: Application of Activity Based Costing

    OpenAIRE

    Vaithiyanathan, Shivany

    2005-01-01

    In this report an analysis has been carried out to study the application of Activity Based Costing (ABC) to the proposed repair and overhaul process at Rolls Royce Group Plc after the implementation of the aftermarket business policies.

  5. Risk Based Optimal Inspection and Repair Planning for Ship Structures Subjected to Corrosion Deterioration

    Institute of Scientific and Technical Information of China (English)

    李典庆; 张圣坤; 唐文勇

    2004-01-01

    A framework of risk based inspection and repair planning was presented to optimize for the ship structures subjected to corrosion deterioration. The planning problem was formulated as an optimization problem where the expected lifetime costs were minimized with a constraint on the minimum acceptable reliability index. The safety margins were established for the inspection events, the repair events and the failure events for ship structures. Moreover, the formulae were derived to calculate failure probabilities and repair probabilities. Based on them, a component subjected to corrosion is investigated for illustration of the process of selecting the optimal inspection and repair strategy. Furthermore, some sensitivity studies were provided. The results show that the optimal inspection instants should take place before the reliability index reaches the minimum acceptable reliability index. The optimal target failure probability is 10-3. In addition, a balance can be achieved between the risk cost and total expected inspection and repair costs by means of the risk-based optimal inspection and repair method, which is very effective in selecting the optimal inspection and repair strategy.

  6. The compatibility of earth-based repair mortars with rammed earth substrates

    OpenAIRE

    Gomes, M. Idália; Gonçalves, Teresa D.; Faria, Paulina

    2013-01-01

    Earth constructions are susceptible to degradation due to natural or human causes. The degradation of the exterior surface of earth walls is very common, either due to lack of maintenance or to the use of incompatible materials, and often requires the application of a repair mortar. This work analyses experimentally the performance of earth-based repair mortars applied on rammed earth surfaces. The mortars are based on earth collected from rammed earth buildings in south Portugal or on a c...

  7. The Impact of Hedgehog Signaling Pathway on DNA Repair Mechanisms in Human Cancer

    Energy Technology Data Exchange (ETDEWEB)

    Meng, Erhong; Hanna, Ann; Samant, Rajeev S.; Shevde, Lalita A., E-mail: lsamant@uab.edu [Department of Pathology, Comprehensive Cancer Center, University of Alabama at Birmingham, WTI320D, 1824 6th Avenue South, Birmingham, AL 35233 (United States)

    2015-07-21

    Defined cellular mechanisms have evolved that recognize and repair DNA to protect the integrity of its structure and sequence when encountering assaults from endogenous and exogenous sources. There are five major DNA repair pathways: mismatch repair, nucleotide excision repair, direct repair, base excision repair and DNA double strand break repair (including non-homologous end joining and homologous recombination repair). Aberrant activation of the Hedgehog (Hh) signaling pathway is a feature of many cancer types. The Hh pathway has been documented to be indispensable for epithelial-mesenchymal transition, invasion and metastasis, cancer stemness, and chemoresistance. The functional transcription activators of the Hh pathway include the GLI proteins. Inhibition of the activity of GLI can interfere with almost all DNA repair types in human cancer, indicating that Hh/GLI functions may play an important role in enabling tumor cells to survive lethal types of DNA damage induced by chemotherapy and radiotherapy. Thus, Hh signaling presents an important therapeutic target to overcome DNA repair-enabled multi-drug resistance and consequently increase chemotherapeutic response in the treatment of cancer.

  8. The Impact of Hedgehog Signaling Pathway on DNA Repair Mechanisms in Human Cancer

    Directory of Open Access Journals (Sweden)

    Erhong Meng

    2015-07-01

    Full Text Available Defined cellular mechanisms have evolved that recognize and repair DNA to protect the integrity of its structure and sequence when encountering assaults from endogenous and exogenous sources. There are five major DNA repair pathways: mismatch repair, nucleotide excision repair, direct repair, base excision repair and DNA double strand break repair (including non-homologous end joining and homologous recombination repair. Aberrant activation of the Hedgehog (Hh signaling pathway is a feature of many cancer types. The Hh pathway has been documented to be indispensable for epithelial-mesenchymal transition, invasion and metastasis, cancer stemness, and chemoresistance. The functional transcription activators of the Hh pathway include the GLI proteins. Inhibition of the activity of GLI can interfere with almost all DNA repair types in human cancer, indicating that Hh/GLI functions may play an important role in enabling tumor cells to survive lethal types of DNA damage induced by chemotherapy and radiotherapy. Thus, Hh signaling presents an important therapeutic target to overcome DNA repair-enabled multi-drug resistance and consequently increase chemotherapeutic response in the treatment of cancer.

  9. The Impact of Hedgehog Signaling Pathway on DNA Repair Mechanisms in Human Cancer

    International Nuclear Information System (INIS)

    Defined cellular mechanisms have evolved that recognize and repair DNA to protect the integrity of its structure and sequence when encountering assaults from endogenous and exogenous sources. There are five major DNA repair pathways: mismatch repair, nucleotide excision repair, direct repair, base excision repair and DNA double strand break repair (including non-homologous end joining and homologous recombination repair). Aberrant activation of the Hedgehog (Hh) signaling pathway is a feature of many cancer types. The Hh pathway has been documented to be indispensable for epithelial-mesenchymal transition, invasion and metastasis, cancer stemness, and chemoresistance. The functional transcription activators of the Hh pathway include the GLI proteins. Inhibition of the activity of GLI can interfere with almost all DNA repair types in human cancer, indicating that Hh/GLI functions may play an important role in enabling tumor cells to survive lethal types of DNA damage induced by chemotherapy and radiotherapy. Thus, Hh signaling presents an important therapeutic target to overcome DNA repair-enabled multi-drug resistance and consequently increase chemotherapeutic response in the treatment of cancer

  10. DNA repair

    International Nuclear Information System (INIS)

    In this chapter a series of DNA repair pathways are discussed which are available to the cell to cope with the problem of DNA damaged by chemical or physical agents. In the case of microorganisms our knowledge about the precise mechanism of each DNA repair pathway and the regulation of it has been improved considerably when mutants deficient in these repair mechanisms became available. In the case of mammalian cells in culture, until recently there were very little repair deficient mutants available, because in almost all mammalian cells in culture at least the diploid number of chromosomes is present. Therefore the frequency of repair deficient mutants in such populations is very low. Nevertheless because replica plating techniques are improving some mutants from Chinese hamsters ovary cells and L5178Y mouse lymphoma cells are now available. In the case of human cells, cultures obtained from patients with certain genetic diseases are available. A number of cells appear to be sensitive to some chemical or physical mutagens. These include cells from patients suffering from xeroderma pigmentosum, Ataxia telangiectasia, Fanconi's anemia, Cockayne's syndrome. However, only in the case of xeroderma pigmentosum cells, has the sensitivity to ultraviolet light been clearly correlated with a deficiency in excision repair of pyrimidine dimers. Furthermore the work with strains obtained from biopsies from man is difficult because these cells generally have low cloning efficiencies and also have a limited lifespan in vitro. It is therefore very important that more repair deficient mutants will become available from established cell lines from human or animal origin

  11. [Ionizing radiation-induced DNA damage and its repair in human cells]. Progress report, [April 1, 1993--February 28, 1994

    Energy Technology Data Exchange (ETDEWEB)

    1994-07-01

    The excision of radiation-induced lesions in DNA by a DNA repair enzyme complex, namely the UvrABC nuclease complex, has been investigated. Irradiated DNA was treated with the enzyme complex. DNA fractions were analyzed by gas chromatography/isotope-dilution mass spectrometry. The results showed that a number pyrimidine- and purine-derived lesions in DNA were excised by the UvrABC nuclease complex and that the enzyme complex does not act on radiation-induced DNA lesions as a glycosylase. This means that it does not excise individual base products, but it excises oligomers containing these lesions. A number of pyrimidine-derived lesions that were no substrates for other DNA repair enzymes investigated in our laboratory were substrates for the UvrABC nuclease complex.

  12. A Feature-based Classification of Model Repair Approaches

    OpenAIRE

    Macedo, Nuno; Jorge, Tiago; Cunha, Alcino

    2015-01-01

    Consistency management, the ability to detect, diagnose and handle inconsistencies, is crucial during the development process in Model-driven Engineering (MDE). As the popularity and application scenarios of MDE expanded, a variety of different techniques were proposed to address these tasks in specific contexts. Of the various stages of consistency management, this work focuses on inconsistency fixing in MDE, where such task is embodied by model repair techniques. This paper proposes a featu...

  13. A novel function of adenomatous polyposis coli (APC) in regulating DNA repair

    OpenAIRE

    Jaiswal, Aruna S.; Narayan, Satya

    2008-01-01

    Prevailing literature suggests diversified cellular functions for the adenomatous polyposis coli (APC) gene. Among them a recently discovered unique role of APC is in DNA repair. The APC gene can modulate the base excision repair (BER) pathway through an interaction with DNA polymerase β (Pol-β) and flap endonuclease 1 (Fen-1). Taken together with the transcriptional activation of APC gene by alkylating agents and modulation of BER activity, APC may play an important role in carcinogenesis an...

  14. Adverse effects of trichothiodystrophy DNA repair and transcription gene disorder on human fetal development

    OpenAIRE

    Moslehi, R; Signore, C; Tamura, D; Mills, JL; DiGiovanna, JJ; Tucker, MA; Troendle, J; Ueda, T; Boyle, J.; Khan, SG; Oh, K-S; Goldstein, AM; Kraemer, KH

    2009-01-01

    The effects of DNA repair and transcription gene abnormalities in human pre-natal life have never been studied. Trichothiodystrophy (TTD) is a rare (affected frequency of 10−6) recessive disorder caused by mutations in genes involved in nucleotide excision repair (NER) pathway and in transcription. Based on our novel clinical observations, we conducted a genetic epidemiologic study to investigate gestational outcomes associated with TTD. We compared pregnancies resulting in TTD-affected offsp...

  15. MD-POR: Multisource and Direct Repair for Network Coding-Based Proof of Retrievability

    OpenAIRE

    Kazumasa Omote; Tran Phuong Thao

    2015-01-01

    When data owners publish their data to a cloud storage, data integrity and availability become typical problems because the cloud servers are never trusted. To address these problems, researchers proposed the Proof of Retrievability (POR) protocol which allows a verifier to check and repair the data stored in the cloud servers. Based on the POR protocol, the network coding technique is commonly applied to increase the efficiency in data transmission and data repair. However, most previous sch...

  16. A cell-free system for DNA repair synthesis using purified enzymes from the Novikoff hepatoma

    International Nuclear Information System (INIS)

    Novikoff DNA polymerase-β and Novikoff DNase V have been used in a cell-free DNA excision repair system for UV-irradiated substrates to determine their DNA repair capabilities. The repair system was shown to depend upon UV-irradiated DNA, incision by phage T4 UV-endonuclease, excision by DNase V and synthesis by DNA polymerase-β; ligation was not included. Highly purified calf thymus DNA was UV-irradiated at 500-750 J/m2 and incised by T4 UV-endonuclease. The repair system was used to follow the purification of DNase V and DNA polymerase-β. For increased specificity, the parameters of UV-irradiation, incision, excision and synthesis were confirmed on highly supercoiled, covalently closed, phage PM2 DNA. Optimal DNA and Mg2+ concentrations were determined for the repair assay, which was shown to be linear with respect to time. Excision of the 3'-apyrimidinic site and the 5'-pyrimidine dimer by bidirectional DNase V, presumed to occur from the above experiments, was studied more thoroughly using lightly UV-irradiated [3H]poly(dT)poly (dA), labeled in both the base and the sugar, and incised with T4 UV-endonuclease

  17. DNA repair in human xeroderma pigmentosum and chinese hamster cells

    International Nuclear Information System (INIS)

    The investigations described were performed to study the genetic heterogeneity of excision repair-deficient XP (xeroderma pigmentosum) strains and the biochemical defects in their repair processes after irradiation with ultraviolet radiation. (Auth.)

  18. DNA repair: Dynamic defenders against cancer and aging

    Energy Technology Data Exchange (ETDEWEB)

    Fuss, Jill O.; Cooper, Priscilla K.

    2006-04-01

    You probably weren't thinking about your body's cellular DNA repair systems the last time you sat on the beach in the bright sunshine. Fortunately, however, while you were subjecting your DNA to the harmful effects of ultraviolet light, your cells were busy repairing the damage. The idea that our genetic material could be damaged by the sun was not appreciated in the early days of molecular biology. When Watson and Crick discovered the structure of DNA in 1953 [1], it was assumed that DNA is fundamentally stable since it carries the blueprint of life. However, over 50 years of research have revealed that our DNA is under constant assault by sunlight, oxygen, radiation, various chemicals, and even our own cellular processes. Cleverly, evolution has provided our cells with a diverse set of tools to repair the damage that Mother Nature causes. DNA repair processes restore the normal nucleotide sequence and DNA structure of the genome after damage [2]. These responses are highly varied and exquisitely regulated. DNA repair mechanisms are traditionally characterized by the type of damage repaired. A large variety of chemical modifications can alter normal DNA bases and either lead to mutations or block transcription if not repaired, and three distinct pathways exist to remove base damage. Base excision repair (BER) corrects DNA base alterations that do not distort the overall structure of the DNA helix such as bases damaged by oxidation resulting from normal cellular metabolism. While BER removes single damaged bases, nucleotide excision repair (NER) removes short segments of nucleotides (called oligonucleotides) containing damaged bases. NER responds to any alteration that distorts the DNA helix and is the mechanism responsible for repairing bulky base damage caused by carcinogenic chemicals such as benzo [a]pyrene (found in cigarette smoke and automobile exhaust) as well as covalent linkages between adjacent pyrimidine bases resulting from the ultraviolet

  19. Repair of ionizing radiation DNA base damage in ataxia-telangiectasia cells

    International Nuclear Information System (INIS)

    Micrococcus luteus endonuclease sensitive sites were measured by alkaline elution in normal human and ataxia-telangiectasia (AT) fibroblasts after ionizing radiation. Due to the sensitivity of this assay, repair of base damage after 3 to 6 kilorads has been measured after oxic or hypoxic radiation. With 5.5 kilorads of oxic radiation, more than 50% of the base damage was removed after 1.5 h of repair incubation in all cells, including exr+ and exr- AT cells, and approximately 75% was removed by 4 h. After 3 or 4.5 kilorads of hypoxic X-irradiation, repair was equivalent in normal and exr- AT cells. This study included three exr- AT strains which have been reported to be deficient in the removal of gamma-ray base damage at higher doses. Since these strains repaired ionizing radiation base damage normally at lower doses, which are more relevant to survival, it is concluded that the X-ray hypersensitivity of AT cells is probably not related to the repair of base damage

  20. The forked flap repair for hypospadias

    Directory of Open Access Journals (Sweden)

    Anil Chadha

    2012-01-01

    Full Text Available Context: Despite the abundance of techniques for the repair of Hypospadias, its problems still persist and a satisfactory design to correct the penile curvature with the formation of neourethra from the native urethral tissue or genital or extragenital tissues, with minimal postoperative complications has yet to evolve. Aim: Persisting with such an endeavor, a new technique for the repair of distal and midpenile hypospadias is described. Materials and Methods: The study has been done in 70 cases over the past 11 years. The "Forked-Flap" repair is a single stage method for the repair of such Hypospadias with chordee. It takes advantage of the rich vascular communication at the corona and capitalizes on the established reliability of the meatal based flip-flap. The repair achieves straightening of the curvature of the penis by complete excision of chordee tissue from the ventral surface of the penis beneath the urethral plate. The urethra is reconstructed using the native plate with forked flap extensions and genital tissue relying on the concept of meatal based flaps. Water proofing by dartos tissue and reinforcement by Nesbit′s prepucial tissue transfer completes the one stage procedure. Statistical Analysis: An analysis of 70 cases of this single stage technique of repair of penile hypospadias with chordee, operated at 3 to 5 years of age over the past 11 years is presented. Results and Conclusion: The Forked Flap gives comparable and replicable results; except for a urethrocutaneous fistula rate of 4% no other complications were observed.

  1. Evidence-based outcomes following inferior alveolar and lingual nerve injury and repair: a systematic review.

    Science.gov (United States)

    Kushnerev, E; Yates, J M

    2015-10-01

    The inferior alveolar nerve (IAN) and lingual (LN) are susceptible to iatrogenic surgical damage. Systematically review recent clinical evidence regarding IAN/LN repair methods and to develop updated guidelines for managing injury. Recent publications on IAN/LN microsurgical repair from Medline, Embase and Cochrane Library databases were screened by title/abstract. Main texts were appraised for exclusion criteria: no treatment performed or results provided, poor/lacking procedural description, cohort injury type, injury timing, neurosensory disturbances and intra-operative findings. Best functional nerve recovery occurred after direct apposition and suturing if nerve ending gaps were nerve grafting (sural/greater auricular nerve). Timing of microneurosurgical repair after injury remains debated. Most authors recommend surgery when neurosensory deficit shows no improvement 90 days post-diagnosis. Nerve transection diagnosed intra-operatively should be repaired in situ; minor nerve injury repair can be delayed. No consensus exists regarding optimal methods and timing for IAN/LN repair. We suggest a schematic guideline for treating IAN/LN injury, based on the most current evidence. We acknowledge that additional RCTs are required to provide definitive confirmation of optimal treatment approaches. PMID:26059454

  2. Value of histopathologic analysis of subcutis excisions by general practitioners

    Directory of Open Access Journals (Sweden)

    Verweij Wim

    2007-01-01

    Full Text Available Abstract Background Only around 60% of skin lesions excised by GPs are referred to a pathologist. Clinical diagnoses of skin excisions by GPs may not be very accurate. Subcutis excisions are rarely done by GPs, and there is hence little information in the literature on the histopathological yield of subcutis excisions by GPs with regard to malignancies. The aim of this study was to evaluate the yield of histopathological investigation of a relatively large group of subcutis excisions by GPs, with special emphasis on discrepancies between clinical and histopathological diagnoses of malignancy. Methods We investigated a series of 90 subcutis excisions, which was derived from a database of consecutive GP submissions from the years 1999–2000 where in the same time period 4595 skin excisions were performed by the same group of GPs. This underlines the apparent reluctance of GPs to perform subcutis excisions. Results The final diagnosis was benign in 88 cases (97.8% and malignant in 2 cases (2.2%. Seven cases had no clinical diagnosis, all of which were benign. Of the 83 clinically benign cases, 81 (97.6% were indeed benign and 2 (2.4% were malignant: one Merkel cell carcinoma and one dermatofibrosarcoma protuberans. The former was clinically thought to be a lipoma, and the latter a trichilemmal cyst. The dermatofibrosarcoma protuberans presented at the age of 27, and the Merkel cell carcinoma at the age of 60. Both were incompletely removed and required re-excision by a surgical oncologist. Conclusion Histopathological investigation of subcutis excisions by GPs yields unexpected and rare malignancies in about 2% of cases that may initially be excised inadequately. Based on these data, and because of the relatively rareness of these type of excisions, it could be argued that it may be worthwhile to have all subcutis excisions by GPs routinely investigated by histopathology.

  3. Acetylation regulates DNA repair mechanisms in human cells.

    Science.gov (United States)

    Piekna-Przybylska, Dorota; Bambara, Robert A; Balakrishnan, Lata

    2016-06-01

    The p300-mediated acetylation of enzymes involved in DNA repair and replication has been previously shown to stimulate or inhibit their activities in reconstituted systems. To explore the role of acetylation on DNA repair in cells we constructed plasmid substrates carrying inactivating damages in the EGFP reporter gene, which should be repaired in cells through DNA mismatch repair (MMR) or base excision repair (BER) mechanisms. We analyzed efficiency of repair within these plasmid substrates in cells exposed to deacetylase and acetyltransferase inhibitors, and also in cells deficient in p300 acetyltransferase. Our results indicate that protein acetylation improves DNA mismatch repair in MMR-proficient HeLa cells and also in MMR-deficient HCT116 cells. Moreover, results suggest that stimulated repair of mismatches in MMR-deficient HCT116 cells is done though a strand-displacement synthesis mechanism described previously for Okazaki fragments maturation and also for the EXOI-independent pathway of MMR. Loss of p300 reduced repair of mismatches in MMR-deficient cells, but did not have evident effects on BER mechanisms, including the long patch BER pathway. Hypoacetylation of the cells in the presence of acetyltransferase inhibitor, garcinol generally reduced efficiency of BER of 8-oxoG damage, indicating that some steps in the pathway are stimulated by acetylation. PMID:27104361

  4. Repair and Strengthening Assessment of Existing Bridges Based on Time-Dependent Reliability Analysis

    Institute of Scientific and Technical Information of China (English)

    孙晓燕; 黄承逵; 孙保沭

    2004-01-01

    The strategy on repair and strengthening of existing bridges based on time-dependent reliability was analyzed with the maximum expected benefit as the objective function. A sample of risk-ranking decision was illustrated based on updated inspection information with 35 survival age. The effect of improvement of live loads and difference of repair methods on time-dependent reliability of existing bridges are considered. The results show that the decision method can be used in real project, with the cost of failure consequence and the risk of failure considered.

