XRCC1 suppresses somatic hypermutation and promotes alternative nonhomologous end joining in Igh genes.

Science.gov (United States)

Saribasak, Huseyin; Maul, Robert W; Cao, Zheng; McClure, Rhonda L; Yang, William; McNeill, Daniel R; Wilson, David M; Gearhart, Patricia J

2011-10-24

Activation-induced deaminase (AID) deaminates cytosine to uracil in immunoglobulin genes. Uracils in DNA can be recognized by uracil DNA glycosylase and abasic endonuclease to produce single-strand breaks. The breaks are repaired either faithfully by DNA base excision repair (BER) or mutagenically to produce somatic hypermutation (SHM) and class switch recombination (CSR). To unravel the interplay between repair and mutagenesis, we decreased the level of x-ray cross-complementing 1 (XRCC1), a scaffold protein involved in BER. Mice heterozygous for XRCC1 showed a significant increase in the frequencies of SHM in Igh variable regions in Peyer's patch cells, and of double-strand breaks in the switch regions during CSR. Although the frequency of CSR was normal in Xrcc1(+/-) splenic B cells, the length of microhomology at the switch junctions decreased, suggesting that XRCC1 also participates in alternative nonhomologous end joining. Furthermore, Xrcc1(+/-) B cells had reduced Igh/c-myc translocations during CSR, supporting a role for XRCC1 in microhomology-mediated joining. Our results imply that AID-induced single-strand breaks in Igh variable and switch regions become substrates simultaneously for BER and mutagenesis pathways.

Association study between XRCC1 gene polymorphisms and sporadic amyotrophic lateral sclerosis.

Science.gov (United States)

Coppedè, Fabio; Migheli, Francesca; Lo Gerfo, Annalisa; Fabbrizi, Maria Rita; Carlesi, Cecilia; Mancuso, Michelangelo; Corti, Stefania; Mezzina, Nicoletta; del Bo, Roberto; Comi, Giacomo P; Siciliano, Gabriele; Migliore, Lucia

2010-01-01

The aim of the present study was to investigate the possible contribution of three common functional polymorphisms in the DNA repair protein X-ray repair cross-complementing group 1 (XRCC1), namely Arg194Trp (rs1799782), Arg280His (rs25489) and Arg399Gln (rs25487), to sporadic amyotrophic lateral sclerosis (SALS). We genotyped 206 Italian SALS patients and 203 matched controls for XRCC1 Arg194Trp, Arg280His and Arg399Gln polymorphisms by means of PCR/RFLP technique, searching for association between any of the studied polymorphisms and disease risk, age and site of onset. We observed a statistically significant difference in XRCC1 Gln399 allele frequencies between SALS cases and controls (0.39/0.28; p=0.001). The present study suggests that the XRCC1 Arg399Gln polymorphism might contribute to SALS risk.

Might there be a link between intron 3 VNTR polymorphism in the XRCC4 DNA repair gene and the etiopathogenesis of rheumatoid arthritis?

Science.gov (United States)

Pehlivan, Sacide; Balci, Sibel Oguzkan; Aydeniz, Ali; Pehlivan, Mustafa; Sever, Tugce; Gursoy, Savas

2015-01-01

DNA repair genes are involved in several diseases such as cancers and autoimmune diseases. Previous studies indicated that a DNA repair system was involved in the development of rheumatoid arthritis (RA). In this study, we aimed to examine whether four polymorphisms in the DNA repair genes (xeroderma pigmentosum complementation group D [XPD], X-ray repair cross-complementing group 1 [XRCC1], and X-ray repair cross-complementing group 4 [XRCC4]) were associated with RA. Sixty-five patients with RA and 70 healthy controls (HCs) were examined for XPD (A-751G), XRCC1 (A399G), and XRCC4 (intron 3 VNTR and G-1394T) polymorphisms. All polymorphisms were genotyped by PCR and/or PCR-RFLP. The association between the polymorphisms and RA was analyzed using the chi-square test and de Finetti program. The intron 3 VNTR polymorphism in the XRCC4 gene showed an association with RA patients. The DI genotype was found lower in RA patients (χ(2)=8.227; p=0.0021), while the II genotype was higher in RA patients (χ(2)=5.285; p=0.010). There were deviations from the Hardy-Weinberg Equilibrium (HWE) in both intron 3 VNTR and G-1394T polymorphisms in the XRCC4 gene and in the polymorphism in the XRCC1 gene, and the observed genotype counts deviated from those expected according to the HWE (p=0.027, 0.004, and 0.002, respectively); however, there was no deviation in the other gene polymorphisms. There is no statistical difference between the RA patients and HCs for XPD (A-751G), XRCC1 (A399G), and XRCC4 (G-1394T) gene polymorphisms (p>0.05). Although XPD (A-751G), XRCC1 (A399G), and XRCC4 (G-1394T) gene polymorphisms have been extensively investigated in different clinical pictures, this is the first study to evaluate the role of these polymorphisms in the genetic etiopathogenesis of RA in Turkish patients. In conclusion, we suggested that the intron 3 VNTR polymorphism in the XRCC4 gene may be associated with the etiopathogenesis of RA as a marker of immune aging.

Modulation of DNA repair capacity and mRNA expression levels of XRCC1, hOGG1 and XPC genes in styrene-exposed workers

International Nuclear Information System (INIS)

Hanova, Monika; Stetina, Rudolf; Vodickova, Ludmila; Vaclavikova, Radka; Hlavac, Pavel; Smerhovsky, Zdenek; Naccarati, Alessio; Polakova, Veronika; Soucek, Pavel; Kuricova, Miroslava; Manini, Paola; Kumar, Rajiv; Hemminki, Kari; Vodicka, Pavel

2010-01-01

Decreased levels of single-strand breaks in DNA (SSBs), reflecting DNA damage, have previously been observed with increased styrene exposure in contrast to a dose-dependent increase in the base-excision repair capacity. To clarify further the above aspects, we have investigated the associations between SSBs, micronuclei, DNA repair capacity and mRNA expression in XRCC1, hOGG1 and XPC genes on 71 styrene-exposed and 51 control individuals. Styrene concentrations at workplace and in blood characterized occupational exposure. The workers were divided into low (below 50 mg/m 3 ) and high (above 50 mg/m 3 ) styrene exposure groups. DNA damage and DNA repair capacity were analyzed in peripheral blood lymphocytes by Comet assay. The mRNA expression levels were determined by qPCR. A significant negative correlation was observed between SSBs and styrene concentration at workplace (R = - 0.38, p = 0.001); SSBs were also significantly higher in men (p = 0.001). The capacity to repair irradiation-induced DNA damage was the highest in the low exposure group (1.34 ± 1.00 SSB/10 9 Da), followed by high exposure group (0.72 ± 0.81 SSB/10 9 Da) and controls (0.65 ± 0.82 SSB/10 9 Da). The mRNA expression levels of XRCC1, hOGG1 and XPC negatively correlated with styrene concentrations in blood and at workplace (p < 0.001) and positively with SSBs (p < 0.001). Micronuclei were not affected by styrene exposure, but were higher in older persons and in women (p < 0.001). In this study, we did not confirm previous findings on an increased DNA repair response to styrene-induced genotoxicity. However, negative correlations of SSBs and mRNA expression levels of XRCC1, hOGG1 and XPC with styrene exposure warrant further highly-targeted study.

XRCC1 and XPD DNA repair gene polymorphisms: a potential risk factor for glaucoma in the Pakistani population

NARCIS (Netherlands)

Yousaf, S.; Khan, M.I.; Micheal, S.; Akhtar, F.; Ali, S.H.; Riaz, M.; Ali, M.; Lall, P.; Waheed, N.K.; Hollander, A.I. den; Ahmed, A.; Qamar, R.

2011-01-01

PURPOSE: The present study was designed to determine the association of polymorphisms of the DNA repair genes X-ray cross-complementing group 1 (XRCC1) (c.1316G>A [rs25487]) and xeroderma pigmentosum complementation group D (XPD) (c.2298A>C [rs13181]) with primary open-angle glaucoma (POAG) and

Role of the XRCC1 - APE1 interaction in the maintenance of genetic stability

International Nuclear Information System (INIS)

Sossou-Becker, M.

2005-09-01

This thesis is divided in four chapters: the first one concerns the genetic instability, the second one is devoted to the DNA repair, the third one is related to the XRCC1 and the chapter four concerns APE1. Then, are defined the objectives and the results. This work fits into the studies of repair mechanisms. The physical and functional characterisation of the interaction between XRCC1 and APE1 allowed to understand its involvement in the prevention of the genetic instability at the origin of cancer. (N.C.)

Association between polymorphisms at promoters of XRCC5 and XRCC6 genes and risk of breast cancer.

Science.gov (United States)

Rajaei, Mehrdad; Saadat, Iraj; Omidvari, Shahpour; Saadat, Mostafa

2014-04-01

Variation in DNA repair genes is one of the mechanisms that may lead to variation in DNA repair capacity. Ku, a heterodimeric DNA-binding complex, is directly involved in repair of DNA double-strand breaks. Ku consists of two subunits, Ku70 and Ku80, which are encoded by the XRCC6 and XRCC5 genes, respectively. In the present study, we investigated whether common genetic variant in variable number of tandem repeats (VNTR) XRCC5 and T-991C XRCC6 was associated with an altered risk of breast cancer. The present study included 407 females with breast cancer and 395 age frequency-matched controls which were randomly selected from the healthy female blood donors. The XRCC5 and XRCC6 polymorphisms were determined using PCR-based methods. For XRCC5 polymorphism, in comparison with the 1R/1R genotype, the 0R/0R genotype increased breast cancer risk (OR 9.55, 95%CI 1.19-76.64, P = 0.034). The 1R/3R genotype compared with 1R/1R genotype decreased the risk of breast cancer (Fisher's exact test P = 0.015). There was no association between T-991C polymorphism of XRCC6 and breast cancer risk. Mean of age at diagnosis of breast cancer for 0, 1, 2, 3, and >4 repeat in XRCC5 were 39.2, 41.9, 44.3, 45.8, and 47.3 years, respectively. The Kaplan-Meier survival analysis revealed that the number of repeat was associated with age at diagnosis of breast cancer (log rank statistic = 13.90, df = 4, P = 0.008). The findings of the present study revealed that either breast cancer risk or age at diagnosis of breast cancer was associated with the VNTR polymorphism at promoter region of XRCC5.

Risk of radiation-induced subcutaneous fibrosis in relation to single nucleotide polymorphisms in TGFB1, SOD2, XRCC1, XRCC3, APEX and ATM - a study based on DNA from formalin fixed paraffin embedded tissue samples

DEFF Research Database (Denmark)

Andreassen, Christian Nicolaj; Alsner, Jan; Overgaard, Marie

2006-01-01

Purpose: In two previously published studies, associations with risk of radiation-induced subcutaneous fibrosis were found for single nucleotide polymorphisms (SNP) in TGFB1 (transforming growth factor beta 1 gene), XRCC1 (X-ray repair cross-complementing 1 gene), XRCC3 (X-ray repair cross...... the influence of genetic variation upon normal tissue radiosensitivity...

Contribution of DNA double-strand break repair gene XRCC3 genotypes to oral cancer susceptibility in Taiwan.

Science.gov (United States)

Tsai, Chia-Wen; Chang, Wen-Shin; Liu, Juhn-Cherng; Tsai, Ming-Hsui; Lin, Cheng-Chieh; Bau, Da-Tian

2014-06-01

The DNA repair gene X-ray repair cross complementing protein 3 (XRCC3) is thought to play a major role in double-strand break repair and in maintaining genomic stability. Very possibly, defective double-strand break repair of cells can lead to carcinogenesis. Therefore, a case-control study was performed to reveal the contribution of XRCC3 genotypes to individual oral cancer susceptibility. In this hospital-based research, the association of XRCC3 rs1799794, rs45603942, rs861530, rs3212057, rs1799796, rs861539, rs28903081 genotypes with oral cancer risk in a Taiwanese population was investigated. In total, 788 patients with oral cancer and 956 age- and gender-matched healthy controls were genotyped. The results showed that there was significant differential distribution among oral cancer and controls in the genotypic (p=0.001428) and allelic (p=0.0013) frequencies of XRCC3 rs861539. As for the other polymorphisms, there was no difference between case and control groups. In gene-lifestyle interaction analysis, we have provided the first evidence showing that there is an obvious joint effect of XRCC3 rs861539 genotype with individual areca chewing habits on oral cancer risk. In conclusion, the T allele of XRCC3 rs861539, which has an interaction with areca chewing habit in oral carcinogenesis, may be an early marker for oral cancer in Taiwanese. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Detection and characterization of polymorphisms in XRCC DNA repair genes in human population

International Nuclear Information System (INIS)

Staynova, A.; Hadjidekova, V.; Savov, A.

2004-01-01

Human population is continuously exposed to low levels of ionizing radiation. The main contribution gives the exposure due to medical applications. Nevertheless, most of the damage induced is repaired shortly after exposure by cellular repair systems. The review is focused on the development and application of methods to estimate the character of polymorphisms in repair genes (XRCC1, APE1), involved in single strand breaks repair which is corresponding mainly to the repair of X-ray induced DNA damage. Since, DSB are major factor for chromosomal aberrations formation, the assays described in this review might be useful for the assessment of the radiation risk for human population. (authors)

Contribution of X-Ray Repair Complementing Defective Repair in Chinese Hamster Cells 3 (XRCC3) Genotype to Leiomyoma Risk.

Science.gov (United States)

Chang, Wen-Shin; Tsai, Chia-Wen; Wang, Ju-Yu; Ying, Tsung-Ho; Hsiao, Tsan-Seng; Chuang, Chin-Liang; Yueh, Te-Cheng; Liao, Cheng-Hsi; Hsu, Chin-Mu; Liu, Shih-Ping; Gong, Chi-Li; Tsai, Chang-Hai; Bau, Da-Tian

2015-09-01

The present study aimed at investigating whether X-ray repair cross complementing protein 3 (XRCC3) genotype may serve as a useful marker for detecting leiomyoma and predicting risk. A total of 640 women (166 patients with leiomyoma and 474 healthy controls) were examined for their XRCC3 rs1799794, rs45603942, rs861530, rs3212057, rs1799796, rs861539, rs28903081 genotype. The distributions of genotypic and allelic frequencies between the two groups were compared. The results showed that the CT and TT genotypes of XRCC3 rs861539 were associated with increased leiomyoma risk (odds ratio=2.19, 95% confidence interval=1.23-3.90; odds ratio=3.72, 95% confidence interval=1.23-11.26, respectively). On allelic frequency analysis, we found a significant difference in the distribution of the T allelic frequency of the XRCC3 rs861539 (p=5.88 × 10(-5)). None of the other six single nucleotide polymorphisms were associated with altered leiomyoma susceptibility. The T allele (CT and TT genotypes) of XRCC3 rs861539 contributes to increased risk of leiomyoma among Taiwanese women and may serve as a early detection and predictive marker. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Repair of radiation-induced heat-labile sites is independent of DNA-PKcs, XRCC1 or PARP

Energy Technology Data Exchange (ETDEWEB)

Stenerl& #246; w, Bo; Karlsson, Karin H.; Radulescu, Irina; Rydberg, Bjorn; Stenerlow, Bo

2008-04-29

Ionizing radiation induces a variety of different DNA lesions: in addition to the most critical DNA damage, the DSB, numerous base alterations, SSBs and other modifications of the DNA double-helix are formed. When several non-DSB lesions are clustered within a short distance along DNA, or close to a DSB, they may interfere with the repair of DSBs and affect the measurement of DSB induction and repair. We have previously shown that a substantial fraction of DSBs measured by pulsed-field gel electrophoresis (PFGE) are in fact due to heat-labile sites (HLS) within clustered lesions, thus reflecting an artifact of preparation of genomic DNA at elevated temperature. To further characterize the influence of HLS on DSB induction and repair, four human cell lines (GM5758, GM7166, M059K, U-1810) with apparently normal DSB rejoining were tested for bi-phasic rejoining after gamma irradiation. When heat-released DSBs were excluded from the measurements the fraction of fast rejoining decreased to less than 50% of the total. However, neither the half-times of the fast (t{sub 1/2} = 7-8 min) or slow (t{sub 1/2} = 2.5 h) DSB rejoining were changed significantly. At t=0 the heat-released DSBs accounted for almost 40% of the DSBs, corresponding to 10 extra DSB/cell/Gy in the initial DSB yield. These heat-released DSBs were repaired within 60-90 min in all tested cells, including M059K cells treated with wortmannin or DNA-PKcs defect M059J cells. Furthermore, cells lacking XRCC1 or Poly(ADP-ribose) polymerase-1 (PARP-1) rejoined both total DSBs and heat-released DSBs similar to normal cells. In summary, the presence of heat-labile sites have a substantial impact on DSB induction yields and DSB rejoining rates measured by pulsed-field gel electrophoresis, and HLS repair is independent of DNA-PKcs, XRCC1 and PARP.

Functional Polymorphisms of Base Excision Repair Genes XRCC1 and APEX1 Predict Risk of Radiation Pneumonitis in Patients With Non-Small Cell Lung Cancer Treated With Definitive Radiation Therapy

International Nuclear Information System (INIS)

Yin Ming; Liao Zhongxing; Liu Zhensheng; Wang, Li-E; Gomez, Daniel; Komaki, Ritsuko; Wei Qingyi

2011-01-01

Purpose: To explore whether functional single nucleotide polymorphisms (SNPs) of base-excision repair genes are predictors of radiation treatment-related pneumonitis (RP), we investigated associations between functional SNPs of ADPRT, APEX1, and XRCC1 and RP development. Methods and Materials: We genotyped SNPs of ADPRT (rs1136410 [V762A]), XRCC1 (rs1799782 [R194W], rs25489 [R280H], and rs25487 [Q399R]), and APEX1 (rs1130409 [D148E]) in 165 patients with non-small cell lung cancer (NSCLC) who received definitive chemoradiation therapy. Results were assessed by both Logistic and Cox regression models for RP risk. Kaplan-Meier curves were generated for the cumulative RP probability by the genotypes. Results: We found that SNPs of XRCC1 Q399R and APEX1 D148E each had a significant effect on the development of Grade ≥2 RP (XRCC1: AA vs. GG, adjusted hazard ratio [HR] = 0.48, 95% confidence interval [CI], 0.24-0.97; APEX1: GG vs. TT, adjusted HR = 3.61, 95% CI, 1.64-7.93) in an allele-dose response manner (Trend tests: p = 0.040 and 0.001, respectively). The number of the combined protective XRCC1 A and APEX1 T alleles (from 0 to 4) also showed a significant trend of predicting RP risk (p = 0.001). Conclusions: SNPs of the base-excision repair genes may be biomarkers for susceptibility to RP. Larger prospective studies are needed to validate our findings.

Lysine 271 but not lysine 210 of XRCC4 is required for the nuclear localization of XRCC4 and DNA ligase IV

Energy Technology Data Exchange (ETDEWEB)

Fukuchi, Mikoto; Wanotayan, Rujira; Liu, Sicheng; Imamichi, Shoji; Sharma, Mukesh Kumar; Matsumoto, Yoshihisa, E-mail: yoshim@nr.titech.ac.jp

2015-06-12

XRCC4 and DNA Ligase IV (LIG4) cooperate to join two DNA ends at the final step of DNA double-strand break (DSB) repair through non-homologous end-joining (NHEJ). However, it is not fully understood how these proteins are localized to the nucleus. Here we created XRCC4{sup K271R} mutant, as Lys271 lies within the putative nuclear localization signal (NLS), and XRCC4{sup K210R} mutant, as Lys210 was reported to undergo SUMOylation, implicated in the nuclear localization of XRCC4. Wild-type and mutated XRCC4 with EGFP tag were introduced into HeLa cell, in which endogenous XRCC4 had been knocked down using siRNA directed to 3′-untranslated region, and tested for the nuclear localization function by fluorescence microscopy. XRCC4{sup K271R} was defective in the nuclear localization of itself and LIG4, whereas XRCC4{sup K210R} was competent for the nuclear localization with LIG4. To examine DSB repair function, wild-type and mutated XRCC4 were introduced into XRCC4-deficient M10. M10-XRCC4{sup K271R}, but not M10-XRCC4{sup K210R}, showed significantly reduced surviving fraction after 2 Gy γ-ray irradiation as compared to M10-XRCC4{sup WT}. The number of γ-H2AX foci remaining 2 h after 2 Gy γ-ray irradiation was significantly greater in M10-XRCC4{sup K271R} than in M10-XRCC4{sup WT}, whereas it was only marginally increased in M10-XRCC4{sup K210R} as compared to M10-XRCC4{sup WT}. The present results collectively indicated that Lys271, but not Lys210, of XRCC4 is required for the nuclear localization of XRCC4 and LIG4 and that the nuclear localizing ability is essential for DSB repair function of XRCC4. - Highlights: • XRCC4{sup K271R} is defective in the nuclear localization of itself and LIG4. • XRCC4{sup K210R} is competent for the nuclear localization of itself and LIG4. • XRCC4{sup K271R} is deficient in DSB repair function. • XRCC4{sup K210R} is mostly normal in DSB repair function.

Recombination activating activity of XRCC1 analogous genes in X-ray sensitive and resistant CHO cell lines

International Nuclear Information System (INIS)

Golubnitchaya-Labudova, O.; Hoefer, M.; Portele, A.; Vacata, V.; Rink, H.; Lubec, G.

1997-01-01

The XRCC1 gene (X-ray repair cross complementing) complements the DNA repair deficiency of the radiation sensitive Chinese hamster ovary (CHO) mutant cell line EM9 but the mechanism of the correction is not elucidated yet. XRCC1 shows substantial homology to the RAG2 gene (recombination activating gene) and we therefore tried to answer the question, whether structural similarities (sequence of a putative recombination activating domain, aa 332-362 for XRCC1 and aa 286-316 in RAG2) would reflect similar functions of the homologous, putative recombination activating domain. PCR experiments revealed that no sequence homologous to the structural part of human XRCC1 was present in cDNA of CHO. Differential display demonstrated two putative recombination activating in the parental CHO line AA8 and one in the radiosensitive mutant EM9. Southern blot experiments showed the presence of several genes with partial homology to human XRCC1. Recombination studies consisted of expressing amplified target domains within chimeric proteins in recA - bacteria and subsequent detection of recombination events by sequencing the recombinant plasmids. Recombination experiments demonstrated recombination activating activity of all putative recombination activating domains amplified from AA8 and EM9 genomes as reflected by deletions within the inserts of the recombinant plasmids. The recombination activating activity of XRCC1 analogues could explain a mechanism responsible for the correction of the DNA repair defect in EM9. (author)

XRCC1 Arg194Trp and Arg399Gln polymorphisms and arsenic methylation capacity are associated with urothelial carcinoma

Energy Technology Data Exchange (ETDEWEB)

Chiang, Chien-I [School of Public Health, College of Public Health and Nutrition, Taipei Medical University, Taipei, Taiwan (China); Huang, Ya-Li [Department of Public Health, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan (China); Chen, Wei-Jen [School of Public Health, College of Public Health and Nutrition, Taipei Medical University, Taipei, Taiwan (China); Shiue, Horng-Sheng [Department of Chinese Medicine, Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Taoyuan, Taiwan (China); Huang, Chao-Yuan; Pu, Yeong-Shiau [Department of Urology, National Taiwan University Hospital, College of Medicine National Taiwan University, Taipei, Taiwan (China); Lin, Ying-Chin [Department of Family Medicine, Shung Ho Hospital, Taipei Medical University, New Taipei, Taiwan (China); Department of Health Examination, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan (China); Division of Family Medicine, School of Medicine, Taipei Medical University, Taipei, Taiwan (China); Hsueh, Yu-Mei, E-mail: ymhsueh@tmu.edu.tw [School of Public Health, College of Public Health and Nutrition, Taipei Medical University, Taipei, Taiwan (China); Department of Public Health, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan (China)

2014-09-15

The association between DNA repair gene polymorphisms and bladder cancer has been widely studied. However, few studies have examined the correlation between urothelial carcinoma (UC) and arsenic or its metabolites. The aim of this study was to examine the association between polymorphisms of the DNA repair genes, XRCC1 Arg194Trp, XRCC1 Arg399Gln, XRCC3 Thr241Met, and XPD Lys751Gln, with urinary arsenic profiles and UC. To this end, we conducted a hospital-based case–control study with 324 UC patients and 647 age- and gender-matched non-cancer controls. Genomic DNA was used to examine the genotype of XRCC1 Arg194Trp, XRCC1 Arg399Gln, XRCC3 Thr241Met, and XPD Lys751Gln by PCR-restriction fragment length polymorphism analysis (PCR-RFLP). Urinary arsenic profiles were measured by high performance liquid chromatography (HPLC) linked with hydride generator and atomic absorption spectrometry. The XRCC1 399 Gln/Gln and 194 Arg/Trp and Trp/Trp genotypes were significantly related to UC, and the odds ratio (OR) and 95% confidence interval (95%CI) were 1.68 (1.03–2.75) and 0.66 (0.48–0.90), respectively. Participants with higher total urinary arsenic levels, a higher percentage of inorganic arsenic (InAs%) and a lower percentage of dimethylarsinic acid (DMA%) had a higher OR of UC. Participants carrying XRCC1 risk diplotypes G-C/G-C, A-C/A-C, and A-T/G-T, and who had higher total arsenic levels, higher InAs%, or lower DMA% compared to those with other XRCC1 diplotypes had a higher OR of UC. Our results suggest that the XRCC1 399 Gln/Gln and 194 Arg/Arg DNA repair genes play an important role in poor arsenic methylation capacity, thereby increasing the risk of UC in non-obvious arsenic exposure areas. - Highlights: • The XRCC1 399Gln/Gln genotype was significantly associated with increased OR of UC. • The XRCC1 194 Arg/Trp and Trp/Trp genotype had a significantly decreased OR of UC. • Combined effect of the XRCC1 genotypes and poor arsenic methylation capacity on

The polymorphism and haplotypes of XRCC1 and survival of non-small-cell lung cancer after radiotherapy

International Nuclear Information System (INIS)

Yoon, Sang Min; Hong, Yun-Chul; Park, Heon Joo; Lee, Jong-Eun; Kim, Sang Yoon; Kim, Jong Hoon; Lee, Sang-Wook; Park, So-Yeon; Lee, Jung Shin; Choi, Eun Kyung

2005-01-01

Purpose: The X-ray repair cross-complementing Group 1 (XRCC1) protein is involved mainly in the base excision repair of the DNA repair process. This study examined the association of 3 polymorphisms (codon 194, 280, and 399) of XRCC1 and lung cancer in terms of whether or not these polymorphisms have an effect on the survival of lung cancer patients who have received radiotherapy. Methods and Materials: Between January 2000 and April 2004, 229 lung cancer patients with non-small-cell lung cancer in Stages I-III were enrolled. Genotyping was performed by single base primer extension assay using the SNP-IT Kit with genomic DNA samples from all patients. The haplotype of the XRCC1 polymorphisms was estimated by PHASE version 2.1. Results: The patients consisted of 191 (83.4%) males and 38 (16.6%) females with a median age of 62 (range, 26-88 years). Sixty percent of the patients were included in Stage I-IIIa. The median progression-free and overall survival was 13 months and 16 months, respectively. The XRCC1 codon 194, histology, and stage were shown to be significant predictors of the progression-free survival. The 6 haplotypes among the XRCC1 polymorphisms (194, 280, and 399) were estimated by PHASE v.2.1. The patients with haplotype pairs other than the homozygous TGG haplotype pairs survived significantly longer (p = 0.04). Conclusions: Polymorphisms of XRCC1 have an effect on the survival of lung cancer patients treated with radiotherapy, and this effect seems to be more significant after the haplotype pairs are considered

The impact of homologous recombination repair deficiency on depleted uranium clastogenicity in Chinese hamster ovary cells: XRCC3 protects cells from chromosome aberrations, but increases chromosome fragmentation

Energy Technology Data Exchange (ETDEWEB)

Holmes, Amie L. [Wise Laboratory of Environmental and Genetic Toxicology, University of Southern Maine, 96 Falmouth St., P.O. Box 9300, Portland, ME 04104-9300, United States of America (United States); Maine Center for Toxicology and Environmental Health, University of Southern Maine, 96 Falmouth St., P.O. Box 9300, Portland, ME 04104-9300, United States of America (United States); Department of Applied Medical Science, University of Southern Maine, 96 Falmouth Street, P.O. Box 9300, Portland, ME 04104-9300, United States of America (United States); Joyce, Kellie [Wise Laboratory of Environmental and Genetic Toxicology, University of Southern Maine, 96 Falmouth St., P.O. Box 9300, Portland, ME 04104-9300, United States of America (United States); Maine Center for Toxicology and Environmental Health, University of Southern Maine, 96 Falmouth St., P.O. Box 9300, Portland, ME 04104-9300, United States of America (United States); Xie, Hong [Wise Laboratory of Environmental and Genetic Toxicology, University of Southern Maine, 96 Falmouth St., P.O. Box 9300, Portland, ME 04104-9300, United States of America (United States); Maine Center for Toxicology and Environmental Health, University of Southern Maine, 96 Falmouth St., P.O. Box 9300, Portland, ME 04104-9300, United States of America (United States); Department of Applied Medical Science, University of Southern Maine, 96 Falmouth Street, P.O. Box 9300, Portland, ME 04104-9300, United States of America (United States); Falank, Carolyne [Wise Laboratory of Environmental and Genetic Toxicology, University of Southern Maine, 96 Falmouth St., P.O. Box 9300, Portland, ME 04104-9300, United States of America (United States); Maine Center for Toxicology and Environmental Health, University of Southern Maine, 96 Falmouth St., P.O. Box 9300, Portland, ME 04104-9300, United States of America (United States); and others

2014-04-15

Highlights: • The role of homologous recombination repair in DU-induced toxicity was examined. • Loss of RAD51D did not affect DU-induced cytotoxicity or genotoxicity. • XRCC3 protects cell from DU-induced chromosome breaks and fusions. • XRCC3 plays a role in DU-induced chromosome fragmentation of the X chromosome. - Abstract: Depleted uranium (DU) is extensively used in both industry and military applications. The potential for civilian and military personnel exposure to DU is rising, but there are limited data on the potential health hazards of DU exposure. Previous laboratory research indicates DU is a potential carcinogen, but epidemiological studies remain inconclusive. DU is genotoxic, inducing DNA double strand breaks, chromosome damage and mutations, but the mechanisms of genotoxicity or repair pathways involved in protecting cells against DU-induced damage remain unknown. The purpose of this study was to investigate the effects of homologous recombination repair deficiency on DU-induced genotoxicity using RAD51D and XRCC3-deficient Chinese hamster ovary (CHO) cell lines. Cells deficient in XRCC3 (irs1SF) exhibited similar cytotoxicity after DU exposure compared to wild-type (AA8) and XRCC3-complemented (1SFwt8) cells, but DU induced more break-type and fusion-type lesions in XRCC3-deficient cells compared to wild-type and XRCC3-complemented cells. Surprisingly, loss of RAD51D did not affect DU-induced cytotoxicity or genotoxicity. DU induced selective X-chromosome fragmentation irrespective of RAD51D status, but loss of XRCC3 nearly eliminated fragmentation observed after DU exposure in wild-type and XRCC3-complemented cells. Thus, XRCC3, but not RAD51D, protects cells from DU-induced breaks and fusions and also plays a role in DU-induced chromosome fragmentation.

The impact of homologous recombination repair deficiency on depleted uranium clastogenicity in Chinese hamster ovary cells: XRCC3 protects cells from chromosome aberrations, but increases chromosome fragmentation

International Nuclear Information System (INIS)

Holmes, Amie L.; Joyce, Kellie; Xie, Hong; Falank, Carolyne

2014-01-01

Highlights: • The role of homologous recombination repair in DU-induced toxicity was examined. • Loss of RAD51D did not affect DU-induced cytotoxicity or genotoxicity. • XRCC3 protects cell from DU-induced chromosome breaks and fusions. • XRCC3 plays a role in DU-induced chromosome fragmentation of the X chromosome. - Abstract: Depleted uranium (DU) is extensively used in both industry and military applications. The potential for civilian and military personnel exposure to DU is rising, but there are limited data on the potential health hazards of DU exposure. Previous laboratory research indicates DU is a potential carcinogen, but epidemiological studies remain inconclusive. DU is genotoxic, inducing DNA double strand breaks, chromosome damage and mutations, but the mechanisms of genotoxicity or repair pathways involved in protecting cells against DU-induced damage remain unknown. The purpose of this study was to investigate the effects of homologous recombination repair deficiency on DU-induced genotoxicity using RAD51D and XRCC3-deficient Chinese hamster ovary (CHO) cell lines. Cells deficient in XRCC3 (irs1SF) exhibited similar cytotoxicity after DU exposure compared to wild-type (AA8) and XRCC3-complemented (1SFwt8) cells, but DU induced more break-type and fusion-type lesions in XRCC3-deficient cells compared to wild-type and XRCC3-complemented cells. Surprisingly, loss of RAD51D did not affect DU-induced cytotoxicity or genotoxicity. DU induced selective X-chromosome fragmentation irrespective of RAD51D status, but loss of XRCC3 nearly eliminated fragmentation observed after DU exposure in wild-type and XRCC3-complemented cells. Thus, XRCC3, but not RAD51D, protects cells from DU-induced breaks and fusions and also plays a role in DU-induced chromosome fragmentation

Role of XRCC4 phosphorylation by DNA-PK in the regulation of NHEJ repair pathway of DNA double strand break

International Nuclear Information System (INIS)

Sharma, Mukesh Kumar; Imamichi, Shoji; Fukuchi, Mikoto; Kamdar, Radhika P.; Sicheng, Liu; Wanotayan, Rujira; Matsumoto, Yoshihisa

2014-01-01

Non-homologous end-joining (NHEJ) is the predominant pathway of DNA double strand breaks in higher eukaryotes and is active throughout the cell cycle. NHEJ repair includes many factors as Ku70/86, DNA-PKcs, XRCC4-Ligase IV complex and XLF (also known as Cernunnos). In these factors, DNA-PKcs acts as central regulator in NHEJ repair. It recruited at the DNA damages site after DNA damage and after association with Ku its kinase activity is activated. It phosphorylates many of important NHEJ proteins in vitro including XRCC4, Ku 70/86, Artemis, and even DNA-PKcs but till now, very less studies have been done to know the role and significance of phosphorylation in the NHEJ repair. Studies by other researchers identified various phosphorylation sites in XRCC4 by DNA-PK using mass spectrometry but these phosphorylation sites were shown to be dispensable for DSB repair. In the present investigation, we identified 3 serine and one new threonine phosphorylation sites in XRCC4 protein by DNA-PK. In vivo phosphorylation at these sites was verified by generating phosphorylation specific antibodies and the requirement for DNA-PK therein was verified by using DNA-PK inhibitor and DNA-PK proficient and deficient cell lines in response to radiation and zeocin treatment. We have also found that phosphorylation at these sites showed dose dependency in response to radiation treatment. The two serine and one threonine phosphorylation site is also biological important as their mutation into alanine significantly elevated radiosensitivity as measured by colony formation assay. Neutral comet assay showed delayed kinetics in DSB repair of these mutants. Furthermore, we have found a protein, with putative DSB repair function, which interacts with domain including the phosphorylation sites.These results indicate that these phosphorylation sites would mediate functional link between XRCC4 and DNA-PK. (author)

Association Between Genetic Polymorphisms in the XRCC1, XRCC3, XPD, GSTM1, GSTT1, MSH2, MLH1, MSH3, and MGMT Genes and Radiosensitivity in Breast Cancer Patients

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Mangoni, Monica; Bisanzi, Simonetta; Carozzi, Francesca; Sani, Cristina; Biti, Giampaolo; Livi, Lorenzo; Barletta, Emanuela; Costantini, Adele Seniori; Gorini, Giuseppe

2011-01-01

Purpose: Clinical radiosensitivity varies considerably among patients, and radiation-induced side effects developing in normal tissue can be therapy limiting. Some single nucleotide polymorphisms (SNPs) have been shown to correlate with hypersensitivity to radiotherapy. We conducted a prospective study of 87 female patients with breast cancer who received radiotherapy after breast surgery. We evaluated the association between acute skin reaction following radiotherapy and 11 genetic polymorphisms in DNA repair genes: XRCC1 (Arg399Gln and Arg194Trp), XRCC3 (Thr241Met), XPD (Asp312Asn and Lys751Gln), MSH2 (gIVS12-6T>C), MLH1 (Ile219Val), MSH3 (Ala1045Thr), MGMT (Leu84Phe), and in damage-detoxification GSTM1 and GSTT1 genes (allele deletion). Methods and Materials: Individual genetic polymorphisms were determined by polymerase chain reaction and single nucleotide primer extension for single nucleotide polymorphisms or by a multiplex polymerase chain reaction assay for deletion polymorphisms. The development of severe acute skin reaction (moist desquamation or interruption of radiotherapy due to toxicity) associated with genetic polymorphisms was modeled using Cox proportional hazards, accounting for cumulative biologically effective radiation dose. Results: Radiosensitivity developed in eight patients and was increased in carriers of variants XRCC3-241Met allele (hazard ratio [HR] unquantifiably high), MSH2 gIVS12-6nt-C allele (HR = 53.36; 95% confidence intervals [95% CI], 3.56-798.98), and MSH3-1045Ala allele (HR unquantifiably high). Carriers of XRCC1-Arg194Trp variant allele in combination with XRCC1-Arg399Gln wild-type allele had a significant risk of radiosensitivity (HR = 38.26; 95% CI, 1.19-1232.52). Conclusions: To our knowledge, this is the first report to find an association between MSH2 and MSH3 genetic variants and the development of radiosensitivity in breast cancer patients. Our findings suggest the hypothesis that mismatch repair mechanisms may be

Effect of APE1 T2197G (Asp148Glu Polymorphism on APE1, XRCC1, PARP1 and OGG1 Expression in Patients with Colorectal Cancer

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Juliana C. Santos

2014-09-01

Full Text Available It has been hypothesized that genetic variation in base excision repair (BER might modify colorectal adenoma risk. Thus, we evaluated the influence of APE1 T2197G (Asp148Glu polymorphism on APE1, XRCC1, PARP1 and OGG1 expression in normal and tumor samples from patients with colorectal cancer. The results indicate a downregulation of OGG1 and an upregulation of XRCC1 expression in tumor tissue. Regarding the anatomical location of APE1, OGG1 and PARP-1, a decrease in gene expression was observed among patients with cancer in the rectum. In patients with or without some degree of tumor invasion, a significant downregulation in OGG1 was observed in tumor tissue. Interestingly, when taking into account the tumor stage, patients with more advanced grades (III and IV showed a significant repression for APE1, OGG1 and PARP-1. XRCC1 expression levels were significantly enhanced in tumor samples and were correlated with all clinical and histopathological data. Concerning the polymorphism T2197G, GG genotype carriers exhibited a significantly reduced expression of genes of the BER repair system (APE1, XRCC1 and PARP1. In summary, our data show that patients with colorectal cancer present expression changes in several BER genes, suggesting a role for APE1, XRCC1, PARP1 and OGG1 and APE1 polymorphism in colorectal carcinogenesis.

Implications of XRCC1, XPD and APE1 gene polymorphism in North Indian population: a comparative approach in different ethnic groups worldwide.

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Gangwar, Ruchika; Manchanda, Parmeet Kaur; Mittal, Rama Devi

2009-05-01

Identifying risk factors for human cancers should consider combinations of genetic variations and environmental exposures. Several polymorphisms in DNA repair genes have impact on repair and cancer susceptibility. We focused on X-ray repair cross-complementing group 1 (XRCC1), Xeroderma pigmentosum D (XPD) and apurinic/apyrimidinic endonuclease (APE1) as these are most extensively studied in cancer. Present study was conducted to determine distribution of XRCC1 C26304T, G27466A, G23591A, APE1 T2197G and XPD A35931C gene polymorphisms in North Indian population and compare with different populations globally. PCR-based analysis was conducted in 209 normal healthy individuals of similar ethnicity. Allelic frequencies in wild type of XRCC1 C26304T were 91.1% C(Arg); G27466A 62.9% G(Arg); G23591A 60.3% G(Arg); APE1 T2197G 75.1% T(Asp) and XPD A35931C 71.8% A(Lys). The variant allele frequency were 8.9% T(Trp) in XRCC1 C26304T; 37.1% A(His) in G27466A; 39.7% A(Gln) in G23591A; 24.9% G(Glu) in APE1 and 28.2% C(Gln) in XPD respectively. We further compared frequency distribution for these genes with various published studies in different ethnicity. Our results suggest that frequency in these DNA repair genes exhibit distinctive pattern in India that could be attributed to ethnicity variation. This could assist in high-risk screening of humans exposed to environmental carcinogens and cancer predisposition in different ethnic groups.

Agrobacterium May Delay Plant Nonhomologous End-Joining DNA Repair via XRCC4 to Favor T-DNA Integration[W

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Vaghchhipawala, Zarir E.; Vasudevan, Balaji; Lee, Seonghee; Morsy, Mustafa R.; Mysore, Kirankumar S.

2012-01-01

Agrobacterium tumefaciens is a soilborne pathogen that causes crown gall disease in many dicotyledonous plants by transfer of a portion of its tumor-inducing plasmid (T-DNA) into the plant genome. Several plant factors that play a role in Agrobacterium attachment to plant cells and transport of T-DNA to the nucleus have been identified, but the T-DNA integration step during transformation is poorly understood and has been proposed to occur via nonhomologous end-joining (NHEJ)–mediated double-strand DNA break (DSB) repair. Here, we report a negative role of X-RAY CROSS COMPLEMENTATION GROUP4 (XRCC4), one of the key proteins required for NHEJ, in Agrobacterium T-DNA integration. Downregulation of XRCC4 in Arabidopsis and Nicotiana benthamiana increased stable transformation due to increased T-DNA integration. Overexpression of XRCC4 in Arabidopsis decreased stable transformation due to decreased T-DNA integration. Interestingly, XRCC4 directly interacted with Agrobacterium protein VirE2 in a yeast two-hybrid system and in planta. VirE2-expressing Arabidopsis plants were more susceptible to the DNA damaging chemical bleomycin and showed increased stable transformation. We hypothesize that VirE2 titrates or excludes active XRCC4 protein available for DSB repair, thus delaying the closure of DSBs in the chromosome, providing greater opportunity for T-DNA to integrate. PMID:23064322

Preliminary Study on the Single Nucleotide Polymorphism (SNP of XRCC1 Gene Identificationto Improve the Outcomes of Radiotherapy for Cervical Cancer

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Devita Tetriana

2015-09-01

Full Text Available Cervical cancer is the most fatal disease among Indonesian women. In recognition of the substantial variation in the intrinsic response of individuals to radiation, an effort had been done to identify the genetic markers, primarily Single Nucleotide polymorphisms (SNPs, which are associated with responsiveness of cancer cells to radiation therapy. One of these SNPs is X-ray repair cross-complementing protein 1 (XRCC1 that is one of the most important genes in deoxyribonucleic acid (DNA repair pathways. Meta-analysis in the determination of the association of XRCC1 polymorphisms with cervical cancer revealed the potential role of XRCC1 polymorphisms in predicting cell response to radiotherapy.Our preliminary study with real-time polymerase chain reaction (RT-PCR showed that radiotherapy affected the XRCC1 gene analyzed in blood of cervical cancer patient. Other published study found three SNPs of XRCC1 (Arg194Trp, Arg280His, and Arg399Gln that cause amino acid substitutions. Arg194Trp is only SNPs that associated with high risk of cervical cancer but not others. Additionally, structure and function of this protein can be altered by functional SNPs, which may lead to the susceptibility of individuals to cancers. Anotherstudy found G399A polymorphisms. We concluded that SNP of this DNA repair genes have been found to be good predictors of efficacy of radiotherapy.Kanker serviks adalah penyakit yang paling fatal pada perempuan di Indonesia. Untuk memahami variasi substansial respon intrinsik individual terhadap radiasi, suatu usaha telah dilakukan untuk mengidentifikasi petanda genetik, terutama Single Nucleotide polymorphism (SNP, yang berkaitan dengan responsel kanker terhadap terapi radiasi. Satu dari SNP tersebut adalah X-ray repair cross-complementing protein 1 (XRCC1 yang merupakan satu dari gen paling penting dalam lajur perbaikan asam deoksiribonukleat (DNA. Meta-analysis dalam penentuan hubungan polimorfisme XRCC1 dengan kanker serviks

Cadmium delays non-homologous end joining (NHEJ) repair via inhibition of DNA-PKcs phosphorylation and downregulation of XRCC4 and Ligase IV

Energy Technology Data Exchange (ETDEWEB)

Li, Weiwei; Gu, Xueyan; Zhang, Xiaoning; Kong, Jinxin [Gansu Key Laboratory of Biomonitoring and Bioremediation for Environmental Pollution, School of Life Sciences, Lanzhou University, Lanzhou 730000 (China); Ding, Nan [Gansu Key laboratory of Space Radiobiology, Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou 730000 (China); Qi, Yongmei; Zhang, Yingmei [Gansu Key Laboratory of Biomonitoring and Bioremediation for Environmental Pollution, School of Life Sciences, Lanzhou University, Lanzhou 730000 (China); Wang, Jufang [Gansu Key laboratory of Space Radiobiology, Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou 730000 (China); Huang, Dejun, E-mail: huangdj@lzu.edu.cn [Gansu Key Laboratory of Biomonitoring and Bioremediation for Environmental Pollution, School of Life Sciences, Lanzhou University, Lanzhou 730000 (China)

2015-09-15

Highlights: • Cadmium (Cd) exposure delayed the repair of DNA damage induced by X-ray. • Cd exposure altered the phosphorylation of DNA-PKcs on Thr-2609 and Ser-2056 sites. • Cd impaired the formation of XRCC4 and Ligase IV foci, and down-regulated their protein expression. • Zinc mitigated the effects of Cd on DDR by regulating pDNA-PKcs (Thr-2609), XRCC4 and Ligase IV. - Abstract: Although studies have shown that cadmium (Cd) interfered with DNA damage repair (DDR), whether Cd could affect non-homologous end joining (NHEJ) repair remains elusive. To further understand the effect of Cd on DDR, we used X-ray irradiation of Hela cells as an in vitro model system, along with γH2AX and 53BP1 as markers for DNA damage. Results showed that X-ray significantly increased γH2AX and 53BP1 foci in Hela cells (p < 0.01), all of which are characteristic of accrued DNA damage. The number of foci declined rapidly over time (1–8 h postirradiation), indicating an initiation of NHEJ process. However, the disappearance of γH2AX and 53BP1 foci was remarkably slowed by Cd pretreatment (p < 0.01), suggesting that Cd reduced the efficiency of NHEJ. To further elucidate the mechanisms of Cd toxicity, several markers of NHEJ pathway including Ku70, DNA-PKcs, XRCC4 and Ligase IV were examined. Our data showed that Cd altered the phosphorylation of DNA-PKcs, and reduced the expression of both XRCC4 and Ligase IV in irradiated cells. These observations are indicative of the impairment of NHEJ-dependent DNA repair pathways. In addition, zinc (Zn) mitigated the effects of Cd on NHEJ, suggesting that the Cd-induced NHEJ alteration may partly result from the displacement of Zn or from an interference with the normal function of Zn-containing proteins by Cd. Our findings provide a new insight into the toxicity of Cd on NHEJ repair and its underlying mechanisms in human cells.

[Association of XRCC1 genetic polymorphism with susceptibility to non-Hodgkin's lymphoma].

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Li, Su-Xia; Zhu, Hong-Li; Guo, Bo; Yang, Yang; Wang, Hong-Yan; Sun, Jing-Fen; Cao, Yong-Bin

2014-08-01

The purpose of this study was to explore the association between X-ray repair cross-complementing group 1 (XRCC1)gene polymorphism and non-Hodgkin's lymphoma risk. A total of 282 non-Hodgkin's lymphoma (NHL) patients and 231 normal controls were used to investigate the effect of three XRCC1 gene polymorphisms (rs25487, rs25489, rs1799782) on susceptibility to non-Hodgkin's lymphoma. Genotyping was performed by using SNaPshot method. All statistical analyses were done with R software. Genotype and allele frequencies of XRCC1 were compared between the patients and controls by using the chi-square test. Crude and adjusted odd ratios and 95% confidence intervals were calculated by using logistic regression on the basis of genetic different models. For four kinds of NHL, subgroup analyses were also conducted. Combined genotype analyses of the three XRCC1 polymorphisms were also done by using logistic regression. The results showed that the variant genotype frequency was not significantly different between the controls and NHL or NHL subtype cases. Combined genotype analyses of XRCC1 399-280-194 results showed that the combined genotype was not associated with risk of NHL overall, but the VT-WT-WT combined genotype was associated with the decreased risk of T-NHL (OR: 0.21; 95%CI (0.06-0.8); P = 0.022), and the WT-VT-WT combined genotype was associated with the increased risk of FL(OR:15.23; 95%CI (1.69-137.39); P = 0.015). It is concluded that any studied polymorphism (rs25487, rs25489, rs1799782) alone was not shown to be rela-ted with the risk of NHL or each histologic subtype of NHL. The combined genotype with mutation of three SNP of XRCC1 was not related to the risk of NHL. However, further large-scale studies would be needed to confirm the association of decreased or increased risk for T-NHL and FL with the risk 3 combined SNP mutants of XRCC1 polymorphism.

Mutations in XRCC4 cause primordial dwarfism without causing immunodeficiency.

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Saito, Shinta; Kurosawa, Aya; Adachi, Noritaka

2016-08-01

In successive reports from 2014 to 2015, X-ray repair cross-complementing protein 4 (XRCC4) has been identified as a novel causative gene of primordial dwarfism. XRCC4 is indispensable for non-homologous end joining (NHEJ), the major pathway for repairing DNA double-strand breaks. As NHEJ is essential for V(D)J recombination during lymphocyte development, it is generally believed that abnormalities in XRCC4 cause severe combined immunodeficiency. Contrary to expectations, however, no overt immunodeficiency has been observed in patients with primordial dwarfism harboring XRCC4 mutations. Here, we describe the various XRCC4 mutations that lead to disease and discuss their impact on NHEJ and V(D)J recombination.

Studies on a role of XRCC4 in human cells

International Nuclear Information System (INIS)

Mori, M.; Itsukaichi, H.; Kanda, R.; Nakamura, A.; Shiomi, N.; Aizawa, S.; Shiomi, T.

2003-01-01

Full text: Ionizing radiation produces a variety of lesions in DNA including single-strand breaks, double-strand breaks and base damage. The repair of DNA double-strand breaks is essential for the maintenance of genomic integrity. Failure to repair DNA double-strand breaks result in loss of genetic information, chromosome translocations, carcinogenesis and cell death. XRCC4 is a member of non-homologous end-joining proteins that functioned in DNA double-strand break repair in eukaryote including human. XRCC4 is a DNA ligase IV accessory factor and required for the rejoining of DNA double-strand breaks. Both XRCC4 and DNA ligase IV deficient mice have been generated. Both deficient mice are not viable because of neuronal degeneration caused by p53-induced apoptosis. Cells obtained from XRCC4 or DNA ligase IV deficient embryo are viable, but show reduced cell proliferation and hypersensitivity to ionizing radiation. To study the role of XRCC4 in human cells, we tried to inactivate XRCC4 gene by using gene targeting technology in human colon cancer cell line, HCT116. We have succeeded to disrupt both alleles of XRCC4 gene. Heterozygous (XRCC4 +/-) cells showed reduced cell proliferation but normal X ray-sensitivity, indicating haploinsufficiency in cell proliferation but not in X ray-sensitivity. Homozygous (XRCC4 -/-) cells show reduced cell proliferation and increased chromosome aberrations, and are highly sensitive to X rays

Susceptibility to gastric cancer and polymorphisms of insertion/deletion at the intron 3 of the XRCC4 and VNTR at the promoter region of the XRCC5.