  5. Effect of surface roughness and adhesive system on repair potential of silorane-based resin composite

    OpenAIRE

    Mobarak, Enas H.

    2012-01-01

    This study was performed to evaluate the influence of surface roughness and adhesive system on the repair strength of silorane-based resin composite. Twenty-four substrate discs from silorane-based FiltekP90 were made and stored for 24 h. Half of the discs were roughened against 320 grit SiC paper while the other half was polished against 4000 grit SiC paper. All discs were etched with phosphoric acid. Repair resin composite, FiltekP90 or FiltekZ250, was bonded to the treated surfaces using t...

  6. DNA Repair Glycosylases with a [4Fe-4S] Cluster: A Redox Cofactor for DNA-mediated Charge Transport?

    OpenAIRE

    Boal, Amie K.; Yavin, Eylon; Barton, Jacqueline K.

    2007-01-01

    The [4Fe-4S] cluster is ubiquitous to a class of base excision repair enzymes, in organisms ranging from bacteria to man, and was first considered as a structural element, owing to its redox stability under physiological conditions. When studied bound to DNA, two of these repair proteins (MutY and Endonuclease III from Escherichia coli) display DNA-dependent reversible electron transfer with characteristics typical of high potential iron proteins. These results have inspired a reexamination o...

  7. Efficient Repair of Abasic Sites in DNA by Mitochondrial Enzymes

    OpenAIRE

    Pinz, Kevin G.; Bogenhagen, Daniel F.

    1998-01-01

    Mutations in mitochondrial DNA (mtDNA) cause a variety of relatively rare human diseases and may contribute to the pathogenesis of other, more common degenerative diseases. This stimulates interest in the capacity of mitochondria to repair damage to mtDNA. Several recent studies have shown that some types of damage to mtDNA may be repaired, particularly if the lesions can be processed through a base excision mechanism that employs an abasic site as a common intermediate. In this paper, we dem...

  8. PARP-1 and Ku compete for repair of DNA double strand breaks by distinct NHEJ pathways

    OpenAIRE

    Wang, Minli; Wu, Weizhong; Wu, Wenqi; Rosidi, Bustanur; Zhang, Lihua; Wang, Huichen; Iliakis, George

    2006-01-01

    Poly(ADP-ribose)polymerase 1 (PARP-1) recognizes DNA strand interruptions in vivo and triggers its own modification as well as that of other proteins by the sequential addition of ADP-ribose to form polymers. This modification causes a release of PARP-1 from DNA ends and initiates a variety of responses including DNA repair. While PARP-1 has been firmly implicated in base excision and single strand break repair, its role in the repair of DNA double strand breaks (DSBs) remains unclear. Here, ...

  9. Role of poly(ADP-ribosepolymerase 2 in DNA repair

    Directory of Open Access Journals (Sweden)

    Lavrik O. I.

    2012-06-01

    Full Text Available Poly(ADP-ribosylation is a posttranslational protein modification significant for the genomic stability and cell survival in response to DNA damage. Poly(ADP-ribosylation is catalyzed by poly(ADP-ribosepolymerases (PARPs, which use NAD+ as a substrate, synthesize polymer of (ADP-ribose (PAR covalently attached to nuclear proteins including PARP themselves. PARPs constitute a large family of proteins, in which PARP1 is the most abundant and best-characterized member. In spite of growing body of PARPs’ role in cellular processes, PARP2, the closest homolog of PARP1, still remains poorly characterized at the level of its contribution to different pathways of DNA repair. An overview summarizes in vivo and in vitro data on PARP2 implication in specialized DNA repair processes, base excision repair and double strand break repair.

  10. Influence of surface treatments to repair recent fillings of silorane-and methacrylate-based composites

    OpenAIRE

    Marina Kaneko; Ricardo Armini Caldas; Victor Pinheiro Feitosa; Rafael Leonardo Xediek Consani; Schneider, Luis Felipe J.; Ataís Bacchi

    2015-01-01

    Purpose: The aim of this study was to evaluate the tensile bond strength (TBS) of repairs in recent fillings of methacrylate- (MBC) or silorane-based composites (SBC) subsequent to different surface treatments. Materials and Methods: Fifty slabs of Filtek P60 (3M ESPE, St Paul, USA) and Filtek P90 (3M ESPE) were stored for 10 days in distilled water at 37°C. The surface of adhesion was abraded with a 600-grit silicone paper and repaired using each respective composite: G1, no treatment (...

  11. ENVIRONMENTAL IMPACT OF REPAIR OF HIGHWAY BRI DGE BASED ON EXAMPLES AND MEASURES OF IMPACT REDUCTION

    Science.gov (United States)

    Kawai, Kenji; Aoki, Yusuke; Iwatani, Yuta; Honjo, Kiyoshi; Nakano, Masahiro; Fukui, Seiji

    In this study, the environm ental impact evaluation for CO2, NOx, SOx and particulate matter emissions was performed based on repair examples of highway bridges. It was shown that the amount of CO2 emission derived from constituent materials, execution and transportation accounts for 40 to 60%, 40 to 60 % and about 5%, respectively, which is different from newly constructed structures. Within this study, in the integrated life-cycle evaluation of the above 4 environmental impact factors, it was found that the environmental impact for repair using pr ecast concrete slabs is smaller than that using reinforced concrete slabs. It was also shown that about 23% of CO2 emission, about 7% of NOx emission, about 39% of SOx emission and about 20% of particulate matter emission can be reduced when a type of binding material and a curing method are changed as a measure for reducing the environmental impact for repair using precast concrete slabs.

  12. Potential of bacteria-based repair solution as healing agent for porous network concrete

    OpenAIRE

    Wiktor, V.A.C.; Sangadji, S.; Jonkers, H.M.; Schlangen, H.E.J.G.

    2013-01-01

    Bacterially induced calcium carbonate precipitation has received considerable attention for its potential application in enforcing or repairing construction material. The mechanism of bacterially mediated calcite precipitation in those studies is primarily based on the enzymatic hydrolysis of urea. Besides calcite precipitation, this reaction mechanism leads also to the production of ammonium ions which may result in excessive environmental pressure. More recently, bacterially mediated calcit...

  13. Temporal-based pericranial flaps for orbitofrontal Dural repair: A technical note and Review of the literature

    Directory of Open Access Journals (Sweden)

    Esther Dupépé

    2016-03-01

    Conclusions: A temporal-based pericranial flap represents an alternative vascularized pedicle flap to the classic frontal-based pericranial flap used in orbitofrontal dural repair. In certain clinical settings, the temporal-based flap may be preferable.

  14. Predictive Value of Excision Repair Cross-complementation Group 1 (ERCC1) Polymorphism on Chemotherapy Response and Survival of Advanced Non-small Cell Lung Cancer Patients Treated with Cisplatin%ERCC1多态性对晚期非小细胞肺癌含铂化疗疗效与生存期的预测价值

    Institute of Scientific and Technical Information of China (English)

    王霖; 戴晓芳; 伍钢; 任精华; 陈卫红; 刘昭

    2012-01-01

    Objective To evaluate the predictive value of excision repair cross-complementation group 1 ( ERCC1 ) codon 118 polymorphism on chemotherapy response and survival of advanced non-small cell lung cancer (NSCLC) patients treated with cisplatin. Methods In 101 advanced NSCLC patients treated with platinum-based chemotherapy, polymerase chain reaction-based restriction fragment length polymorphism ( PCR-RFLP) assay was used to detect ERCC1 codon 118 C>T polymorphism. Results We found codon 118 polymorphism was not correlated with sex, age, family history of malignant cancer, smoking, pathology type and stage, and chemotherapy response. Median survival lime and one-year survival rate of C/C genotype carriers were higher than those of C/T and T/T genotype carriers (13. 7 m vs. 10. 6 m; 67% vs. 35% , both PT) C allele can be a prediction factor for better survival of advanced NSCLC patients treated with risplalin.%目的 探讨ERCC1第118位密码子C>T多态性对晚期非小细胞肺癌(NSCLC)一线含铂化疗疗效及生存期的预测价值.方法 采用聚合酶链式反应-限制性内切酶片段长度多态性(PCR-RFLP)的方法,对101例接受铂刺为基础化疗的晚期NSCLC患者的ERCC1第118位密码子C>T的多态性进行检测,分析其与化疗反应率及生存期的相关性.结果 NSCLC患者ERCC1第118位密码子多态性分布与患者性别、年龄、恶性肿瘤家族史、吸烟习惯、病理类型、分期、疗效评价均无显著相关性.C/C基因型携带者的中位生存时间和一年生存率均优于C/T和T/T基因型携带者(13.7个月,10.6个月,P<0.01;67%,35%,P<0.01).Cox多因素分析显示,性别为男性,病理类型为鳞癌,ERCC1基因型为C/T或T/T均是本组患者总体生存率低的独立预后因素(Hazard ratio=1.907,P=0.022;Hazard ratio=1.980,P=0.032;Hazard ratio=2.536,P<0.01).结论 ERCC1第118位密码子C>T位点C等位基因是接受顺铂为基础化疗的NSCLC患者生存获益的潜在预测因素.

  15. Surgical technique for repair of complex anterior skull base defects

    Directory of Open Access Journals (Sweden)

    Kevin Reinard

    2015-01-01

    Conclusion: The layered reconstruction of large anterior cranial fossa defects resulted in postoperative CSF leak in only 5% of the patients and represents a simple and effective closure option for skull base surgeons.

  16. Surgical anatomy and utility of pedicled vascularized tissue flaps for multilayered repair of skull base defects.

    Science.gov (United States)

    Safavi-Abbasi, Sam; Komune, Noritaka; Archer, Jacob B; Sun, Hai; Theodore, Nicholas; James, Jeffrey; Little, Andrew S; Nakaji, Peter; Sughrue, Michael E; Rhoton, Albert L; Spetzler, Robert F

    2016-08-01

    OBJECT The objective of this study was to describe the surgical anatomy and technical nuances of various vascularized tissue flaps. METHODS The surgical anatomy of various tissue flaps and their vascular pedicles was studied in 5 colored silicone-injected anatomical specimens. Medical records were reviewed of 11 consecutive patients who underwent repair of extensive skull base defects with a combination of various vascularized flaps. RESULTS The supraorbital, supratrochlear, superficial temporal, greater auricular, and occipital arteries contribute to the vascular supply of the pericranium. The pericranial flap can be designed based on an axial blood supply. Laterally, various flaps are supplied by the deep or superficial temporal arteries. The nasoseptal flap is a vascular pedicled flap based on the nasoseptal artery. Patients with extensive skull base defects can undergo effective repair with dual flaps or triple flaps using these pedicled vascularized flaps. CONCLUSIONS Multiple pedicled flaps are available for reconstitution of the skull base. Knowledge of the surgical anatomy of these flaps is crucial for the skull base surgeon. These vascularized tissue flaps can be used effectively as single or combination flaps. Multilayered closure of cranial base defects with vascularized tissue can be used safely and may lead to excellent repair outcomes. PMID:26613175

  17. SNF2H interacts with XRCC1 and is involved in repair of H2O2-induced DNA damage.

    Science.gov (United States)

    Kubota, Yoshiko; Shimizu, Shinji; Yasuhira, Shinji; Horiuchi, Saburo

    2016-07-01

    The protein XRCC1 has no inherent enzymatic activity, and is believed to function in base excision repair as a dedicated scaffold component that coordinates other DNA repair factors. Repair foci clearly represent the recruitment and accumulation of DNA repair factors at sites of damage; however, uncertainties remain regarding their organization in the context of nuclear architecture and their biological significance. Here we identified the chromatin remodeling factor SNF2H/SMARCA5 as a novel binding partner of XRCC1, with their interaction dependent on the casein kinase 2-mediated constitutive phosphorylation of XRCC1. The proficiency of repairing H2O2-induced damage was strongly impaired by SNF2H knock-down, and similar impairment was observed with knock-down of both XRCC1 and SNF2H simultaneously, suggesting their role in a common repair pathway. Most SNF2H exists in the nuclear matrix fraction, forming salt extraction-resistant foci-like structures in unchallenged nuclei. Remarkably, damage-induced formation of both PAR and XRCC1 foci depended on SNF2H, and the PAR and XRCC1 foci co-localized with the SNF2H foci. We propose a model in which a base excision repair complex containing damaged chromatin is recruited to specific locations in the nuclear matrix for repair, with this recruitment mediated by XRCC1-SNF2H interaction. PMID:27268481

  18. Generalized renewal process for repairable systems based on finite Weibull mixture

    Energy Technology Data Exchange (ETDEWEB)

    Veber, B. [Faculty of Mechanical Engineering, University of Ljubljana, Askerceva 6, SI-1000 Ljubljana (Slovenia)], E-mail: bostjan.veber@fs.uni-lj.si; Nagode, M.; Fajdiga, M. [Faculty of Mechanical Engineering, University of Ljubljana, Askerceva 6, SI-1000 Ljubljana (Slovenia)

    2008-10-15

    Repairable systems can be brought to one of possible states following a repair. These states are: 'as good as new', 'as bad as old' and 'better than old but worse than new'. The probabilistic models traditionally used to estimate the expected number of failures account for the first two states, but they do not properly apply to the last one, which is more realistic in practice. In this paper, a probabilistic model that is applicable to all of the three after-repair states, called generalized renewal process (GRP), is applied. Simplistically, GRP addresses the repair assumption by introducing the concept of virtual age into the stochastic point processes to enable them to represent the full spectrum of repair assumptions. The shape of measured or design life distributions of systems can vary considerably, and therefore frequently cannot be approximated by simple distribution functions. The scope of the paper is to prove that a finite Weibull mixture, with positive component weights only, can be used as underlying distribution of the time to first failure (TTFF) of the GRP model, on condition that the unknown parameters can be estimated. To support the main idea, three examples are presented. In order to estimate the unknown parameters of the GRP model with m-fold Weibull mixture, the EM algorithm is applied. The GRP model with m mixture components distributions is compared to the standard GRP model based on two-parameter Weibull distribution by calculating the expected number of failures. It can be concluded that the suggested GRP model with Weibull mixture with an arbitrary but finite number of components is suitable for predicting failures based on the past performance of the system.

  19. Generalized renewal process for repairable systems based on finite Weibull mixture

    International Nuclear Information System (INIS)

    Repairable systems can be brought to one of possible states following a repair. These states are: 'as good as new', 'as bad as old' and 'better than old but worse than new'. The probabilistic models traditionally used to estimate the expected number of failures account for the first two states, but they do not properly apply to the last one, which is more realistic in practice. In this paper, a probabilistic model that is applicable to all of the three after-repair states, called generalized renewal process (GRP), is applied. Simplistically, GRP addresses the repair assumption by introducing the concept of virtual age into the stochastic point processes to enable them to represent the full spectrum of repair assumptions. The shape of measured or design life distributions of systems can vary considerably, and therefore frequently cannot be approximated by simple distribution functions. The scope of the paper is to prove that a finite Weibull mixture, with positive component weights only, can be used as underlying distribution of the time to first failure (TTFF) of the GRP model, on condition that the unknown parameters can be estimated. To support the main idea, three examples are presented. In order to estimate the unknown parameters of the GRP model with m-fold Weibull mixture, the EM algorithm is applied. The GRP model with m mixture components distributions is compared to the standard GRP model based on two-parameter Weibull distribution by calculating the expected number of failures. It can be concluded that the suggested GRP model with Weibull mixture with an arbitrary but finite number of components is suitable for predicting failures based on the past performance of the system

  20. Tailored approach in inguinal hernia repair – Decision tree based on the guidelines

    Directory of Open Access Journals (Sweden)

    FerdinandKöckerling

    2014-06-01

    Full Text Available The endoscopic procedures TEP and TAPP and the open techniques Lichtenstein, Plug and Patch and PHS currently represent the gold standard in inguinal hernia repair recommended in the guidelines of the European Hernia Society, the International Endohernia Society and the European Association of Endoscopic Surgery. 82 % of experienced hernia surgeons use the "tailored approach", the differentiated use of the several inguinal hernia repair techniques depending on the findings of the patient, trying to minimize the risks. The following differential therapeutic situations must be distinguished in inguinal hernia repair: unilateral in men, unilateral in women, bilateral, scrotal, after previous pelvic and lower abdominal surgery, no general anaesthesia possible, recurrence and emergency surgery. Evidence-based guidelines and consensus conferences of experts give recommendations for the best approach in the individual situation of a patient. This review tries to summarized the recommendations of the various guidelines and to transfer them into a practical dicision tree for the daily work of surgeons performing inguinal hernia repair.

  1. Mechanical properties of masonry repair dolomitic lime-based mortars

    OpenAIRE

    Lanas, J.; Perez, J. L.; Bello, M.A. (Miguel Ángel); Alvarez, J.I. (José Ignacio)

    2006-01-01

    180 different mortars made with a dolomitic lime and different aggregates were prepared in order to be used in restoration works. This paper focuses on the effect of technological variables on pore structure and mechanical properties of magnesian lime-based mortars. Compressive and flexural strengths of the specimens were discussed according to curing time, binder : aggregate ratios, attributes of the aggregates and porosity, at long-term tests. A strong increase in the strength of mortar...

  2. Helicobacter pylori infection affects mitochondrial function and DNA repair, thus, mediating genetic instability in gastric cells

    DEFF Research Database (Denmark)

    Machado, Ana Manuel Dantas; Madsen, Claus Desler; Bøggild, Cecilie Sisse Line;

    2013-01-01

    causes mtDNA mutations and a decrease of mtDNA content. Consequently, we show a decrease of respiration coupled ATP turnover and respiratory capacity and accordingly a lower level and activity of complex I of the electron transport chain. We wanted to investigate if the increased mutational load in the...... mitochondrial genome was caused by down-regulation of mitochondrial DNA repair pathways. We lowered the expression of APE-1 and YB-1, which are believed to be involved in mitochondrial base excision repair and mismatch repair. Our results suggest that both APE-1 and YB-1 are involved in mtDNA repair during H....... pylori infection, furthermore, the results demonstrate that multiple DNA repair activities are involved in protecting mtDNA during infection....

  3. Repair of ultraviolet irradiation damage in mouse neuroblasts cells

    International Nuclear Information System (INIS)

    It was demonstrated, using hydroxyurea inhibition of DNA replication and CsCl density centrifugation, that excision repair occurs both in the differentiated state, and when cells have been restored to growing conditions. since the ability to remove photodamage is present, we postulated that sensitivity was due to failure to remove damage from critical regions of the genome or alternatively a deficiency in another mechanism of repair, such as post replication repair, which has been demonstrated in rodent cells. The first possibility was examined by comparing excision repair in pyrmidine tracts, in which preferential formation of dimers occurs at lower UV doses, and nontract regions. Excision repair was also determined in satellite and main band DNA. The results indicate that in the particular regions of the genome examined no preference for excision repair is detected. Post replication repair (i.e. the ability to elongate DNA which is synthesized in low molecular weight pieces after UV irradiation) was determined in growing and differentiated cells. The results show that postreplication repair is normal in differentiated cells which have entered the first S phase after serum return. since excision repair and postreplication repair appear to be normal it is possible that an additional repair process is involved or that some other cell function is irreversible damage. (author)

  4. Present status and applications of bacterial cellulose-based materials for skin tissue repair.

    Science.gov (United States)

    Fu, Lina; Zhang, Jin; Yang, Guang

    2013-02-15

    Bacterial cellulose (BC, also known as microbial cellulose, MC) is a promising natural polymer which is biosynthesized by certain bacteria. This review focused on BC-based materials which can be utilized for skin tissue repair. Firstly, it is illustrated that BC has unique structural and mechanical properties as compared with higher plant cellulose, and is thus expected to become a commodity material. Secondly, we summarized the basic properties and different types of BC, including self-assembled, oriented BC, and multiform BC. Thirdly, composites prepared by using BC in conjunction with other polymers are explored, and the research on BC for application in skin tissue engineering is addressed. Finally, experimental results and clinical treatments assessing the performance of wound healing materials based on BC were examined. With its superior mechanical properties, as well as its excellent biocompatibility, BC was shown to have great potential for biomedical application and very high clinical value for skin tissue repair. PMID:23399174

  5. Condition-based maintenance under an imperfect repair policy for a deteriorating production system

    OpenAIRE

    Do Van, Phuc; Bérenguer, Christophe

    2011-01-01

    This paper presents a condition-based maintenance policy taking into account both maintenance cost and production capacity aspects for single unit deterioratin production system whose condition is periodically monitored. Imperfect preventive maintenance activities, which restore the production system to better states and might not be as good as new, are considered. We assume however that the system can be repaired only a limited number of times. In order to access the performance of the propo...