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Saadat, Mostafa; Pashaei, Samira; Amerizade, Foroozan

2015-07-01

The genes encoding X-ray repair cross-complementing group 4 (XRCC4; OMIM: 194363) and 5 (XRCC5; OMIM: 194364) are involved in repair of DNA double-strand breaks. To investigating the associations between polymorphisms of Insertion/Deletion (I/D, rs28360071) in the intron 3 of the XRCC4 and VNTR in the promoter region of the XRCC5 and risk of gastric cancer, the present study was carried out. We included 159 (56 females, 103 males) with gastric cancer and 242 (75 females, 167 males) healthy blood donors frequency matched for age and gender. Using PCR-based methods, the genotypes of the study polymorphisms were determined. The alleles of VNTR XRCC5 polymorphism divided into two groups: L (0 and 1 repeats) and H (2 and 3 repeats) alleles. For the I/D XRCC4 polymorphism, after stratification of the subjects according to their family history (FH) of cancer, either the ID (OR = 3.19, 95%CI: 1.35-7.50, P = 0.008) or the DD genotypes (OR = 4.62, 95%CI: 1.63-13.0, P = 0.004) among positive FH persons, increased the risk of gastric cancer compared with the reference group (persons who have negative FH and II genotype). For the VNTR XRCC5 polymorphism, the LH + HH genotypes among positive FH persons, increased the risk of gastric cancer compared with the reference group (persons who have negative FH and LL genotype) (OR = 2.88, 95%CI: 1.34-6.18, P = 0.006). Sensitivity analysis showed that the above mentioned associations were not occurred due to the maldistribution of the genotypes among missing data. The present study suggests that both polymorphisms of the XRCC4 and XRCC5 might be risk factors for gastric cancer development especially among persons with positive FH.

Search for novel remedies to augment radiation resistance of inhabitants of Fukushima and Chernobyl disasters: identifying DNA repair protein XRCC4 inhibitors.

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Sun, Mao-Feng; Chen, Hsin-Yi; Tsai, Fuu-Jen; Lui, Shu-Hui; Chen, Chih-Yi; Chen, Calvin Yu-Chian

2011-10-01

Two nuclear plant disasters occurring within a span of 25 years threaten health and genome integrity both in Fukushima and Chernobyl. Search for remedies capable of enhancing DNA repair efficiency and radiation resistance in humans appears to be a urgent problem for now. XRCC4 is an important enhancer in promoting repair pathway triggered by DNA double-strand break (DSB). In the context of radiation therapy, active XRCC4 could reduce DSB-mediated apoptotic effect on cancer cells. Hence, developing XRCC4 inhibitors could possibly enhance radiotherapy outcomes. In this study, we screened traditional Chinese medicine (TCM) database, TCM Database@Taiwan, and have identified three potent inhibitor agents against XRCC4. Through molecular dynamics simulation, we have determined that the protein-ligand interactions were focused at Lys188 on chain A and Lys187 on chain B. Intriguingly, the hydrogen bonds for all three ligands fluctuated frequently but were held at close approximation. The pi-cation interactions and ionic interactions mediated by o-hydroxyphenyl and carboxyl functional groups respectively have been demonstrated to play critical roles in stabilizing binding conformations. Based on these results, we reported the identification of potential radiotherapy enhancers from TCM. We further characterized the key binding elements for inhibiting the XRCC4 activities.

Radiation-induced XRCC4 association with chromatin DNA analyzed by biochemical fractionation

International Nuclear Information System (INIS)

Kamdar, R.P.; Matsumoto, Yoshihisa

2010-01-01

XRCC4, in association with DNA ligase IV, is thought to play a critical role in the ligation of two DNA ends in DNA double-strand break (DSB) repair through non-homologous end-joining (NHEJ) pathway. In the present study, we captured radiation-induced chromatin-recruitment of XRCC4 by biochemical fractionation using detergent Nonidet P-40. A subpopulation of XRCC4 changed into a form that is resistant to the extraction with 0.5% Nonidet P-40-containing buffer after irradiation. This form of XRCC4 was liberated by micrococcal nuclease treatment, indicating that it had been tethered to chromatin DNA. This chromatin-recruitment of XRCC4 could be seen immediately (<0.1 hr) after irradiation and remained up to 4 hr after 20 Gy irradiation. It was seen even after irradiation of small doses, id est (i.e.), 2 Gy, but the residence of XRCC4 on chromatin was very transient after 2 Gy irradiation, returning to near normal level in 0.2-0.5 hr after irradiation. The chromatin-bound XRCC4 represented only -1% of total XRCC4 molecules even after 20 Gy irradiation and the quantitative analysis using purified protein as the reference suggested that only a few XRCC4-DNA ligase IV complexes were recruited to each DNA end. We further show that the chromatin-recruitment of XRCC4 was not attenuated by wortmannin, an inhibitor of DNA-PK, or siRNA-mediated knockdown of the DNA-PK catalytic subunit (DNA-PKcs), indicating that this process does not require DNA-PKcs. These results would provide us with useful experimental tools and important insights to understand the DNA repair process through NHEJ pathway. (author)

Polymorphisms of XRCC1 genes and risk of nasopharyngeal carcinoma in the Cantonese population

International Nuclear Information System (INIS)

Cao, Yun; Miao, Xiao-Ping; Huang, Ma-Yan; Deng, Ling; Hu, Li-Fu; Ernberg, Ingemar; Zeng, Yi-Xin; Lin, Dong-Xin; Shao, Jian-Yong

2006-01-01

Nasopharyngeal carcinoma (NPC) is one of the most common cancers in southern China. In addition to environmental factors such as Epstein-Barr virus infection and diet, genetic susceptibility has been reported to play a key role in the development of this disease. The x-ray repair cross-complementing group 1 (XRCC1) gene is important in DNA base excision repair. We hypothesized that two common single nucleotide polymorphisms of XRCC1 (codons 194 Arg→Trp and 399 Arg→Gln) are related to the risk of NPC and interact with tobacco smoking. We sought to determine whether these genetic variants of the XRCC1 gene were associated with the risk of NPC among the Cantonese population in a hospital-based case control study using polymerase chain reaction-restriction fragment length polymorphism analysis. We conducted this study in 462 NPC patients and 511 healthy controls. After adjustment for sex and age, we found a reduced risk of developing NPC in individuals with the Trp194Trp genotype (OR = 0.48; 95% CI, 0.27–0.86) and the Arg194Trp genotype (OR = 0.79; 95% CI, 0.60–1.05) compared with those with the Arg194Arg genotype. Compared with those with the Arg399Arg genotype, the risk for NPC was not significantly different in individuals with the Arg399Gln genotype (OR = 0.82; 95% CI, 0.62–1.08) and the Gln399Gln genotype (OR = 1.20; 95% CI, 0.69–2.06). Further analyses stratified by gender and smoking status revealed a significantly reduced risk of NPC among males (OR = 0.32; 95% CI, 0.14–0.70) and smokers (OR = 0.34; 95% CI, 0.14–0.82) carrying the XRCC1 194Trp/Trp genotype compared with those carrying the Arg/Arg genotype. No association was observed between Arg399Gln variant genotypes and the risk of NPC combined with smoking and gender. Our findings suggest that the XRCC1 Trp194Trp variant genotype is associated with a reduced risk of developing NPC in Cantonese population, particularly in males and smokers. Larger studies are needed to confirm our findings

PARP inhibition versus PARP-1 silencing: different outcomes in terms of single-strand break repair and radiation susceptibility

International Nuclear Information System (INIS)

Godon, C.; Cordelieres, F.P.; Giocanti, N.; Megnin-Chanet, F.; Hall, J.; Favaudon, V.; Godon, C.; Giocanti, N.; Megnin-Chanet, F.; Hall, J.; Favaudon, V.; Cordelieres, F.P.; Cordelieres, F.P.; Biard, D.

2008-01-01

The consequences of PARP-1 disruption or inhibition on DNA single-strand break repair (SSBR) and radio-induced lethality were determined in synchronized, iso-genic HeLa cells stably silenced or not for poly(ADP-ribose) polymerase-1 (PARP-1) (PARP-1(KD)) or XRCC1 (XRCC1(KD)). PARP-1 inhibition prevented XRCC1-YFP recruitment at sites of 405 nm laser micro irradiation, slowed SSBR 10-fold and triggered the accumulation of large persistent foci of GFP-PARP-1 and GFP-PCNA at photo damaged sites. These aggregates are presumed to hinder the recruitment of other effectors of the base excision repair (BER) pathway.PARP-1 silencing also prevented XRCC1-YFP recruitment but did not lengthen the lifetime of GFP-PCNA foci. Moreover, PARP-1(KD) and XRCC1(KD) cells in S phase completed SSBR as rapidly as controls, while SSBR was delayed in G1. Taken together, the data demonstrate that a PARP-1- and XRCC1-independent SSBR pathway operates when the short patch repair branch of the BER is deficient. Long patch repair is the likely mechanism, as GFP-PCNA recruitment at photo-damaged sites was normal in PARP-1(KD) cells. PARP-1 silencing elicited hyper-radiosensitivity, while radiosensitization by a PARP inhibitor reportedly occurs only in those cells treated in S phase. PARP-1 inhibition and deletion thus have different outcomes in terms of SSBR and radiosensitivity. (authors)

Genetic polymorphisms in homologous recombination repair genes in healthy Slovenian population and their influence on DNA damage

International Nuclear Information System (INIS)

Goricar, Katja; Erculj, Nina; Zadel, Maja; Dolzan, Vita

2012-01-01

Homologous recombination (HR) repair is an important mechanism involved in repairing double-strand breaks in DNA and for maintaining genomic stability. Polymorphisms in genes coding for enzymes involved in this pathway may influence the capacity for DNA repair. The aim of this study was to select tag single nucleotide polymorphisms (SNPs) in specific genes involved in HR repair, to determine their allele frequencies in a healthy Slovenian population and their influence on DNA damage detected with comet assay. In total 373 individuals were genotyped for nine tag SNPs in three genes: XRCC3 722C>T, XRCC3 -316A>G, RAD51 -98G>C, RAD51 -61G>T, RAD51 1522T>G, NBS1 553G>C, NBS1 1197A>G, NBS1 37117C>T and NBS1 3474A>C using competitive allele-specific amplification (KASPar assay). Comet assay was performed in a subgroup of 26 individuals to determine the influence of selected SNPs on DNA damage. We observed that age significantly affected genotype frequencies distribution of XRCC3 -316A>G (P = 0.039) in healthy male blood donors. XRCC3 722C>T (P = 0.005), RAD51 -61G>T (P = 0.023) and NBS1 553G>C (P = 0.008) had a statistically significant influence on DNA damage. XRCC3 722C>T, RAD51 -61G>T and NBS1 553G>C polymorphisms significantly affect the repair of damaged DNA and may be of clinical importance as they are common in Slovenian population

Prognostic significance of XRCC4 expression in hepatocellular carcinoma

Science.gov (United States)

Huang, Xiao-Ying; Yao, Jin-Guang; Wang, Chao; Wei, Zhong-Hong; Ma, Yun; Wu, Xue-Min; Luo, Chun-Ying; Xia, Qiang; Long, Xi-Dai

2017-01-01

Background Our previous investigations have shown that the variants of X-ray repair complementing 4 (XRCC4) may be involved in hepatocellular carcinoma (hepatocarcinoma) tumorigenesis. This study aimed to investigate the possible prognostic significance of XRCC4 expression for hepatocarcinoma patients and possible value for the selection of transarterial chemoembolization (TACE) treatment. Materials and Methods We conducted a hospital-based retrospective analysis (including 421 hepatocarcinoma cases) to analyze the effects of XRCC4 on hepatocarcinoma prognosis and TACE. The levels of XRCC4 expression were tested using immunohistochemistry. The sensitivity of cancer cells to anti-cancer drug doxorubicin was evaluated using the half-maximal inhibitory concentration (IC50). Results XRCC4 expression was significantly correlated with pathological features including tumor stage, liver cirrhosis, and micro-vessel density. XRCC4 expression was an independent prognostic factor of hepatocarcinoma, and TACE treatments had no effects on prognosis of hepatocarcinoma patients with high XRCC4 expression. More intriguingly, TACE improved the prognosis of hepatocarcinoma patients with low XRCC4 expression. Functionally, XRCC4 overexpression increased while XRCC4 knockdown reduced the IC50 of cancer cells to doxorubicin. Conclusions These results suggest that XRCC4 may be an independent prognostic factor for hepatocarcinoma patients, and that decreasing XRCC4 expression may be beneficial for post-operative adjuvant TACE treatment in hepatocarcinoma. PMID:29152133

XRCC3 Thr241Met Polymorphism is not Associated with Lung Cancer Risk in a Romanian Population.

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Catana, Andreea; Pop, Monica; Marginean, Dragos Horea; Blaga, Ioana Cristina; Porojan, Mihai Dumitru; Popp, Radu Anghel; Pop, Ioan Victor

2016-01-01

Deoxyribonucleic Acid (DNA) repair mechanisms play a critical role in protecting the cellular genome against carcinogens. X-ray cross-complementing gene 3 (XRCC3) is involved in DNA repair and therefore certain genetic polymorphisms that occur in DNA repair genes may affect the ability to repair DNA defects and may represent a risk factor in carcinogenesis. The purpose of our study was to investigate the association between XRCC3 gene substitution of Threonine with Methionine in codon 241 of XRCC3 gene (Thr241Met) polymorphism and the risk of lung cancer, in a Romanian population. We recruited 93 healthy controls and 85 patients with lung cancer, all smokers. Thr241Met, XRCC3 gene genotyping was determined by multiplex Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP). Statistical analysis (OR, recessive model), did not revealed an increased risk for lung cancer, for the variant 241Met allele and Thr241Met genotypes (p=0.138, OR=0.634, CI=0.348-1.157; p=0.023, OR=0.257, CI=0.085-6.824). Also, there were no positive statistical associations between Thr241Met polymorphism of XRCC3 gene, gender, tobacco and various histopathological tumor type of lung cancer. In conclusion, the results of the study suggest that the XRCC3 gene Thr241Met polymorphism is not associated with an increased risk for the development of lung cancer in this Romanian group.

Association between XRCC1 polymorphism 399 G->A and glioma among Caucasians: a systematic review and meta-analysis

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Jacobs Daniel I

2012-10-01

Full Text Available Abstract Background The x-ray cross complementing group 1 gene (XRCC1 is crucial to proper repair of DNA damage such as single-strand DNA breaks. A non-synonymous polymorphism in XRCC1, 399 G → A, has been shown to reduce effectiveness of such DNA repair and has been associated with the risk of certain cancers. The known risk for glioma from high dose ionizing radiation makes associations between this polymorphism and glioma of particular interest. Methods A systematic literature review and meta-analysis was conducted to explore the association between XRCC1 399 G → A and glioma. Subgroup analyses by grade, gender, genotyping method, country in which study was conducted, and study size were conducted when data were available and validity of the results were assessed by influence analyses and exploration of potential publication bias. Results Six studies were eligible for meta-analysis including data on 2,362 Caucasian glioma cases and 3,085 Caucasian controls. Pooled analysis yielded a significant association between the variant of interest and risk of glioma (OR = 1.17, 95% CI: 1.05-1.30 which was found to be disproportionately driven by a single study. Exclusion of this study, in an influence analysis, produced no statistically significant evidence of association with glioma (OR = 1.10, 95% CI: 0.98-1.23, and no evidence of publication bias. Conclusions This meta-analysis does not suggest a major role of the XRCC1 399 G → A polymorphism in influencing risk of glioma among Caucasians. Future studies should report data separately for glioma subtypes to permit stratified analyses for Grade III and Grade IV glioma and examine other polymorphisms in this gene.

Radiation-induced damage to normal tissues after radiotherapy in patients treated for gynecologic tumors: Association with single nucleotide polymorphisms in XRCC1, XRCC3, and OGG1 genes and in vitro chromosomal radiosensitivity in lymphocytes

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Ruyck, Kim de; Eijkeren, Marc van; Claes, Kathleen; Morthier, Rudy; Paepe, Anne de; Vral, Anne; Ridder, Leo de; Thierens, Hubert

2005-01-01

Purpose: To examine the association of polymorphisms in XRCC1 (194Arg/Trp, 280Arg/His, 399Arg/Gln, 632Gln/Gln), XRCC3 (5' UTR 4.541A>G, IVS5-14 17.893A>G, 241Thr/Met), and OGG1 (326Ser/Cys) with the development of late radiotherapy (RT) reactions and to assess the correlation between in vitro chromosomal radiosensitivity and clinical radiosensitivity. Methods and Materials: Sixty-two women with cervical or endometrial cancer treated with RT were included in the study. According to the Common Terminology Criteria for Adverse Events, version 3.0, scale, 22 patients showed late adverse RT reactions. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assays were performed to examine polymorphic sites, the G2 assay was used to measure chromosomal radiosensitivity, and patient groups were compared using actuarial methods. Results: The XRCC3 IVS5-14 polymorphic allele was significantly associated with the risk of developing late RT reactions (odds ratio 3.98, p = 0.025), and the XRCC1 codon 194 variant showed a significant protective effect (p = 0.028). Patients with three or more risk alleles in XRCC1 and XRCC3 had a significantly increased risk of developing normal tissue reactions (odds ratio 10.10, p = 0.001). The mean number of chromatid breaks per cell was significantly greater in patients with normal tissue reactions than in patients with no reactions (1.16 and 1.34, respectively; p = 0.002). Patients with high chromosomal radiosensitivity showed a 9.2-fold greater annual risk of complications than patients with intermediate chromosomal radiosensitivity. Combining the G2 analysis with the risk allele model allowed us to identify 23% of the patients with late normal tissue reactions, without false-positive results. Conclusion: The results of the present study showed that clinical radiosensitivity is associated with an enhanced G2 chromosomal radiosensitivity and is significantly associated with a combination of different polymorphisms in

Genetic Polymorphisms in XRCC1, CD3EAP, PPP1R13L, XPB, XPC, and XPF and the Risk of Chronic Benzene Poisoning in a Chinese Occupational Population.

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Ping Xue

Full Text Available Individual variations in the capacity of DNA repair machinery to relieve benzene-induced DNA damage may be the key to developing chronic benzene poisoning (CBP, an increasingly prevalent occupational disease in China. ERCC1 (Excision repair cross complementation group 1 is located on chromosome 19q13.2-3 and participates in the crucial steps of Nucleotide Excision Repair (NER; moreover, we determined that one of its polymorphisms, ERCC1 rs11615, is a biomarker for CBP susceptibility in our previous report. Our aim is to further explore the deeper association between some genetic variations related to ERCC1 polymorphisms and CBP risk.Nine single nucleotide polymorphisms (SNPs of XRCC1 (X-ray repair cross-complementing 1, CD3EAP (CD3e molecule, epsilon associated protein, PPP1R13L (protein phosphatase 1, regulatory subunit 13 like, XPB (Xeroderma pigmentosum group B, XPC (Xeroderma pigmentosum group C and XPF (Xeroderma pigmentosum group F were genotyped by the Snapshot and TaqMan-MGB® probe techniques, in a study involving 102 CBP patients and 204 controls. The potential interactions between these SNPs and lifestyle factors, such as smoking and drinking, were assessed using a stratified analysis.An XRCC1 allele, rs25487, was related to a higher risk of CBP (P<0.001 even after stratifying for potential confounders. Carriers of the TT genotype of XRCC1 rs1799782 who were alcohol drinkers (OR = 8.000; 95% CI: 1.316-48.645; P = 0.022, male (OR = 9.333; 95% CI: 1.593-54.672; P = 0.019, and had an exposure of ≤12 years (OR = 2.612; 95% CI: 1.048-6.510; P = 0.035 had an increased risk of CBP. However, the T allele in PPP1R13L rs1005165 (P<0.05 and the GA allele in CD3EAP rs967591 (OR = 0.162; 95% CI: 0039~0.666; P = 0.037 decreased the risk of CBP in men. The haplotype analysis of XRCC1 indicated that XRCC1 rs25487A, rs25489G and rs1799782T (OR = 15.469; 95% CI: 5.536-43.225; P<0.001 were associated with a high risk of CBP.The findings showed that

Tetratricopeptide-motif-mediated interaction of FANCG with recombination proteins XRCC3 and BRCA2.

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Hussain, Shobbir; Wilson, James B; Blom, Eric; Thompson, Larry H; Sung, Patrick; Gordon, Susan M; Kupfer, Gary M; Joenje, Hans; Mathew, Christopher G; Jones, Nigel J

2006-05-10

Fanconi anaemia is an inherited chromosomal instability disorder characterised by cellular sensitivity to DNA interstrand crosslinkers, bone-marrow failure and a high risk of cancer. Eleven FA genes have been identified, one of which, FANCD1, is the breast cancer susceptibility gene BRCA2. At least eight FA proteins form a nuclear core complex required for monoubiquitination of FANCD2. The BRCA2/FANCD1 protein is connected to the FA pathway by interactions with the FANCG and FANCD2 proteins, both of which co-localise with the RAD51 recombinase, which is regulated by BRCA2. These connections raise the question of whether any of the FANC proteins of the core complex might also participate in other complexes involved in homologous recombination repair. We therefore tested known FA proteins for direct interaction with RAD51 and its paralogs XRCC2 and XRCC3. FANCG was found to interact with XRCC3, and this interaction was disrupted by the FA-G patient derived mutation L71P. FANCG was co-immunoprecipitated with both XRCC3 and BRCA2 from extracts of human and hamster cells. The FANCG-XRCC3 and FANCG-BRCA2 interactions did not require the presence of other FA proteins from the core complex, suggesting that FANCG also participates in a DNA repair complex that is downstream and independent of FANCD2 monoubiquitination. Additionally, XRCC3 and BRCA2 proteins co-precipitate in both human and hamster cells and this interaction requires FANCG. The FANCG protein contains multiple tetratricopeptide repeat motifs (TPRs), which function as scaffolds to mediate protein-protein interactions. Mutation of one or more of these motifs disrupted all of the known interactions of FANCG. We propose that FANCG, in addition to stabilising the FA core complex, may have a role in building multiprotein complexes that facilitate homologous recombination repair.

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

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

2009-01-01

predisposition to cancer risk. To address this question in more detail, we conducted a hospital-based case-control study consisting of 55 lung cancer cases and 74 cancer-free controls matched on age and ethnicity among nonsmoking Chinese women. We analyzed five coding single-nucleotide polymorphisms in the XRCC1...

DNA Repair Gene (XRCC1 Polymorphism (Arg399Gln Associated with Schizophrenia in South Indian Population: A Genotypic and Molecular Dynamics Study.

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S P Sujitha

Full Text Available This paper depicts the first report from an Indian population on the association between the variant Arg399Gln of XRCC1 locus in the DNA repair system and schizophrenia, the debilitating disease that affects 1% of the world population. Genotypic analysis of a total of 523 subjects (260 patients and 263 controls revealed an overwhelming presence of Gln399Gln in the case subjects against the controls (P < 0.0068, indicating significant level of association of this nsSNP with schizophrenia; the Gln399 allele frequency was also perceptibly more in cases than in controls (p < 0.003; OR = 1.448. The results of the genotypic studies were further validated using pathogenicity and stability prediction analysis employing computational tools [I-Mutant Suite, iStable, PolyPhen2, SNAP, and PROVEAN], with a view toassess the magnitude of deleteriousness of the mutation. The pathogenicity analysis reveals that the nsSNP could be deleterious inasmuch as it could affect the functionality of the gene, and interfere with protein function. Molecular dynamics simulation of 60ns was performed using GROMACS to analyse structural change due to a mutation (Arg399Gln that was never examined before. RMSD, RMSF, hydrogen bonds, radius of gyration and SASA analysis showedthe existence of asignificant difference between the native and the mutant protein. The present study gives astrong indication that the XRCC1 locus deserves serious attention, as it could be a potential candidatecontributing to the etio-pathogenesis of the disease.

DNA repair and cyclin D1 polymorphisms and styrene-induced genotoxicity and immunotoxicity

International Nuclear Information System (INIS)

Kuricova, M.; Naccarati, A.; Kumar, R.; Koskinen, M.; Sanyal, S.; Dusinska, M.; Tulinska, J.; Vodickova, L.; Liskova, A.; Jahnova, E.; Fuortes, L.; Haufroid, V.; Hemminki, K.; Vodicka, P.

2005-01-01

1-SO-adenine DNA adducts, DNA single-strand breaks (SBs), chromosomal aberrations (CAs), mutant frequency (MF) at the HPRT gene, and immune parameters (hematological and of humoral immunity) were studied in styrene-exposed human subjects and controls. Results were correlated with genetic polymorphisms in DNA repair genes (XPD, exon 23, XPG, exon 15, XPC, exon 15, XRCC1, exon 10, XRCC3, exon 7) and cell cycle gene cyclin D1. Results for biomarkers of genotoxicity after stratification for the different DNA repair genetic polymorphisms showed that the polymorphism in exon 23 of the XPD gene modulates levels of chromosomal and DNA damage, HPRT MF, and moderately affects DNA adduct levels. The highest levels of biomarkers were associated with the wild-type homozygous AA genotype. The exposed individuals with the wild-type GG genotype for XRCC1 gene exhibited the lowest CA frequencies, compared to those with an A allele (P < 0.05). Cyclin D1 polymorphism seems to modulate the number of leukocytes and lymphocytes in the analyzed subjects. The number of eosinophiles was positively associated with XPD variant C allele and negatively with XRCC1 variant A allele (P < 0.05) and XPC variant C allele (P < 0.05). Immunoglobulin IgA was positively associated with an XRCC3 variant T allele (P < 0.01) and negatively with XPC variant C allele (P < 0.05). Both C3- and C4-complement components were lower in individuals with XRCC3 CT (P < 0.05) and TT genotypes (P < 0.01). Adhesion molecules sL-selectin and sICAM-1 were associated with XPC genotype (P < 0.05). Individual susceptibility may be reflected in genotoxic and immunotoxic responses to environmental and occupational exposures to xenobiotics

C-terminal region of DNA ligase IV drives XRCC4/DNA ligase IV complex to chromatin

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Liu, Sicheng; Liu, Xunyue; Kamdar, Radhika Pankaj; Wanotayan, Rujira; Sharma, Mukesh Kumar; Adachi, Noritaka; Matsumoto, Yoshihisa

2013-01-01

Highlights: •Chromatin binding of XRCC4 is dependent on the presence of DNA ligase IV. •C-terminal region of DNA ligase IV alone can recruit itself and XRCC4 to chromatin. •Two BRCT domains of DNA ligase IV are essential for the chromatin binding of XRCC4. -- Abstract: DNA ligase IV (LIG4) and XRCC4 form a complex to ligate two DNA ends at the final step of DNA double-strand break (DSB) repair through non-homologous end-joining (NHEJ). It is not fully understood how these proteins are recruited to DSBs. We recently demonstrated radiation-induced chromatin binding of XRCC4 by biochemical fractionation using detergent Nonidet P-40. In the present study, we examined the role of LIG4 in the recruitment of XRCC4/LIG4 complex to chromatin. The chromatin binding of XRCC4 was dependent on the presence of LIG4. The mutations in two BRCT domains (W725R and W893R, respectively) of LIG4 reduced the chromatin binding of LIG4 and XRCC4. The C-terminal fragment of LIG4 (LIG4-CT) without N-terminal catalytic domains could bind to chromatin with XRCC4. LIG4-CT with W725R or W893R mutation could bind to chromatin but could not support the chromatin binding of XRCC4. The ability of C-terminal region of LIG4 to interact with chromatin might provide us with an insight into the mechanisms of DSB repair through NHEJ

Dynamic regulation of cerebral DNA repair genes by psychological stress

DEFF Research Database (Denmark)

Forsberg, Kristin; Aalling, Nadia; Wörtwein, Gitta

2015-01-01

Neuronal genotoxic insults from oxidative stress constitute a putative molecular link between stress and depression on the one hand, and cognitive dysfunction and dementia risk on the other. Oxidative modifications to DNA are repaired by specific enzymes; a process that plays a critical role...... restraint stress (6h/day) or daily handling (controls), and sacrificed after 1, 7 or 21 stress sessions. The mRNA expression of seven genes (Ogg1, Ape1, Ung1, Neil1, Xrcc1, Ercc1, Nudt1) involved in the repair of oxidatively damaged DNA was determined by quantitative real time polymerase chain reaction...

ERCC1 Cys8092Ala and XRCC1 Arg399Gln polymorphisms predict progression-free survival after curative radiotherapy for nasopharyngeal carcinoma.

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Hekun Jin

Full Text Available BACKGROUND: Single nucleotide polymorphisms (SNPs in DNA repair genes can alter gene expression and activity and affect response to cancer treatment and, correspondingly, survival. The present study was designed to evaluate the utility of the XRCC1 Arg399Gln and ERCC1 Cys8092Ala SNPs, measured in pretreatment biopsy samples, as predictors of response to radiotherapy in patients with non-metastatic nasopharyngeal carcinoma (NPC. MATERIALS AND METHODS: The study included 75 consecutive patients with stage II-IVA-B NPC. XRCC1 Arg399Glu and ERCC1 Cys8092Ala SNPs were identified from paraffin-embedded biopsy specimens via Sanger sequencing. Expression of p53 and pAkt protein was analyzed by immunohistochemical staining. Potential relationships between genetic polymorphisms and progression-free survival (PFS were analyzed by using a Cox proportional hazards model, the Kaplan-Meier method, and the log-rank test. RESULTS: Multivariate analysis showed that carriers of the ERCC1 8092 Ala/Ala genotype [hazard ratio (HR 1.882; 95% confidence interval (CI 1.031-3.438; P = 0.039] and heavy smokers (≥20 pack-years carrying the XRCC1 Arg/Arg genotype (HR 2.019; 95% CI 1.010-4.036; P = 0.047 had significantly lower PFS rates. Moreover, combined positive expression of p53 and pAkt led to significantly increased PFS in subgroups carrying the XRCC1 Gln allele (HR 7.057; 95% CI 2.073-24.021; P = 0.002 or the ERCC1 Cys allele (HR 2.568; 95% CI 1.056-6.248; P = 0.038. CONCLUSIONS: The ERCC1 Cys8092Ala polymorphism is an independent predictor of response to radiotherapy for NPC, and the XRCC1 Arg399Glu mutation combined with smoking status seems to predict PFS as well. Our results further suggest a possible correlation between these genetic polymorphisms and p53 protein status on survival.

Association between Single Nucleotide Polymorphisms in XRCC3 and Radiation-Induced Adverse Effects on Normal Tissue: A Meta-Analysis.

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Yu-Zhe Song

Full Text Available The X-ray repair cross-complementing group 3 (XRCC3 protein plays an important role in the repair of DNA double-strand breaks. The relationship between XRCC3 polymorphisms and the risk of radiation-induced adverse effects on normal tissue remains inconclusive. Thus, we performed a meta-analysis to elucidate the association between XRCC3 polymorphisms and radiation-induced adverse effects on normal tissue. All eligible studies up to December 2014 were identified through a search of the PubMed, Embase and Web of Science databases. Seventeen studies involving 656 cases and 2193 controls were ultimately included in this meta-analysis. The pooled odds ratios (ORs with corresponding 95% confidence intervals (CIs were calculated to evaluate the association between XRCC3 polymorphisms and the risk of radiation-induced normal tissue adverse effects. We found that the XRCC3 p.Thr241Met (rs861539 polymorphism was significantly associated with early adverse effects induced by radiotherapy (OR = 1.99, 95%CI: 1.31-3.01, P = 0.001. A positive association lacking statistical significance with late adverse effects was also identified (OR = 1.28, 95%CI: 0.97-1.68, P = 0.08. In addition, the rs861539 polymorphism was significantly correlated with a higher risk of adverse effects induced by head and neck area irradiation (OR = 2.41, 95%CI: 1.49-3.89, p = 0.0003 and breast irradiation (OR = 1.41, 95%CI: 1.02-1.95, p = 0.04, whereas the correlation was not significant for lung irradiation or pelvic irradiation. Furthermore, XRCC3 rs1799794 polymorphism may have a protective effect against late adverse effects induced by radiotherapy (OR = 0.47, 95%CI: 0.26-0.86, P = 0.01. Well-designed large-scale clinical studies are required to further validate our results.

Rad51C deficiency destabilizes XRCC3, impairs recombination and radiosensitizes S/G2-phase cells

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Lio, Yi-Ching; Schild, David; Brenneman, Mark A.; Redpath, J. Leslie; Chen, David J.

2004-05-01

The highly conserved Rad51 protein plays an essential role in repairing DNA damage through homologous recombination. In vertebrates, five Rad51 paralogs (Rad51B, Rad51C, Rad51D, XRCC2, XRCC3) are expressed in mitotically growing cells, and are thought to play mediating roles in homologous recombination, though their precise functions remain unclear. Here we report the use of RNA interference to deplete expression of Rad51C protein in human HT1080 and HeLa cells. In HT1080 cells, depletion of Rad51C by small interfering RNA caused a significant reduction of frequency in homologous recombination. The level of XRCC3 protein was also sharply reduced in Rad51C-depleted HeLa cells, suggesting that XRCC3 is dependent for its stability upon heterodimerization with Rad51C. In addition, Rad51C-depleted HeLa cells showed hypersensitivity to the DNA cross-linking agent mitomycin C, and moderately increased sensitivity to ionizing radiation. Importantly, the radiosensitivity of Rad51C-deficient HeLa cells was evident in S and G{sub 2}/M phases of the cell cycle but not in G{sub 1} phase. Together, these results provide direct cellular evidence for the importance of human Rad51C in homologous recombinational repair.

In cellulo phosphorylation of XRCC4 Ser320 by DNA-PK induced by DNA damage

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Sharma, Mukesh Kumar; Imamichi, Shoji; Fukuchi, Mikoto; Samarth, Ravindra Mahadeo; Tomita, Masanori; Matsumoto, Yoshihisa

2016-01-01

XRCC4 is a protein associated with DNA Ligase IV, which is thought to join two DNA ends at the final step of DNA double-strand break repair through non-homologous end joining. In response to treatment with ionizing radiation or DNA damaging agents, XRCC4 undergoes DNA-PK-dependent phosphorylation. Furthermore, Ser260 and Ser320 (or Ser318 in alternatively spliced form) of XRCC4 were identified as the major phosphorylation sites by purified DNA-PK in vitro through mass spectrometry. However, it has not been clear whether these sites are phosphorylated in vivo in response to DNA damage. In the present study, we generated an antibody that reacts with XRCC4 phosphorylated at Ser320 and examined in cellulo phosphorylation status of XRCC4 Ser320. The phosphorylation of XRCC4 Ser320 was induced by γ-ray irradiation and treatment with Zeocin. The phosphorylation of XRCC4 Ser320 was detected even after 1 Gy irradiation and increased in a manner dependent on radiation dose. The phosphorylation was observed immediately after irradiation and remained mostly unchanged for up to 4 h. The phosphorylation was inhibited by DNA-PK inhibitor NU7441 and was undetectable in DNA-PKcs-deficient cells, indicating that the phosphorylation was mainly mediated by DNA-PK. These results suggested potential usefulness of the phosphorylation status of XRCC4 Ser320 as an indicator of DNA-PK functionality in living cells

The relationship between XRCC1 and XRCC3 gene polymorphisms and lung cancer risk in northeastern Chinese.

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Shujie Guo

Full Text Available BACKGROUND: The prevalence of lung cancer in China will be the world's highest if allowed to proceed uncurbed. To unravel its genetic underpinnings, we sought to investigate the association of three well-characterized nonsynonymous polymorphisms in XRCC1 (Arg194Trp and Arg399Gln and XRCC3 (Thr241Met genes with lung cancer risk in northeastern Chinese. METHODOLOGY/PRINCIPAL FINDINGS: This study was hospital-based in design, encompassing 684 patients with lung cancer and 604 cancer-free controls. Genotyping was performed using the PCR-LDR (ligase detection reactions method. Data were analyzed by R language and multifactor dimensionality reduction (MDR software. Single-locus analysis identified significance in genotype distributions of polymorphism Arg194Trp (P = 0.002 and Arg399Gln (P = 0.017, and in allele distributions of Thr241Met (P = 0.005. Carriers of 399Gln/Gln genotype conferred a 147% increased risk relative to the non-carriers (odds ratio (OR: 2.47; 95% confidence interval (95% CI: 1.48-4.13; P<0.001. For Thr241Met, significance persisted under allelic (OR = 1.63; 95% CI: 1.14-2.33; P = 0.005, additive (OR = 1.64; 95% CI: 1.16-2.32; P = 0.005 and dominant (OR = 1.67; 95% CI: 1.17-2.38; P = 0.004 models. However, common allele combinations were comparable in frequency between patients and controls. In interaction analysis, the overall best MDR model included Arg399Gln and Thr241Met polymorphisms, with a maximal testing accuracy of 63.18% and a maximal cross-validation consistency of 10 out of 10 (P = 0.0175. CONCLUSIONS: Our study significantly demonstrated an independent and synergistic contribution of XRCC1 Arg399Gln and XRCC3 Thr241Met polymorphisms to lung cancer susceptibility in northeastern Chinese.

Relationship between XRCC1 polymorphism and acute complication of chemoradiation therapy in the patients with colorectal cancer

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Kim, Woo Chul; Choi, Sun Keun [Inha University College of Medicine, Incheon (Korea, Republic of); Hong, Yun Chul [Seoul National University College of Medicine, Seoul (Korea, Republic of)] (and others)

2006-03-15

It is well known from clinical experience that acute complications of chemoradiation therapy vary from patients to patients. However, there are no known factors to predict these acute complications before treatment starts. The human XRCC1 gene is known as a DNA base excision repair gene. We investigated the possibilities of XRCC1 gene polymorphisms as a predictor for the acute complications of chemoradiation therapy in colorectal cancer patients. From July 1997 to June 2003, 86 colorectal cancer patients (71 rectal cancer, 13 sigmoid colon cancer and 2 colon cancer patients) were treated with chemoradiation therapy at the Department of Radiation Oncology, Inha University Hospital. Twenty-two patients were in stage B, 50 were in stage C, 8 were in stage D and 6 patients were unresectable cases. External radiation therapy was delivered with 10MV X-ray at a 1.8 Gy fraction per day for a total dose of radiation of 30.6 {approx} 59.4 Gy (median: 54 Gy). All the patients received 5-FU based chemotherapy regimen. We analyzed the acute complications of upper and lower gastrointestinal tract based on the RTOG complication scale. The initial and lowest WBC and platelet count were recorded during both the RT period and the whole treatment period. Allelic variants of the XRCC1 gene at codons 194, 280 and 399 were analyzed in the lymphocyte DNA by performing PCR-RFLP. Statistical analyses were carried out with the SAS (version 6.12) statistical package. When all the variables were assessed on the multivariate analysis, recurrent disease revealed the factors that significantly correlated with upper gastrointestinal acute complications. Arg399Gln polymorphisms of the XRCC1 gene, the radiation dose and the frequencies of chemotherapy during radiation therapy were significantly correlated with lower gastrointestinal complications. Arg399Gln polymorphisms also affected the decrease of the WBC and platelet count during radiation therapy. Although the present sample size was too small

Relationship between XRCC1 polymorphism and acute complication of chemoradiation therapy in the patients with colorectal cancer

International Nuclear Information System (INIS)

Kim, Woo Chul; Choi, Sun Keun; Hong, Yun Chul

2006-01-01

It is well known from clinical experience that acute complications of chemoradiation therapy vary from patients to patients. However, there are no known factors to predict these acute complications before treatment starts. The human XRCC1 gene is known as a DNA base excision repair gene. We investigated the possibilities of XRCC1 gene polymorphisms as a predictor for the acute complications of chemoradiation therapy in colorectal cancer patients. From July 1997 to June 2003, 86 colorectal cancer patients (71 rectal cancer, 13 sigmoid colon cancer and 2 colon cancer patients) were treated with chemoradiation therapy at the Department of Radiation Oncology, Inha University Hospital. Twenty-two patients were in stage B, 50 were in stage C, 8 were in stage D and 6 patients were unresectable cases. External radiation therapy was delivered with 10MV X-ray at a 1.8 Gy fraction per day for a total dose of radiation of 30.6 ∼ 59.4 Gy (median: 54 Gy). All the patients received 5-FU based chemotherapy regimen. We analyzed the acute complications of upper and lower gastrointestinal tract based on the RTOG complication scale. The initial and lowest WBC and platelet count were recorded during both the RT period and the whole treatment period. Allelic variants of the XRCC1 gene at codons 194, 280 and 399 were analyzed in the lymphocyte DNA by performing PCR-RFLP. Statistical analyses were carried out with the SAS (version 6.12) statistical package. When all the variables were assessed on the multivariate analysis, recurrent disease revealed the factors that significantly correlated with upper gastrointestinal acute complications. Arg399Gln polymorphisms of the XRCC1 gene, the radiation dose and the frequencies of chemotherapy during radiation therapy were significantly correlated with lower gastrointestinal complications. Arg399Gln polymorphisms also affected the decrease of the WBC and platelet count during radiation therapy. Although the present sample size was too small for

ERCC1 and XRCC1 but not XPA single nucleotide polymorphisms correlate with response to chemotherapy in endometrial carcinoma

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Chen L

2016-11-01

Full Text Available Liang Chen,1 Mei-Mei Liu,1 Hui Liu,1 Dan Lu,2 Xiao-Dan Zhao,3 Xue-Jing Yang4 1Department of Gynecology and Obstetrics, 2Department of Oncology, 3Department of Clinical Laboratory, The 2nd Affiliated Hospital, Harbin Medical University, 4Nursing Department, Harbin Chest Hospital, Harbin, People’s Republic of China Abstract: Our study aimed to investigate the correlation between single nucleotide polymorphisms of ERCC1/XRCC1/XPA genes and postoperative chemotherapy efficacy and prognosis of endometrial carcinoma. Our study included 108 patients with endometrial carcinoma and 100 healthy participants. ERCC1 rs11615/XRCC1 rs25487/XPA rs1800975 gene polymorphisms were detected by polymerase chain reaction–restriction fragment length polymorphism. Then the chemotherapy efficacy and toxic effects of the patients were assessed. The genotype and allele frequency of ERCC1 rs11615/XRCC1 rs25487 in the case group were significantly different from that in the control group (all P<0.05. The patients with AA + GA in ERCC1 rs11615 had an increased risk of endometrial carcinoma than those with GG, and the risk of endometrial carcinoma for patients with AA + GA was also higher in comparison with patients with GG genotype in XRCC1 rs25487 (all P<0.05. GG on both ERCC1 rs11615/XRCC1 rs25487 had a higher effective rate of chemotherapy than GA + AA (all P<0.05. ERCC1 rs11615/XRCC1 rs25487 gene polymorphisms were linked with toxic effects in liver, kidney, and nervous system. ERCC1 rs11615/XRCC1 rs25487, muscular invasion, and tumor stage were independent risk factors for the prognosis of endometrial carcinoma (all P<0.05. However, no significant associations were observed between XPA rs1800975 polymorphism and chemotherapy efficacy and prognosis of endometrial carcinoma (all P>0.05. These results indicated that ERCC1 and XRCC1 but not XPA polymorphisms correlate with response to chemotherapy in endometrial carcinoma. Keywords: ERCC1, XRCC1, XPA, single nucleotide

Mutations in the NHEJ component XRCC4 cause primordial dwarfism.

Science.gov (United States)

Murray, Jennie E; van der Burg, Mirjam; IJspeert, Hanna; Carroll, Paula; Wu, Qian; Ochi, Takashi; Leitch, Andrea; Miller, Edward S; Kysela, Boris; Jawad, Alireza; Bottani, Armand; Brancati, Francesco; Cappa, Marco; Cormier-Daire, Valerie; Deshpande, Charu; Faqeih, Eissa A; Graham, Gail E; Ranza, Emmanuelle; Blundell, Tom L; Jackson, Andrew P; Stewart, Grant S; Bicknell, Louise S

2015-03-05

Non-homologous end joining (NHEJ) is a key cellular process ensuring genome integrity. Mutations in several components of the NHEJ pathway have been identified, often associated with severe combined immunodeficiency (SCID), consistent with the requirement for NHEJ during V(D)J recombination to ensure diversity of the adaptive immune system. In contrast, we have recently found that biallelic mutations in LIG4 are a common cause of microcephalic primordial dwarfism (MPD), a phenotype characterized by prenatal-onset extreme global growth failure. Here we provide definitive molecular genetic evidence supported by biochemical, cellular, and immunological data for mutations in XRCC4, encoding the obligate binding partner of LIG4, causing MPD. We report the identification of biallelic mutations in XRCC4 in five families. Biochemical and cellular studies demonstrate that these alterations substantially decrease XRCC4 protein levels leading to reduced cellular ligase IV activity. Consequently, NHEJ-dependent repair of ionizing-radiation-induced DNA double-strand breaks is compromised in XRCC4 cells. Similarly, immunoglobulin junctional diversification is impaired in cells. However, immunoglobulin levels are normal, and individuals lack overt signs of immunodeficiency. Additionally, in contrast to individuals with LIG4 mutations, pancytopenia leading to bone marrow failure has not been observed. Hence, alterations that alter different NHEJ proteins give rise to a phenotypic spectrum, from SCID to extreme growth failure, with deficiencies in certain key components of this repair pathway predominantly exhibiting growth deficits, reflecting differential developmental requirements for NHEJ proteins to support growth and immune maturation. Copyright © 2015 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Genetic polymorphisms of XRCC1 (codon 399) and susceptibility to breast cancer in Iranian women, a case-control study.

Science.gov (United States)

Saadat, Mostafa; Kohan, Leila; Omidvari, Shahpour

2008-10-01

The X-ray repair cross-complementation group 1 (XRCC1) protein plays an important role in base excision repair. In the present study, we specifically investigated whether common genetic variant in XRCC1 (exon 10, codon Arg399Gln) was associated with an altered risk of breast cancer. The eligible cases were patients at chemotherapy unit of Nemazi hospital, Shiraz Iran, from October 1999 to August 2000 and from July 2004 to July 2005. The present study included 186 females with breast cancer. Age frequency-matched controls were randomly selected from the healthy females blood donor, according to the age distribution of the cases. A total of 187 healthy females included in the study. Using PCR-based method, the XRCC1 Arg399Gln polymorphism was determined. In control group the 399Gln allele frequency was 32.6%. The genotypic frequencies of the control subjects did not show significant deviation from Hardy-Weinberg equilibrium (chi(2) = 1.683, df = 1, P > 0.05). A statistically significant association was observed in comparison between Gln/Gln and Arg/Arg genotype (OR = 2.01, 95% CI:1.02-3.94, P = 0.041). There was no linear trend for presence of 0, 1, and 2 of the 399Gln allele and risk of breast cancer (chi(2) = 1.212, P = 0.271). In the recessive effect of the Gln allele (comparison between Gln/Gln vs. Arg/Arg+Arg/Gln), Gln/Gln genotype significantly increased the risk of breast cancer (OR = 2.31, 95% CI:1.21-4.35, P = 0.010). In the dominant effect of the Gln allele (comparison between Gln/Gln+Arg/Gln vs. Arg/Arg), Gln/Gln+Arg/Gln genotypes was not associated with the risk of breast cancer (OR = 0.95, 95% CI:0.63-1.42, P = 0.799). It is concluded that 399Gln allele may act as a recessive allele and increase the breast cancer risk.

DNA Repair and Ethnic Differences in Prostate Cancer Risk

National Research Council Canada - National Science Library

Goldman, Radoslav

2008-01-01

.... To evaluate this hypothesis we quantify DNA repair capacity in blood cells using comet assay and evaluate how this repair capacity is related to genetic variants in OGG1 and XRCC1 DNA repair genes...

DNA Repair and Ethnic Differences in Prostate Cancer Risk

National Research Council Canada - National Science Library

Goldman, Radoslav

2007-01-01

.... To evaluate this hypothesis we quantify DNA repair capacity in blood cells using comet assay and evaluate how this repair capacity is related to genetic variants in OGG1 and XRCC1 DNA repair genes...

DNA Repair and Ethnic Differences in Prostate Cancer Risk

National Research Council Canada - National Science Library

Goldman, Radoslav

2006-01-01

.... To evaluate this hypothesis, we quantify DNA repair capacity in blood cells using comet assay and evaluate how this repair capacity is related to genetic variants in OGG1 and XRCC1 DNA repair genes...

Association between polymorphisms in XRCC1 gene and treatment outcomes of patients with advanced gastric cancer: a systematic review and meta-analysis.

Directory of Open Access Journals (Sweden)

Zhuo Cao

Full Text Available BACKGROUND: Many reports have shown inconsistent results on the relationship between single nucleotide polymorphisms (SNPs of X-ray repair cross complementing protein (XRCC1 gene and platinum-based chemotherapeutic efficacy. This meta-analysis aimed to summarize published data about the association between two SNPs of XRCC1 (Arg194Trp and Arg399Gln and treatment outcomes of patients with advanced gastric cancer. METHODOLOGY/PRINCIPAL FINDINGS: We retrieved the relevant articles from MEDLINE, Web of Knowledge, and the China National Knowledge Infrastructure (CNKI databases. Studies were selected according to specific inclusion and exclusion criteria. Study quality was assessed according to the guidelines outlined by Hayden, et al. and PRISMA guidelines. We estimated the odds ratio (OR for response rate versus no response after platinum-based chemotherapy. Progression-free survival (PFS and overall survival (OS were evaluated by pooled Cox proportional hazard ratios (HRs and 95% confidence intervals (CIs. We found that none of the XRCC1 Arg194Trp and Arg399Gln polymorphisms was significantly associated with tumor response. Stratified analysis by ethnicity or sensitivity analysis also showed that XRCC1 SNPs were not related with chemotherapy response. Patients with minor variant A allele were likely to have poorer 2-year survival rate than those with G/G genotype. However, in the group of 5-year follow up, there was no significant association between the A allele and OS yet. CONCLUSIONS/SIGNIFICANCE: There is no evidence to support the use of XRCC1 Arg194Trp and Arg399Gln polymorphisms as prognostic predictors of TR and PFS in gastric patients treated with platinum-based chemotherapy. The relationship between minor variant A allele and OS requires further verification.

XRCC1 Polymorphism Associated With Late Toxicity After Radiation Therapy in Breast Cancer Patients

Energy Technology Data Exchange (ETDEWEB)

Seibold, Petra; Behrens, Sabine [Division of Cancer Epidemiology, German Cancer Research Center, Heidelberg (Germany); Schmezer, Peter [Division of Epigenomics and Cancer Risk Factors, German Cancer Research Center, Heidelberg (Germany); Helmbold, Irmgard [Division of Cancer Epidemiology, German Cancer Research Center, Heidelberg (Germany); Barnett, Gillian; Coles, Charlotte [Department of Oncology, Oncology Centre, Cambridge University Hospital NHS Foundation Trust, United Kingdom (UK) (United Kingdom); Yarnold, John [Institute of Cancer Research and Royal Marsden NHS Foundation Trust, London (United Kingdom); Talbot, Christopher J. [Department of Genetics, University of Leicester, Leicester (United Kingdom); Imai, Takashi [Advanced Radiation Biology Research Program, National Institute of Radiological Sciences, Chiba (Japan); Azria, David [Department of Radiation Oncology and Medical Physics, I.C.M. – Institut regional du Cancer Montpellier, Montpellier (France); Koch, C. Anne [Radiation Medicine Program, Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario (Canada); Dunning, Alison M. [Centre for Cancer Genetic Epidemiology, University of Cambridge, Strangeways Research Laboratory, Cambridge (United Kingdom); Burnet, Neil [Department of Oncology, Oncology Centre, Cambridge University Hospital NHS Foundation Trust, University of Cambridge, Cambridge (United Kingdom); Bliss, Judith M. [The Institute of Cancer Research, Clinical Trials and Statistics Unit, Sutton (United Kingdom); Symonds, R. Paul; Rattay, Tim [Department of Cancer Studies and Molecular Medicine, University of Leicester, Leicester (United Kingdom); Suga, Tomo [Advanced Radiation Biology Research Program, National Institute of Radiological Sciences, Chiba (Japan); Kerns, Sarah L. [Department of Radiation Oncology, Icahn School of Medicine at Mount Sinai, New York, NH (United States); and others

2015-08-01

Purpose: To identify single-nucleotide polymorphisms (SNPs) in oxidative stress–related genes associated with risk of late toxicities in breast cancer patients receiving radiation therapy. Methods and Materials: Using a 2-stage design, 305 SNPs in 59 candidate genes were investigated in the discovery phase in 753 breast cancer patients from 2 prospective cohorts from Germany. The 10 most promising SNPs in 4 genes were evaluated in the replication phase in up to 1883 breast cancer patients from 6 cohorts identified through the Radiogenomics Consortium. Outcomes of interest were late skin toxicity and fibrosis of the breast, as well as an overall toxicity score (Standardized Total Average Toxicity). Multivariable logistic and linear regression models were used to assess associations between SNPs and late toxicity. A meta-analysis approach was used to summarize evidence. Results: The association of a genetic variant in the base excision repair gene XRCC1, rs2682585, with normal tissue late radiation toxicity was replicated in all tested studies. In the combined analysis of discovery and replication cohorts, carrying the rare allele was associated with a significantly lower risk of skin toxicities (multivariate odds ratio 0.77, 95% confidence interval 0.61-0.96, P=.02) and a decrease in Standardized Total Average Toxicity scores (−0.08, 95% confidence interval −0.15 to −0.02, P=.016). Conclusions: Using a stage design with replication, we identified a variant allele in the base excision repair gene XRCC1 that could be used in combination with additional variants for developing a test to predict late toxicities after radiation therapy in breast cancer patients.