  6. DNA repair in neurons: So if they don't divide what's to repair?

    Energy Technology Data Exchange (ETDEWEB)

    Fishel, Melissa L. [Department of Pediatrics (Section of Hematology/Oncology), Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, 1044 W. Walnut, Room 302C, Indianapolis, IN 46202 (United States); Vasko, Michael R. [Department of Pharmacology and Toxicology, Indiana University School of Medicine, 1044 W. Walnut St., Indianapolis, IN 46202 (United States); Kelley, Mark R. [Department of Pediatrics (Section of Hematology/Oncology), Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, 1044 W. Walnut, Room 302C, Indianapolis, IN 46202 (United States) and Department of Pharmacology and Toxicology, Indiana University School of Medicine, 1044 W. Walnut St., Indianapolis, IN 46202 (United States) and Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, 1044 W. Walnut, Room 302C, Indianapolis, IN 46202 (United States)]. E-mail: mkelley@iupui.edu

    2007-01-03

    Neuronal DNA repair remains one of the most exciting areas for investigation, particularly as a means to compare the DNA repair response in mitotic (cancer) vs. post-mitotic (neuronal) cells. In addition, the role of DNA repair in neuronal cell survival and response to aging and environmental insults is of particular interest. DNA damage caused by reactive oxygen species (ROS) such as generated by mitochondrial respiration includes altered bases, abasic sites, and single- and double-strand breaks which can be prevented by the DNA base excision repair (BER) pathway. Oxidative stress accumulates in the DNA of the human brain over time especially in the mitochondrial DNA (mtDNA) and is proposed to play a critical role in aging and in the pathogenesis of several neurological disorders including Parkinson's disease, ALS, and Alzheimer's diseases. Because DNA damage accumulates in the mtDNA more than nuclear DNA, there is increased interest in DNA repair pathways and the consequence of DNA damage in the mitochondria of neurons. The type of damage that is most likely to occur in neuronal cells is oxidative DNA damage which is primarily removed by the BER pathway. Following the notion that the bulk of neuronal DNA damage is acquired by oxidative DNA damage and ROS, the BER pathway is a likely area of focus for neuronal studies of DNA repair. BER variations in brain aging and pathology in various brain regions and tissues are presented. Therefore, the BER pathway is discussed in greater detail in this review than other repair pathways. Other repair pathways including direct reversal, nucleotide excision repair (NER), mismatch repair (MMR), homologous recombination and non-homologous end joining are also discussed. Finally, there is a growing interest in the role that DNA repair pathways play in the clinical arena as they relate to the neurotoxicity and neuropathy associated with cancer treatments. Among the numerous side effects of cancer treatments, major

  7. Analysis of resynthesis tracts in repaired Escherichia coli deoxyribonucleic acid.

    OpenAIRE

    Kuemmerle, N; R. Ley; Masker, W

    1981-01-01

    Excision repair of ultraviolet radiation-induced damage in a wild-type strain of Escherichia coli has been examined, using two methods for characterizing the resynthesis step of the repair process. Comparison of data obtained after both isopycnic analysis of repaired deoxyribonucleic acid and sedimentation velocity analysis of deoxyribonucleic acid after selective photolysis of bromouracil-containing repaired regions has shown that the repaired deoxyribonucleic acid molecules contain a semico...

  8. Photoreversal-dependent release of thymidine and thymidine monophosphate from pyrimidine dimer-containing DNA excision fragments isolated from ultraviolet-damaged human fibroblasts

    International Nuclear Information System (INIS)

    To elucidate the enzymatic excision-repair process operative on cyclobutane-type pyrimidine photodimers in human dermal fibroblasts, we have examined excised dimer-containing material recovered in the trichloroacetic acid soluble fraction from far-ultraviolet-irradiated (254 nm, 40 J m-2) and incubated (24 h) cell cultures. The excised DNA photoproducts were found in oligonucleotide fragments with an estimated mean chain length of approximately 3.7 bases. Exposure of these isolated excision fragments, labeled with [3H]thymidine (dT), to a secondary, dimer-photoreversing fluence of far-UV (5.5 kJ m-2) resulted in the release of free dT and thymidine monophosphate (TMP). Photorelease of these two radioactive species was measured by high-performance liquid chromatography, with TMP being detected as the increase in dT following bacterial alkaline phosphatase treatment. These data imply that the photoliberated dT and TMP moieties were attached to the excision fragments solely by the cyclobutane ring of the dimer. No evidence was obtained for the photoliberation of free thymine, thus corroborating a conclusion reached by others that the excision of dimers in human cells is not initiated by scission of an intradimer N-glycosyl bond. The sum of the tritium label recovered in dT plus TMP corresponded to approximately 40% of that disappearing from thymine-containing dimers on photoreversal, suggesting that in about 80% of the isolated excision fragments the dimer is located at one end of the oligonucleotide and contains a break in its internal phosphodiester bond

  9. Is Early Nasal Shaping With a Limited Alar Base Incision Possible in the Repair of Cleft Lips?

    Science.gov (United States)

    Kapi, Emin; Bozkurt, Mehmet; Ozer, Torun; Celik, Mehmet Yusuf

    2016-05-01

    The repair of cleft lips has an important place in plastic and reconstructive surgery. In the treatment of these deformities, the aim was to restore the normal lip morphology in the cleft area as well as repairing any coexisting nasal deformities. Various methods are in use for this purpose. One of the most commonly employed surgical methods is Millard repair. However, this method may lead to additional scarring in the alar base on the cleft side subsequent to the incision. In this study, the results obtained from a group who have been applied alar base incisions during the modified Millard repair are compared to a group who have undergone intranasal wide dissections.The patients enrolled in the study were randomized into 2 groups. The first group were applied the modified Millard repair. In the second group, the rotation, advancement, and C-flaps were prepared according to Millard surgical repair technique; however, instead of an alar base and nasofacial groove incision on the cleft side, a nasal and maxillary supraperiosteal wide dissection was made through the incision in the mucosa of the nostril.The intranasal dissection performed during the study was observed to provide the targeted outcome and a more satisfactory cosmetic result through the modified Millard repair. In conclusion, the authors are of the opinion that the limited alar base incision and the wide supraperiosteal dissection performed in patients with cleft lips is an alternative method that can be employed in a wide range of patients. PMID:27092908

  10. Impact of Age-Associated Cyclopurine Lesions on DNA Repair Helicases

    OpenAIRE

    Khan, Irfan; Suhasini, Avvaru N.; Banerjee, Taraswi; Sommers, Joshua A.; Kaplan, Daniel L.; Kuper, Jochen; Kisker, Caroline; Brosh, Jr, Robert M

    2014-01-01

    8,5' cyclopurine deoxynucleosides (cPu) are locally distorting DNA base lesions corrected by nucleotide excision repair (NER) and proposed to play a role in neurodegeneration prevalent in genetically defined Xeroderma pigmentosum (XP) patients. In the current study, purified recombinant helicases from different classifications based on sequence homology were examined for their ability to unwind partial duplex DNA substrates harboring a single site-specific cPu adduct. Superfamily (SF) 2 RecQ ...

  11. Characterization of DNA repair phenotypes of Xeroderma pigmentosum cell lines by a paralleled in vitro test; Phenotypage de la reparation de l'ADN de lignees Xeroderma pigmentosum, par un test in vitro multiparametrique

    Energy Technology Data Exchange (ETDEWEB)

    Raffin, A.L.

    2009-06-15

    DNA is constantly damaged modifying the genetic information for which it encodes. Several cellular mechanisms as the Base Excision Repair (BER) and the Nucleotide Excision Repair (NER) allow recovering the right DNA sequence. The Xeroderma pigmentosum is a disease characterised by a deficiency in the NER pathway. The aim of this study was to propose an efficient and fast test for the diagnosis of this disease as an alternative to the currently available UDS test. DNA repair activities of XP cell lines were quantified using in vitro miniaturized and paralleled tests in order to establish DNA repair phenotypes of XPA and XPC deficient cells. The main advantage of the tests used in this study is the simultaneous measurement of excision or excision synthesis (ES) of several lesions by only one cellular extract. We showed on one hand that the relative ES of the different lesions depend strongly on the protein concentration of the nuclear extract tested. Working at high protein concentration allowed discriminating the XP phenotype versus the control one, whereas it was impossible under a certain concentration's threshold. On the other hand, while the UVB irradiation of control cells stimulated their repair activities, this effect was not observed in XP cells. This study brings new information on the XPA and XPC protein roles during BER and NER and underlines the complexity of the regulations of DNA repair processes. (author)

  12. Single-step scaffold-based cartilage repair in the knee: A systematic review.

    Science.gov (United States)

    Fischer, Stefan; Kisser, Agnes

    2016-12-01

    Chondral lesions are difficult-to-treat entities that often affect young and active people. Moreover, cartilage has limited intrinsic healing potential. The purpose of this systematic literature review was to analyse whether the single-step scaffold-based cartilage repair in combination with microfracturing (MFx) is more effective and safe in comparison to MFx alone. From the three identified studies, it seems that the single-step scaffold-assisted cartilage repair in combination with MFx leads to similar short- to medium-term (up to five years follow-up) results, compared to MFx alone. All of the studies have shown improvements regarding joint functionality, pain and partly quality of life. PMID:27408497

  13. Uncertain multiobjective redundancy allocation problem of repairable systems based on artificial bee colony algorithm

    Institute of Scientific and Technical Information of China (English)

    Guo Jiansheng; Wang Zutong; Zheng Mingfa; Wang Ying

    2014-01-01

    Based on the uncertainty theory, this paper is devoted to the redundancy allocation problem in repairable parallel-series systems with uncertain factors, where the failure rate, repair rate and other relative coefficients involved are considered as uncertain variables. The availability of the system and the corresponding designing cost are considered as two optimization objectives. A crisp multiobjective optimization formulation is presented on the basis of uncertainty theory to solve this resultant problem. For solving this problem efficiently, a new multiobjective artificial bee colony algorithm is proposed to search the Pareto efficient set, which introduces rank value and crowding distance in the greedy selection strategy, applies fast non-dominated sort procedure in the exploitation search and inserts tournament selection in the onlooker bee phase. It shows that the proposed algorithm outperforms NSGA-II greatly and can solve multiobjective redundancy allocation problem efficiently. Finally, a numerical example is provided to illustrate this approach.

  14. Arthroscopic excision of heterotopic calcification in a chronic rectus femoris origin injury: a case report

    OpenAIRE

    El-Husseiny, M; Sukeik, M.; Haddad, FS

    2012-01-01

    Rectus femoris origin injuries in adult athletes are uncommon. In the acute phase, conservative treatment seems to have a favourable outcome, with surgical repair reserved for unsuccessful cases only. However, a group of patients may develop chronic pain and disability after recovery from the acute phase due to heterotopic calcification occurring at the site of injury. Open and arthroscopic excision of such calcifications has been described in the literature although arthroscopic excision of ...

  15. Optimization and translation of MSC-based hyaluronic acid hydrogels for cartilage repair

    Science.gov (United States)

    Erickson, Isaac E.

    2011-12-01

    Traumatic injury and disease disrupt the ability of cartilage to carry joint stresses and, without an innate regenerative response, often lead to degenerative changes towards the premature development of osteoarthritis. Surgical interventions have yet to restore long-term mechanical function. Towards this end, tissue engineering has been explored for the de novo formation of engineered cartilage as a biologic approach to cartilage repair. Research utilizing autologous chondrocytes has been promising, but clinical limitations in their yield have motivated research into the potential of mesenchymal stem cells (MSCs) as an alternative cell source. MSCs are multipotent cells that can differentiate towards a chondrocyte phenotype in a number of biomaterials, but no combination has successfully recapitulated the native mechanical function of healthy articular cartilage. The broad objective of this thesis was to establish an MSC-based tissue engineering approach worthy of clinical translation. Hydrogels are a common class of biomaterial used for cartilage tissue engineering and our initial work demonstrated the potential of a photo-polymerizable hyaluronic acid (HA) hydrogel to promote MSC chondrogenesis and improved construct maturation by optimizing macromer and MSC seeding density. The beneficial effects of dynamic compressive loading, high MSC density, and continuous mixing (orbital shaker) resulted in equilibrium modulus values over 1 MPa, well in range of native tissue. While compressive properties are crucial, clinical translation also demands that constructs stably integrate within a defect. We utilized a push-out testing modality to assess the in vitro integration of HA constructs within artificial cartilage defects. We established the necessity for in vitro pre-maturation of constructs before repair to achieve greater integration strength and compressive properties in situ. Combining high MSC density and gentle mixing resulted in integration strength over 500 k

  16. Radiation- and drug-induced DNA repair in mammalian oocytes and embryos

    Energy Technology Data Exchange (ETDEWEB)

    Pedersen, R A; Brandriff, B

    1979-01-01

    A review of studies showing ultraviolet- or drug-induced unscheduled DNA synthesis in mammalian oocytes and embryos suggests that the female gamete has an excision repair capacity from the earliest stages of oocyte growth. The oocyte's demonstrable excision repair capacity decreases at the time of meiotic maturation for unknown reasons, but the fully mature oocyte maintans a repair capacity, in contrast to the mature sperm, and contributes this to the zygote. Early embryo cells maintain relatively constant levels of excision repair until late fetal stages, when they lose their capacity for excision repair. These apparent changes in excision repair capacity do not have a simple relationship to known differences in radiation sensitivity of germ cells and embryos.

  17. Radiation- and drug-induced DNA repair in mammalian oocytes and embryos

    International Nuclear Information System (INIS)

    A review of studies showing ultraviolet- or drug-induced unscheduled DNA synthesis in mammalian oocytes and embryos suggests that the female gamete has an excision repair capacity from the earliest stages of oocyte growth. The oocyte's demonstrable excision repair capacity decreases at the time of meiotic maturation for unknown reasons, but the fully mature oocyte maintans a repair capacity, in contrast to the mature sperm, and contributes this to the zygote. Early embryo cells maintain relatively constant levels of excision repair until late fetal stages, when they lose their capacity for excision repair. These apparent changes in excision repair capacity do not have a simple relationship to known differences in radiation sensitivity of germ cells and embryos

  18. Arsenic exposure disrupts the normal function of the FA/BRCA repair pathway.

    Science.gov (United States)

    Peremartí, Jana; Ramos, Facundo; Marcos, Ricard; Hernández, Alba

    2014-11-01

    Chronic arsenic exposure is known to enhance the genotoxicity/carcinogenicity of other DNA-damaging agents by inhibiting DNA repair activities. Interference with nucleotide excision repair and base excision repair are well documented, but interactions with other DNA repair pathways are poorly explored so far. The Fanconi anemia FA/BRCA pathway is a DNA repair mechanism required for maintaining genomic stability and preventing cancer. Here, interactions between arsenic compounds and the FA/BRCA pathway were explored by using isogenic FANCD2(-/-) (FA/BRCA-deficient) and FANCD2(+/+) (FA/BRCA-corrected) human fibroblasts. To study whether arsenic disrupts the normal FA/BRCA function, FANCD2(+/+) cells were preexposed to subtoxic concentrations of the trivalent arsenic compounds methylarsonous acid (MMA(III)) and arsenic trioxide (ATO) for 2 weeks. The cellular response to mitomicin-C, hydroxyurea, or diepoxybutane, typical inducers of the studied pathway, was then evaluated and compared to that of FANCD2(-/-) cells. Our results show that preexposure to the trivalent arsenicals MMA(III) and ATO induces in corrected cells, a cellular FA/BRCA-deficient phenotype characterized by hypersensitivity, enhanced accumulation in the G2/M compartment and increased genomic instability--measured as micronuclei. Overall, our data demonstrate that environmentally relevant arsenic exposures disrupt the normal function of the FA/BRCA activity, supporting a novel source of arsenic co- and carcinogenic effects. This is the first study linking arsenic exposure with the FA/BRCA DNA repair pathway. PMID:25092648

  19. Molecular Mechanisms of Ultraviolet Radiation-Induced DNA Damage and Repair

    Directory of Open Access Journals (Sweden)

    Rajesh P. Rastogi

    2010-01-01

    Full Text Available DNA is one of the prime molecules, and its stability is of utmost importance for proper functioning and existence of all living systems. Genotoxic chemicals and radiations exert adverse effects on genome stability. Ultraviolet radiation (UVR (mainly UV-B: 280–315 nm is one of the powerful agents that can alter the normal state of life by inducing a variety of mutagenic and cytotoxic DNA lesions such as cyclobutane-pyrimidine dimers (CPDs, 6-4 photoproducts (6-4PPs, and their Dewar valence isomers as well as DNA strand breaks by interfering the genome integrity. To counteract these lesions, organisms have developed a number of highly conserved repair mechanisms such as photoreactivation, base excision repair (BER, nucleotide excision repair (NER, and mismatch repair (MMR. Additionally, double-strand break repair (by homologous recombination and nonhomologous end joining, SOS response, cell-cycle checkpoints, and programmed cell death (apoptosis are also operative in various organisms with the expense of specific gene products. This review deals with UV-induced alterations in DNA and its maintenance by various repair mechanisms.

  20. 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

    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......, candidate proteins in extracts can be inhibited or depleted in a controlled way, making defined extracts an important source for mechanistic studies. The major drawback is that there is no standardized method of preparing nuclear extract for BER studies, and it does not appear to be a topic given much...... 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...

  1. Decrease in Abundance of Apurinic/Apyrimidinic Endonuclease Causes Failure of Base Excision Repair in Culture-Adapted Human Embryonic Stem Cells

    Czech Academy of Sciences Publication Activity Database

    Krutá, M.; Bálek, L.; Hejnová, R.; Dobšáková, Z.; Eiselleová, L.; Matulka, K.; Bárta, T.; Fojtík, P.; Fajkus, Jiří; Hampl, A.; Dvořák, P.; Rotrekl, V.

    2013-01-01

    Roč. 31, č. 4 (2013), s. 693-702. ISSN 1066-5099 R&D Projects: GA ČR(CZ) GBP302/12/G157 Grant ostatní: GA MŠk(CZ) ED1.100/02/0123 Institutional support: RVO:68081707 Keywords : DNA-DAMAGE * GENOMIC INSTABILITY * HETEROZYGOUS MICE Subject RIV: BO - Biophysics Impact factor: 7.133, year: 2013

  2. The catalytic mechanism of hOGG1 base-excision repair enzyme; the theoretical modeling of reaction channels and substrate activation

    Czech Academy of Sciences Publication Activity Database

    Šebera, Jakub; Nencka, Radim; Trantírek, L.; Tanaka, Y.; Sychrovský, Vladimír

    Nové Hrady : -, 2014. [Visegrad Symposium on Structural Systems Biology /4./. 17.06.2014-20.06.2014, Nové Hrady] R&D Projects: GA ČR GA13-27676S Institutional support: RVO:61388963 Keywords : hOGG1 * enzyme catalysis * DFT Subject RIV: CF - Physical ; Theoretical Chemistry

  3. Rethinking Transcription Coupled DNA Repair

    OpenAIRE

    Kamarthapu, Venu; Nudler, Evgeny

    2015-01-01

    Nucleotide excision repair (NER) is an evolutionarily conserved, multistep process that can detect a wide variety of DNA lesions. Transcription coupled repair (TCR) is a sub-pathway of NER that repairs the transcribed DNA strand faster than the rest of the genome. RNA polymerase (RNAP) stalled at DNA lesions mediates the recruitment of NER enzymes to the damage site. In this review we focus on a newly identified bacterial TCR pathway in which the NER enzyme UvrD, in conjunction with NusA, pla...