XRCC1 Polymorphism Associated With Late Toxicity After Radiation Therapy in Breast Cancer Patients

International Nuclear Information System (INIS)

Seibold, Petra; Behrens, Sabine; Schmezer, Peter; Helmbold, Irmgard; Barnett, Gillian; Coles, Charlotte; Yarnold, John; Talbot, Christopher J.; Imai, Takashi; Azria, David; Koch, C. Anne; Dunning, Alison M.; Burnet, Neil; Bliss, Judith M.; Symonds, R. Paul; Rattay, Tim; Suga, Tomo; Kerns, Sarah L.

2015-01-01

Purpose: To identify single-nucleotide polymorphisms (SNPs) in oxidative stress–related genes associated with risk of late toxicities in breast cancer patients receiving radiation therapy. Methods and Materials: Using a 2-stage design, 305 SNPs in 59 candidate genes were investigated in the discovery phase in 753 breast cancer patients from 2 prospective cohorts from Germany. The 10 most promising SNPs in 4 genes were evaluated in the replication phase in up to 1883 breast cancer patients from 6 cohorts identified through the Radiogenomics Consortium. Outcomes of interest were late skin toxicity and fibrosis of the breast, as well as an overall toxicity score (Standardized Total Average Toxicity). Multivariable logistic and linear regression models were used to assess associations between SNPs and late toxicity. A meta-analysis approach was used to summarize evidence. Results: The association of a genetic variant in the base excision repair gene XRCC1, rs2682585, with normal tissue late radiation toxicity was replicated in all tested studies. In the combined analysis of discovery and replication cohorts, carrying the rare allele was associated with a significantly lower risk of skin toxicities (multivariate odds ratio 0.77, 95% confidence interval 0.61-0.96, P=.02) and a decrease in Standardized Total Average Toxicity scores (−0.08, 95% confidence interval −0.15 to −0.02, P=.016). Conclusions: Using a stage design with replication, we identified a variant allele in the base excision repair gene XRCC1 that could be used in combination with additional variants for developing a test to predict late toxicities after radiation therapy in breast cancer patients

Assessment of genetic mutations in the XRCC2 coding region by high resolution melting curve analysis and the risk of differentiated thyroid carcinoma in Iran

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Shima Fayaz

2012-01-01

Full Text Available Homologous recombination (HR is the major pathway for repairing double strand breaks (DSBs in eukaryotes and XRCC2 is an essential component of the HR repair machinery. To evaluate the potential role of mutations in gene repair by HR in individuals susceptible to differentiated thyroid carcinoma (DTC we used high resolution melting (HRM analysis, a recently introduced method for detecting mutations, to examine the entire XRCC2 coding region in an Iranian population. HRM analysis was used to screen for mutations in three XRCC2 coding regions in 50 patients and 50 controls. There was no variation in the HRM curves obtained from the analysis of exons 1 and 2 in the case and control groups. In exon 3, an Arg188His polymorphism (rs3218536 was detected as a new melting curve group (OR: 1.46; 95%CI: 0.432-4.969; p = 0.38 compared with the normal melting curve. We also found a new Ser150Arg polymorphism in exon 3 of the control group. These findings suggest that genetic variations in the XRCC2 coding region have no potential effects on susceptibility to DTC. However, further studies with larger populations are required to confirm this conclusion.

DNA repair gene polymorphisms in relation to chromosome aberration frequencies in retired radiation workers

International Nuclear Information System (INIS)

Wilding, Craig S.; Relton, Caroline L.; Rees, Gwen S.; Tarone, Robert E.; Whitehouse, Caroline A.; Tawn, E. Janet

2005-01-01

Polymorphic variation in DNA repair genes was examined in a group of retired workers from the British Nuclear Fuels plc facility at Sellafield in relation to previously determined translocation frequencies in peripheral blood lymphocytes. Variation at seven polymorphisms in four genes involved in the base excision repair (XRCC1 R194W, R399Q and a [AC] n microsatellite in the 3' UTR) and double strand break repair (XRCC3 T241M and a [AC] n microsatellite in intron 3 of XRCC3, XRCC4 I134T, and a GACTAn microsatellite located 120kb 5' of XRCC5) pathways was determined for 291 retired radiation workers who had received cumulative occupational external radiation doses of between 0 and 1873mSv. When the interaction between radiation dose and each DNA repair gene polymorphism was examined in relation to translocation frequency there was no evidence for any of the polymorphisms studied influencing the response to occupational exposure. A positive interaction observed between genotype (individuals with at least one allele >=20 repeat units) at a microsatellite locus in the XRCC3 gene and smoking status should be interpreted cautiously because interactions were investigated for seven polymorphisms and two exposures. Nonetheless, further research is warranted to examine whether this DNA repair gene variant might be associated with a sub-optimal repair response to smoking-induced DNA damage and hence an increased frequency of translocations

Genetic polymorphisms of DNA double-strand break repair pathway genes and glioma susceptibility

International Nuclear Information System (INIS)

Zhao, Peng; Zou, Peng; Zhao, Lin; Yan, Wei; Kang, Chunsheng; Jiang, Tao; You, Yongping

2013-01-01

Genetic variations in DNA double-strand break repair genes can influence the ability of a cell to repair damaged DNA and alter an individual’s susceptibility to cancer. We studied whether polymorphisms in DNA double-strand break repair genes are associated with an increased risk of glioma development. We genotyped 10 potentially functional single nucleotide polymorphisms (SNPs) in 7 DNA double-strand break repair pathway genes (XRCC3, BRCA2, RAG1, XRCC5, LIG4, XRCC4 and ATM) in a case–control study including 384 glioma patients and 384 cancer-free controls in a Chinese Han population. Genotypes were determined using the OpenArray platform. In the single-locus analysis there was a significant association between gliomas and the LIG4 rs1805388 (Ex2 +54C>T, Thr9Ile) TT genotype (adjusted OR, 3.27; 95% CI, 1.87-5.71), as well as the TC genotype (adjusted OR, 1.62; 95% CI, 1.20-2.18). We also found that the homozygous variant genotype (GG) of XRCC4 rs1805377 (IVS7-1A>G, splice-site) was associated with a significantly increased risk of gliomas (OR, 1.77; 95% CI, 1.12-2.80). Interestingly, we detected a significant additive and multiplicative interaction effect between the LIG4 rs1805388 and XRCC4 rs1805377 polymorphisms with an increasing risk of gliomas. When we stratified our analysis by smoking status, LIG4 rs1805388 was associated with an increased glioma risk among smokers. These results indicate for the first time that LIG4 rs1805388 and XRCC4 rs1805377, alone or in combination, are associated with a risk of gliomas

Evaluation of the frequency of polymorphisms in XRCC1 (Arg399Gln) and XPD (Lys751Gln) genes related to the genome stability maintenance in individuals of the resident population from Monte Alegre, PA/Brazil municipality; Avaliacao da frequencia de polimorfismos nos genes XRCC1 (Arg399Gln) e XPD (Lys751Gln) relacionados a manutencao da estabilidade do genoma em individuos da populacao residente no municipio de Monte Alegre, PA

Energy Technology Data Exchange (ETDEWEB)

Duarte, Isabelle Magliano

2010-07-01

The human exposure to ionizing radiation coming from natural sources is an inherent feature of human life on Earth. Ionizing radiation is a known genotoxic agent, which can affect biological molecules, causing DNA damage and genomic instability. The cellular system of DNA repair plays an important role in maintaining genomic stability by repairing DNA damage caused by genotoxic agents. However, genes related to DNA repair may have their role committed when presenting a certain polymorphism. This study intended to analyze the frequency of single nucleotide polymorphisms (SNPs) in genes of DNA repair XRCC1 (Arg39-9Gln) and XPD (Lys751Gln) in a: population of the city of Monte Alegre, that resides in an area of high exposure to natural radioactivity. Samples of saliva were collected from individuals of the population of Monte Alegre, in which 40 samples were of male and 46 female. Through the use of RFLP (length polymorphism restriction fragment) the frequency of homozygous genotypes and / or heterozygous was determined for polymorphic genes. The XRCC1 gene had 65.4% of the presence of the allele 399Gln and XPD gene had 32.9% of the 751Gln allele. These values are similar to those found in previous studies for the XPD gene, whereas XRCC1 showed a frequency much higher than described in the literature. The. influence of these polymorphisms, which are involved in DNA repair and consequent genotoxicity induced by radiation depends on dose and exposure factors such as smoking, statistically a factor in public health surveillance in the region. This study gathered information and molecular epidemiology for risk assessment of cancer in the population of Monte Alegre. (author)

Polymorphisms in the genes ERCC2, XRCC3 and CD3EAP influence treatment outcome in multiple myeloma patients undergoing autologous bone marrow transplantation

DEFF Research Database (Denmark)

Vangsted, Annette; Gimsing, Peter; Klausen, Tobias W

2007-01-01

) of polymorphism in the DNA repair genes ERCC1, ERCC2 and XRCC3, and in the apoptotic genes PPP1R13L and CD3EAP in 348 patients with multiple myeloma undergoing autologous bone marrow transplantation. Carriers of the variant C-allele of ERCC2 K751Q, the variant T-allele of XRCC3 T241M and the variant A...... the outcome for patients treated with autologous stem cell transplantation. Udgivelsesdato: 2007-Mar-1...

Asparagine 326 in the extremely C-terminal region of XRCC4 is essential for the cell survival after irradiation

Energy Technology Data Exchange (ETDEWEB)

Wanotayan, Rujira; Fukuchi, Mikoto; Imamichi, Shoji; Sharma, Mukesh Kumar; Matsumoto, Yoshihisa, E-mail: yoshim@nr.titech.ac.jp

2015-02-20

XRCC4 is one of the crucial proteins in the repair of DNA double-strand break (DSB) through non-homologous end-joining (NHEJ). As XRCC4 consists of 336 amino acids, N-terminal 200 amino acids include domains for dimerization and for association with DNA ligase IV and XLF and shown to be essential for XRCC4 function in DSB repair and V(D)J recombination. On the other hand, the role of the remaining C-terminal region of XRCC4 is not well understood. In the present study, we noticed that a stretch of ∼20 amino acids located at the extreme C-terminus of XRCC4 is highly conserved among vertebrate species. To explore its possible importance, series of mutants in this region were constructed and assessed for the functionality in terms of ability to rescue radiosensitivity of M10 cells lacking XRCC4. Among 13 mutants, M10 transfectant with N326L mutant (M10-XRCC4{sup N326L}) showed elevated radiosensitivity. N326L protein showed defective nuclear localization. N326L sequence matched the consensus sequence of nuclear export signal. Leptomycin B treatment accumulated XRCC4{sup N326L} in the nucleus but only partially rescued radiosensitivity of M10-XRCC4{sup N326L}. These results collectively indicated that the functional defects of XRCC4{sup N326L} might be partially, but not solely, due to its exclusion from nucleus by synthetic nuclear export signal. Further mutation of XRCC4 Asn326 to other amino acids, i.e., alanine, aspartic acid or glutamine did not affect the nuclear localization but still exhibited radiosensitivity. The present results indicated the importance of the extremely C-terminal region of XRCC4 and, especially, Asn326 therein. - Highlights: • Extremely C-terminal region of XRCC4 is highly conserved among vertebrate species. • XRCC4 C-terminal point mutants, R325F and N326L, are functionally deficient in terms of survival after irradiation. • N326L localizes to the cytoplasm because of synthetic nuclear export signal. • Leptomycin B restores the

Distinct kinetics of human DNA ligases I, IIIalpha, IIIbeta, and IV reveal direct DNA sensing ability and differential physiological functions in DNA repair

Energy Technology Data Exchange (ETDEWEB)

Chen, Xi; Ballin, Jeff D.; Della-Maria, Julie; Tsai, Miaw-Sheue; White, Elizabeth J.; Tomkinson, Alan E.; Wilson, Gerald M.

2009-05-11

The three human LIG genes encode polypeptides that catalyze phosphodiester bond formation during DNA replication, recombination and repair. While numerous studies have identified protein partners of the human DNA ligases (hLigs), there has been little characterization of the catalytic properties of these enzymes. In this study, we developed and optimized a fluorescence-based DNA ligation assay to characterize the activities of purified hLigs. Although hLigI joins DNA nicks, it has no detectable activity on linear duplex DNA substrates with short, cohesive single-strand ends. By contrast, hLigIII{beta} and the hLigIII{alpha}/XRCC1 and hLigIV/XRCC4 complexes are active on both nicked and linear duplex DNA substrates. Surprisingly, hLigIV/XRCC4, which is a key component of the major non-homologous end joining (NHEJ) pathway, is significantly less active than hLigIII on a linear duplex DNA substrate. Notably, hLigIV/XRCC4 molecules only catalyze a single ligation event in the absence or presence of ATP. The failure to catalyze subsequent ligation events reflects a defect in the enzyme-adenylation step of the next ligation reaction and suggests that, unless there is an in vivo mechanism to reactivate DNA ligase IV/XRCC4 following phosphodiester bond formation, the cellular NHEJ capacity will be determined by the number of adenylated DNA ligaseIV/XRCC4 molecules.

Interaction between lifestyle factors and the XRCC1, XPD, and XRCC3 genetic variations modulates the risk for sporadic colorectal cancer

Directory of Open Access Journals (Sweden)

Procopciuc Lucia Maria

2014-03-01

Full Text Available Introducere: Variațiile genetice, cum ar fi cele care influențează sistemele de reparare a defectelor de replicare a ADN, pot reprezenta factori de susceptibilitate în cancerul colorectal sporadic (CCR ca urmare a interacțiunii cu factori de mediu. Material și metodă: 80 de femei și 70 de bărbați, pacienți diagnosticați cu CCR sporadic în Clinica Chirurgie III Cluj au fost genotipați pentru Arg399Gln-XRCC1, Lys751Gln-XPD și Met241Thr-XRCC3 utilizând metodele PCR-RFLP. Am determinat de asemenea, genotipurile pentru 100 femei și 62 bărbați , care au format grupul de control. Rezultatele au fost analizate din punct de vedere al relației cu factorii de risc de mediu, fumatul și dieta. Rezultate: Bărbații fumători purtători ai variațiilor genetice Arg399Gln, Lys751Gln, Met241Thr au avut un risc semnificativ crescut de 4.09 (95%IC[0.96-19.98],p=0.05, 5.95(95%IC[1.08-43.22],p=0.03 și respectiv 3.73(95%IC[0.86-18.53],p=0.05 de a dezvolta cancer colorectal sporadic. Un risc semnificativ crescut de a dezvolta cancer colorectal sporadic a fost observat în cazul femeilor și bărbaților cu o dietă bogată în carne roșie prăjită purtători ai variațiilor genetice Arg399Gln (OR 2.77 95%IC [1.34-6.82],p=0.015 și OR 8.64 95%IC[2.67-29.14],p<0.001, Lys751Gln (OR 4.12 95%IC[1.37-12.74],p=0.007 și OR 5.06 95%IC[1.4- 19.02],p=0.006, Met241Thr (OR5.92 95%IC[2.21-16.23],p<0.001 și OR 5.64 95%IC[1.52-21.7],p=0.022. Femeile a căror dietă a inclus cantități mari de carne roșie prăjită au avut un risc semnificativ crescut de a dezvolta timpuriu cancer colorectal sporadic dacă au fost purtătoare a variațiilor genetice Arg399Gln-XRCC1 (OR 5.14 95%IC[0.99-28.3],p=0.047, Thr241Met-XRCC3 (OR 6.67 95%IC[1.05-46.67],p=0.025 și Lys751Gln-XPD (OR 4.7 95%IC[0.99-23.32],p=0.034. Concluzii: În cazul populației de origine română, asocierea genotipurilor mutante cu factori de mediu modulează riscul pentru CCR sporadic. La femei

Expression and relevant research of MGMT and XRCC1 gene in differentgrades of brain glioma and normal brain tissues

Institute of Scientific and Technical Information of China (English)

Ya-Fei Zhang

2015-01-01

Objective: To explore and analyze expression and relevant research of MGMT and XRCC1 gene in different grades of brain glioma and normal brain tissues. Methods: 52 cases of patients with brain glioma treated in our hospital from December 2013 to December 2014, and 50 cases of normal brain-tissue patients with intracranial hypertension were selected, and proceeding test to the surgical resection of brain tissue of the above patients to determine its MGMT and XRCC1 protein content, sequentially to record the expression of MGMT and XRCC1 of both groups. Grading of tumors to brain glioma after operation was carried out, and the expression of MGMT and XRCC1 gene in brain tissues of different patients was analyzed and compared;finally the contingency tables of X2 test was used to analyze the correlation of XRCC1and MGMT. Results:Positive rate of MGMT expression in normal brain tissue was 2%,while positive rate of MGMT expression in brain glioma was 46.2%,which was obviously higher than that in normal brain tissues (χ2=26.85, P0.05), which had no statistical significance. There were 12 cases of patients whose MGMT protein expression was positive and XRCC1 protein expression was positive; there were 18 cases of patients whose MGMT protein expression was negative and XRCC1 protein expression was negative. Contingency tables of X2 test was used to analyze the correlation of XRCC1 and MGMT, which indicated that the expression of XRCCI and MGMT in brain glioma had no correlation (r=0.9%, P=0.353), relevancy of both was r=0.9%. Conclusions: Positive rate of the expression of MGMT and XRCC1 in brain glioma was obviously higher than that in normal brain tissues, but the distribution of different grades of brain glioma had no obvious difference, and MGMT and XRCC1 expression had no obvious correlation, which needed further research.

Direct Involvement of Retinoblastoma Family Proteins in DNA Repair by Non-homologous End-Joining

Directory of Open Access Journals (Sweden)

Rebecca Cook

2015-03-01

Full Text Available Deficiencies in DNA double-strand break (DSB repair lead to genetic instability, a recognized cause of cancer initiation and evolution. We report that the retinoblastoma tumor suppressor protein (RB1 is required for DNA DSB repair by canonical non-homologous end-joining (cNHEJ. Support of cNHEJ involves a mechanism independent of RB1’s cell-cycle function and depends on its amino terminal domain with which it binds to NHEJ components XRCC5 and XRCC6. Cells with engineered loss of RB family function as well as cancer-derived cells with mutational RB1 loss show substantially reduced levels of cNHEJ. RB1 variants disabled for the interaction with XRCC5 and XRCC6, including a cancer-associated variant, are unable to support cNHEJ despite being able to confer cell-cycle control. Our data identify RB1 loss as a candidate driver of structural genomic instability and a causative factor for cancer somatic heterogeneity and evolution.

Evaluation of the frequency of polymorphisms in XRCC1 (Arg399Gln) and XPD (Lys751Gln) genes related to the genome stability maintenance in individuals of the resident population from Monte Alegre, PA/Brazil municipality

International Nuclear Information System (INIS)

Duarte, Isabelle Magliano

2010-01-01

The human exposure to ionizing radiation coming from natural sources is an inherent feature of human life on Earth. Ionizing radiation is a known genotoxic agent, which can affect biological molecules, causing DNA damage and genomic instability. The cellular system of DNA repair plays an important role in maintaining genomic stability by repairing DNA damage caused by genotoxic agents. However, genes related to DNA repair may have their role committed when presenting a certain polymorphism. This study intended to analyze the frequency of single nucleotide polymorphisms (SNPs) in genes of DNA repair XRCC1 (Arg39-9Gln) and XPD (Lys751Gln) in a: population of the city of Monte Alegre, that resides in an area of high exposure to natural radioactivity. Samples of saliva were collected from individuals of the population of Monte Alegre, in which 40 samples were of male and 46 female. Through the use of RFLP (length polymorphism restriction fragment) the frequency of homozygous genotypes and / or heterozygous was determined for polymorphic genes. The XRCC1 gene had 65.4% of the presence of the allele 399Gln and XPD gene had 32.9% of the 751Gln allele. These values are similar to those found in previous studies for the XPD gene, whereas XRCC1 showed a frequency much higher than described in the literature. The. influence of these polymorphisms, which are involved in DNA repair and consequent genotoxicity induced by radiation depends on dose and exposure factors such as smoking, statistically a factor in public health surveillance in the region. This study gathered information and molecular epidemiology for risk assessment of cancer in the population of Monte Alegre. (author)

Susceptibility to Colorectal Cancer and Two Genetic Polymorphisms of XRCC4.

Science.gov (United States)

Emami, Naghmeh; Saadat, Iraj; Omidvari, Shahpour

2015-09-01

The X-ray complementing group 4 (XRCC4, OMIM: 194363) plays a key role in non-homologous end-joining DNA repair pathway in mammalian cells. This pathway is believed to help maintain genomic stability. In the present study, it is hypothesized that genetic polymorphisms in the NHEJ repair XRCC4 gene may be associated with an increased risk in developing colorectal cancer (CRC). We genotyped two polymorphisms of XRCC4, G-1394T (rs6869366) and intron 3 insertion/deletion (I/D; rs28360071) in 200 colorectal cancer patients as well as 256 healthy individuals, and evaluated their association with CRC. We found that in G-1394T polymorphism, neither the TG nor the GG genotypes (versus the TT genotype) were associated with the risk of developing CRC. The results of our study indicate that in comparison with the II genotype, ID and DD genotypes had no significant association with the risk of developing CRC. Subjects with TT genotype and positive family history in colorectal cancer were found to be at a much lower risk of developing CRC in comparison with the reference group (OR = 0.31, 95%CI: 0.11-0.85, P =  .023). It should be noted that participants having at least one G allele (TG+GG genotypes) were at a significantly higher risk to develop the disease compared with the reference group (OR = 9.10, 95%CI: 2.00-41.3, P = 0.004). In relation to I/D polymorphism, among participants, those with positive family history, either with ID (OR =  .78, 95%CI: 2.26-10.0, P < 0.001) or DD genotypes (OR = 5.73, 95%CI: 1.99-16.4, P = 0.001) had a significantly association with the disease. Among participants with a positive family history in CRC, the haplotype GD dramatically increased the risk of developing CRC (OR = 10.2, 95%CI: 2.28-46, P = 0.002). The results of this study indicate that G-1394T and I/D polymorphisms of XRCC4 among individuals with positive family history for colorectal cancer substantially increase the risk factor for developing colorectal cancers.

XRCC1 codon 399Gln polymorphism is associated with radiotherapy-induced acute dermatitis and mucositis in nasopharyngeal carcinoma patients

International Nuclear Information System (INIS)

Li, Haijun; You, Yanjie; Lin, Canfeng; Zheng, Mingzhang; Hong, Chaoqun; Chen, Jiongyu; Li, Derui; Au, William W; Chen, Zhijian

2013-01-01

To evaluate the association between single nucleotide polymorphisms (SNPs) at the 194 and 399 codons of XRCC1, and the risk of severe acute skin and oral mucosa reactions in nasopharyngeal carcinoma patients in China. 114 patients with nasopharyngeal carcinoma were sequentially recruited in this study. Heparinized peripheral blood samples were taken for SNPs analysis before the start of radiation treatment. SNPs in XRCC1 (194Arg/Trp and 399Arg/Gln) gene were analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Dermatitis at upper neck and oral mucositis were clinically recorded according to the Common Terminology Criteria for Adverse Events v.3.0. The variant allele frequencies were 0.289 for XRCC1 194Trp and 0.263 for XRCC1 399Gln. Of the 114 patients, 24 experienced grade 3 acute dermatitis and 48 had grade 3 acute mucositis. The XRCC1 399Arg/Gln was significantly associated with the development of grade 3 dermatitis (Odds Ratio, 2.65; 95% CI, 1.04–6.73; p = 0.037, χ2 = 4.357). In addition, it was also associated with higher incidence of grade 3 mucositis with a borderline statistical significance (Odds Ratio, 2.11; 95% CI, 0.951–4.66; p = 0.065, χ2 = 3.411). The relationship between XRCC1 194Arg/Trp and acute dermatitis, and mucositis was not found. Our investigation shows, for the first time, that patients with the XRCC1 399Arg/Gln genotype were more likely to experience severe acute dermatitis and oral mucositis. With further validation, the information can be used to determine personalized radiotherapy strategy

The association of XRCC3 Thr241Met genetic variant with risk of ...

African Journals Online (AJOL)

Background: Previous studies suggest that the X-ray repair cross-complementing group 3 gene (XRCC3) Thr241Met genetic variant could be potentially associated with the risk of prostate cancer. However, results from these published studies were conflicting rather than conclusive. Objectives:This meta-analysis aimed to ...

Structure-Based Virtual Ligand Screening on the XRCC4/DNA Ligase IV Interface

Science.gov (United States)

Menchon, Grégory; Bombarde, Oriane; Trivedi, Mansi; Négrel, Aurélie; Inard, Cyril; Giudetti, Brigitte; Baltas, Michel; Milon, Alain; Modesti, Mauro; Czaplicki, Georges; Calsou, Patrick

2016-03-01

The association of DNA Ligase IV (Lig4) with XRCC4 is essential for repair of DNA double-strand breaks (DSBs) by Non-homologous end-joining (NHEJ) in humans. DSBs cytotoxicity is largely exploited in anticancer therapy. Thus, NHEJ is an attractive target for strategies aimed at increasing the sensitivity of tumors to clastogenic anticancer treatments. However the high affinity of the XRCC4/Lig4 interaction and the extended protein-protein interface make drug screening on this target particularly challenging. Here, we conducted a pioneering study aimed at interfering with XRCC4/Lig4 assembly. By Molecular Dynamics simulation using the crystal structure of the complex, we first delineated the Lig4 clamp domain as a limited suitable target. Then, we performed in silico screening of ~95,000 filtered molecules on this Lig4 subdomain. Hits were evaluated by Differential Scanning Fluorimetry, Saturation Transfer Difference - NMR spectroscopy and interaction assays with purified recombinant proteins. In this way we identified the first molecule able to prevent Lig4 binding to XRCC4 in vitro. This compound has a unique tripartite interaction with the Lig4 clamp domain that suggests a starting chemotype for rational design of analogous molecules with improved affinity.

Cloning and characterization of human DNA repair genes

International Nuclear Information System (INIS)

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

1987-01-01

The isolation of two addition human genes that give efficient restoration of the repair defects in other CHO mutant lines is reported. The gene designated ERCC2 (Excision Repair Complementing Chinese hamster) corrects mutant UV5 from complementation group 1. They recently cloned this gene by first constructing a secondary transformant in which the human gene was shown to have become physically linked to the bacterial gpt dominant-marker gene by cotransfer in calcium phosphate precipitates in the primary transfection. Transformants expressing both genes were recovered by selecting for resistance to both UV radiation and mycophenolic acid. Using similar methods, the human gene that corrects CHO mutant EM9 was isolated in cosmids and named XRCC1 (X-ray Repair Complementing Chinese hamster). In this case, transformants were recovered by selecting for resistance to CldUrd, which kills EM9 very efficiently. In both genomic and cosmid transformants, the XRCC1 gene restored resistance to the normal range. DNA repair was studied using the kinetics of strand-break rejoining, which was measured after exposure to 137 Cs γ-rays

Chromosomal damage and polymorphisms of DNA repair genes XRCC1 and XRCC3 in workers exposed to chromium

Czech Academy of Sciences Publication Activity Database

Halasová, E.; Mataková, T.; Mušák, L.; Poláková, Veronika; Vodička, Pavel

2008-01-01

Roč. 29, č. 5 (2008), s. 658-662 ISSN 0172-780X Institutional research plan: CEZ:AV0Z50390703 Keywords : Chromosomal aberrations * Polymorphisms * Repair genes Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.359, year: 2008

De novo CNV formation in mouse embryonic stem cells occurs in the absence of Xrcc4-dependent nonhomologous end joining.

Directory of Open Access Journals (Sweden)

Martin F Arlt

2012-09-01

Full Text Available Spontaneous copy number variant (CNV mutations are an important factor in genomic structural variation, genomic disorders, and cancer. A major class of CNVs, termed nonrecurrent CNVs, is thought to arise by nonhomologous DNA repair mechanisms due to the presence of short microhomologies, blunt ends, or short insertions at junctions of normal and de novo pathogenic CNVs, features recapitulated in experimental systems in which CNVs are induced by exogenous replication stress. To test whether the canonical nonhomologous end joining (NHEJ pathway of double-strand break (DSB repair is involved in the formation of this class of CNVs, chromosome integrity was monitored in NHEJ-deficient Xrcc4(-/- mouse embryonic stem (ES cells following treatment with low doses of aphidicolin, a DNA replicative polymerase inhibitor. Mouse ES cells exhibited replication stress-induced CNV formation in the same manner as human fibroblasts, including the existence of syntenic hotspot regions, such as in the Auts2 and Wwox loci. The frequency and location of spontaneous and aphidicolin-induced CNV formation were not altered by loss of Xrcc4, as would be expected if canonical NHEJ were the predominant pathway of CNV formation. Moreover, de novo CNV junctions displayed a typical pattern of microhomology and blunt end use that did not change in the absence of Xrcc4. A number of complex CNVs were detected in both wild-type and Xrcc4(-/- cells, including an example of a catastrophic, chromothripsis event. These results establish that nonrecurrent CNVs can be, and frequently are, formed by mechanisms other than Xrcc4-dependent NHEJ.

shRNA-Mediated XRCC2 Gene Knockdown Efficiently Sensitizes Colon Tumor Cells to X-ray Irradiation in Vitro and in Vivo

Directory of Open Access Journals (Sweden)

Qin Wang

2014-01-01

Full Text Available Colon cancer is one of the most common tumors of the digestive tract. Resistance to ionizing radiation (IR decreased therapeutic efficiency in these patients’ radiotherapy. XRCC2 is the key protein of DNA homologous recombination repair, and its high expression is associated with enhanced resistance to DNA damage induced by IR. Here, we investigated the effect of XRCC2 silencing on colon tumor cells’ growth and sensitivity to X-radiation in vitro and in vivo. Colon tumor cells (T84 cell line were cultivated in vitro and tumors originated from the cell line were propagated as xenografts in nude mice. The suppression of XRCC2 expression was achieved by using vector-based short hairpin RNA (shRNA in T84 cells. We found that the knockdown of XRCC2 expression effectively decreased T84 cellular proliferation and colony formation, and led to cell apoptosis and cell cycle arrested in G2/M phase induced by X-radiation in vitro. In addition, tumor xenograft studies suggested that XRCC2 silencing inhibited tumorigenicity after radiation treatment in vivo. Our data suggest that the suppression of XRCC2 expression rendered colon tumor cells more sensitive to radiation therapy in vitro and in vivo, implying XRCC2 as a promising therapeutic target for the treatment of radioresistant human colon cancer.

Polymorphic Variation in Double Strand Break Repair Gene in Indian Population: A Comparative Approach with Worldwide Ethnic Group Variations.

Science.gov (United States)

Mandal, Raju Kumar; Mittal, Rama Devi

2018-04-01

DNA repair capacity is essential in maintaining cellular functions and homeostasis. Identification of genetic polymorphisms responsible for reduced DNA repair capacity may allow better cancer prevention. Double strand break repair pathway plays critical roles in maintaining genome stability. Present study was conducted to determine distribution of XRCC3 Exon 7 (C18067T, rs861539) and XRCC7 Intron 8 (G6721T, rs7003908) gene polymorphisms in North Indian population and compare with different populations globally. The genotype assays were performed in 224 normal healthy individuals of similar ethnicity using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Allelic frequencies of wild type were 79% (C) in XRCC3 Exon 7 C > T and 57% (G) in XRCC7 Intron 8 (G > T) 57% (G) observed. On the other hand, the variant allele frequency were 21% (T) in XRCC3 Exon 7 C > T and 43% (T) in XRCC7 Intron 8 G > T respectively. Major differences from other ethnic populations were observed. Our results suggest that frequency in these DNA repair genes exhibit distinctive pattern in India that could be attributed to ethnicity variation. This could assist in high-risk screening of humans exposed to environmental carcinogens and cancer predisposition in different ethnic groups.

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

International Nuclear Information System (INIS)

Bajinskis, Ainars; Olsson, Gunilla; Harms-Ringdahl, Mats

2012-01-01

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 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.

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.

Science.gov (United States)

Bajinskis, Ainars; Olsson, Gunilla; Harms-Ringdahl, Mats

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(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. Copyright © 2011 Elsevier B.V. All rights reserved.

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.

Emerging roles of the nucleolus in regulating the DNA damage response: the noncanonical DNA repair enzyme APE1/Ref-1 as a paradigmatical example.

Science.gov (United States)

Antoniali, Giulia; Lirussi, Lisa; Poletto, Mattia; Tell, Gianluca

2014-02-01

An emerging concept in DNA repair mechanisms is the evidence that some key enzymes, besides their role in the maintenance of genome stability, display also unexpected noncanonical functions associated with RNA metabolism in specific subcellular districts (e.g., nucleoli). During the evolution of these key enzymes, the acquisition of unfolded domains significantly amplified the possibility to interact with different partners and substrates, possibly explaining their phylogenetic gain of functions. After nucleolar stress or DNA damage, many DNA repair proteins can freely relocalize from nucleoli to the nucleoplasm. This process may represent a surveillance mechanism to monitor the synthesis and correct assembly of ribosomal units affecting cell cycle progression or inducing p53-mediated apoptosis or senescence. A paradigm for this kind of regulation is represented by some enzymes of the DNA base excision repair (BER) pathway, such as apurinic/apyrimidinic endonuclease 1 (APE1). In this review, the role of the nucleolus and the noncanonical functions of the APE1 protein are discussed in light of their possible implications in human pathologies. A productive cross-talk between DNA repair enzymes and proteins involved in RNA metabolism seems reasonable as the nucleolus is emerging as a dynamic functional hub that coordinates cell growth arrest and DNA repair mechanisms. These findings will drive further analyses on other BER proteins and might imply that nucleic acid processing enzymes are more versatile than originally thought having evolved DNA-targeted functions after a previous life in the early RNA world.

Function of Rad51 paralogs in eukaryotic homologous recombinational repair

International Nuclear Information System (INIS)

Liu, N.; Skowronek, K.

2003-01-01

Full text: Homologous recombinational repair (HRR) is an important mechanism for maintaining genetic integrity and cancer prevention by accurately repair of DNA double strand breaks induced by environmental insults or occurred in DNA replication. A critical step in HRR is the polymerization of Rad51 on single stranded DNA to form nuclear protein filaments, the later conduct DNA strand paring and exchange between homologous strands. A number of proteins, including replication protein A (RPA), Rad52 and Rad51 paralogs, are suggested to modulate or facilitate the process of Rad51 filament formation. Five Rad51 paralogs, namely XRCC2, XRCC3, Rad51B, Rad51C and Rad51D have been identified in eucaryotic cells. These proteins show distant protein sequence identity to Rad51, to yeast Rad51 paralogs (Rad55 and Rad57) and to each other. Hamster or chicken mutants of Rad51 paralogs exhibit hypersensitivity to a variety of DNA damaging agents, especially cross-linking agents, and are defective in assembly of Rad51 onto HRR site after DNA damage. Recent data from our and other labs showed that Rad51 paralogs constitute two distinct complexes in cell extracts, one contains XRCC2, Rad51B, Rad51C and Rad51D, and the other contains Rad51C and XRCC3. Rad51C is involved in both complexes. Our results also showed that XRCC3-Rad51C complex interacts with Rad51 in vivo. Furthermore, overexpression of Rad52 can partially suppress the hypersensitivity of XRCC2 mutant irs1 to ionizing radiation and corrected the defects in Rad51 focus formation. These results suggest that XRCC2 and other Rad51 paralogs play a mediator function to Rad51 in the early stage of HRR

Cloning human DNA repair genes

International Nuclear Information System (INIS)

Jeggo, P.A.; Carr, A.M.; Lehmann, A.R.

1994-01-01

Many human genes involved in the repair of UV damage have been cloned using different procedures and they have been of great value in assisting the understanding of the mechanism of nucleotide excision-repair. Genes involved in repair of ionizing radiation damage have proved more difficult to isolate. Positional cloning has localized the XRCC5 gene to a small region of chromosome 2q33-35, and a series of yeast artificial chromosomes covering this region have been isolated. Very recent work has shown that the XRCC5 gene encodes the 80 kDa subunit of the Ku DNA-binding protein. The Ku80 gene also maps to this region. Studies with fission yeast have shown that radiation sensitivity can result not only from defective DNA repair but also from abnormal cell cycle control following DNA damage. Several genes involved in this 'check-point' control in fission yeast have been isolated and characterized in detail. It is likely that a similar checkpoint control mechanism exists in human cells. (author)

Post-irradiation inactivation, protection, and repair of the sulfhydryl enzyme malate synthase

International Nuclear Information System (INIS)

Durchschlag, H.; Zipper, P.

1985-01-01

Malate synthase from baker's yeast, a trimeric sulfhydryl enzyme with one essential sulfhydryl group per subunit, was inactivated by 2 kGy X-irradiation in air-saturated aqueous solution (enzyme concentration: 0.5 mg/ml). The radiation induced changes of enzymic activity were registered at about 0,30,60 h after irradiation. To elucidate the role of OH - , O 2 , and H 2 O 2 in the X-ray inactivation of the enzyme, experiments were performed in the absence of presence of different concentrations of specific additives (formate, superoxide dismutase, catalase). These additives were added to malate synthase solutions before or after X-irradiation. Moreover, repairs of inactivated malate synthase were initiated at about 0 or 30 h after irradiation by means of the sulfhydryl agent dithiothreitol. Experiments yielded the following results: 1. Irradiation of malate synthase in the absence of additives inactivated the enzyme immediately to a residual activity Asub(r)=3% (corresponding to a D 37 =0.6 kGy), and led to further slow inactivation in the post-irradiation phase. Repairs, initiated at different times after irradiation, restored enzymic activity considerably. The repair initiated at t=0 led to Asub(r)=21%; repairs started later on resulted in somewhat lower activities. The decay of reparability, however, was found to progress more slowly than post-irradiation inactivation itself. After completion of repair the activities of repaired samples did not decrease significantly. 2. The presence of specific additives during irradiation caused significant protective effects against primary inactivation. The protection by formate was very pronounced (e.g., Asub(r)=72% and D 37 =6 kGy for 100 mM formate). The presence of catalytic amounts of superoxide dismutase and/or catalase exhibited only minor effects, depending on the presence and concentration of formate. (orig.)

Prediction of radiotherapy induced normal tissue adverse reactions: the role of double-strand break repair

International Nuclear Information System (INIS)

Rao, B.S. Satish; Mumbrekar, K.D.; Goutham, H.V.; Donald, J.F.; Vadhiraja, M.B.; Satyamoorthy, K.

2016-01-01

We aimed at evaluating the predictive potential of DSB repair kinetics (using γH2AX foci assay) in lymphocytes and analysed the genetic variants in the selected radioresponsive candidate genes like XRCC3, LIG4, NBN, CD44, RAD9A, LIG3, SH3GL1, BAXS, XRCC1, MAD2L2 on the individual susceptibility to radiotherapy (RT) induced acute skin reactions among the head and neck cancer (HNC), and breast cancer (BC) patients. All the 183 HNC and 132 BC patients were treated by a 3-dimensional conformal RT technique

Molecular dynamics simulations of deoxyribonucleic acids and repair enzyme T4 endonuclease V

International Nuclear Information System (INIS)

Pinak, Miroslav

1999-01-01

This report describes the results of molecular dynamics (MD) simulation of deoxyribonucleic acids (DNA) and specific repair enzyme T4 endonuclease V. Namely research described here is focused on the examination of specific recognition process, in which this repair enzyme recognizes the damaged site on the DNA molecule-thymine dimer (TD). TD is frequent DNA damage induced by UV radiation in sun light and unless properly repaired it may be mutagenic or lethal for cell, and is also considered among the major causes of skin cancer. T4 endonuclease V is a DNA specific repair enzyme from bacteriophage T4 that catalyzes the first reaction step of TD repair pathway. MD simulations of three molecules - native DNA dodecamer (12 base pairs), DNA of the same sequence of nucleotides as native one but with TD, and repair enzyme T4 endonuclease V - were performed for 1 ns individually for each molecule. Simulations were analyzed to determine the role of electrostatic interaction in the recognition process. It is found that electrostatic energies calculated for amino acids of the enzyme have positive values of around +15 kcal/mol. The electrostatic energy of TD site has negative value of approximately -9 kcal/mol, different from the nearly neutral value of the respective thymines site of the native DNA. The electrostatic interaction of TD site with surrounding water environment differs from the electrostatic interaction of other nucleotides. Differences found between TD site and respective thymines site of native DNA indicate that the electrostatic energy is an important factor contributing to proper recognition of TD site during scanning process in which enzyme scans the DNA. In addition to the electrostatic energy, the important factor in recognition process might be structural complementarity of enzyme and bent DNA with TD. There is significant kink formed around TD site, that is not observed in native DNA. (author)

The studies of DNA double-strand break (DSB) rejoining and mRNA expression of repair gene XRCCs in malignant transformed cell lines of human bronchial epithelial cells generated by α-particles

International Nuclear Information System (INIS)

Sun Jingfen; Sui Jianli; Geng Yu; Zhou Pingkun; Wu Dechang

2002-01-01

Objective: To investigate the efficiency of γ-ray-induced DNA DSB rejoining and the mRNA expression of DNA repair genes in malignantly transformed cell lines of human bronchial epithelial cells generated by exposure to a-particles. Methods: Pulsed field gel electrophoresis (PFGE) was used to detect DNA. DSBs mRNA expression was analyzed by RT-PCR. Results: The residual DNA DSB damage level after 4hrs repair following 0-150 Gy of γ-irradiation in the malignantly transformed cell lines BERP35T-1 and BERP35T-4 was significantly higher than that in their parental BEP2D cells. The analysis of mRNA level revealed a 2.5-to 6.5-fold down-regulated expression of the DNA repair genes XRCC-2, XRCC-3 and Ku80 (XRCC-5) in BERP35T-1 and BERP35T-4 cells as compared with the parental BEP2D cells. In contrast, the expression of DNA-PKcs(XRCC7) was 2.4-fold up-regulated in the transformed cell line BERP35T-4, in which there was a significantly higher proportion of polyploid cells. Conclusion: This study results show that the deficiency of DNA DSB rejoining and depressed mRNA expression of DNA repair genes could be involved in the malignant transformation process of BEP2D cells induced by exposure to α-particles

Genetic variation in a DNA double strand break repair gene in saudi population: a comparative study with worldwide ethnic groups.

Science.gov (United States)

Areeshi, Mohammed Yahya

2013-01-01

DNA repair capacity is crucial in maintaining cellular functions and homeostasis. However, it can be altered based on DNA sequence variations in DNA repair genes and this may lead to the development of many diseases including malignancies. Identification of genetic polymorphisms responsible for reduced DNA repair capacity is necessary for better prevention. Homologous recombination (HR), a major double strand break repair pathway, plays a critical role in maintaining the genome stability. The present study was performed to determine the frequency of the HR gene XRCC3 Exon 7 (C18067T, rs861539) polymorphisms in Saudi Arabian population in comparison with epidemiological studies by "MEDLINE" search to equate with global populations. The variant allelic (T) frequency of XRCC3 (C>T) was found to be 39%. Our results suggest that frequency of XRCC3 (C>T) DNA repair gene exhibits distinctive patterns compared with the Saudi Arabian population and this might be attributed to ethnic variation. The present findings may help in high-risk screening of humans exposed to environmental carcinogens and cancer predisposition in different ethnic groups.

FANCG promotes formation of a newly identified protein complex containing BRCA2, FANCD2 and XRCC3.

Science.gov (United States)

Wilson, J B; Yamamoto, K; Marriott, A S; Hussain, S; Sung, P; Hoatlin, M E; Mathew, C G; Takata, M; Thompson, L H; Kupfer, G M; Jones, N J

2008-06-12

Fanconi anemia (FA) is a human disorder characterized by cancer susceptibility and cellular sensitivity to DNA crosslinks and other damages. Thirteen complementation groups and genes are identified, including BRCA2, which is defective in the FA-D1 group. Eight of the FA proteins, including FANCG, participate in a nuclear core complex that is required for the monoubiquitylation of FANCD2 and FANCI. FANCD2, like FANCD1/BRCA2, is not part of the core complex, and we previously showed direct BRCA2-FANCD2 interaction using yeast two-hybrid analysis. We now show in human and hamster cells that expression of FANCG protein, but not the other core complex proteins, is required for co-precipitation of BRCA2 and FANCD2. We also show that phosphorylation of FANCG serine 7 is required for its co-precipitation with BRCA2, XRCC3 and FANCD2, as well as the direct interaction of BRCA2-FANCD2. These results argue that FANCG has a role independent of the FA core complex, and we propose that phosphorylation of serine 7 is the signalling event required for forming a discrete complex comprising FANCD1/BRCA2-FANCD2-FANCG-XRCC3 (D1-D2-G-X3). Cells that fail to express either phospho-Ser7-FANCG, or full length BRCA2 protein, lack the interactions amongst the four component proteins. A role for D1-D2-G-X3 in homologous recombination repair (HRR) is supported by our finding that FANCG and the RAD51-paralog XRCC3 are epistatic for sensitivity to DNA crosslinking compounds in DT40 chicken cells. Our findings further define the intricate interface between FANC and HRR proteins in maintaining chromosome stability.

XRCC3 polymorphisms are associated with the risk of developing radiation-induced late xerostomia in nasopharyngeal carcinoma patients treated with intensity modulation radiated therapy.

Science.gov (United States)

Zou, Yan; Song, Tao; Yu, Wei; Zhao, Ruping; Wang, Yong; Xie, Ruifei; Chen, Tian; Wu, Bo; Wu, Shixiu

2014-03-01

The incidence of radiation-induced late xerostomia varies greatly in nasopharyngeal carcinoma patients treated with radiotherapy. The single-nucleotide polymorphisms in genes involved in DNA repair and fibroblast proliferation may be correlated with such variability. The purpose of this paper was to evaluate the association between the risk of developing radiation-induced late xerostomia and four genetic polymorphisms: TGFβ1 C-509T, TGFβ1 T869C, XRCC3 722C>T and ATM 5557G>A in nasopharyngeal carcinoma patients treated with Intensity Modulation Radiated Therapy. The severity of late xerostomia was assessed using a patient self-reported validated xerostomia questionnaire. Polymerase chain reaction-ligation detection reaction methods were performed to determine individual genetic polymorphism. The development of radiation-induced xerostomia associated with genetic polymorphisms was modeled using Cox proportional hazards, accounting for equivalent uniform dose. A total of 43 (41.7%) patients experienced radiation-induced late xerostomia. Univariate Cox proportional hazard analyses showed a higher risk of late xerostomia for patients with XRCC3 722 TT/CT alleles. In multivariate analysis adjusted for clinical and dosimetric factors, XRCC3 722C>T polymorphisms remained a significant factor for higher risk of late xerostomia. To our knowledge, this is the first study that demonstrated an association between genetic polymorphisms and the risk of radiation-induced late xerostomia in nasopharyngeal carcinoma patients treated with Intensity Modulation Radiated Therapy. Our findings suggest that the polymorphisms in XRCC3 are significantly associated with the risk of developing radiation-induced late xerostomia.

Lycopene intake and prostate cancer risk : Effect modification by plasma antioxidants and the XRCC1 genotype

NARCIS (Netherlands)

Goodman, Michael; Bostick, Roberd M.; Ward, Kevin C.; Terry, Paul D.; van Gils, Carla H.; Taylor, Jack A.; Mandel, Jack S.

2006-01-01

Lycopene has been associated with reduced prostate cancer risk, although the results ofepidemiological studies have varied We hypothesize that an effect of lycopene may be modified by XRCC1 genotype and other antioxidants. We used a food-frequency questionnaire to assess lycopene intake in a

Isolating human DNA repair genes using rodent-cell mutants

International Nuclear Information System (INIS)

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

1987-01-01

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

Pharmacogenetic Study in Rectal Cancer Patients Treated With Preoperative Chemoradiotherapy: Polymorphisms in Thymidylate Synthase, Epidermal Growth Factor Receptor, GSTP1, and DNA Repair Genes

International Nuclear Information System (INIS)

Páez, David; Salazar, Juliana; Paré, Laia; Pertriz, Lourdes; Targarona, Eduardo; Rio, Elisabeth del; Barnadas, Agusti; Marcuello, Eugenio; Baiget, Montserrat

2011-01-01

Purpose: Several studies have been performed to evaluate the usefulness of neoadjuvant treatment using oxaliplatin and fluoropyrimidines for locally advanced rectal cancer. However, preoperative biomarkers of outcome are lacking. We studied the polymorphisms in thymidylate synthase, epidermal growth factor receptor, glutathione S-transferase pi 1 (GSTP1), and several DNA repair genes to evaluate their usefulness as pharmacogenetic markers in a cohort of 128 rectal cancer patients treated with preoperative chemoradiotherapy. Methods and Materials: Blood samples were obtained from 128 patients with Stage II-III rectal cancer. DNA was extracted from the peripheral blood nucleated cells, and the genotypes were analyzed by polymerase chain reaction amplification and automated sequencing techniques or using a 48.48 dynamic array on the BioMark system. The germline polymorphisms studied were thymidylate synthase, (VNTR/5′UTR, 2R G>C single nucleotide polymorphism [SNP], 3R G>C SNP), epidermal growth factor receptor (Arg497Lys), GSTP1 (Ile105val), excision repair cross-complementing 1 (Asn118Asn, 8092C>A, 19716G>C), X-ray repair cross-complementing group 1 (XRCC1) (Arg194Trp, Arg280His, Arg399Gln), and xeroderma pigmentosum group D (Lys751Gln). The pathologic response, pathologic regression, progression-free survival, and overall survival were evaluated according to each genotype. Results: The ∗3/∗3 thymidylate synthase genotype was associated with a greater response rate (pathologic complete remission and microfoci residual tumor, 59% in ∗3/∗3 vs. 35% in ∗2/∗2 and ∗2/∗3; p = .013). For the thymidylate synthase genotype, the median progression-free survival was 103 months for the ∗3/∗3 patients and 84 months for the ∗2/∗2 and ∗2/∗3 patients (p = .039). For XRCC1 Arg399Gln SNP, the median progression-free survival was 101 months for the G/G, 78 months for the G/A, and 31 months for the A/A patients (p = .048). Conclusions: The thymidylate

Pharmacogenetic Study in Rectal Cancer Patients Treated With Preoperative Chemoradiotherapy: Polymorphisms in Thymidylate Synthase, Epidermal Growth Factor Receptor, GSTP1, and DNA Repair Genes

Energy Technology Data Exchange (ETDEWEB)

Paez, David, E-mail: dpaez@santpau.cat [Department of Medical Oncology, Hospital de la Santa Creu i Sant Pau, Universitat Autonoma de Barcelona, Barcelona (Spain); Salazar, Juliana; Pare, Laia [Centre for Biomedical Network Research on Rare Diseases, Barcelona (Spain); Department of Genetics, Hospital de la Santa Creu i Sant Pau, Universitat Autonoma de Barcelona, Barcelona (Spain); Pertriz, Lourdes [Department of Radiotherapy, Hospital de la Santa Creu i Sant Pau, Universitat Autonoma de Barcelona, Barcelona (Spain); Targarona, Eduardo [Department of Surgery, Hospital de la Santa Creu i Sant Pau, Universitat Autonoma de Barcelona, Barcelona (Spain); Rio, Elisabeth del [Centre for Biomedical Network Research on Rare Diseases, Barcelona (Spain); Department of Genetics, Hospital de la Santa Creu i Sant Pau, Universitat Autonoma de Barcelona, Barcelona (Spain); Barnadas, Agusti; Marcuello, Eugenio [Department of Medical Oncology, Hospital de la Santa Creu i Sant Pau, Universitat Autonoma de Barcelona, Barcelona (Spain); Baiget, Montserrat [Centre for Biomedical Network Research on Rare Diseases, Barcelona (Spain); Department of Genetics, Hospital de la Santa Creu i Sant Pau, Universitat Autonoma de Barcelona, Barcelona (Spain)

2011-12-01

Purpose: Several studies have been performed to evaluate the usefulness of neoadjuvant treatment using oxaliplatin and fluoropyrimidines for locally advanced rectal cancer. However, preoperative biomarkers of outcome are lacking. We studied the polymorphisms in thymidylate synthase, epidermal growth factor receptor, glutathione S-transferase pi 1 (GSTP1), and several DNA repair genes to evaluate their usefulness as pharmacogenetic markers in a cohort of 128 rectal cancer patients treated with preoperative chemoradiotherapy. Methods and Materials: Blood samples were obtained from 128 patients with Stage II-III rectal cancer. DNA was extracted from the peripheral blood nucleated cells, and the genotypes were analyzed by polymerase chain reaction amplification and automated sequencing techniques or using a 48.48 dynamic array on the BioMark system. The germline polymorphisms studied were thymidylate synthase, (VNTR/5 Prime UTR, 2R G>C single nucleotide polymorphism [SNP], 3R G>C SNP), epidermal growth factor receptor (Arg497Lys), GSTP1 (Ile105val), excision repair cross-complementing 1 (Asn118Asn, 8092C>A, 19716G>C), X-ray repair cross-complementing group 1 (XRCC1) (Arg194Trp, Arg280His, Arg399Gln), and xeroderma pigmentosum group D (Lys751Gln). The pathologic response, pathologic regression, progression-free survival, and overall survival were evaluated according to each genotype. Results: The Asterisk-Operator 3/ Asterisk-Operator 3 thymidylate synthase genotype was associated with a greater response rate (pathologic complete remission and microfoci residual tumor, 59% in Asterisk-Operator 3/ Asterisk-Operator 3 vs. 35% in Asterisk-Operator 2/ Asterisk-Operator 2 and Asterisk-Operator 2/ Asterisk-Operator 3; p = .013). For the thymidylate synthase genotype, the median progression-free survival was 103 months for the Asterisk-Operator 3/ Asterisk-Operator 3 patients and 84 months for the Asterisk-Operator 2/ Asterisk-Operator 2 and Asterisk-Operator 2/ Asterisk

Regulatory mechanisms of RNA function: emerging roles of DNA repair enzymes.