  4. The indirect effect of radiation reduces the repair fidelity of NHEJ as verified in repair deficient CHO cell lines exposed to different radiation qualities and potassium bromate

    Energy Technology Data Exchange (ETDEWEB)

    Bajinskis, Ainars, E-mail: ainars.bajinskis@gmt.su.se [Centre for Radiation Protection Research, Department of Genetics, Microbiology and Toxicology, Stockholm University, S-10691 Stockholm (Sweden); Olsson, Gunilla; Harms-Ringdahl, Mats [Centre for Radiation Protection Research, Department of Genetics, Microbiology and Toxicology, Stockholm University, S-10691 Stockholm (Sweden)

    2012-03-01

    The complexity of DNA lesions induced by ionizing radiation is mainly dependent on radiation quality, where the indirect action of radiation may contribute to different extent depending on the type of radiation under study. The effect of indirect action of radiation can be investigated by using agents that induce oxidative DNA damage or by applying free radical scavengers. The aim of this study was to investigate the role of the indirect effect of radiation for the repair fidelity of non-homologous end-joining (NHEJ), homologous recombination repair (HRR) and base excision repair (BER) when DNA damage of different complexity was induced by gamma radiation, alpha particles or from base damages (8-oxo-dG) induced by potassium bromate (KBrO{sub 3}). CHO cells lines deficient in XRCC3 (HRR) irs1SF, XRCC7 (NHEJ) V3-3 and XRCC1 (BER) EM9 were irradiated in the absence or presence of the free radical scavenger dimethyl sulfoxide (DMSO). The endpoints investigated included rate of cell proliferation by the DRAG assay, clonogenic cell survival and the level of primary DNA damage by the comet assay. The results revealed that the indirect effect of low-LET radiation significantly reduced the repair fidelity of both NHEJ and HRR pathways. For high-LET radiation the indirect effect of radiation also significantly reduced the repair fidelity for the repair deficient cell lines. The results suggest further that the repair fidelity of the error prone NHEJ repair pathway is more impaired by the indirect effect of high-LET radiation relative to the other repair pathways studied. The response to bromate observed for the two DSB repair deficient cell lines strongly support earlier studies that bromate induces complex DNA damages. The significantly reduced repair fidelity of irs1SF and V3-3 suggests that NHEJ as well as HRR are needed for the repair, and that complex DSBs are formed after bromate exposure.

  5. Defective repair of uracil causes telomere defects in mouse hematopoietic cells.

    Science.gov (United States)

    Vallabhaneni, Haritha; Zhou, Fang; Maul, Robert W; Sarkar, Jaya; Yin, Jinhu; Lei, Ming; Harrington, Lea; Gearhart, Patricia J; Liu, Yie

    2015-02-27

    Uracil in the genome can result from misincorporation of dUTP instead of dTTP during DNA synthesis, and is primarily removed by uracil DNA glycosylase (UNG) during base excision repair. Telomeres contain long arrays of TTAGGG repeats and may be susceptible to uracil misincorporation. Using model telomeric DNA substrates, we showed that the position and number of uracil substitutions of thymine in telomeric DNA decreased recognition by the telomere single-strand binding protein, POT1. In primary mouse hematopoietic cells, uracil was detectable at telomeres, and UNG deficiency further increased uracil loads and led to abnormal telomere lengthening. In UNG-deficient cells, the frequencies of sister chromatid exchange and fragility in telomeres also significantly increased in the absence of telomerase. Thus, accumulation of uracil and/or UNG deficiency interferes with telomere maintenance, thereby underscoring the necessity of UNG-initiated base excision repair for the preservation of telomere integrity. PMID:25572391

  6. A child with xeroderma pigmentosum for excision of basal cell carcinoma

    Directory of Open Access Journals (Sweden)

    Sridevi M Mulimani

    2013-01-01

    Full Text Available Xeroderma pigmentosum (XP is characterized by hypersensitivity to sunlight, ocular involvement, and progressive neurological complications. These manifestations are due to a cellular hypersensitivity to ultraviolet radiation leading to a defect in repair of DNA by the process of nucleotide excision repair. Basal cell carcinoma which is rare in children can occur with XP. Though the XP induced changes are predominately dermatologic, pose several challenges in anaesthetic management. Hence, we are reporting a 9-year-old child with XP scheduled for excision of basal cell carcinoma under general anaesthesia.

  7. Investigation of Phosphate Cement-based Binder with Super High Early Strength for Repair of Concrete

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Magnesium phosphate cement-based binder (MPB) for repair of concrete was prepared by proportionally mixing over burned MgO powder (M) with NHH2PO4 powder (P) and set modifying admixtures. It is characteristic by excellent properties such as rapid setting,high strength and high bond strength to old concrete.. The study is focused on the key factors influencing the setting time and strength of MPB, the bond property of MPB to old concrete and the kinetic feature of the hydration of MPB.

  8. 'Failure limited' data and TTT-based trend tests in multiple repairable systems

    International Nuclear Information System (INIS)

    Tests for trend in multiple repairable systems based on the total time on test (introduced by Kvaloy and Lindqvist) are valid for data that end at a failure time only if data collection was planned to continue until a predetermined number of failures had occurred. They are not valid for a commoner form of data that end with the last of a random number of failures within a predetermined observation period. The necessary modification to the analysis is given here and appropriate terminology is suggested.

  9. Alkaline gel electrophoresis assay to detect DNA strand breaks and repair mechanisms in Escherichia coli

    OpenAIRE

    José Carlos Pelielo De Mattos; Ellen Serri da Motta; Márcia Betania Nunes de Oliveira; Flávio José da Silva Dantas; Adriano Caldeira de Araujo

    2008-01-01

    Reactive oxygen species (ROS) can induce lesions in different cellular targets, including DNA. Stannous chloride (SnCl2) is a ROS generator, leading to lethality in Escherichia coli (E. coli), with the base excision repair (BER) mechanism playing a role in this process. Many techniques have been developed to detect genotoxicity, as comet assay, in eukaryotic cells, and plasmid DNA agarose gel electrophoresis. In this study, an adaptation of the alkaline gel electrophoresis method was carried ...

  10. Purification of mammalian DNA repair protein XRCC1

    Energy Technology Data Exchange (ETDEWEB)

    Chen, I. [Univ. of California, Berkeley, CA (United States)

    1995-11-01

    Malfunctioning DNA repair systems lead to cancer mutations, and cell death. XRCC1 (X-ray Repair Cross Complementing) is a human DNA repair gene that has been found to fully correct the x-ray repair defect in Chinese hamster ovary (CHO) cell mutant EM9. The corresponding protein (XRCC1) encoded by this gene has been linked to a DNA repair pathway known as base excision repair, and affects the activity of DNA ligase III. Previously, an XRCC1 cDNA minigene (consisting of the uninterrupted coding sequence for XRCC1 protein followed by a decahistidine tag) was constructed and cloned into vector pET-16b for the purpose of: (1) overproduction of XRCC1 in both prokaryotic and eukaryotic cells; and (2) to facilitate rapid purification of XRCC1 from these systems. A vector is basically a DNA carrier that allows recombinant protein to be cloned and overexpressed in host cells. In this study, XRCC1 protein was overexpressed in E. coli and purified by immobilized metal affinity chromatography. Currently, the XRCC1 minigene is being inserted into a new vector [pET-26b(+)] in hopes to increase overexpression and improve purification. Once purified XRCC1 can be crystallized for structural studies, or studied in vitro for its biological function.

  11. Mechanisms of DNA Repair in Mammalian Cells

    International Nuclear Information System (INIS)

    The authors examined DNA synthesis in cultured mammalian cells after irradiation with X-rays or ultraviolet light, using equilibrium density gradient and autoradiographic techniques. Unscheduled DNA synthesis (the synthesis of DNA by cells not in S phase of the cell cycle) occurs at doses of u.v. where survival is greater than 90% and at doses of X-rays where survival is of the order of 50%. At higher doses it was established that repair replication (insertion of precursors into parental strands of DNA ) occurs in these cells, and it is presumed that these two phenomena (unscheduled DNA synthesis and repair replication) are manifestations of the same repair process. During the time that these phenomena occur, very little degradation of DNA takes place, as measured by appearance of prelabelled components of DNA in the medium or in the acid soluble portion of the cell. This is in direct contrast to the situation in bacteria, in which extensive degradation of DNA occurs after irradiation, presumably as a result of enzymatic processes that remove many undamaged bases in addition to the ones injured by the irradiation. A small amount of radioactivity does appear in the acid soluble portion and in the media from prelabelled cells from both irradiated and control mammalian cell cultures. The amount in the medium from irradiated cultures is slightly, but significantly, greater than that from controls; thus there does appear to be a very low level of degradation of DNA in irradiated mammalian cells. These data indicate that the repair of DNA in mammalian cells does not involve the same steps as those that occur in bacteria. The results suggest instead that mammalian cells have a much more specific system for repair, which involves excision of only the damaged portion of the DNA. Possible mechanisms of repair of mammalian DNA are considered. (author)

  12. Interference by toxic metal ions with DNA repair processes and cell cycle control: molecular mechanisms.

    OpenAIRE

    Hartwig, A.; Asmuss, M; Ehleben, I; Herzer, U; Kostelac, D; Pelzer, A; Schwerdtle, T.; Bürkle, A

    2002-01-01

    Nickel, cadmium, cobalt, and arsenic compounds are well-known carcinogens to humans and experimental animals. Even though their DNA-damaging potentials are rather weak, they interfere with the nucleotide and base excision repair at low, noncytotoxic concentrations. For example, both water-soluble Ni(II) and particulate black NiO greatly reduced the repair of DNA adducts induced by benzo[a]pyrene, an important environmental pollutant. Furthermore, Ni(II), As(III), and Co(II) interfered with ce...

  13. Postreplication repair of ultraviolet-irradiated transforming deoxyribonucleic acid in Bacillus subtilis

    International Nuclear Information System (INIS)

    Repair of ultraviolet-irradiated transforming deoxyribonucleic acid (DNA) in several strains of Bacillus subtilis was studied in order to determine the effects of excision repair and postreplication repair on transformation. Two mutations that cause a Uvr- phenotype (uvr-1 and uvr-42) were shown to have strikingly different effects on repair of ultraviolet-irradiated transforming DNA

  14. Rethinking transcription coupled DNA repair.

    Science.gov (United States)

    Kamarthapu, Venu; Nudler, Evgeny

    2015-04-01

    Nucleotide excision repair (NER) is an evolutionarily conserved, multistep process that can detect a wide variety of DNA lesions. Transcription coupled repair (TCR) is a subpathway of NER that repairs the transcribed DNA strand faster than the rest of the genome. RNA polymerase (RNAP) stalled at DNA lesions mediates the recruitment of NER enzymes to the damage site. In this review we focus on a newly identified bacterial TCR pathway in which the NER enzyme UvrD, in conjunction with NusA, plays a major role in initiating the repair process. We discuss the tradeoff between the new and conventional models of TCR, how and when each pathway operates to repair DNA damage, and the necessity of pervasive transcription in maintaining genome integrity. PMID:25596348

  15. 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 cancer.

  16. Alkaline gel electrophoresis assay to detect DNA strand breaks and repair mechanisms in Escherichia coli

    International Nuclear Information System (INIS)

    Reactive oxygen species (ROS) can induce lesions in different cellular targets, including DNA. Stannous chloride (SnCl2) is a ROS generator, leading to lethality in Escherichia coli (E. coli), with the base excision repair (BER) mechanism playing a role in this process. Many techniques have been developed to detect genotoxicity, as comet assay, in eukaryotic cells, and plasmid DNA agarose gel electrophoresis. In this study, an adaptation of the alkaline gel electrophoresis method was carried out to ascertain the induction of strand breaks by SnCl2 in bacterial DNA, from E. coli BER mutants, and its repair pathway. Results obtained show that SnCl2 was able to induce DNA strand breaks in all strains tested. Moreover, endonuclease IV and exonuclease III play a role in DNA repair. On the whole, data has shown that the alkaline gel electrophoresis assay could be used both for studying DNA strand breaks induction and for associated repair mechanisms. (author)

  17. Dual roles of DNA repair enzymes in RNA biology/post-transcriptional control.

    Science.gov (United States)

    Vohhodina, Jekaterina; Harkin, D Paul; Savage, Kienan I

    2016-09-01

    Despite consistent research into the molecular principles of the DNA damage repair pathway for almost two decades, it has only recently been found that RNA metabolism is very tightly related to this pathway, and the two ancient biochemical mechanisms act in alliance to maintain cellular genomic integrity. The close links between these pathways are well exemplified by examining the base excision repair pathway, which is now well known for dual roles of many of its members in DNA repair and RNA surveillance, including APE1, SMUG1, and PARP1. With additional links between these pathways steadily emerging, this review aims to provide a summary of the emerging roles for DNA repair proteins in the post-transcriptional regulation of RNAs. WIREs RNA 2016, 7:604-619. doi: 10.1002/wrna.1353 For further resources related to this article, please visit the WIREs website. PMID:27126972

  18. A child with xeroderma pigmentosum for excision of basal cell carcinoma

    OpenAIRE

    Mulimani, Sridevi M; Talikoti, Dayanand G

    2013-01-01

    Xeroderma pigmentosum (XP) is characterized by hypersensitivity to sunlight, ocular involvement, and progressive neurological complications. These manifestations are due to a cellular hypersensitivity to ultraviolet radiation leading to a defect in repair of DNA by the process of nucleotide excision repair. Basal cell carcinoma which is rare in children can occur with XP. Though the XP induced changes are predominately dermatologic, pose several challenges in anaesthetic management. Hence, we...

  19. Application of a Pattern-based Classification System for Invasive Endocervical Adenocarcinoma in Cervical Biopsy, Cone and Loop Electrosurgical Excision (LEEP) Material: Pattern on Cone and LEEP is Predictive of Pattern in the Overall Tumor.

    Science.gov (United States)

    Djordjevic, Bojana; Parra-Herran, Carlos

    2016-09-01

    A pattern-based classification system has been recently proposed for invasive endocervical adenocarcinoma, which is predictive of the risk of nodal metastases. Identifying cases at risk of nodal involvement is most relevant at the time of biopsy and loop electrosurgical excision procedure (LEEP) to allow for optimal surgical planning, and, most importantly, consideration of lymphadenectomy. This study aims to determine the topography of patterns of stromal invasion in invasive endocervical adenocarcinoma with emphasis on patterns in biopsy, cone, and LEEP. Invasive pattern was assessed following the pattern-based classification (Patterns A, B, and C) in 47 invasive endocervical adenocarcinomas treated with hysterectomy or trachelectomy and correlated with pattern of invasion at the tumor surface (2 mm of tumor depth) and on preoperative biopsy and cone/LEEP. Patterns A, B, and C were present in 21.3%, 36.2%, and 42.5% of cases, respectively. Most pattern A cases were Stage IA (90%), whereas most Pattern B and C cases were Stage IB (76.5% and 80%, respectively). Horizontal spread was on average larger in Pattern C (24.1 mm) than in Patterns A and B (7.7 and 12.3 mm, respectively). Pattern at the tumor surface correlated with the overall pattern in 95.7% of cases. Concordance between patterns at cone/LEEP and hysterectomy was 92.8%; the only discrepant case was upgraded from Pattern A on LEEP to C on final excision. Agreement between patterns in biopsy and the overall tumor, however, was only 37.5%. In all discrepant cases, biopsy failed to reveal destructive invasion, which was evident on excision. All discrepant biopsies with pattern A showed glandular complexity resembling exophytic papillary growth but did not meet criteria for destructive invasion. On excision, marked gland confluence with papillary architecture was evident. We conclude that the pattern of invasion on cone/LEEP is a good predictor of pattern of invasion on hysterectomy, particularly if there is

  20. Effect of coating repair on microstructure and mechanical properties of Ni3Al based alloy IC6

    Institute of Scientific and Technical Information of China (English)

    李树索; 韩雅芳; 肖程波; 宋尽霞; 李建平

    2002-01-01

    The effect of coating repair on microstr ucture and mechanical properties of a directionally solidified nickel base alloy IC6 was studied. The experime ntal results show that after coating repair treatment, the substrate/coating interface keeps well, and the combination of coating and substrate is very good , as well as very slight mutual diffusion of alloying elements between substrate and coating occurred. Although coating repair treatment make diffusion time double, only a little amount of Mo diffused into coating, and the elements Cr and Co of NiCoCrAlYHf coating only exist in a thin layer of influence region of the substrate, which has no obvious effect on the microstructure of alloy IC6. Tensile tests at room temperature and stress rupture tests under 1100 ℃, 90 Mpa of the coating-repaired sample were conducted. The results show that the coating repair has no evident effect on mechanical properties. Therefore, it may be considered that when the coating is unexpectedly destroyed, it can be repaired.

  1. Performance analysis of complex repairable industrial systems using PSO and fuzzy confidence interval based methodology.

    Science.gov (United States)

    Garg, Harish

    2013-03-01

    The main objective of the present paper is to propose a methodology for analyzing the behavior of the complex repairable industrial systems. In real-life situations, it is difficult to find the most optimal design policies for MTBF (mean time between failures), MTTR (mean time to repair) and related costs by utilizing available resources and uncertain data. For this, the availability-cost optimization model has been constructed for determining the optimal design parameters for improving the system design efficiency. The uncertainties in the data related to each component of the system are estimated with the help of fuzzy and statistical methodology in the form of the triangular fuzzy numbers. Using these data, the various reliability parameters, which affects the system performance, are obtained in the form of the fuzzy membership function by the proposed confidence interval based fuzzy Lambda-Tau (CIBFLT) methodology. The computed results by CIBFLT are compared with the existing fuzzy Lambda-Tau methodology. Sensitivity analysis on the system MTBF has also been addressed. The methodology has been illustrated through a case study of washing unit, the main part of the paper industry. PMID:23098922

  2. Radiation damage and its repair in non-sporulating bacteria

    International Nuclear Information System (INIS)

    A review is given of radiation damage and its repair in non-sporulating bacteria. The identification and measurement of radiation damage in the DNA of the bacteria after exposure to ultraviolet radiation and ionizing radiation is described. Measuring the extent of DNA repair and ways of isolating repair mutants are also described. The DNA repair mechanisms for UV-induced damage are discussed including photoreactivation repair, excision repair, post-replication recombination repair and induced error-prone repair. The DNA repair mechanisms for ionizing radiation damage are also discussed including the repair of both single and double-strand breaks. Other aspects discussed include the effects of growth, irradiation medium and recovery medium on survival, DNA repair in humans, the commercial use of UV and ionizing radiations and the future of ionizing irradiation as a food treatment process. (U.K.)

  3. Use of Preputial Skin as Cutaneous Graft after Nevus Excision

    Directory of Open Access Journals (Sweden)

    A. D'Alessio

    2010-01-01

    Full Text Available We report a four-year-old boy with a nevus covering all the plantar side of his second finger on the left foot. He was also affected by congenital phimosis. Surgical excision of the nevus was indicated, but the skin defect would have been too large to be directly closed. The foreskin was taken as a full-thickness skin graft to cover the cutaneous defect of the finger. The graft intake was favourable and provided a functional repair with good aesthetic characteristic.

  4. Use of Preputial Skin as Cutaneous Graft after Nevus Excision

    OpenAIRE

    A. D'Alessio; Piro, E; Brugnoni, M.; L. Abati

    2010-01-01

    We report a four-year-old boy with a nevus covering all the plantar side of his second finger on the left foot. He was also affected by congenital phimosis. Surgical excision of the nevus was indicated, but the skin defect would have been too large to be directly closed. The foreskin was taken as a full-thickness skin graft to cover the cutaneous defect of the finger. The graft intake was favourable and provided a functional repair with good aesthetic characteristic.

  5. DNA-mediated charge transport for DNA repair

    OpenAIRE

    Boon, Elizabeth M; Livingston, Alison L.; Chmiel, Nikolas H.; David, Sheila S.; Barton, Jacqueline K.

    2003-01-01

    MutY, like many DNA base excision repair enzymes, contains a [4Fe4S](2+) cluster of undetermined function. Electrochemical studies of MutY bound to a DNA-modified gold electrode demonstrate that the [4Fe4S] cluster of MutY can be accessed in a DNA-mediated redox reaction. Although not detectable without DNA, the redox potential of DNA-bound MutY is approximate to275 mV versus NHE, which is characteristic of HiPiP iron proteins. Binding to DNA is thus associated with a change in [4Fe4S](3+/2+)...