Science.gov (United States)

Jobert, Laure; Nilsen, Hilde

2014-07-01

The acquisition of an appropriate set of chemical modifications is required in order to establish correct structure of RNA molecules, and essential for their function. Modification of RNA bases affects RNA maturation, RNA processing, RNA quality control, and protein translation. Some RNA modifications are directly involved in the regulation of these processes. RNA epigenetics is emerging as a mechanism to achieve dynamic regulation of RNA function. Other modifications may prevent or be a signal for degradation. All types of RNA species are subject to processing or degradation, and numerous cellular mechanisms are involved. Unexpectedly, several studies during the last decade have established a connection between DNA and RNA surveillance mechanisms in eukaryotes. Several proteins that respond to DNA damage, either to process or to signal the presence of damaged DNA, have been shown to participate in RNA quality control, turnover or processing. Some enzymes that repair DNA damage may also process modified RNA substrates. In this review, we give an overview of the DNA repair proteins that function in RNA metabolism. We also discuss the roles of two base excision repair enzymes, SMUG1 and APE1, in RNA quality control.

Molecular dynamics of formation of TD lesioned DNA complexed with repair enzyme - onset of the enzymatic repair process

Energy Technology Data Exchange (ETDEWEB)

Pinak, Miroslav [Japan Atomic Energy Research Inst., Tokai, Ibaraki (Japan). Tokai Research Establishment

1999-12-01

To describe the first step of the enzymatic repair process (formation of complex enzyme-DNA), in which the thymine dimer (TD) part is removed from DNA, the 500 picosecond (ps) molecular dynamics (MD) simulation of TD lesioned DNA and part of repair enzyme cell (inclusive of catalytic center - Arg-22, Glu-23, Arg-26 and Thr-2) was performed. TD is UV originated lesion in DNA and T4 Endonuclease V is TD specific repair enzyme. Both molecules were located in the same simulation cell and their relative movement was examined. During the simulation the research was focused on the role of electrostatic energy in formation of complex enzyme-DNA. It is found, that during the first 100 ps of MD, the part of enzyme approaches the DNA surface at the TD lesion, interacts extensively by electrostatic and van der Walls interactions with TD part of DNA and forms complex that lasts stabile for 500 ps of MD. In the beginning of MD, the positive electrostatic interaction energy between part of enzyme and TD ({approx} +10 kcal/mol) drives enzyme towards the DNA molecule. Water-mediated hydrogen bonds between enzyme and DNA help to keep complex stabile. As a reference, the MD simulation of the identical system with native DNA molecule (two native thymines (TT) instead of TD) was performed. In this system the negative electrostatic interaction energy between part of enzyme and TT ({approx} -11 kcal/mol), in contrary to the positive one in the system with TD, doesn't drive enzyme towards DNA and complex is not formed. (author)

Molecular dynamics of formation of TD lesioned DNA complexed with repair enzyme - onset of the enzymatic repair process

International Nuclear Information System (INIS)

Pinak, Miroslav

1999-12-01

To describe the first step of the enzymatic repair process (formation of complex enzyme-DNA), in which the thymine dimer (TD) part is removed from DNA, the 500 picosecond (ps) molecular dynamics (MD) simulation of TD lesioned DNA and part of repair enzyme cell (inclusive of catalytic center - Arg-22, Glu-23, Arg-26 and Thr-2) was performed. TD is UV originated lesion in DNA and T4 Endonuclease V is TD specific repair enzyme. Both molecules were located in the same simulation cell and their relative movement was examined. During the simulation the research was focused on the role of electrostatic energy in formation of complex enzyme-DNA. It is found, that during the first 100 ps of MD, the part of enzyme approaches the DNA surface at the TD lesion, interacts extensively by electrostatic and van der Walls interactions with TD part of DNA and forms complex that lasts stabile for 500 ps of MD. In the beginning of MD, the positive electrostatic interaction energy between part of enzyme and TD (∼ +10 kcal/mol) drives enzyme towards the DNA molecule. Water-mediated hydrogen bonds between enzyme and DNA help to keep complex stabile. As a reference, the MD simulation of the identical system with native DNA molecule (two native thymines (TT) instead of TD) was performed. In this system the negative electrostatic interaction energy between part of enzyme and TT (∼ -11 kcal/mol), in contrary to the positive one in the system with TD, doesn't drive enzyme towards DNA and complex is not formed. (author)

ABCC5, ERCC2, XPA and XRCC1 transcript abundance levels correlate with cisplatin chemoresistance in non-small cell lung cancer cell lines

Directory of Open Access Journals (Sweden)

Khuder Sadik A

2005-05-01

Full Text Available Abstract Background Although 40–50% of non-small cell lung cancer (NSCLC tumors respond to cisplatin chemotherapy, there currently is no way to prospectively identify potential responders. The purpose of this study was to determine whether transcript abundance (TA levels of twelve selected DNA repair or multi-drug resistance genes (LIG1, ERCC2, ERCC3, DDIT3, ABCC1, ABCC4, ABCC5, ABCC10, GTF2H2, XPA, XPC and XRCC1 were associated with cisplatin chemoresistance and could therefore contribute to the development of a predictive marker. Standardized RT (StaRT-PCR, was employed to assess these genes in a set of NSCLC cell lines with a previously published range of sensitivity to cisplatin. Data were obtained in the form of target gene molecules relative to 106 β-actin (ACTB molecules. To cancel the effect of ACTB variation among the different cell lines individual gene expression values were incorporated into ratios of one gene to another. Each two-gene ratio was compared as a single variable to chemoresistance for each of eight NSCLC cell lines using multiple regression. In an effort to validate these results, six additional lines then were evaluated. Results Following validation, single variable models best correlated with chemoresistance (p ERCC2/XPC, ABCC5/GTF2H2, ERCC2/GTF2H2, XPA/XPC and XRCC1/XPC. All single variable models were examined hierarchically to achieve two variable models. The two variable model with the highest correlation was (ABCC5/GTF2H2, ERCC2/GTF2H2 with an R2 value of 0.96 (p Conclusion These results provide markers suitable for assessment of small fine needle aspirate biopsies in an effort to prospectively identify cisplatin resistant tumors.

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.

Computational studies of radiation and oxidative damage to DNA and its recognition by repair enzyme

Energy Technology Data Exchange (ETDEWEB)

Pinak, M. [Center for Promotion of Computational Science and Engineering, Tokai Research Establishment, Japan Atomic Energy Research Inst., Tokai, Ibaraki (Japan)

2000-03-01

Molecular dynamics (MD) simulation is used to study the time evolution of the recognition processes and to construct a model of the specific DNA-repair enzyme' complexes. MD simulations of the following molecules were performed: DNA dodecamer with thymine dimer (TD), DNA 30-mer with thymine glycol (TG), and respective specific repair enzymes T4 Endonuclease V and Endonuclease III. Both DNA lesions are experimentally suggested to be mutagenic and carcinogenic unless properly recognized and repaired by repair enzymes. In the case of TD, there is detected a strong kink around the TD site, that is not observed in native DNA. In addition there is observed a different value of electrostatic energy at the TD site - negative '-9 kcal/mol', in contrast to the nearly neutral value of the native thymine site. These two factors - structural changes and specific electrostatic energy - seem to be important for proper recognition of a TD damaged site and for formation of DNA-enzyme complex. Formation of this complex is the onset of the repair of DNA. In the case of TG damaged DNA the structural characteristics of the TG were calculated (charges, bond lengths, bond angles, etc.). The formed TG was used to replace the native thymine and then submitted to the simulation in the system with a repair enzyme with Endonuclease III for the purpose of the study of the formation of the DNA-enzyme complex. (author)

Molecular dynamics simulation studies of radiation damaged DNA. Molecules and repair enzymes

International Nuclear Information System (INIS)

Pinak, Miroslav

2004-12-01

Molecular dynamics (MD) studies on several radiation damages to DNA and their recognition by repair enzymes are introduced in order to describe the stepwise description of molecular process observed at radiation lesion sites. MD studies were performed on pyrimidine (thymine dimer, thymine glycol) and purine (8-oxoguanine) lesions using an MD simulation code AMBER 5.0. The force field was modified for each lesion. In all cases the significant structural changes in the DNA double helical structure were observed; a) the breaking of hydrogen bond network between complementary bases and resulting opening of the double helix (8-oxoguanine); b) the sharp bending of the DNA helix centered at the lesion site (thymine dimer, thymine glycol); and c) the flipping-out base on the strand complementary to the lesion (8-oxoguanine). These changes were related to the overall collapsing double helical structure around the lesion and might facilitate the docking of the repair enzyme into the DNA and formation of DNA-enzyme complex. In addition to the structural changes, at lesion sites there were found electrostatic interaction energy values different from those at native sites (thymine dimer -10 kcal/mol, thymine glycol -26 kcal/mol, 8-oxoguanine -48 kcal/mol). These values of electrostatic energy may discriminate lesion from values at native sites (thymine 0 kcal/mol, guanine -37 kcal/mol) and enable a repair enzyme to recognize a lesion during scanning DNA surface. The observed specific structural conformation and energetic properties at the lesions sites are factors that guide a repair enzyme to discriminate lesions from non-damaged native DNA segments. (author)

Role of XPC, XPD, XRCC1, GSTP genetic polymorphisms and Barrett’s esophagus in a cohort of Italian subjects. A neural network analysis

Directory of Open Access Journals (Sweden)

Tarlarini C

2012-08-01

Full Text Available Claudia Tarlarini,1 Silvana Penco,1 Massimo Conio,2 Enzo Grossi3 On behalf of the Barrett Italian Study Group 1Department of Laboratory Medicine, Medical Genetics, Niguarda Ca’ Granda Hospital, Milan, Italy; 2Department of Gastroenterology, General Hospital, San Remo, Italy; 3Medical Department, Bracco Imaging SpA, Milan, ItalyBackground: Barrett’s esophagus (BE, a metaplastic premalignant disorder, represents the primary risk factor for the development of esophageal adenocarcinoma. Chronic gastroesophageal reflux disease and central obesity have been associated with BE and esophageal adenocarcinoma, but relatively little is known about the specific genes that confer susceptibility to BE carcinogenesis.Methods: A total of 74 patients with BE and 67 controls coming from six gastrointestinal Italian units were evaluated for six polymorphisms in four genes: XPC, XPD nucleotide excision repair (NER genes, XRCC1 (BER gene, and glutathione S-transferase P1. Smoking status was analyzed together with the genetic data. Statistical analysis was performed through Artificial Neural Networks.Results: Distributions of sex, smoking history, and polymorphisms among BE cases and controls did not show statistically significant differences. The r-value from linear correlation allowed us to identify possible protective factors as well as possible risk factors. The application of advanced intelligent systems allowed for the selection of a subgroup of nine variables. Artificial Neural Networks applied on the final data set reached mean global accuracy of 60%, reaching as high as 65.88%.Conclusion: We report here results from an exploratory study. Results from this study failed to find an association among the tested single nucleotide polymorphisms and BE phenotype through classical statistical methods. On the contrary, advanced intelligent systems are really able to handle the disease complexity, not treating the data with reductionist approaches unable to detect

Oxidative Stress, DNA Damage and DNA Repair in Female Patients with Diabetes Mellitus Type 2.

Directory of Open Access Journals (Sweden)

Annemarie Grindel

Full Text Available Diabetes mellitus type 2 (T2DM is associated with oxidative stress which in turn can lead to DNA damage. The aim of the present study was to analyze oxidative stress, DNA damage and DNA repair in regard to hyperglycemic state and diabetes duration.Female T2DM patients (n = 146 were enrolled in the MIKRODIAB study and allocated in two groups regarding their glycated hemoglobin (HbA1c level (HbA1c≤7.5%, n = 74; HbA1c>7.5%, n = 72. In addition, tertiles according to diabetes duration (DD were created (DDI = 6.94±3.1 y, n = 49; DDII = 13.35±1.1 y, n = 48; DDIII = 22.90±7.3 y, n = 49. Oxidative stress parameters, including ferric reducing ability potential, malondialdehyde, oxidized and reduced glutathione, reduced thiols, oxidized LDL and F2-Isoprostane as well as the activity of antioxidant enzymes superoxide dismutase, catalase and glutathione peroxidase were measured. Damage to DNA was analyzed in peripheral blood mononuclear cells and whole blood with single cell gel electrophoresis. DNA base excision repair capacity was tested with the modified comet repair assay. Additionally, mRNA expressions of nine genes related to base excision repair were analyzed in a subset of 46 matched individuals.No significant differences in oxidative stress parameters, antioxidant enzyme activities, damage to DNA and base excision repair capacity, neither between a HbA1c cut off />7.5%, nor between diabetes duration was found. A significant up-regulation in mRNA expression was found for APEX1, LIG3 and XRCC1 in patients with >7.5% HbA1c. Additionally, we observed higher total cholesterol, LDL-cholesterol, LDL/HDL-cholesterol, triglycerides, Framingham risk score, systolic blood pressure, BMI and lower HDL-cholesterol in the hyperglycemic group.BMI, blood pressure and blood lipid status were worse in hyperglycemic individuals. However, no major disparities regarding oxidative stress, damage to DNA and DNA repair were present which might be due to good medical

Journal of Genetics | Indian Academy of Sciences

Indian Academy of Sciences (India)

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

Cobalt-induced genotoxicity in male zebrafish (Danio rerio), with implications for reproduction and expression of DNA repair genes

Energy Technology Data Exchange (ETDEWEB)

Reinardy, Helena C.; Syrett, James R. [School of Biomedical and Biological Sciences, University of Plymouth (United Kingdom); Jeffree, Ross A. [Faculty of Science, University of Technology, Sydney, PO Box 123, Broadway, NSW 2007 (Australia); Henry, Theodore B., E-mail: ted.henry@plymouth.ac.uk [School of Biomedical and Biological Sciences, University of Plymouth (United Kingdom); Center for Environmental Biotechnology, University of Tennessee, Knoxville, TN 37996 (United States); Department of Forestry, Wildlife and Fisheries, University of Tennessee, Knoxville, TN 37996. USA (United States); Jha, Awadhesh N. [School of Biomedical and Biological Sciences, The University of Plymouth (United Kingdom)

2013-01-15

Although cobalt (Co) is an environmental contaminant of surface waters in both radioactive (e.g. {sup 60}Co) and non-radioactive forms, there is relatively little information about Co toxicity in fishes. The objective of this study was to investigate acute and chronic toxicity of Co in zebrafish, with emphasis on male genotoxicity and implications for reproductive success. The lethal concentration for 50% mortality (LC{sub 50}) in larval zebrafish exposed (96 h) to 0-50 mg l{sup -1} Co was 35.3 {+-} 1.1 (95% C.I.) mg l{sup -1} Co. Adult zebrafish were exposed (13 d) to sub-lethal (0-25 mg l{sup -1}) Co and allowed to spawn every 4 d and embryos were collected. After 12-d exposure, fertilisation rate was reduced (6% total eggs fertilised, 25 mg l{sup -1}) and embryo survival to hatching decreased (60% fertilised eggs survived, 25 mg l{sup -1}). A concentration-dependent increase in DNA strand breaks was detected in sperm from males exposed (13 d) to Co, and DNA damage in sperm returned to control levels after males recovered for 6 d in clean water. Induction of DNA repair genes (rad51, xrcc5, and xrcc6) in testes was complex and not directly related to Co concentration, although there was significant induction in fish exposed to 15 and 25 mg l{sup -1} Co relative to controls. Induction of 4.0 {+-} 0.9, 2.5 {+-} 0.7, and 3.1 {+-} 0.7-fold change (mean {+-} S.E.M. for rad51, xrcc5, and xrcc6, respectively) was observed in testes at the highest Co concentration (25 mg l{sup -1}). Expression of these genes was not altered in offspring (larvae) spawned after 12-d exposure. Chronic exposure to Co resulted in DNA damage in sperm, induction of DNA repair genes in testes, and indications of reduced reproductive success.

Clustering of double strand break-containing chromosome domains is not inhibited by inactivation of major repair proteins

International Nuclear Information System (INIS)

Krawczyk, P. M.; Stap, C.; Van Oven, C.; Hoebe, R.; Aten, J. A.

2006-01-01

For efficient repair of DNA double strand breaks (DSBs) cells rely on a process that involves the Mre11/Rad50/Nbs1 complex, which may help to protect non-repaired DNA ends from separating until they can be rejoined by DNA repair proteins. It has been observed that as a secondary effect, this process can lead to unintended clustering of multiple, initially separate, DSB-containing chromosome domains. This work demonstrates that neither inactivation of the major repair proteins XRCC3 and the DNA-dependent protein kinase (DNA-PK) nor inhibition of DNA-PK by vanillin influences the aggregation of DSB-containing chromosome domains. (authors)

Human DNA repair and recombination genes

International Nuclear Information System (INIS)

Thompson, L.H.; Weber, C.A.; Jones, N.J.

1988-09-01

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

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

Association of thymine glycol lesioned DNA with repair enzyme endonuclease III-molecular dynamics study

International Nuclear Information System (INIS)

Pinak, Miroslav

2001-07-01

The 2 nanoseconds molecular dynamics (MD) simulation has been performed for the system consisting of repair enzyme and DNA 30-mer with native thymine at position 16 replaced by thymine glycol (TG) solvated in water environment. After 950 picoseconds of MD the enzyme and DNA associated together to form complex that lasted stable up to 2 ns when simulation was terminated. At the contact area of enzyme and DNA there is glutamic acid located as close as 1.6 A to the C3' atom of phosphodiester bond of TG. Initial B-DNA molecule was bent and kinked at the TG during MD. This distortion caused that phosphodiester bond was easier accessible by amino acids of enzyme. The negative value of electrostatic energy (-26 kcal/mol) discriminates TG from nearly neutral native thymine and contributes to the specific recognition of this lesion. Higher number of close water molecules at TG site before formation of complex (compared with other nucleotides) indicates that glycosyl bond of the lesion is easily approached by repair enzyme during scanning of DNA surface and suggests the importance of specific hydration at the lesion during recognition process. (author)

Association of thymine glycol lesioned DNA with repair enzyme endonuclease III-molecular dynamics study

Energy Technology Data Exchange (ETDEWEB)

Pinak, Miroslav [Japan Atomic Energy Research Inst., Tokai, Ibaraki (Japan). Tokai Research Establishment

2001-07-01

The 2 nanoseconds molecular dynamics (MD) simulation has been performed for the system consisting of repair enzyme and DNA 30-mer with native thymine at position 16 replaced by thymine glycol (TG) solvated in water environment. After 950 picoseconds of MD the enzyme and DNA associated together to form complex that lasted stable up to 2 ns when simulation was terminated. At the contact area of enzyme and DNA there is glutamic acid located as close as 1.6 A to the C3' atom of phosphodiester bond of TG. Initial B-DNA molecule was bent and kinked at the TG during MD. This distortion caused that phosphodiester bond was easier accessible by amino acids of enzyme. The negative value of electrostatic energy (-26 kcal/mol) discriminates TG from nearly neutral native thymine and contributes to the specific recognition of this lesion. Higher number of close water molecules at TG site before formation of complex (compared with other nucleotides) indicates that glycosyl bond of the lesion is easily approached by repair enzyme during scanning of DNA surface and suggests the importance of specific hydration at the lesion during recognition process. (author)

Rubisco Activases: AAA+ Chaperones Adapted to Enzyme Repair.

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Bhat, Javaid Y; Thieulin-Pardo, Gabriel; Hartl, F Ulrich; Hayer-Hartl, Manajit

2017-01-01

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), the key enzyme of the Calvin-Benson-Bassham cycle of photosynthesis, requires conformational repair by Rubisco activase for efficient function. Rubisco mediates the fixation of atmospheric CO 2 by catalyzing the carboxylation of the five-carbon sugar ribulose-1,5-bisphosphate (RuBP). It is a remarkably inefficient enzyme, and efforts to increase crop yields by bioengineering Rubisco remain unsuccessful. This is due in part to the complex cellular machinery required for Rubisco biogenesis and metabolic maintenance. To function, Rubisco must undergo an activation process that involves carboxylation of an active site lysine by a non-substrate CO 2 molecule and binding of a Mg 2+ ion. Premature binding of the substrate RuBP results in an inactive enzyme. Moreover, Rubisco can also be inhibited by a range of sugar phosphates, some of which are "misfire" products of its multistep catalytic reaction. The release of the inhibitory sugar molecule is mediated by the AAA+ protein Rubisco activase (Rca), which couples hydrolysis of ATP to the structural remodeling of Rubisco. Rca enzymes are found in the vast majority of photosynthetic organisms, from bacteria to higher plants. They share a canonical AAA+ domain architecture and form six-membered ring complexes but are diverse in sequence and mechanism, suggesting their convergent evolution. In this review, we discuss recent advances in understanding the structure and function of this important group of client-specific AAA+ proteins.

Repair of abasic sites in DNA

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Dianov, Grigory L.; Sleeth, Kate M.; Dianova, Irina I.; Allinson, Sarah L

2003-10-29

Repair of both normal and reduced AP sites is activated by AP endonuclease, which recognizes and cleaves a phosphodiester bond 5' to the AP site. For a short period of time an incised AP site is occupied by poly(ADP-ribose) polymerase and then DNA polymerase {beta} adds one nucleotide into the repair gap and simultaneously removes the 5'-sugar phosphate. Finally, the DNA ligase III/XRCC1 complex accomplishes repair by sealing disrupted DNA ends. However, long-patch BER pathway, which is involved in the removal of reduced abasic sites, requires further DNA synthesis resulting in strand displacement and the generation of a damage-containing flap that is later removed by the flap endonuclease. Strand-displacement DNA synthesis is accomplished by DNA polymerase {delta}/{epsilon} and DNA ligase I restores DNA integrity. DNA synthesis by DNA polymerase {delta}/{epsilon} is dependent on proliferating cell nuclear antigen, which also stimulates the DNA ligase I and flap endonuclease. These repair events are supported by multiple protein-protein interactions.

Islet expression of the DNA repair enzyme 8-oxoguanosine DNA glycosylase (Ogg1 in human type 2 diabetes

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Yoon Kun-Ho

2002-04-01

Full Text Available Abstract Background It has become increasingly clear that β-cell failure plays a critical role in the pathogenesis of type 2 diabetes. Free-radical mediated β-cell damage has been intensively studied in type 1 diabetes, but not in human type 2 diabetes. Therefore, we studied the protein expression of the DNA repair enzyme Ogg1 in pancreases from type 2 diabetics. Ogg1 was studied because it is the major enzyme involved in repairing 7,8-dihydro-8-oxoguanosine DNA adducts, a lesion previously observed in a rat model of type 2 diabetes. Moreover, in a gene expression screen, Ogg1 was over-expressed in islets from a human type 2 diabetic. Methods Immunofluorescent staining of Ogg1 was performed on pancreatic specimens from healthy controls and patients with diabetes for 2–23 years. The intensity and islet area stained for Ogg1 was evaluated by semi-quantitative scoring. Results Both the intensity and the area of islet Ogg1 staining were significantly increased in islets from the type 2 diabetic subjects compared to the healthy controls. A correlation between increased Ogg1 fluorescent staining intensity and duration of diabetes was also found. Most of the staining observed was cytoplasmic, suggesting that mitochondrial Ogg1 accounts primarily for the increased Ogg1 expression. Conclusion We conclude that oxidative stress related DNA damage may be a novel important factor in the pathogenesis of human type 2 diabetes. An increase of Ogg1 in islet cell mitochondria is consistent with a model in which hyperglycemia and consequent increased β-cell oxidative metabolism lead to DNA damage and the induction of Ogg1 expression.