  6. Enzymic excision of ultraviolet-induced cytosine hydrates from left-handed DNA

    International Nuclear Information System (INIS)

    Ultraviolet irradiation of DNA produces a variety of pyrimidine modifications. These include cytosine hydrate (5,6-dihydro-6-hydroxycytosine), released as a free base by E. coli endonuclease III. The enzymic excision of cytosine hydrate by this purified enzyme was investigated by assaying release of labeled photoproduct from DNA into the ethanol-soluble fraction. Ultraviolet-irradiated poly(dG-dC):poly(dG-dC), radio-labeled in cytosines, was used as substrate. Shifts between the right-handed B-conformation and the left-handed Z-conformation were effected by heating the polymer in the presence of either Ni[II] or Co[II]. Conformational states were determined by ultraviolet circular dichroism. Rates of enzymic cytosine hydrate release did not differ between the different substrate conformations, B-DNA and Z-DNA. Irradiation of left-handed poly(dG-dC):poly(dG-dC) resulted in cytosine hydrate formation. Therefore, neither formation nor enzymic excision of ultraviolet-induced cytosine hydrates are substantially affected by the DNA conformational state. Cytosine hydrates are most likely to occur in alternating purine:pyrimidine sequences. Such segments can adopt the Z-conformation as a result of reactions with chemical carcinogens, the presence of metal ions, or negative superhelicity. These results indicate repair of cytosine hydrates to be likely, regardless of the DNA conformational state

  7. Repair deficient and hypersensitive diseases of man

    International Nuclear Information System (INIS)

    Many diseases are hypersensitive to DNA damaging agents but only xeroderma pigmentosum is consistently DNA repair deficient. Groups A to G exhibit reduction in excision of pyrimidine dimers and other lesions, the variant and alteration in polymerization or ligation. Pulse chase experiments indicate that excision defective and variant cells suffer increased blocking of replication forks during semiconservative replication after irradiation; but later bypass of such blocks is completely normal in all cell types. Thus, the variant is not defective in ''post-replication repair,'' and even the definition of such a system comes under question. (author)

  8. 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.

  9. Moving black holes via singularity excision

    International Nuclear Information System (INIS)

    We present a singularity excision algorithm appropriate for numerical simulations of black holes moving throughout the computational domain. The method is an extension of the excision procedure previously used to obtain stable simulations of single, non-moving black holes. The excision procedure also shares elements used in recent work to study the dynamics of a scalar field in the background of a single, boosted black hole. The excision method is tested with single black-hole evolutions using a coordinate system in which the coordinate location of the black hole, and thus the excision boundary, moves throughout the computational domain

  10. Radioimmunoassay studies on repair of ultraviolet damaged DNA in cultured animal cells

    International Nuclear Information System (INIS)

    UV (ultraviolet) damaged DNA and its repair of various cultured animal cells were observed by radioimmunoassay using anti-serum against the UV irradiation induced heat-degenerated DNA. There is some difference among the cells of used animals according to their DNA repairabilities. The cells were divided into four groups according to the existence or strength of their repairabilities. 1) excision repair type: cells of men and chimpanzees. 2) photoreactivation type: cells derived from Tachydromus tachydromoides and chicks. 3) photoreactivation with excision repair: cells of rats, kangaroos and mosquitos. 4) non-excision repair type: cells of mice, Meriones and rats. Animal cells have plural types of repair. Main types of repair will differ according to the kind of animals. (Ichikawa, K.)

  11. Low-level laser irradiation alters mRNA expression from genes involved in DNA repair and genomic stabilization in myoblasts

    Science.gov (United States)

    Trajano, L. A. S. N.; Sergio, L. P. S.; Silva, C. L.; Carvalho, L.; Mencalha, A. L.; Stumbo, A. C.; Fonseca, A. S.

    2016-07-01

    Low-level lasers are used for the treatment of diseases in soft and bone tissues, but few data are available regarding their effects on genomic stability. In this study, we investigated mRNA expression from genes involved in DNA repair and genomic stabilization in myoblasts exposed to low-level infrared laser. C2C12 myoblast cultures in different fetal bovine serum concentrations were exposed to low-level infrared laser (10, 35 and 70 J cm‑2), and collected for the evaluation of DNA repair gene expression. Laser exposure increased gene expression related to base excision repair (8-oxoguanine DNA glycosylase and apurinic/apyrimidinic endonuclease 1), nucleotide excision repair (excision repair cross-complementation group 1 and xeroderma pigmentosum C protein) and genomic stabilization (ATM serine/threonine kinase and tumor protein p53) in normal and low fetal bovine serum concentrations. Results suggest that genomic stability could be part of a biostimulation effect of low-level laser therapy in injured muscles.

  12. Complex DNA repair pathways as possible therapeutic targets to overcome temozolomide resistance in glioblastoma

    Directory of Open Access Journals (Sweden)

    Koji eYoshimoto

    2012-12-01

    Full Text Available Many conventional chemotherapeutic drugs exert their cytotoxic function by inducing DNA damage in the tumor cell. Therefore, a cell-inherent DNA repair pathway, which reverses the DNA-damaging effect of the cytotoxic drugs, can mediate therapeutic resistance to chemotherapy. The monofunctional DNA-alkylating agent temozolomide (TMZ is a commonly used chemotherapeutic drug and the gold standard treatment for glioblastoma. Although the activity of DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT has been described as the main modulator to determine the sensitivity of glioblastoma to TMZ, a subset of glioblastoma does not respond despite MGMT inactivation, suggesting that another DNA repair mechanism may also modulate the tolerance to TMZ. Considerable interest has focused on MGMT, mismatch repair (MMR, and the base-excision repair (BER pathway in the mechanism of mediating TMZ resistance, but emerging roles for the DNA strand-break repair pathway have been demonstrated. In the first part of this review article, we briefly review the significant role of MGMT, MMR, and the BER pathway in the tolerance to TMZ; in the last part, we review the recent publications that demonstrate possible roles of DNA strand-break repair pathways, such as single-strand break (SSB repair and double-strand break (DSB repair, as well as the Fanconi anemia pathway in the repair process after alkylating agent-based therapy. It is possible that all of these repair pathways have a potential to modulate the sensitivity to TMZ and aid in overcoming the therapeutic resistance in the clinic.

  13. Prediction of the cumulative number of failures for a repairable system based on past performance

    OpenAIRE

    Veber, Boštjan; Fajdiga, Matija; Nagode, Marko

    2015-01-01

    The prediction of the cumulative number of failures for a repairable system isan important topic in reliability theory. A repairable system may end up in one of the three possible states after a repair: 'as good as new', 'as bad as old' and 'better than old but worse than new'. Current probabilistic models used in repairable system analysis account for the first two states, but they do not properly apply to the last one, which is more common in practice. In this paper, a robust solution to a ...

  14. Effect of radiotherapy on survival of women with locally excised ductal carcinoma in situ of the breast: a Surveillance, Epidemiology, and End Results population-based analysis

    Directory of Open Access Journals (Sweden)

    Qian GW

    2015-06-01

    Full Text Available Guo-Wei Qian,1,* Xiao-Jian Ni,1,* Zheng Wang,2 Yi-Zhou Jiang,1 Ke-Da Yu,1 Zhi-Ming Shao1 1Department of Breast Surgery, 2Department of Radiation Oncology, Shanghai Cancer Center and Cancer Institute, Fudan University, Shanghai, People’s Republic of China *These authors contributed equally to this work Background: Although it has been previously reported that radiotherapy (RT effectively reduced the incidence of local recurrence of ductal carcinoma in situ (DCIS following breast-conserving surgery (BCS, little is known about the effect of RT on survival of patients with locally excised DCIS. Patients and methods: Using Surveillance, Epidemiology, and End Results registry data, we selected 56,968 female DCIS patients treated with BCS between 1998 and 2007. Overall survival (OS and breast cancer-specific survival (BCSS were compared among patients who received RT or no RT using the Kaplan–Meier methods and Cox proportional hazards regression models. Results: Median follow-up was 91 months. In the multivariable model, patients receiving postoperative RT had better OS than those undergoing BCS alone (hazard ratio [HR] 0.59, 95% confidence interval [CI] 0.53–0.67, P<0.001. This pattern remained after stratification by estrogen receptor (ER status and age. In contrast, RT delivery was not significantly associated with improved BCSS (HR 0.71, 95% CI 0.48–1.03, P=0.073. However, after stratifying by the above two variables, RT contributed to better BCSS in ER-negative/borderline patients (HR 0.41, 95% CI 0.19–0.88, P=0.023 and younger patients (≤50 years old; HR 0.37, 95% CI 0.15–0.91, P=0.030. Conclusion: Our analysis confirms the beneficial effect of RT on OS in women with locally excised DCIS and reveals the specific protective effect of RT on BCSS in ER-negative/borderline and younger patients. Keywords: ductal carcinoma in situ, breast cancer, breast-conserving surgery, radiotherapy, survival

  15. A eukaryotic gene encoding an endonuclease that specifically repairs DNA damaged by ultraviolet light.

    OpenAIRE

    Yajima, H; Takao, M; Yasuhira, S; Zhao, J. H.; Ishii, C.; Inoue, H; Yasui, A

    1995-01-01

    Many eukaryotic organisms, including humans, remove ultraviolet (UV) damage from their genomes by the nucleotide excision repair pathway, which requires more than 10 separate protein factors. However, no nucleotide excision repair pathway has been found in the filamentous fungus Neurospora crassa. We have isolated a new eukaryotic DNA repair gene from N.crassa by its ability to complement UV-sensitive Escherichia coli cells. The gene is altered in a N.crassa mus-18 mutant and responsible for ...

  16. Mitochondrial DNA repair: a novel therapeutic target for heart failure.

    Science.gov (United States)

    Marín-García, José

    2016-09-01

    Mitochondria play a crucial role in a variety of cellular processes ranging from energy metabolism, generation of reactive oxygen species (ROS) and Ca(2+) handling to stress responses, cell survival and death. Malfunction of the organelle may contribute to the pathogenesis of neuromuscular, cancer, premature aging and cardiovascular diseases (CVD), including myocardial ischemia, cardiomyopathy and heart failure (HF). Mitochondria contain their own genome organized into DNA-protein complexes, called "mitochondrial nucleoids," along with multiprotein machineries, which promote mitochondrial DNA (mtDNA) replication, transcription and repair. Although the mammalian organelle possesses almost all known nuclear DNA repair pathways, including base excision repair, mismatch repair and recombinational repair, the proximity of mtDNA to the main sites of ROS production and the lack of protective histones may result in increased susceptibility to various types of mtDNA damage. These include accumulation of mtDNA point mutations and/or deletions and decreased mtDNA copy number, which will impair mitochondrial function and finally, may lead to CVD including HF. PMID:26940911

  17. Preferential DNA repair in expressed genes.

    Science.gov (United States)

    Hanawalt, P C

    1987-01-01

    Potentially deleterious alterations to DNA occur nonrandomly within the mammalian genome. These alterations include the adducts produced by many chemical carcinogens, but not the UV-induced cyclobutane pyrimidine dimer, which may be an exception. Recent studies in our laboratory have shown that the excision repair of pyrimidine dimers and certain other lesions is nonrandom in the mammalian genome, exhibiting a distinct preference for actively transcribed DNA sequences. An important consequence of this fact is that mutagenesis and carcinogenesis may be determined in part by the activities of the relevant genes. Repair may also be processive, and a model is proposed in which excision repair is coupled to transcription at the nuclear matrix. Similar but freely diffusing repair complexes may account for the lower overall repair efficiencies in the silent domains of the genome. Risk assessment in relation to chemical carcinogenesis requires assays that determine effective levels of DNA damage for producing malignancy. The existence of nonrandom repair in the genome casts into doubt the reliability of overall indicators of DNA binding and lesion repair for such determinations. Furthermore, some apparent differences between the intragenomic repair heterogeneity in rodent cells and that in human cells mandate a reevaluation of rodent test systems for human risk assessment. Tissue-specific and cell-specific differences in the coordinate regulation of gene expression and DNA repair may account for corresponding differences in the carcinogenic response. Images FIGURE 1. FIGURE 1. PMID:3447906

  18. Factors that influence telomeric oxidative base damage and repair by DNA glycosylase OGG1

    DEFF Research Database (Denmark)

    Rhee, David B; Ghosh, Avik; Lu, Jian; Bohr, Vilhelm A; Liu, Yie

    2011-01-01

    -telomeric double-stranded substrates. In addition, telomere repeat binding factors TRF1 and TRF2 do not impair OGG1 incision activity. Yet, 8-oxodG in some telomere structures (e.g., fork-opening, 3'-overhang, and D-loop) were less effectively excised by OGG1, depending upon its position in these substrates...

  19. 75 FR 13127 - Lead-Based Paint Renovation, Repair and Painting Activities in Target Housing and Child Occupied...

    Science.gov (United States)

    2010-03-18

    ... Exposure Reduction. In the Federal Register dated April 22, 2008 (73 FR 21692), EPA promulgated final TSCA... are conducted in target housing and child-occupied facilities: 1. Establish the discipline of lead... AGENCY Lead-Based Paint Renovation, Repair and Painting Activities in Target Housing and Child...

  20. 75 FR 51808 - Lead-Based Paint Renovation, Repair and Painting Activities in Target Housing and Child Occupied...

    Science.gov (United States)

    2010-08-23

    ... April 22, 2008, (73 FR 21692), EPA promulgated final TSCA section 402(c)(3) regulations governing... in target housing and child-occupied facilities. These rules: 1. Establish the discipline of lead... AGENCY Lead-Based Paint Renovation, Repair and Painting Activities in Target Housing and Child...

  1. A soft self-repairing for FBG sensor network in SHM system based on PSO-SVR model reconstruction

    Science.gov (United States)

    Zhang, Xiaoli; Wang, Peng; Liang, Dakai; Fan, Chunfeng; Li, Cailing

    2015-05-01

    Structural health monitoring (SHM) system takes advantage of an array of sensors to continuously monitor a structure and provide an early prediction such as the damage position and damage degree etc. Such a system requires monitoring the structure in any conditions including bad condition. Therefore, it must be robust and survivable, even has the self-repairing ability. In this study, a model reconstruction predicting algorithm based on particle swarm optimization-support vector regression (PSO-SVR) is proposed to achieve the self-repairing of the Fiber Bragg Grating (FBG) sensor network in SHM system. Furthermore, an eight-point FBG sensor SHM system is experimented in an aircraft wing box. For the damage loading position prediction on the aircraft wing box, six kinds of disabled modes are experimentally studied to verify the self-repairing ability of the FBG sensor network in the SHM system, and the predicting performance are compared with non-reconstruction based on PSO-SVR model. The research results indicate that the model reconstruction algorithm has more excellence than that of non-reconstruction model, if partial sensors are invalid in the FBG-based SHM system, the predicting performance of the model reconstruction algorithm is almost consistent with that no sensor is invalid in the SHM system. In this way, the self-repairing ability of the FBG sensor is achieved for the SHM system, such the reliability and survivability of the FBG-based SHM system is enhanced if partial FBG sensors are invalid.

  2. Repair of gamma-ray-induced DNA base damage in xeroderma pigmentosum cells

    International Nuclear Information System (INIS)

    The repair of DNA damage produced by 137Cs gamma irradiation was measured with a preparation from Micrococcus luteus containing DNA damage-specific endonucleases in combination with alkaline elution. The frequency of these endonuclease sensitive sites (ESS) was determined after 54 or 110 Gy of oxic irradiation in normal and xeroderma pigmentosum (XP) fibroblasts from complementation groups A, C, D, and G. Repair was rapid in all cell strains with greater than 50% repair after 1.5 h of repair incubation. At later repair times, 12-17 h, more ESS remained in XP than in normal cells. The frequency of excess ESS in XP cells was approximately 0.04 per 10(9) Da of DNA per Gy which was equivalent to 10% of the initial ESS produced. The removal of ESS was comparable in XP cells with normal radiosensitivity and XP3BR cells which have been reported to be moderately radiosensitive

  3. p21CDKN1A Regulates the Binding of Poly(ADP-Ribose) Polymerase-1 to DNA Repair Intermediates

    OpenAIRE

    Dutto, Ilaria; Sukhanova, Maria; Tillhon, Micol; Cazzalini, Ornella; Stivala, Lucia A.; Scovassi, A. Ivana; Lavrik, Olga; Prosperi, Ennio

    2016-01-01

    The cell cycle inhibitor p21CDKN1A was previously found to interact directly with DNA nick-sensor poly(ADP-ribose) polymerase-1 (PARP-1) and to promote base excision repair (BER). However, the molecular mechanism responsible for this BER-related association of p21 with PARP-1 remains to be clarified. In this study we investigate the capability of p21 to influence PARP-1 binding to DNA repair intermediates in a reconstituted BER system in vitro. Using model photoreactive BER substrates contain...

  4. Surgical repair of the isolated incomplete median cleft lip.

    Science.gov (United States)

    Topkara, A; Özkan, A; Özcan, R H; Öksüz, M

    2016-02-01

    Median cleft lip refers to a vertical cleft on the midline of the upper lip. It is a rare congenital craniofacial anomaly brought about by a fusion failure in the medial nasal prominence. A novel surgical approach to median cleft lips and their repair is reported herein, with reference to a clinical case. The patient had a cleft in the lower half of the upper lip. There were no other craniofacial anomalies in this patient other than the cleft. Within the framework of the patient's surgical treatment, a functional and cosmetically satisfactory result was achieved by performing a V-Y advancement flap on the columella base, Z-plasty in the vermillion zone, and Z pattern muscular tissue repair, without having to resort to any tissue excisions. PMID:26364580

  5. Android Platform Based Smartphones for a Logistical Remote Association Repair Framework

    Directory of Open Access Journals (Sweden)

    Shao-Fan Lien

    2014-06-01

    Full Text Available The maintenance of large-scale systems is an important issue for logistics support planning. In this paper, we developed a Logistical Remote Association Repair Framework (LRARF to aid repairmen in keeping the system available. LRARF includes four subsystems: smart mobile phones, a Database Management System (DBMS, a Maintenance Support Center (MSC and wireless networks. The repairman uses smart mobile phones to capture QR-codes and the images of faulty circuit boards. The captured QR-codes and images are transmitted to the DBMS so the invalid modules can be recognized via the proposed algorithm. In this paper, the Linear Projective Transform (LPT is employed for fast QR-code calibration. Moreover, the ANFIS-based data mining system is used for module identification and searching automatically for the maintenance manual corresponding to the invalid modules. The inputs of the ANFIS-based data mining system are the QR-codes and image features; the output is the module ID. DBMS also transmits the maintenance manual back to the maintenance staff. If modules are not recognizable, the repairmen and center engineers can obtain the relevant information about the invalid modules through live video. The experimental results validate the applicability of the Android-based platform in the recognition of invalid modules. In addition, the live video can also be recorded synchronously on the MSC for later use.

  6. Low frequency of defective mismatch repair in a population-based series of upper urothelial carcinoma

    Directory of Open Access Journals (Sweden)

    Isfoss Björn L

    2005-03-01

    Full Text Available Abstract Background Upper urothelial cancer (UUC, i.e. transitional cell carcinomas of the renal pelvis and the ureter, occur at an increased frequency in patients with hereditary nonpolyposis colorectal cancer (HNPCC. Defective mismatch repair (MMR specifically characterizes HNPCC-associated tumors, but also occurs in subsets of some sporadic tumors, e.g. in gastrointestinal cancer and endometrial cancer. Methods We assessed the contribution of defective MMR to the development of UUC in a population-based series from the southern Swedish Cancer Registry, through microsatellite instability (MSI analysis and immunohistochemical evaluation of expression of the MMR proteins MLH1, PMS2, MSH2, and MSH6. Results A MSI-high phenotype was identified in 9/216 (4% successfully analyzed patients and a MSI-low phenotype in 5/216 (2%. Loss of MMR protein immunostaining was found in 11/216 (5% tumors, and affected most commonly MSH2 and MSH6. Conclusion This population-based series indicates that somatic MMR inactivation is a minor pathway in the development of UUC, but tumors that display defective MMR are, based on the immunohistochemical expression pattern, likely to be associated with HNPCC.

  7. Android platform based smartphones for a logistical remote association repair framework.

    Science.gov (United States)

    Lien, Shao-Fan; Wang, Chun-Chieh; Su, Juhng-Perng; Chen, Hong-Ming; Wu, Chein-Hsing

    2014-01-01

    The maintenance of large-scale systems is an important issue for logistics support planning. In this paper, we developed a Logistical Remote Association Repair Framework (LRARF) to aid repairmen in keeping the system available. LRARF includes four subsystems: smart mobile phones, a Database Management System (DBMS), a Maintenance Support Center (MSC) and wireless networks. The repairman uses smart mobile phones to capture QR-codes and the images of faulty circuit boards. The captured QR-codes and images are transmitted to the DBMS so the invalid modules can be recognized via the proposed algorithm. In this paper, the Linear Projective Transform (LPT) is employed for fast QR-code calibration. Moreover, the ANFIS-based data mining system is used for module identification and searching automatically for the maintenance manual corresponding to the invalid modules. The inputs of the ANFIS-based data mining system are the QR-codes and image features; the output is the module ID. DBMS also transmits the maintenance manual back to the maintenance staff. If modules are not recognizable, the repairmen and center engineers can obtain the relevant information about the invalid modules through live video. The experimental results validate the applicability of the Android-based platform in the recognition of invalid modules. In addition, the live video can also be recorded synchronously on the MSC for later use. PMID:24967603

  8. Local excision for selected colorectal carcinomas.

    Science.gov (United States)

    Lawrence, M A; Goldberg, S M

    1989-07-01

    In summary, local excision is a useful tool in the management of selected colorectal carcinomas. The advent of the fibreoptic colonoscope has revised the concept of local excision when dealing with carcinoma-containing polyps of the colon. The clinician now has the means of locally excising certain carcinomas which would have required laparotomy in the not so distant past. In dealing with carcinoma of the rectum, local excision is not advocated for all rectal carcinomas. In fact, when the previously discussed tumour related factors are considered, local excision should be the ultimate procedure in less than 5% of operations performed for rectal carcinomas. However, when appropriately used, local excision provides a less morbid alternative to more radical procedures without compromising patient survival rates or local recurrence rates. PMID:2692739

  9. PARP-1: Friend or Foe of DNA Damage and Repair in Tumorigenesis?

    Energy Technology Data Exchange (ETDEWEB)

    Swindall, Amanda F.; Stanley, Jennifer A. [Department of Radiation Oncology Comprehensive Cancer Center, University of Alabama at Birmingham School of Medicine, 176F HSROC Suite 2232B, 1700 6th Avenue South, Birmingham, AL 35249 (United States); Yang, Eddy S., E-mail: eyang@uab.edu [Department of Radiation Oncology Comprehensive Cancer Center, University of Alabama at Birmingham School of Medicine, 176F HSROC Suite 2232B, 1700 6th Avenue South, Birmingham, AL 35249 (United States); Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, AL 35249 (United States); Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, AL 35249 (United States)

    2013-07-26

    Oxidative stress induced by reactive oxygen species can result in DNA damage within cells and subsequently increase risk for carcinogenesis. This may be averted by repair of DNA damage through the base or nucleotide excision repair (BER/NER) pathways. PARP, a BER protein, is known for its role in DNA-repair. However, multiple lesions can occur within a small range of DNA, known as oxidative clustered DNA lesions (OCDLs), which are difficult to repair and may lead to the more severe DNA double-strand break (DSB). Inefficient DSB repair can then result in increased mutagenesis and neoplastic transformation. OCDLs occur more frequently within a variety of tumor tissues. Interestingly, PARP is highly expressed in several human cancers. Additionally, chronic inflammation may contribute to tumorigenesis through ROS-induced DNA damage. Furthermore, PARP can modulate inflammation through interaction with NFκB and regulating the expression of inflammatory signaling molecules. Thus, the upregulation of PARP may present a double-edged sword. PARP is needed to repair ROS-induced DNA lesions, but PARP expression may lead to increased inflammation via upregulation of NFκB signaling. Here, we discuss the role of PARP in the repair of oxidative damage versus the formation of OCDLs and speculate on the feasibility of PARP inhibition for the treatment and prevention of cancers by exploiting its role in inflammation.

  10. PARP-1 and Ku compete for repair of DNA double strand breaks by distinct NHEJ pathways

    Science.gov (United States)

    Wang, Minli; Wu, Weizhong; Wu, Wenqi; Rosidi, Bustanur; Zhang, Lihua; Wang, Huichen; Iliakis, George

    2006-01-01

    Poly(ADP-ribose)polymerase 1 (PARP-1) recognizes DNA strand interruptions in vivo and triggers its own modification as well as that of other proteins by the sequential addition of ADP-ribose to form polymers. This modification causes a release of PARP-1 from DNA ends and initiates a variety of responses including DNA repair. While PARP-1 has been firmly implicated in base excision and single strand break repair, its role in the repair of DNA double strand breaks (DSBs) remains unclear. Here, we show that PARP-1, probably together with DNA ligase III, operates in an alternative pathway of non-homologous end joining (NHEJ) that functions as backup to the classical pathway of NHEJ that utilizes DNA-PKcs, Ku, DNA ligase IV, XRCC4, XLF/Cernunnos and Artemis. PARP-1 binds to DNA ends in direct competition with Ku. However, in irradiated cells the higher affinity of Ku for DSBs and an excessive number of other forms of competing DNA lesions limit its contribution to DSB repair. When essential components of the classical pathway of NHEJ are absent, PARP-1 is recruited for DSB repair, particularly in the absence of Ku and non-DSB lesions. This form of DSB repair is sensitive to PARP-1 inhibitors. The results define the function of PARP-1 in DSB repair and characterize a candidate pathway responsible for joining errors causing genomic instability and cancer. PMID:17088286

  11. PARP-1 and Ku compete for repair of DNA double strand breaks by distinct NHEJ pathways.

    Science.gov (United States)

    Wang, Minli; Wu, Weizhong; Wu, Wenqi; Rosidi, Bustanur; Zhang, Lihua; Wang, Huichen; Iliakis, George

    2006-01-01

    Poly(ADP-ribose)polymerase 1 (PARP-1) recognizes DNA strand interruptions in vivo and triggers its own modification as well as that of other proteins by the sequential addition of ADP-ribose to form polymers. This modification causes a release of PARP-1 from DNA ends and initiates a variety of responses including DNA repair. While PARP-1 has been firmly implicated in base excision and single strand break repair, its role in the repair of DNA double strand breaks (DSBs) remains unclear. Here, we show that PARP-1, probably together with DNA ligase III, operates in an alternative pathway of non-homologous end joining (NHEJ) that functions as backup to the classical pathway of NHEJ that utilizes DNA-PKcs, Ku, DNA ligase IV, XRCC4, XLF/Cernunnos and Artemis. PARP-1 binds to DNA ends in direct competition with Ku. However, in irradiated cells the higher affinity of Ku for DSBs and an excessive number of other forms of competing DNA lesions limit its contribution to DSB repair. When essential components of the classical pathway of NHEJ are absent, PARP-1 is recruited for DSB repair, particularly in the absence of Ku and non-DSB lesions. This form of DSB repair is sensitive to PARP-1 inhibitors. The results define the function of PARP-1 in DSB repair and characterize a candidate pathway responsible for joining errors causing genomic instability and cancer. PMID:17088286

  12. PARP-1: Friend or Foe of DNA Damage and Repair in Tumorigenesis?

    International Nuclear Information System (INIS)

    Oxidative stress induced by reactive oxygen species can result in DNA damage within cells and subsequently increase risk for carcinogenesis. This may be averted by repair of DNA damage through the base or nucleotide excision repair (BER/NER) pathways. PARP, a BER protein, is known for its role in DNA-repair. However, multiple lesions can occur within a small range of DNA, known as oxidative clustered DNA lesions (OCDLs), which are difficult to repair and may lead to the more severe DNA double-strand break (DSB). Inefficient DSB repair can then result in increased mutagenesis and neoplastic transformation. OCDLs occur more frequently within a variety of tumor tissues. Interestingly, PARP is highly expressed in several human cancers. Additionally, chronic inflammation may contribute to tumorigenesis through ROS-induced DNA damage. Furthermore, PARP can modulate inflammation through interaction with NFκB and regulating the expression of inflammatory signaling molecules. Thus, the upregulation of PARP may present a double-edged sword. PARP is needed to repair ROS-induced DNA lesions, but PARP expression may lead to increased inflammation via upregulation of NFκB signaling. Here, we discuss the role of PARP in the repair of oxidative damage versus the formation of OCDLs and speculate on the feasibility of PARP inhibition for the treatment and prevention of cancers by exploiting its role in inflammation

  13. PARP-1: Friend or Foe of DNA Damage and Repair in Tumorigenesis?

    Directory of Open Access Journals (Sweden)

    Eddy S. Yang

    2013-07-01

    Full Text Available Oxidative stress induced by reactive oxygen species can result in DNA damage within cells and subsequently increase risk for carcinogenesis. This may be averted by repair of DNA damage through the base or nucleotide excision repair (BER/NER pathways. PARP, a BER protein, is known for its role in DNA-repair. However, multiple lesions can occur within a small range of DNA, known as oxidative clustered DNA lesions (OCDLs, which are difficult to repair and may lead to the more severe DNA double-strand break (DSB. Inefficient DSB repair can then result in increased mutagenesis and neoplastic transformation. OCDLs occur more frequently within a variety of tumor tissues. Interestingly, PARP is highly expressed in several human cancers. Additionally, chronic inflammation may contribute to tumorigenesis through ROS-induced DNA damage. Furthermore, PARP can modulate inflammation through interaction with NFκB and regulating the expression of inflammatory signaling molecules. Thus, the upregulation of PARP may present a double-edged sword. PARP is needed to repair ROS-induced DNA lesions, but PARP expression may lead to increased inflammation via upregulation of NFκB signaling. Here, we discuss the role of PARP in the repair of oxidative damage versus the formation of OCDLs and speculate on the feasibility of PARP inhibition for the treatment and prevention of cancers by exploiting its role in inflammation.

  14. 顺势疗法药物山金车30C通过上调核苷酸切除修复基因的表达减少紫外线照射后大肠杆菌的DNA损伤%Potential of the homeopathic remedy, Arnica Montana 30C,to reduce DNA damage in Escherichia coli exposed to ultraviolet irradiation through up-regulation of nucleotide excision repair genes

    Institute of Scientific and Technical Information of China (English)

    Sreemanti Das; Santu Kumar Saha; Arnab De; Durba Das; Anisur Rahman Khuda-Bukhs

    2012-01-01

    目的:检测高度稀释的顺势疗法药物山金车30C是否能够调节暴露于紫外线照射下的大肠杆菌的核苷酸切除修复基因的表达.方法:大肠杆菌在标准培养基中培养至对数阶段,然后接受亚致死剂量的紫外线照射(25和50 J/m2分别照射22.5和45 s).接受不同剂量紫外线照射的大肠杆菌分别与山金车30C及安慰剂30C共同培养,90 min后检测其DNA损伤情况及氧化应激状态.采用多种方法及指标如彗星实验、梯度凝胶电泳、细胞内活性氧生成及测量其他生物活性指标如过氧化物歧化酶、过氧化氢酶及谷胱甘肽衡量DNA损伤情况及细胞氧化应激状态.逆转录聚合酶链反应检测大肠杆菌细胞紫外线损伤修复基因uvrA、B、C(核苷酸切除修复基因)mRNA的表达情况.结果:接受照射后的大肠杆菌出现了DNA损伤及氧化应激反应,表现为细胞内活性氧生成增加及过氧化物歧化酶、过氧化氢酶和谷胱甘肽活性降低.与安慰剂组相比,山金车30C降低了大肠杆菌的DNA损伤及氧化应激反应,表现为细胞内活性氧生成减少及过氧化物歧化酶、过氧化氢酶和谷胱甘肽活性增强.与对照组相比,山金车30C上调了大肠杆菌细胞紫外线损伤修复基因的表达.结论:山金车30C能够通过上调紫外线损伤修复基因的表达修复紫外线引起的大肠杆菌细胞的DNA损伤,并通过减少细胞内活性氧的生成及调节抗氧化酶活性降低细胞的氧化应激反应.%OBJECTIVE:To examine to what degree an ultra-highly diluted homeopathic remedy,Arnica Montana 30C (AM-30C),used in the treatment of shock and injury,can modulate the expression of nucleotide excision repair genes in Escherichia coii exposed to ultraviolet (UV) irradiation.METHODS:E.coli were cultured to their log phase in a standard Luria-Bertani medium and then exposed to sublethal doses of UV irradiation at 25 and 50 J/m2 for 22.5 and 45 s,respectively.The UV

  15. Developing a Model for Optimizing Inventory of Repairable Items at Single Operating Base

    OpenAIRE

    Le, Tin

    2016-01-01

    The use of EOQ model in inventory management is popular. However, EOQ models has many disadvantages, especially, when the model is applied to manage repairable items. In order to deal with high-cost and repairable items, Craig C. Sherbrooke introduced a model in his book “Optimal Inventory Modeling of Systems: Multi-Echelon Techniques”. The research focus is to implement and develop a program to execute the single-site in-ventory model for repairable items. The model helps to significantl...

  16. Repair of Ischemic Injury by Pluripotent Stem Cell Based Cell Therapy without Teratoma through Selective Photosensitivity

    Directory of Open Access Journals (Sweden)

    Seung-Ju Cho

    2015-12-01

    Full Text Available Stem-toxic small molecules have been developed to induce selective cell death of pluripotent stem cells (PSCs to lower the risk of teratoma formation. However, despite their high efficacies, chemical-based approaches may carry unexpected toxicities on specific differentiated cell types. Herein, we took advantage of KillerRed (KR as a suicide gene, to selectively induce phototoxicity using visible light via the production of reactive oxygen species. PSCs in an undifferentiated state that exclusively expressed KR (KR-PSCs were eliminated by a single exposure to visible light. This highly selective cell death in KR-PSCs was exploited to successfully inhibit teratoma formation. In particular, endothelial cells from KR-mPSCs remained fully functional in vitro and sufficient to repair ischemic injury in vivo regardless of light exposure, suggesting that a genetic approach in which KR is expressed in a tightly controlled manner would be a viable strategy to inhibit teratoma formation for future safe PSC-based therapies.

  17. Repair pathways in mammalian cells: Incision-ligation kinetics after UV, x-rays and mutagenic chemicals. Effects of 3-aminobenzamide and hydroxyurea + cytosine arabinoside

    International Nuclear Information System (INIS)

    Repair of DNA-damage involves the combined action of several enzyme functions. With regard to the incision step different pathways exist. In nucleotide excision repair an endonuclease recognizes the lesion, cuts the DNA strand and the faulty nucleotide is excised together with a number of undamaged nucleotides. This seems to be the process which operates in mammalian cells after exposure to UV and a number of chemicals which introduce so called bulky lesions in the DNA. After exposure to X-rays and simple alkylating chemicals such as methyl methanesulfonate. The first step involves the action of a glycosylase which only removes the damaged base. The incision is exercised by an endonuclease which recognises abasic sites. In this process, i.e. base excision repair, probably only the damaged nucleotide is exchanged. When the damaged piece of DNA has been removed one would imagine that, independently of the type of lesion initially present, the same types of enzymes could be used to close the gap in the strand, this seems, however, not to be the case. In this investigation the authors used 3-aminobenzamide (BA) or hydroxy urea (HU) combined with cytosine arabinoside (ara C) to study repair of damage induced by UV and a number of mutagenic chemicals. DNA strand breaks have been measured by the DNA-unwinding technique

  18. Biomarkers of oxidative damage to DNA and repair.

    Science.gov (United States)

    Loft, Steffen; Høgh Danielsen, Pernille; Mikkelsen, Lone; Risom, Lotte; Forchhammer, Lykke; Møller, Peter

    2008-10-01

    Oxidative-stress-induced damage to DNA includes a multitude of lesions, many of which are mutagenic and have multiple roles in cancer and aging. Many lesions have been characterized by MS-based methods after extraction and digestion of DNA. These preparation steps may cause spurious base oxidation, which is less likely to occur with methods such as the comet assay, which are based on nicking of the DNA strand at modified bases, but offer less specificity. The European Standards Committee on Oxidative DNA Damage has concluded that the true levels of the most widely studied lesion, 8-oxodG (8-oxo-7,8-dihydro-2'-deoxyguanosine), in cellular DNA is between 0.5 and 5 lesions per 10(6) dG bases. Base excision repair of oxidative damage to DNA can be assessed by nicking assays based on oligonucleotides with lesions or the comet assay, by mRNA expression levels or, in the case of, e.g., OGG1 (8-oxoguanine DNA glycosylase 1), responsible for repair of 8-oxodG, by genotyping. Products of repair in DNA or the nucleotide pool, such as 8-oxodG, excreted into the urine can be assessed by MS-based methods and generally reflects the rate of damage. Experimental and population-based studies indicate that many environmental factors, including particulate air pollution, cause oxidative damage to DNA, whereas diets rich in fruit and vegetables or antioxidant supplements may reduce the levels and enhance repair. Urinary excretion of 8-oxodG, genotype and expression of OGG1 have been associated with risk of cancer in cohort settings, whereas altered levels of damage, repair or urinary excretion in case-control settings may be a consequence rather than the cause of the disease. PMID:18793191

  19. Distally Based Dorsal Digital Fasciocutaneous Flap for the Repair of Digital Terminal Amputation Defects

    OpenAIRE

    Hou, Ruixing; Ju, Jihui; Zhao, Qiang; Liu, Yuefei

    2012-01-01

    The preferred plastic surgery regimen for distal digital segment wounds remains unknown, although multiple options are available for the repair. The purpose of this investigation is to study its anatomic rationale and clinical outcomes, in addition to the role of dorsal digital veins in digital reconstruction. Patients (n  =  765) suffering from digital terminal segment traumatic wounds (823 digits) were identified and reviewed in a retrospective manner. The wounds were repaired using distall...

  20. Real-time observation of DNA repair: 2-aminopurine as a molecular probe

    Science.gov (United States)

    Krishnan, Rajagopal; Butcher, Christina E.; Oh, Dennis H.

    2008-02-01

    Triplex forming oligos (TFOs) that target psoralen photoadducts to specific DNA sequences have generated interest as a potential agent in gene therapy. TFOs also offer an opportunity to study the mechanism of DNA repair in detail. In an effort to understand the mechanism of DNA repair at a specific DNA sequence in real-time, we have designed a plasmid containing a psoralen reaction site adjacent to a TFO binding site corresponding to a sequence within the human interstitial collagenase gene. Two 2-aminopurine residues incorporated into the purine-rich strand of the TFO binding site and located within six nucleotides of the psoralen reaction site serve as molecular probes for excision repair events involving the psoralen photoadducts on that DNA strand. In duplex DNA, the 2-aminopurine fluorescence is quenched. However, upon thermal or formamide-induced denaturation of duplex DNA to single stranded DNA, the 2-aminopurine fluorescence increases by eight fold. These results suggest that monitoring 2-aminopurine fluorescence from plasmids damaged by psoralen TFOs may be a method for measuring excision of single-stranded damaged DNA from the plasmid in cells. A fluorescence-based molecular probe to the plasmid may significantly simplify the real-time observation of DNA repair in both populations of cells as well as single cells.

  1. Role of polynucleotide kinase/phosphatase in mitochondrial DNA repair

    Science.gov (United States)

    Tahbaz, Nasser; Subedi, Sudip; Weinfeld, Michael

    2012-01-01

    Mutations in mitochondrial DNA (mtDNA) are implicated in a broad range of human diseases and in aging. Compared to nuclear DNA, mtDNA is more highly exposed to oxidative damage due to its proximity to the respiratory chain and the lack of protection afforded by chromatin-associated proteins. While repair of oxidative damage to the bases in mtDNA through the base excision repair pathway has been well studied, the repair of oxidatively induced strand breaks in mtDNA has been less thoroughly examined. Polynucleotide kinase/phosphatase (PNKP) processes strand-break termini to render them chemically compatible for the subsequent action of DNA polymerases and ligases. Here, we demonstrate that functionally active full-length PNKP is present in mitochondria as well as nuclei. Downregulation of PNKP results in an accumulation of strand breaks in mtDNA of hydrogen peroxide-treated cells. Full restoration of repair of the H2O2-induced strand breaks in mitochondria requires both the kinase and phosphatase activities of PNKP. We also demonstrate that PNKP contains a mitochondrial-targeting signal close to the C-terminus of the protein. We further show that PNKP associates with the mitochondrial protein mitofilin. Interaction with mitofilin may serve to translocate PNKP into mitochondria. PMID:22210862

  2. The inhibition of DNA repair by aphidicolin or cytosine arabinoside in X-irradiated normal and xeroderma pigmentosum fibroblasts

    International Nuclear Information System (INIS)

    Normal and excision-deficient xeroderma pigmentosum fibroblasts were X-irradiated and the influence on DNA repair of either the repair inhibitor cytosine arabinoside or the specific inhibitor of DNA polymerase α, aphidicolin, investigated. The data indicated that the repair of a certain fraction of X-ray-induced lesions can be inhibited in both cell lines by both compounds. Thus, as aphidicolin blocks the operation of polymerase α, this enzyme must be involved in an excision repair pathway operating in both normal and excision-deficient xeroderma pigmentosum cells. (orig.)

  3. Inhibition of DNA repair by aphidicolin or cytosine arabinoside in X-irradiated normal and xeroderma pigmentosum fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Waters, R.; Crocombe, K.; Mirzayans, R. (University Coll. of Swansea (UK). Dept. of Genetics)

    1981-01-01

    Normal and excision-deficient xeroderma pigmentosum fibroblasts were X-irradiated and the influence on DNA repair of either the repair inhibitor cytosine arabinoside or the specific inhibitor of DNA polymerase ..cap alpha.., aphidicolin, investigated. The data indicated that the repair of a certain fraction of X-ray-induced lesions can be inhibited in both cell lines by both compounds. Thus, as aphidicolin blocks the operation of polymerase ..cap alpha.., this enzyme must be involved in an excision repair pathway operating in both normal and excision-deficient xeroderma pigmentosum cells.

  4. The inhibition of DNA repair by aphidicolin or cytosine arabinoside in X-irradiated normal and xeroderma pigmentosum fibroblasts.

    Science.gov (United States)

    Waters, R; Crocombe, K; Mirzayans, R

    1982-05-01

    Normal and excision-deficient xeroderma pigmentosum fibroblasts were X-irradiated and the influence on DNA repair of either the repair inhibitor cytosine arabinoside or the specific inhibitor of Dna polymerase alpha, aphidicolin, investigated. The data indicated that the repair of a certain fraction of X-ray-induced lesions can be inhibited in both cell lines by both compounds. Thus, as aphidicolin blocks the operation of polymerase alpha, this enzyme must be involved in an excision repair pathway operating in both normal and excision-deficient xeroderma pigmentosum cells. PMID:6808389

  5. Tissue Engineering for Rotator Cuff Repair: An Evidence-Based Systematic Review

    Directory of Open Access Journals (Sweden)

    Nicola Maffulli

    2012-01-01

    Full Text Available The purpose of this systematic review was to address the treatment of rotator cuff tears by applying tissue engineering approaches to improve tendon healing, specifically platelet rich plasma (PRP augmentation, stem cells, and scaffolds. Our systematic search was performed using the combination of the following terms: “rotator cuff”, “shoulder”, “PRP”, “platelet rich plasma”, “stemcells”, “scaffold”, “growth factors”, and “tissue engineering”. No level I or II studies were found on the use of scaffolds and stem cells for rotator cuff repair. Three studies compared rotator cuff repair with or without PRP augmentation. All authors performed arthroscopic rotator cuff repair with different techniques of suture anchor fixation and different PRP augmentation. The three studies found no difference in clinical rating scales and functional outcomes between PRP and control groups. Only one study showed clinical statistically significant difference between the two groups at the 3-month followup. Any statistically significant difference in the rates of tendon rerupture between the control group and the PRP group was found using the magnetic resonance imaging. The current literature on tissue engineering application for rotator cuff repair is scanty. Comparative studies included in this review suggest that PRP augmented repair of a rotator cuff does not yield improved functional and clinical outcome compared with non-augmented repair at a medium and long-term followup.

  6. DNA repair processes and the shape of dose-response curves for induced genetic changes in yeast

    International Nuclear Information System (INIS)

    Gene conversion is a genetic change which requires a functional recombinational repair system. Dose-response curves for induced gene conversion in D7 strains of diploid S. cerevisiae appear to be one-hit (effect proportional to dose) for ionizing radiation or mitomycin C, but are closer to two-hit characteristics (effect proportional to square of dose) for 254 nm UV radiation or nitrous acid at sublethal doses of these agents. These data are suggest that recombinational repair in normal cells is stimulated by coincident or double-strand damage in the DNA: for example, a double-strand break produced by ionizing radiation, an interstrand cross-link produced by mitomycin C, or a double strand break produced by ''overlapping'' excision repair of two pyrimidine dimers located on opposite DNA strands within about 100 base pairs of each other. Other initiating mechanisms for UV-induced conversion may predominate when the excision repair pathway is not available (as in rad 3 mutants). It is of interest that the shapes of the dose-response curves for back mutations affecting the iIvl locus in repair - proficient D7 strains are similar to those observed for gene conversions induced by γ-rays, UV light and nitrous acid, even though the relative yields of convertants and back mutations vary considerably from one agent to another

  7. PLGA-based microcarriers induce mesenchymal stem cell chondrogenesis and stimulate cartilage repair in osteoarthritis.

    Science.gov (United States)

    Morille, Marie; Toupet, Karine; Montero-Menei, Claudia N; Jorgensen, Christian; Noël, Danièle

    2016-05-01

    In the present study, we aimed at evaluating the ability of novel PLGA-P188-PLGA-based microspheres to induce the differentiation of mesenchymal stem/stromal cells (MSC) into chondrocytes. To this aim, we tested microspheres releasing TGFβ3 (PAM-T) in vitro and in situ, in a pathological osteoarthritic (OA) environment. We first evaluated the chondrogenic differentiation of human MSCs seeded onto PAM-T in vitro and confirmed the up-regulation of chondrogenic markers while the secretome of the cells was not changed by the 3D environment. We then injected human MSC seeded onto PAM-T in the knee joints of mice with collagenase-induced OA. After 6 weeks, histological analysis revealed that formation of a cartilage-like tissue occurred at the vicinity of PAM-T that was not observed when MSCs were seeded onto PAM. We also noticed that the endogenous articular cartilage was less degraded. The extent of cartilage protection was further analysed by confocal laser microscopy. When MSCs seeded onto PAM-T were injected early after OA induction, protection of cartilage against degradation was evidenced and this effect was associated to a higher survival of MSCs in presence of TGFβ3. This study points to the interest of using MSCs seeded onto PAM for cartilage repair and stimulation of endogenous cartilage regeneration. PMID:26945456

  8. AN A ESTHETIC MANAGEMENT OF A CASE OF XERODERMA PIGMENTOSUM WITH SQUAMOUS CELL CARCINOMA POSTED FOR WIDE EXCISION AND GRAFTING PRE SENTING WITH A DIFFICULT AIRWAY

    OpenAIRE

    Shahedha; Santhisree; Krishna Prasad; Sowbaghya Lakshmi

    2015-01-01

    BACKGROUND : Xeroderma Pigmentosum (XP) is a rare autosomal recessive disorder characterized by hypersensitivity of the skin to UV radiation. These patients show a failure to repair UV induced DNA lesions caused by Nucleotide Excision Repair mechanism. They develop neoplasms at an early age and require repeated surgeries. METHODOLOGY : We rep...

  9. Functional capacity of XRCC1 protein variants identified in DNA repair-deficient Chinese hamster ovary cell lines and the human population

    DEFF Research Database (Denmark)

    Berquist, Brian R; Singh, Dharmendra Kumar; Fan, Jinshui;

    2010-01-01

    XRCC1 operates as a scaffold protein in base excision repair, a pathway that copes with base and sugar damage in DNA. Studies using recombinant XRCC1 proteins revealed that: a C389Y substitution, responsible for the repair defects of the EM-C11 CHO cell line, caused protein instability; a V86R...... mutation abolished the interaction with POLbeta, but did not disrupt the interactions with PARP-1, LIG3alpha and PCNA; and an E98K substitution, identified in EM-C12, reduced protein integrity, marginally destabilized the POLbeta interaction, and slightly enhanced DNA binding. Two rare (P161L and Y576S...

  10. Analysis of resynthesis tracts in repaired Escherichia coli deoxyribonucleic acid

    International Nuclear Information System (INIS)

    Excision repair of ultraviolet radiation-induced damage in a wild-type strain of Escherichia coli has been examined, using two methods for characterizing the resynthesis step of the repair process. Comparison of data obtained after both isopycnic analysis of repaired deoxyribonucleic acid and sedimentation velocity analysis of deoxyribonucleic acid after selective photolysis of bromouracil-containing repaired regions has shown that the repaired deoxyribonucleic acid molecules contain a semicontinuous distribution of sizes of repair tracts. Further analysis of our data suggests two major classes of repair patches, one about 20 to 40 nucleotides in length, and the other containing 1600 to 2000 nucleotides. Under the conditions employed, approximately 2 to 10% of the fully repaired regions are long repair patches

  11. Functional evaluation of DNA repair in human biopsies and their relation to other cellular biomarkers

    Directory of Open Access Journals (Sweden)

    Jana eSlyskova

    2014-05-01

    Full Text Available Thousands of DNA lesions are estimated to occur in each cell every day and almost all are recognized and repaired. DNA repair is an essential system that prevents accumulation of mutations which can lead to serious cellular malfunctions. Phenotypic evaluation of DNA repair activity of individuals is a relatively new approach. Methods to assess base and nucleotide excision repair pathways (BER and NER in peripheral blood cells based on modified comet assay protocols have been widely applied in human epidemiological studies. These provided some interesting observations of individual DNA repair activity being suppressed among cancer patients. However, extension of these results to cancer target tissues requires a different approach. Here we describe the evaluation of BER and NER activities in extracts from deep-frozen colon biopsies using an upgraded version of the in vitro comet-based DNA repair assay in which twelve reactions on one microscope slide can be performed. The aim of this report is to provide a detailed, easy-to-follow protocol together with results of optimization experiments. Additionally, results obtained by functional assays were analysed in the context of other cellular biomarkers, namely single nucleotide polymorphisms and gene expressions. We have shown that measuring DNA repair activity is not easily replaceable by genomic or transcriptomic approaches, but should be applied with the latter techniques in a complementary manner. The ability to measure DNA repair directly in cancer target tissues might finally answer questions about the tissue-specificity of DNA repair processes and their real involvement in the process of carcinogenesis.

  12. THE EFFECTS OF STRESS ON DNA REPAIR CAPACITY

    OpenAIRE

    Forlenza, Michael J; Latimer, Jean J.; Baum, Andrew

    2000-01-01

    Research has shown that lymphocytes of high-distress patients have reduced DNA repair relative to that of low-distress patients and healthy controls. Furthermore, deficits in repair are associated with an increased risk of cancer. Using and academic stress model, we hypothesized that students would exhibit lower levels of Nucleotide Excision Repair (NER) during a stressful exam period when compared to a lower stress period. Participants were 19 healthy graduate level students. NER was measure...

  13. 27 CFR 26.30 - Excise taxes.

    Science.gov (United States)

    2010-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Excise taxes. 26.30 Section 26.30 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT... Into the United States From Areas Other Than Puerto Rico and the Virgin Islands § 26.30 Excise...

  14. 27 CFR 70.412 - Excise taxes.

    Science.gov (United States)

    2010-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 2 2010-04-01 2010-04-01 false Excise taxes. 70.412 Section 70.412 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT... Beer § 70.412 Excise taxes. (a) Collection. Taxes on distilled spirits, wines, and beer are paid...

  15. 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.

  16. 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

    International Nuclear Information System (INIS)

    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

  17. Repair of damaged DNA in vivo: Final technical report

    International Nuclear Information System (INIS)

    This contract was initiated in 1962 with the US Atomic Energy Commission to carry out basic research on the effects of radiation on the process of DNA replication in bacteria. Within the first contract year we discovered repair replication at the same time that Setlow and Carrier discovered pyrimidine dimer excision. These discoveries led to the elucidation of the process of excision-repair, one of the most important mechanisms by which living systems, including humans, respond to structural damage in their genetic material. We improved methodology for distinguishing repair replication from semiconservative replication and instructed others in these techniques. Painter then was the first to demonstrate repair replication in ultraviolet irradiated human cells. He, in turn, instructed James Cleaver who discovered that skin fibroblasts from patients with xeroderma pigmentosum were defective in excision-repair. People with this genetic defect are extremely sensitive to sunlight and they develop carcinomas and melanomas of the skin with high frequency. The existence of this hereditary disease attests to the importance of DNA repair in man. We certainly could not survive in the normal ultraviolet flux from the sun if our DNA were not continuously monitored for damage and repaired. Other hereditary diseases such as ataxia telangiectasia, Cockayne's syndrome, Blooms syndrome and Fanconi's anemia also involve deficiencies in DNA damage processing. The field of DNA repair has developed rapidly as we have learned that most environmental chemical carcinogens as well as radiation produce repairable damage in DNA. 251 refs

  18. Concise review: Insights from normal bone remodeling and stem cell-based therapies for bone repair.

    Science.gov (United States)

    Khosla, Sundeep; Westendorf, Jennifer J; Mödder, Ulrike I

    2010-12-01

    There is growing interest in the use of mesenchymal stem cells for bone repair. As a major reason for normal bone remodeling is the removal of fatigue microcracks, advances in our understanding of this process may inform approaches to enhance fracture healing. Increasing evidence now indicates that physiological bone remodeling occurs in close proximity to blood vessels and that these vessels carry perivascular stem cells that differentiate into osteoblasts. Similarly, fracture healing is critically dependent on the ingrowth of blood vessels not only for a nutrient supply but also for the influx of osteoblasts. A number of animal and human studies have now shown the potential benefit of bone marrow-derived mesenchymal stem cells in enhancing bone repair. However, as in other tissues, the question of whether these cells improve fracture healing directly by differentiating into osteoblasts or indirectly by secreting paracrine factors that recruit blood vessels and the accompanying perivascular stem cells remains a major unresolved issue. Moreover, CD34+ cells, which are enriched for endothelial/hematopoietic cells, have also shown efficacy in various bone repair models, at least in part due to the induction of angiogenesis and recruitment of host progenitor cells. Thus, mesenchymal and nonmesenchymal stem/progenitor cells are attractive options for bone repair. It is possible that they contribute directly to bone repair, but it is also likely that they express paracrine factors in the appropriate amounts and combinations that promote and sustain the healing process. PMID:20960512

  19. An age-based replacement model for repairable systems with attention to high-speed marine diesel engines

    OpenAIRE

    Harlem, Alf

    2008-01-01

    The background of this thesis is the high number of severe failures of high-speed propulsion engines in high-speed vessels. When number of failures is high, the preventive maintenance comes into particular focus. Hence, this study will consider the interval (age), at which to carry out scheduled engine replacement or renewal by overhaul.The main objective shall be to develop a new age-based replacement model for repairable systems that combines system reliability theory with observed system f...

  20. Risk-based reliability allocation at component level in non-repairable systems by using evolutionary algorithm

    OpenAIRE

    Hussain, Syed Adeel

    2007-01-01

    The approach for setting system reliability in the risk-based reliability allocation (RBRA) method is driven solely by the amount of ‘total losses’ (sum of reliability investment and risk of failure) associated with a non-repairable system failure. For a system consisting of many components, reliability allocation by RBRA method becomes a very complex combinatorial optimisation problem particularly if large numbers of alternatives, with different levels of reliability and assoc...

  1. Distribution of DNA repair-related ESTs in sugarcane

    Directory of Open Access Journals (Sweden)

    W.C. Lima

    2001-12-01

    Full Text Available DNA repair pathways are necessary to maintain the proper genomic stability and ensure the survival of the organism, protecting it against the damaging effects of endogenous and exogenous agents. In this work, we made an analysis of the expression patterns of DNA repair-related genes in sugarcane, by determining the EST (expressed sequence tags distribution in the different cDNA libraries of the SUCEST transcriptome project. Three different pathways - photoreactivation, base excision repair and nucleotide excision repair - were investigated by employing known DNA repair proteins as probes to identify homologous ESTs in sugarcane, by means of computer similarity search. The results showed that DNA repair genes may have differential expressions in tissues, depending on the pathway studied. These in silico data provide important clues on the potential variation of gene expression, to be confirmed by direct biochemical analysis.As vias de reparo de DNA são requeridas para manter a necessária estabilidade genômica e garantir a sobrevivência do organismo, frente aos efeitos deletérios causados por fatores endógenos e exógenos. Neste trabalho, realizamos a análise dos padrões de expressão dos genes de reparo de DNA encontrados na cana-de-açúcar, pela determinação da distribuição de ESTs nas diferentes bibliotecas de cDNA no projeto de transcriptoma SUCEST. Três vias de reparo - fotorreativação, reparo por excisão de bases e reparo por excisão de nucleotídeos - foram estudadas através do uso de proteínas de reparo como sondas para identificação de ESTs homólogos em cana-de-açúcar, com base na procura computacional de similaridade. Os resultados indicam que os genes de reparo de DNA possuem uma expressão diferencial nos tecidos, dependendo da via de reparo analisada. Esses dados in silico fornecem importantes indícios da expressão diferencial, a qual deve ser confirmada por análises bioquímicas diretas.

  2. An unprecedented nucleic acid capture mechanism for excision of DNA damage

    Energy Technology Data Exchange (ETDEWEB)

    Rubinson, Emily H.; Prakasha Gowda, A.S.; Spratt, Thomas E.; Gold, Barry; Eichmanbrand, Brandt F. (Pitt); (Vanderbilt); (Penn)

    2010-11-18

    DNA glycosylases that remove alkylated and deaminated purine nucleobases are essential DNA repair enzymes that protect the genome, and at the same time confound cancer alkylation therapy, by excising cytotoxic N3-methyladenine bases formed by DNA-targeting anticancer compounds. The basis for glycosylase specificity towards N3- and N7-alkylpurines is believed to result from intrinsic instability of the modified bases and not from direct enzyme functional group chemistry. Here we present crystal structures of the recently discovered Bacillus cereus AlkD glycosylase in complex with DNAs containing alkylated, mismatched and abasic nucleotides. Unlike other glycosylases, AlkD captures the extrahelical lesion in a solvent-exposed orientation, providing an illustration for how hydrolysis of N3- and N7-alkylated bases may be facilitated by increased lifetime out of the DNA helix. The structures and supporting biochemical analysis of base flipping and catalysis reveal how the HEAT repeats of AlkD distort the DNA backbone to detect non-Watson-Crick base pairs without duplex intercalation.

  3. POLD1: Central mediator of DNA replication and repair, and implication in cancer and other pathologies.

    Science.gov (United States)

    Nicolas, Emmanuelle; Golemis, Erica A; Arora, Sanjeevani

    2016-09-15

    The evolutionarily conserved human polymerase delta (POLD1) gene encodes the large p125 subunit which provides the essential catalytic activities of polymerase δ (Polδ), mediated by 5'-3' DNA polymerase and 3'-5' exonuclease moieties. POLD1 associates with three smaller subunits (POLD2, POLD3, POLD4), which together with Replication Factor C and Proliferating Nuclear Cell Antigen constitute the polymerase holoenzyme. Polδ function is essential for replication, with a primary role as the replicase for the lagging strand. Polδ also has an important proofreading ability conferred by the exonuclease activity, which is critical for ensuring replicative fidelity, but also serves to repair DNA lesions arising as a result of exposure to mutagens. Polδ has been shown to be important for multiple forms of DNA repair, including nucleotide excision repair, double strand break repair, base excision repair, and mismatch repair. A growing number of studies in the past decade have linked germline and sporadic mutations in POLD1 and the other subunits of Polδ with human pathologies. Mutations in Polδ in mice and humans lead to genomic instability, mutator phenotype and tumorigenesis. The advent of genome sequencing techniques has identified damaging mutations in the proofreading domain of POLD1 as the underlying cause of some inherited cancers, and suggested that mutations in POLD1 may influence therapeutic management. In addition, mutations in POLD1 have been identified in the developmental disorders of mandibular hypoplasia, deafness, progeroid features and lipodystrophy and atypical Werner syndrome, while changes in expression or activity of POLD1 have been linked to senescence and aging. Intriguingly, some recent evidence suggests that POLD1 function may also be altered in diabetes. We provide an overview of critical Polδ activities in the context of these pathologic conditions. PMID:27320729

  4. Photodynamic DNA damage induced by phycocyanin and its repair in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    M. Pádula

    1999-09-01

    Full Text Available In the present study, we analyzed DNA damage induced by phycocyanin (PHY in the presence of visible light (VL using a set of repair endonucleases purified from Escherichia coli. We demonstrated that the profile of DNA damage induced by PHY is clearly different from that induced by molecules that exert deleterious effects on DNA involving solely singlet oxygen as reactive species. Most of PHY-induced lesions are single strand breaks and, to a lesser extent, base oxidized sites, which are recognized by Nth, Nfo and Fpg enzymes. High pressure liquid chromatography coupled to electrochemical detection revealed that PHY photosensitization did not induce 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo at detectable levels. DNA repair after PHY photosensitization was also investigated. Plasmid DNA damaged by PHY photosensitization was used to transform a series of Saccharomyces cerevisiae DNA repair mutants. The results revealed that plasmid survival was greatly reduced in rad14 mutants, while the ogg1 mutation did not modify the plasmid survival when compared to that in the wild type. Furthermore, plasmid survival in the ogg1 rad14 double mutant was not different from that in the rad14 single mutant. The results reported here indicate that lethal lesions induced by PHY plus VL are repaired differently by prokaryotic and eukaryotic cells. Morever, nucleotide excision repair seems to play a major role in the recognition and repair of these lesions in Saccharomyces cerevisiae.

  5. Simulation-based Optimization of a Real-world Travelling Salesman Problem Using an Evolutionary Algorithm with a Repair Function

    Directory of Open Access Journals (Sweden)

    Anna Syberfeldt

    2015-10-01

    Full Text Available This paper presents a real-world case study of optimizing waste collection in Sweden. The problem, involving approximately 17,000 garbage bins served by three bin lorries, is approached as a travelling salesman problem and solved using simulation-based optimization and an evolutionary algorithm. To improve the performance of the evolutionary algorithm, it is enhanced with a repair function that adjusts its genome values so that shorter routes are found more quickly. The algorithm is tested using two crossover operators, i.e., the order crossover and heuristic crossover, combined with different mutation rates. The results indicate that the order crossover is superior to the heuristics crossover, but that the driving force of the search process is the mutation operator combined with the repair function.

  6. Computer-based assessment of right ventricular regional ejection fraction in patients with repaired Tetralogy of Fallot

    Science.gov (United States)

    Teo, S.-K.; Wong, S. T.; Tan, M. L.; Su, Y.; Zhong, L.; Tan, Ru-San

    2015-03-01

    After surgical repair for Tetralogy of Fallot (TOF), most patients experience long-term complications as the right ventricle (RV) undergoes progressive remodeling that eventually affect heart functions. Thus, post-repair surgery is required to prevent further deterioration of RV functions that may result in malignant ventricular arrhythmias and mortality. The timing of such post-repair surgery therefore depends crucially on the quantitative assessment of the RV functions. Current clinical indices for such functional assessment measure global properties such as RV volumes and ejection fraction. However, these indices are less than ideal as regional variations and anomalies are obscured. Therefore, we sought to (i) develop a quantitative method to assess RV regional function using regional ejection fraction (REF) based on a 13-segment model, and (ii) evaluate the effectiveness of REF in discriminating 6 repaired TOF patients and 6 normal control based on cardiac magnetic resonance (CMR) imaging. We observed that the REF for the individual segments in the patient group is significantly lower compared to the control group (P < 0.05 using a 2-tail student t-test). In addition, we also observed that the aggregated REF at the basal, mid-cavity and apical regions for the patient group is significantly lower compared to the control group (P < 0.001 using a 2-tail student t-test). The results suggest that REF could potentially be used as a quantitative index for assessing RV regional functions. The computational time per data set is approximately 60 seconds, which demonstrates our method's clinical potential as a real-time cardiac assessment tool.

  7. Robotic excision of aortic valve papillary fibroelastoma and concomitant Maze procedure

    OpenAIRE

    Murphy, Edward T

    2012-01-01

    Abstract: Cardiothoracic surgeons have utilized the surgical robot to provide a minimally invasive approach to a number of intracardiac operations, including tumor resection, valve repair, and ablation of atrial arrhythmia. We report the case of a 58 year-old woman who was found to have a mobile mass on her aortic valve during evaluation of atrial fibrillation. Both of these conditions were addressed when she underwent a combined robotic biatrial Maze procedure and excision of the mass, which...

  8. DNA repair pathways involved in determining the level of cytotoxicity of environmentally relevant UV radiation

    International Nuclear Information System (INIS)

    The sensitivity of cell lines with defects in various DNA repair processes to different wavelengths of UV has been assessed in order to determine the importance of these repair pathways to the cytotoxicity of UV light. The cell lines used in this work were xrs-6 (a Chinese Hamster Ovary (CHO) cell line) mutant for XRCC5/Ku80, EM9 a CHO cell line mutant for XRCC1, UV61 a CHO cell line mutant for ERCC6/CSB, and E3p53-/-, a mouse embryonic fibroblast cell line null for p53. Xrs-6 (defective in Non Homologous End-Joining) was found to be sensitive to the cytotoxic effects of broadband UVA, but not narrowband UVA or narrowband UVB. EM9 (defective in Base Excision Repair/Single-Strand Break Repair) was not sensitive to the cytotoxic effects of both broadband and narrowband UVA, narrowband UVB or narrowband UVC. UV61 (defective in the Transcription Coupled Repair branch of Nucleotide Excision Repair) was sensitive to the cytotoxic effects of narrowband UVA, UVB and UVC. E3p53-/- was sensitive to the cytotoxic effects of narrowband UVA and UVB. Broadband UVA was found to induce high levels of chromosomal damage in xrs-6, as quantified by the micronucleus assay, most likely as a result of this cell lines inability to repair DNA double strand breaks. EM9 was found to be defective in the repair of broadband UVA-induced single strand breaks, as measured by the alkaline gel electrophoresis ('comet') assay. UV61 was unable to repair broadband UVB-induced DNA damage as measured by the alkaline gel electrophoresis ('comet') assay. These results provide evidence that: 1. DNA double-strand breaks contribute to the cytotoxicity of UVA to a greater extent than single-strand breaks. 2. Repair mechanisms that operate in response to UVA may be coupled to transcription. 3. UVB may directly induce SSBs. 4. P53 is involved in the response of the cell to both UVA and UVB radiation. (author)

  9. Relationship between the genetic polymorphisms in nucleotide excision repair genes of ERCC1, XPD, XPC and the arsenism caused by coal-burning%核苷酸切除修复基因ERCC1、XPD和XPC多态性与燃煤污染型地方性砷中毒的关系研究

    Institute of Scientific and Technical Information of China (English)

    魏绍峰; 张爱华; 梁冰; 黄晓欣

    2011-01-01

    Objective To investigate the relationship between genetic polymorphisms in excision repair cross-complementing 1 (ERCC 1 ),xeroderma pigmentosum group D (XPD),xeroderma pigmentosum group C (XPC) and the risk of arsenism caused by coal-burning.Methods Two hundred and twenty-nine patients with arsenism in the endemic area of Jiaole village Xingren county Guizhou province were selected into experimental group.One hundred and ninety-eight inhabitants who had similar living habits but did no burning coal with high arsenic in Dagnoduo village were selected into control group.Two milliliters vein blood samples were taken and analyzed with polymerase chain reaction-restriction frgment length polymorphism technique (PCR-RFLP) to measure the gene polymorphisms of ERCC1 C8092A,XPD Lys751Gln,XPD Asp312Asn,XPD Arg156Arg,and XPC P(AT +/-).Relationship between genotype and the risk of arsenism was also analyzed.Results The frequency of ERCC1 8092CA/AA geno-type in case group [ CA:29.78% (67/225),AA:10.67% (24/225) ] was significantly higher than that of control group[CA:23.08%(45/195),AA:5.13%(10/195),x2 =8.116,P < 0.05].The frequency difference of other gene polymorphisms between case and control group was not statistically significant,respectively (x2 =5.649,4.394,0.865,1.490,all P > 0.05).There were 1.780(95%CI:1.174 - 2.698),1.681(95%CI:1.081 - 2.615),and 1.790(95%CI:1.014 - 3.158)-fold increase in risk of arsenism for individuals carrying ERCC1 8092CA + AA,XPD Lys751Gln + Gln751Gln,and XPD Asp312Asn + Asn312Asn genotypes compared respectively with individuals canying ERCC1 8092CC,XPD Lys751Lys,and XPD Asp312Asp(all P < 0.05).The sufferers only with XPD Arg156Arg or XPC P(AT +/-) didn't have higher risk of arsenism(all P > 0.05).Conclusion The results of this study suggest that the gene polymorphisms of ERCC1 C8092A,XPD Lys751Gln,and Asp312Asn are related to the arsenism caused by coal-burning.%目的 探讨核苷酸切除修复基因ERCCI、XPD、XPC

  10. Simple immunochemical method for measuring DNA repair rate in u.v.-irradiated bacteria

    International Nuclear Information System (INIS)

    A straightforward immunochemical method is reported to determine the repair rate of DNA in u.v. (ultraviolet)-irradiated bacteria based on use of antibodies specifically interacting with u.v.-irradiated DNA. The method involves quantitative precipitation of labeled 3H-DNA on nitrocellulose filters in the presence of specific antiserum. The method is justified by the evaluation of thymine dimers excision in various strains of E. coli K12. It was found that the dimers are almost completely eliminated in cells of wild strain during postradiation incubation after u.v.-irradiation with 400 ergs/mm2. In the cells of u.v.-sensitive uvr A6 mutant the dimers are practically not affected, whereas in those of uvr 502 strain excision of the dimers does take place, though more slowly than in wild strain bacteria. The reported approach gives results in good accord with those obtained by others chromatographically. (author)

  11. RNA polymerase II transcription inhibits DNA repair by photolyase in the transcribed strand of active yeast genes.

    OpenAIRE

    Livingstone-Zatchej, M; Meier, A; Suter, B.; Thoma, F

    1997-01-01

    Yeast uses nucleotide excision repair (NER) and photolyase (photoreactivation) to repair cyclobutane pyrimidine dimers (CPDs) generated by ultraviolet light. In active genes, NER preferentially repairs the transcribed strand (TS). In contrast, we recently showed that photolyase preferentially repairs the non-transcribed strands (NTS) of the URA3 and HIS3 genes in minichromosomes. To test whether photoreactivation depends on transcription, repair of CPDs was investigated in the transcriptional...

  12. INTERNAL REPAIR OF PIPELINES

    Energy Technology Data Exchange (ETDEWEB)

    Bill Bruce; Nancy Porter; George Ritter; Matt Boring; Mark Lozev; Ian Harris; Bill Mohr; Dennis Harwig; Robin Gordon; Chris Neary; Mike Sullivan

    2005-07-20

    The two broad categories of fiber-reinforced composite liner repair and deposited weld metal repair technologies were reviewed and evaluated for potential application for internal repair of gas transmission pipelines. Both are used to some extent for other applications and could be further developed for internal, local, structural repair of gas transmission pipelines. Principal conclusions from a survey of natural gas transmission industry pipeline operators can be summarized in terms of the following performance requirements for internal repair: (1) Use of internal repair is most attractive for river crossings, under other bodies of water, in difficult soil conditions, under highways, under congested intersections, and under railway crossings. (2) Internal pipe repair offers a strong potential advantage to the high cost of horizontal direct drilling when a new bore must be created to solve a leak or other problem. (3) Typical travel distances can be divided into three distinct groups: up to 305 m (1,000 ft.); between 305 m and 610 m (1,000 ft. and 2,000 ft.); and beyond 914 m (3,000 ft.). All three groups require pig-based systems. A despooled umbilical system would suffice for the first two groups which represents 81% of survey respondents. The third group would require an onboard self-contained power unit for propulsion and welding/liner repair energy needs. (4) The most common size range for 80% to 90% of operators surveyed is 508 mm (20 in.) to 762 mm (30 in.), with 95% using 558.8 mm (22 in.) pipe. Evaluation trials were conducted on pipe sections with simulated corrosion damage repaired with glass fiber-reinforced composite liners, carbon fiber-reinforced composite liners, and weld deposition. Additional un-repaired pipe sections were evaluated in the virgin condition and with simulated damage. Hydrostatic failure pressures for pipe sections repaired with glass fiber-reinforced composite liner were only marginally greater than that of pipe sections without

  13. Functional interactions and signaling properties of mammalian DNA mismatch repair proteins.

    Science.gov (United States)

    Bellacosa, A

    2001-11-01

    The mismatch repair (MMR) system promotes genomic fidelity by repairing base-base mismatches, insertion-deletion loops and heterologies generated during DNA replication and recombination. This function is critically dependent on the assembling of multimeric complexes involved in mismatch recognition and signal transduction to downstream repair events. In addition, MMR proteins coordinate a complex network of physical and functional interactions that mediate other DNA transactions, such as transcription-coupled repair, base excision repair and recombination. MMR proteins are also involved in activation of cell cycle checkpoint and induction of apoptosis when DNA damage overwhelms a critical threshold. For this reason, they play a role in cell death by alkylating agents and other chemotherapeutic drugs, including cisplatin. Inactivation of MMR genes in hereditary and sporadic cancer is associated with a mutator phenotype and inhibition of apoptosis. In the future, a deeper understanding of the molecular mechanisms and functional interactions of MMR proteins will lead to the development of more effective cancer prevention and treatment strategies. PMID:11687886

  14. 基于排队论的可修件维修模型%Repairing Model for Repairable Items Based on Queuing Theory

    Institute of Scientific and Technical Information of China (English)

    林健; 马振书; 黄刚; 贺鑫

    2011-01-01

    Queuing theory and method is applied to analyze repairing queuing system, and a model of repairing queuing system is established, which provides a theoretic basis for evaluating system efficiency and improving repairing system, and finally the example data is used to analyze and validated feasibility of the model.%通过运用排队论的理论和方法对可修件的维修排队系统进行分析,建立了维修排队系统模型,为评估系统效能以及调整改进系统提供了理论依据,最后通过实例分析对模型的可行性进行了验证.

  15. Intraoral excision of large submental dermoid

    OpenAIRE

    Ankur Bhatnagar; Vinay Kumar Verma; Pankaj Kumar Patel

    2013-01-01

    Sublingual dermoids are the rarest forms of craniofacial dermoids mostly seen in young individuals. Excision of large and deep submental dermoid is generally done via extraoral approach scarring the most prominent part of the face, which can lead to post operative scar hypertrophy and hyperpigmentation especially in non-Caucasian races. Presence of such scars leads to adverse psychological effects in young individuals. Excision via intraoral route, although technically demanding, can be simpl...

  16. Giant rhinophyma: Excision with coblation assisted surgery

    OpenAIRE

    Caner Sahin; Mesut Turker; Bulent Celasun

    2014-01-01

    An 83-year-old man presented with an unusually severe case of rhinophyma. Giant rhinopyhma is very rare in literature. The giant lesion was widely excised using sharp surgical incision and coblation assisted surgery. Using direct coblation to the nasal dorsum may cause edema in the surrounding tissue. There was minimal edema in surrounding tissue using this technique. A full thickness-skin graft was applied after excision. Cosmetic and functional postoperative results were satisfactory.

  17. Efficient Excise Taxation: The Evidence from Cigarettes.

    OpenAIRE

    Benjamin, Daniel K; William R. Dougan

    1997-01-01

    The authors develop a model in which optimizing policy makers in adjacent government jurisdictions levy excise taxes on a commodity that has a unique point of production from which all shipments emanate. From this model the authors derive an unusually specific predicted geographical pattern of those excise taxes in which these taxes rise at specific decreasing rate as one moves outward from the point of production. The authors compare these predictions to the observed pattern of cigarette tax...

  18. Reduced repair capacity of a DNA clustered damage site comprised of 8-oxo-7,8-dihydro-2′-deoxyguanosine and 2-deoxyribonolactone results in an increased mutagenic potential of these lesions

    Energy Technology Data Exchange (ETDEWEB)

    Cunniffe, Siobhan [CRUK-MRC Gray Institute for Radiation Oncology and Biology, Department of Oncology, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford OX3 7DQ (United Kingdom); O’Neill, Peter, E-mail: peter.oneill@oncology.ox.ac.uk [CRUK-MRC Gray Institute for Radiation Oncology and Biology, Department of Oncology, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford OX3 7DQ (United Kingdom); Greenberg, Marc M. [Johns Hopkins University, Department of Chemistry, 3400 N. Charles St. , Baltimore, MD 21218 (United States); Lomax, Martine E. [CRUK-MRC Gray Institute for Radiation Oncology and Biology, Department of Oncology, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford OX3 7DQ (United Kingdom)

    2014-04-15

    Highlights: • A dL lesion is not repaired as effectively as an AP site. • The repair of a cluster with dL and 8-oxodGuo lesions is compromised. • Delayed repair of the cluster leads to an increase in mutation frequency. - Abstract: A signature of ionizing radiation is the induction of DNA clustered damaged sites. Non-double strand break (DSB) clustered damage has been shown to compromise the base excision repair pathway, extending the lifetimes of the lesions within the cluster, compared to isolated lesions. This increases the likelihood the lesions persist to replication and thus increasing the mutagenic potential of the lesions within the cluster. Lesions formed by ionizing radiation include 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) and 2-deoxyribonolactone (dL). dL poses an additional challenge to the cell as it is not repaired by the short-patch base excision repair pathway. Here we show recalcitrant dL repair is reflected in mutations observed when DNA containing it and a proximal 8-oxodGuo is replicated in Escherichia coli. 8-oxodGuo in close proximity to dL on the opposing DNA strand results in an enhanced frequency of mutation of the lesions within the cluster and a 20 base sequence flanking the clustered damage site in an E. coli based plasmid assay. In vitro repair of a dL lesion is reduced when compared to the repair of an abasic (AP) site and a tetrahydrofuran (THF), and this is due mainly to a reduction in the activity of polymerase β, leading to retarded FEN1 and ligase 1 activities. This study has given insights in to the biological effects of clusters containing dL.

  19. Reduced repair capacity of a DNA clustered damage site comprised of 8-oxo-7,8-dihydro-2′-deoxyguanosine and 2-deoxyribonolactone results in an increased mutagenic potential of these lesions

    International Nuclear Information System (INIS)

    Highlights: • A dL lesion is not repaired as effectively as an AP site. • The repair of a cluster with dL and 8-oxodGuo lesions is compromised. • Delayed repair of the cluster leads to an increase in mutation frequency. - Abstract: A signature of ionizing radiation is the induction of DNA clustered damaged sites. Non-double strand break (DSB) clustered damage has been shown to compromise the base excision repair pathway, extending the lifetimes of the lesions within the cluster, compared to isolated lesions. This increases the likelihood the lesions persist to replication and thus increasing the mutagenic potential of the lesions within the cluster. Lesions formed by ionizing radiation include 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) and 2-deoxyribonolactone (dL). dL poses an additional challenge to the cell as it is not repaired by the short-patch base excision repair pathway. Here we show recalcitrant dL repair is reflected in mutations observed when DNA containing it and a proximal 8-oxodGuo is replicated in Escherichia coli. 8-oxodGuo in close proximity to dL on the opposing DNA strand results in an enhanced frequency of mutation of the lesions within the cluster and a 20 base sequence flanking the clustered damage site in an E. coli based plasmid assay. In vitro repair of a dL lesion is reduced when compared to the repair of an abasic (AP) site and a tetrahydrofuran (THF), and this is due mainly to a reduction in the activity of polymerase β, leading to retarded FEN1 and ligase 1 activities. This study has given insights in to the biological effects of clusters containing dL

  20. UvrD facilitates DNA repair by pulling RNA polymerase backwards

    OpenAIRE

    Epshtein, Vitaly; Kamarthapu, Venu; McGary, Katelyn; Svetlov, Vladimir; Ueberheide, Beatrix; Proshkin, Sergey; Mironov, Alexander; Nudler, Evgeny

    2014-01-01

    UvrD helicase is required for nucleotide excision repair, although its role in this process is not well defined. Here we show that Escherichia coli UvrD binds RNA polymerase during transcription elongation and, using its helicase/translocase activity, forces RNA polymerase to slide backward along DNA. By inducing backtracking, UvrD exposes DNA lesions shielded by blocked RNA polymerase, allowing nucleotide excision repair enzymes to gain access to sites of damage. Our results establish UvrD a...