Effects of expression level of DNA repair-related genes involved in the NHEJ pathway on radiation-induced cognitive impairment

International Nuclear Information System (INIS)

Zhang Liyuan; Chen Liesong; Sun Rui; Ji Shengjun; Ding Yanyan; Wu Jia; Tian Ye

2013-01-01

Cranial radiation therapy can induce cognitive decline. Impairments of hippocampal neurogenesis are thought to be a paramountly important mechanism underlying radiation-induced cognitive dysfunction. In the mature nervous system, DNA double-strand breaks (DSBs) are mainly repaired by non-homologous end-joining (NHEJ) pathways. It has been demonstrated that NHEJ deficiencies are associated with impaired neurogenesis. In our study, rats were randomly divided into five groups to be irradiated by single doses of 0 (control), 0 (anesthesia control), 2, 10, and 20 Gy, respectively. The cognitive function of the irradiated rats was measured by open field, Morris water maze and passive avoidance tests. Real-time PCR was also used to detect the expression level of DNA DSB repair-related genes involved in the NHEJ pathway, such as XRCC4, XRCC5 and XRCC6, in the hippocampus. The influence of different radiation doses on cognitive function in rats was investigated. From the results of the behavior tests, we found that rats receiving 20 Gy irradiation revealed poorer learning and memory, while no significant loss of learning and memory existed in rats receiving irradiation from 0-10 Gy. The real-time PCR and Western blot results showed no significant difference in the expression level of DNA repair-related genes between the 10 and 20 Gy groups, which may help to explain the behavioral results, id est (i.e.) DNA damage caused by 0-10 Gy exposure was appropriately repaired, however, damage induced by 20 Gy exceeded the body's maximum DSB repair ability. Ionizing radiation-induced cognitive impairments depend on the radiation dose, and more directly on the body's own ability to repair DNA DSBs via the NHEJ pathway. (author)

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

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Saeed Rezapoor

2017-01-01

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

Polymorphisms of Selected DNA Repair Genes and Lung Cancer in Chromium Exposure.

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Halasova, E; Matakova, T; Skerenova, M; Krutakova, M; Slovakova, P; Dzian, A; Javorkova, S; Pec, M; Kypusova, K; Hamzik, J

2016-01-01

Chromium is a well-known mutagen and carcinogen involved in lung cancer development. DNA repair genes play an important role in the elimination of genetic changes caused by chromium exposure. In the present study, we investigated the polymorphisms of the following DNA repair genes: XRCC3, participating in the homologous recombination repair, and hMLH1 and hMSH2, functioning in the mismatch repair. We focused on the risk the polymorphisms present in the development of lung cancer regarding the exposure to chromium. We analyzed 106 individuals; 45 patients exposed to chromium with diagnosed lung cancer and 61 healthy controls. Genotypes were determined by a PCR-RFLP method. We unravelled a potential for increased risk of lung cancer development in the hMLH1 (rs1800734) AA genotype in the recessive model. In conclusion, gene polymorphisms in the DNA repair genes underscores the risk of lung cancer development in chromium exposed individuals.

The association of folate pathway and DNA repair polymorphisms with susceptibility to childhood acute lymphoblastic leukemia.

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Goričar, Katja; Erčulj, Nina; Faganel Kotnik, Barbara; Debeljak, Maruša; Hovnik, Tinka; Jazbec, Janez; Dolžan, Vita

2015-05-15

Genetic factors may play an important role in susceptibility to childhood acute lymphoblastic leukemia (ALL). The aim of our study was to evaluate the associations of genetic polymorphisms in folate pathway and DNA repair genes with susceptibility to ALL. In total, 121 children with ALL and 184 unrelated healthy controls of Slovenian origin were genotyped for 14 polymorphisms in seven genes of folate pathway, base excision repair and homologous recombination repair (TYMS, MTHFR, OGG1, XRCC1, NBN, RAD51, and XRCC3). In addition, the exon 6 of NBN was screened for the presence of mutations using denaturing high performance liquid chromatography. Twelve polymorphisms were in Hardy-Weinberg equilibrium in controls and their genotype frequencies were in agreement with those reported in other Caucasian populations. Among the investigated polymorphisms and mutations, NBN Glu185Gln significantly decreased susceptibility to B-cell ALL (p=0.037), while TYMS 3R allele decreased susceptibility to T-cell ALL (p=0.011). Moreover, significantly decreased susceptibility to ALL was observed for MTHFR TA (p=0.030) and RAD51 GTT haplotypes (p=0.016). Susceptibility to ALL increased with the increasing number of risk alleles (ptrend=0.007). We also observed significant influence of hOGG-RAD51 and NBN-RAD51 interactions on susceptibility to ALL. Our results suggest that combination of several polymorphisms in DNA repair and folate pathways may significantly affect susceptibility to childhood ALL. Copyright © 2015 Elsevier B.V. All rights reserved.

DNA repair pathways involved in determining the level of cytotoxicity of environmentally relevant UV radiation

International Nuclear Information System (INIS)

Carpenter, L.

2000-01-01

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)

Exploring the deleterious SNPs in XRCC4 gene using computational approach and studying their association with breast cancer in the population of West India.

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Singh, Preety K; Mistry, Kinnari N; Chiramana, Haritha; Rank, Dharamshi N; Joshi, Chaitanya G

2018-05-20

Non-homologous end joining (NHEJ) pathway has pivotal role in repair of double-strand DNA breaks that may lead to carcinogenesis. XRCC4 is one of the essential proteins of this pathway and single-nucleotide polymorphisms (SNPs) of this gene are reported to be associated with cancer risks. In our study, we first used computational approaches to predict the damaging variants of XRCC4 gene. Tools predicted rs79561451 (S110P) nsSNP as the most deleterious SNP. Along with this SNP, we analysed other two SNPs (rs3734091 and rs6869366) to study their association with breast cancer in population of West India. Variant rs3734091 was found to be significantly associated with breast cancer while rs6869366 variant did not show any association. These SNPs may influence the susceptibility of individuals to breast cancer in this population. Copyright © 2018 Elsevier B.V. All rights reserved.

DNA Repair Mechanism Gene, XRCC1A (Arg194Trp) but not XRCC3 (Thr241Met) Polymorphism Increased the Risk of Breast Cancer in Premenopausal Females: A Case–Control Study in Northeastern Region of India

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Ahmed, Jishan; Narain, Kanwar; Mukherjee, Kaustab; Majumdar, Gautam; Chenkual, Saia; Zonunmawia, Jason C.

2017-01-01

X-ray repair cross complementary group gene is one of the most studied candidate gene involved in different types of cancers. Studies have shown that X-ray repair cross complementary genes are significantly associated with increased risk of breast cancer in females. Moreover, studies have revealed that X-ray repair cross complementary gene polymorphism significantly varies between and within different ethnic groups globally. The present case–control study was aimed to investigate the association of X-ray repair cross complementary 1A (Arg194Trp) and X-ray repair cross complementary 3 (Thr241Met) polymorphism with the risk of breast cancer in females from northeastern region of India. The present case–control study includes histopathologically confirmed and newly diagnosed 464 cases with breast cancer and 534 apparently healthy neighborhood community controls. Information on sociodemographic factors and putative risk factors were collected from each study participant by conducting face-to-face interviews. Genotyping of X-ray repair cross complementary 1A (Arg194Trp) and X-ray repair cross complementary 3 (Thr241Met) was carried out by polymerase chain reaction-restriction fragment length polymorphism. For statistical analysis, both univariate and multivariate logistic regression analyses were performed. We also performed stratified analysis to find out the association of X-ray repair cross complementary genes with the risk of breast cancer stratified based on menstrual status. This study revealed that tryptophan allele (R/W-W/W genotype) in X-ray repair cross complementary 1A (Arg194Trp) gene significantly increased the risk of breast cancer (adjusted odds ratio = 1.44, 95% confidence interval = 1.06-1.97, P India which may be beneficial for prognostic purposes. PMID:29332455

Differential recruitment of DNA Ligase I and III to DNA repair sites

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Mortusewicz, Oliver; Rothbauer, Ulrich; Cardoso, M. Cristina; Leonhardt, Heinrich

2006-01-01

DNA ligation is an essential step in DNA replication, repair and recombination. Mammalian cells contain three DNA Ligases that are not interchangeable although they use the same catalytic reaction mechanism. To compare the recruitment of the three eukaryotic DNA Ligases to repair sites in vivo we introduced DNA lesions in human cells by laser microirradiation. Time lapse microscopy of fluorescently tagged proteins showed that DNA Ligase III accumulated at microirradiated sites before DNA Ligase I, whereas we could detect only a faint accumulation of DNA Ligase IV. Recruitment of DNA Ligase I and III to repair sites was cell cycle independent. Mutational analysis and binding studies revealed that DNA Ligase I was recruited to DNA repair sites by interaction with PCNA while DNA Ligase III was recruited via its BRCT domain mediated interaction with XRCC1. Selective recruitment of specialized DNA Ligases may have evolved to accommodate the particular requirements of different repair pathways and may thus enhance efficiency of DNA repair. PMID:16855289

Polymorphisms of XRCC4 are involved in reduced colorectal cancer risk in Chinese schizophrenia patients

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Li Tao

2010-10-01

Full Text Available Abstract Background Genetic factors related to the regulation of apoptosis in schizophrenia patients may be involved in a reduced vulnerability to cancer. XRCC4 is one of the potential candidate genes associated with schizophrenia which might induce colorectal cancer resistance. Methods To examine the genetic association between colorectal cancer and schizophrenia, we analyzed five SNPs (rs6452526, rs2662238, rs963248, rs35268, rs2386275 covering ~205.7 kb in the region of XRCC4. Results We observed that two of the five genetic polymorphisms showed statistically significant differences between 312 colorectal cancer subjects without schizophrenia and 270 schizophrenia subjects (rs6452536, p = 0.004, OR 0.61, 95% CI 0.44-0.86; rs35268, p = 0.028, OR 1.54, 95% CI 1.05-2.26. Moreover, the haplotype which combined all five markers was the most significant, giving a global p = 0.0005. Conclusions Our data firstly indicate that XRCC4 may be a potential protective gene towards schizophrenia, conferring reduced susceptibility to colorectal cancer in the Han Chinese population.

Comparative Study between topical applications liposomally entrapped DNA repair enzymes and thymidine dinucleotide as radioprotectors

International Nuclear Information System (INIS)

Shabon, M.H.; El-Bedewi, A.F.

2005-01-01

The delivery of active agents to the skin by liposome carriers received great interest during the last three decades. This is based on their potential to enclose various types of biological materials and to deliver them to diverse cell types. Recent work suggests that liposomes as vehicles for topical drug delivery may be superior to conventional preparations. Also, topical application of DNA repair enzymes to irradiated skin increases the rate of repair of DNA potentially damaged cells. Moreover, thymidine dinucleotide is a new skin photo-protective agent against non-ionizing radiation through induction of DNA repair. Gamma irradiation can produce DNA damage in human skin. DNA mutations have an important role in the development of skin cancer and precancerous skin lesions. Albino rats were irradiated with Cobalt-60 gamma radiation with different doses (0.5, 1.5, 3 Gy), and were treated by either thymidine dinucleotide or liposomally entrapped DNA repair enzymes topically 24 hours before irradiation. Evaluation was done histopathologically by H and E stain. Computerized image analyzer using Masson's trichrome stain was also done. Gamma radiation produced epidermal thinning and dermal inflammatory cells together with collagen fragmentation and clumping in a dose-dependent manner. Comparing between both thymidine dinucleotide and liposomally entrapped DNA repair enzymes pretreated and irradiated rats. Low dose irradiation (0.5 Gy) together with previous drugs showed preservation of epidermis with no inflammatory cells and also it maintained the normal architecture of collagen bundles. However, they were ineffective with higher doses. In conclusion our results may suggest that the effects of gamma radiation on the skin at low dose could be minimized by the use of these drugs before exposure

To Investigate the Association of Thr241Met Polymorphisms of the XRCC3 Gene with the Risk of Breast Cancer in Women in Markazi Province

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Ahmad Hamta

2018-03-01

Full Text Available Abstract Background: Biological and epidemiological data suggest that damage induced by endogenous and exogenous factors affects the integrity and stability of DNA and associated with susceptibility to breast cancer. The XRCC3 protein participates in DNA double-strand breaks and recombination repair. The aim of the present study was to evaluate associations between the risk of breast cancer and Thr241Met polymorphism in the XRCC3 gene. Materials and Methods: In this study, the effects of Thr241Met polymorphism of the XRCC3 gene and the risk of breast cancer in a population-based case-control study inclusive 80 patients and 80 healthy individuals of women in Markazi province were evaluated. Genomic DNA was extracted from blood samples using the kit procedure. The genotypes of samples were determined by PCR-RFLP technique. Statistical analysis was done using SPSS software (estimation of χ2 and p-value and the final results were determined. Results: Statistically significant difference was observed between the two groups of patients and controls for three genotypes of the site rs861539 (p= 0.000. Genotype CT (p= 0.000, OR=2.352, CI= 95%; 2.431 - 39.948 and TT (p = 0.003, OR= 2.352, CI=95%; 0.611 - 9.049 significant associations were showed with risk of breast cancer. Instead, the genotype CC (p= 0.000 showed a protective role against susceptibility to breast cancer. Conclusion: This study identified that there is significant association between Thr241Met polymorphisms of the XRCC3 and the risk of susceptibility to breast cancer, which is in accordance to some of researchers' studies.

Mechanism of Enzyme Repair by the AAA+ Chaperone Rubisco Activase.

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Bhat, Javaid Y; Miličić, Goran; Thieulin-Pardo, Gabriel; Bracher, Andreas; Maxwell, Andrew; Ciniawsky, Susanne; Mueller-Cajar, Oliver; Engen, John R; Hartl, F Ulrich; Wendler, Petra; Hayer-Hartl, Manajit

2017-09-07

How AAA+ chaperones conformationally remodel specific target proteins in an ATP-dependent manner is not well understood. Here, we investigated the mechanism of the AAA+ protein Rubisco activase (Rca) in metabolic repair of the photosynthetic enzyme Rubisco, a complex of eight large (RbcL) and eight small (RbcS) subunits containing eight catalytic sites. Rubisco is prone to inhibition by tight-binding sugar phosphates, whose removal is catalyzed by Rca. We engineered a stable Rca hexamer ring and analyzed its functional interaction with Rubisco. Hydrogen/deuterium exchange and chemical crosslinking showed that Rca structurally destabilizes elements of the Rubisco active site with remarkable selectivity. Cryo-electron microscopy revealed that Rca docks onto Rubisco over one active site at a time, positioning the C-terminal strand of RbcL, which stabilizes the catalytic center, for access to the Rca hexamer pore. The pulling force of Rca is fine-tuned to avoid global destabilization and allow for precise enzyme repair. Copyright © 2017 Elsevier Inc. All rights reserved.

Phosphoramide mustard exposure induces DNA adduct formation and the DNA damage repair response in rat ovarian granulosa cells

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Ganesan, Shanthi, E-mail: shanthig@iastate.edu; Keating, Aileen F., E-mail: akeating@iastate.edu

2015-02-01

Phosphoramide mustard (PM), the ovotoxic metabolite of the anti-cancer agent cyclophosphamide (CPA), destroys rapidly dividing cells by forming NOR-G-OH, NOR-G and G-NOR-G adducts with DNA, potentially leading to DNA damage. A previous study demonstrated that PM induces ovarian DNA damage in rat ovaries. To investigate whether PM induces DNA adduct formation, DNA damage and induction of the DNA repair response, rat spontaneously immortalized granulosa cells (SIGCs) were treated with vehicle control (1% DMSO) or PM (3 or 6 μM) for 24 or 48 h. Cell viability was reduced (P < 0.05) after 48 h of exposure to 3 or 6 μM PM. The NOR-G-OH DNA adduct was detected after 24 h of 6 μM PM exposure, while the more cytotoxic G-NOR-G DNA adduct was formed after 48 h by exposure to both PM concentrations. Phosphorylated H2AX (γH2AX), a marker of DNA double stranded break occurrence, was also increased by PM exposure, coincident with DNA adduct formation. Additionally, induction of genes (Atm, Parp1, Prkdc, Xrcc6, and Brca1) and proteins (ATM, γH2AX, PARP-1, PRKDC, XRCC6, and BRCA1) involved in DNA repair were observed in both a time- and dose-dependent manner. These data support that PM induces DNA adduct formation in ovarian granulosa cells, induces DNA damage and elicits the ovarian DNA repair response. - Highlights: • PM forms ovarian DNA adducts. • DNA damage marker γH2AX increased by PM exposure. • PM induces ovarian DNA double strand break repair.

Genetic variants in DNA double-strand break repair genes and risk of salivary gland carcinoma: a case-control study.

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Li Xu

Full Text Available DNA double strand break (DSB repair is the primary defense mechanism against ionizing radiation-induced DNA damage. Ionizing radiation is the only established risk factor for salivary gland carcinoma (SGC. We hypothesized that genetic variants in DSB repair genes contribute to individual variation in susceptibility to SGC. To test this hypothesis, we conducted a case-control study in which we analyzed 415 single nucleotide polymorphisms (SNPs in 45 DSB repair genes in 352 SGC cases and 598 controls. Multivariate logistic regression analysis was performed to calculate odds ratios (ORs and 95% confidence intervals (CIs. Rs3748522 in RAD52 and rs13180356 in XRCC4 were significantly associated with SGC after Bonferroni adjustment; ORs (95% CIs for the variant alleles of these SNPs were 1.71 (1.40-2.09, P = 1.70 × 10(-7 and 0.58 (0.45-0.74, P = 2.00 × 10(-5 respectively. The genetic effects were modulated by histological subtype. The association of RAD52-rs3748522 with SGC was strongest for mucoepidermoid carcinoma (OR = 2.21, 95% CI: 1.55-3.15, P = 1.25 × 10(-5, n = 74, and the association of XRCC4-rs13180356 with SGC was strongest for adenoid cystic carcinoma (OR = 0.60, 95% CI: 0.42-0.87, P = 6.91 × 10(-3, n = 123. Gene-level association analysis revealed one gene, PRKDC, with a marginally significant association with SGC risk in non-Hispanic whites. To our knowledge, this study is the first to comprehensively evaluate the genetic effect of DSB repair genes on SGC risk. Our results indicate that genetic variants in the DSB repair pathways contribute to inter-individual differences in susceptibility to SGC and show that the impact of genetic variants differs by histological subtype. Independent studies are warranted to confirm these findings.

Thyroid nodules, polymorphic variants in DNA repair and RET-related genes, and interaction with ionizing radiation exposure from nuclear tests in Kazakhstan

Science.gov (United States)

Sigurdson, Alice J.; Land, Charles E.; Bhatti, Parveen; Pineda, Marbin; Brenner, Alina; Carr, Zhanat; Gusev, Boris I.; Zhumadilov, Zhaxibay; Simon, Steven L.; Bouville, Andre; Rutter, Joni L.; Ron, Elaine; Struewing, Jeffery P.

2010-01-01

Risk factors for thyroid cancer remain largely unknown except for ionizing radiation exposure during childhood and a history of benign thyroid nodules. Because thyroid nodules are more common than thyroid cancers and are associated with thyroid cancer risk, we evaluated several polymorphisms potentially relevant to thyroid tumors and assessed interaction with ionizing radiation exposure to the thyroid gland. Thyroid nodules were detected in 1998 by ultrasound screening of 2997 persons who lived near the Semipalatinsk nuclear test site in Kazakhstan when they were children (1949-62). Cases with thyroid nodules (n=907) were frequency matched (1:1) to those without nodules by ethnicity (Kazakh or Russian), gender, and age at screening. Thyroid gland radiation doses were estimated from fallout deposition patterns, residence history, and diet. We analyzed 23 polymorphisms in 13 genes and assessed interaction with ionizing radiation exposure using likelihood ratio tests (LRT). Elevated thyroid nodule risks were associated with the minor alleles of RET S836S (rs1800862, p = 0.03) and GFRA1 -193C>G (rs not assigned, p = 0.05) and decreased risk with XRCC1 R194W (rs1799782, p-trend = 0.03) and TGFB1 T263I (rs1800472, p = 0.009). Similar patterns of association were observed for a small number of papillary thyroid cancers (n=25). Ionizing radiation exposure to the thyroid gland was associated with significantly increased risk of thyroid nodules (age and gender adjusted excess odds ratio/Gy = 0.30, 95% confidence interval 0.05-0.56), with evidence for interaction by genotype found for XRCC1 R194W (LRT p value = 0.02). Polymorphisms in RET signaling, DNA repair, and proliferation genes may be related to risk of thyroid nodules, consistent with some previous reports on thyroid cancer. Borderline support for gene-radiation interaction was found for a variant in XRCC1, a key base excision repair protein. Other pathways, such as genes in double strand break repair, apoptosis, and

The effect of caffeine on repair in chlamydomonas reinhardtii. Pt. 1

International Nuclear Information System (INIS)

Rosen, H.; Rehn, M.M.; Johnson, B.A.

1980-01-01

The effect of caffeine on repair was studied in the green alga Chlamydomonas reinhardtii. Treatment of UV-irradiated wild-type (UVS + ) cells with a sublethal level of caffeine caused a significant increase in survival compared to untreated UV-irradiated cells. Caffeine did not affect survival in the repair-deficient strain UVSE1, which is deficient in repair of UV-induced damage carried out by enzymes associated with recombination during meiosis. A significant increase in survival in the presence of caffeine was observed in the repair-deficient strain UVSE4 in which recombination during meiosis is not affected. Treatment of zygotes homozygous for UVS + , UVSE1, or UVSE4 with sublethal levels of caffeine caused marked increases in recombination frequency in UVS + and UVSE4 zygotes and no increase in recombination in UVSE1 zygotes. These results indicate that caffeine increases recombination in normal strains. Increased opportunity for recombination caused by caffeine would not result in increased recombination frequency in the UVSE1 strain, assuming limited-recombination enzyme activity in this strain. The observed increase in survival following UV-irradiation in the presence of caffeine in strains having normal recombination would therefore be associated with a caffeine-induced increase in opportunities for recombination repair. (orig.)

Influence of XRCC1 Genetic Polymorphisms on Ionizing Radiation-Induced DNA Damage and Repair

OpenAIRE

Sterpone, Silvia; Cozzi, Renata

2010-01-01

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 de...

Caffeine inhibits homology-directed repair of I-SceI-induced DNA double-strand breaks.

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Wang, Huichen; Boecker, Wilfried; Wang, Hongyan; Wang, Xiang; Guan, Jun; Thompson, Larry H; Nickoloff, Jac A; Iliakis, George

2004-01-22

We recently reported that two Chinese hamster mutants deficient in the RAD51 paralogs XRCC2 and XRCC3 show reduced radiosensitization after treatment with caffeine, thus implicating homology-directed repair (HDR) of DNA double-strand breaks (DSBs) in the mechanism of caffeine radiosensitization. Here, we investigate directly the effect of caffeine on HDR initiated by DSBs induced by a rare cutting endonuclease (I-SceI) into one of two direct DNA repeats. The results demonstrate a strong inhibition by caffeine of HDR in wild-type cells, and a substantial reduction of this effect in HDR-deficient XRCC3 mutant cells. Inhibition of HDR and cell radiosensitization to killing shows similar dependence on caffeine concentration suggesting a cause-effect relationship between these effects. UCN-01, a kinase inhibitor that effectively abrogates checkpoint activation in irradiated cells, has only a small effect on HDR, indicating that similar to radiosensitization, inhibition of checkpoint signaling is not sufficient for HDR inhibition. Recombination events occurring during treatment with caffeine are characterized by rearrangements reminiscent to those previously reported for the XRCC3 mutant, and immunofluorescence microscopy demonstrates significantly reduced formation of IR-specific RAD51 foci after caffeine treatment. In summary, our results identify inhibition of HDR as a significant contributor to caffeine radiosensitization.

Live cell imaging combined with high-energy single-ion microbeam

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Guo, Na; Du, Guanghua; Liu, Wenjing; Guo, Jinlong; Wu, Ruqun; Chen, Hao; Wei, Junzhe

2016-03-01

DNA strand breaks can lead to cell carcinogenesis or cell death if not repaired rapidly and efficiently. An online live cell imaging system was established at the high energy microbeam facility at the Institute of Modern Physics to study early and fast cellular response to DNA damage after high linear energy transfer ion radiation. The HT1080 cells expressing XRCC1-RFP were irradiated with single high energy nickel ions, and time-lapse images of the irradiated cells were obtained online. The live cell imaging analysis shows that strand-break repair protein XRCC1 was recruited to the ion hit position within 20 s in the cells and formed bright foci in the cell nucleus. The fast recruitment of XRCC1 at the ion hits reached a maximum at about 200 s post-irradiation and then was followed by a slower release into the nucleoplasm. The measured dual-exponential kinetics of XRCC1 protein are consistent with the proposed consecutive reaction model, and the measurements obtained that the reaction rate constant of the XRCC1 recruitment to DNA strand break is 1.2 × 10-3 s-1 and the reaction rate constant of the XRCC1 release from the break-XRCC1 complex is 1.2 × 10-2 s-1.

N-Butyrate alters chromatin accessibility to DNA repair enzymes

International Nuclear Information System (INIS)

Smith, P.J.

1986-01-01

Current evidence suggests that the complex nature of mammalian chromatin can result in the concealment of DNA damage from repair enzymes and their co-factors. Recently it has been proposed that the acetylation of histone proteins in chromatin may provide a surveillance system whereby damaged regions of DNA become exposed due to changes in chromatin accessibility. This hypothesis has been tested by: (i) using n-butyrate to induce hyperacetylation in human adenocarcinoma (HT29) cells; (ii) monitoring the enzymatic accessibility of chromatin in permeabilised cells; (iii) measuring u.v. repair-associated nicking of DNA in intact cells and (iv) determining the effects of n-butyrate on cellular sensitivity to DNA damaging agents. The results indicate that the accessibility of chromatin to Micrococcus luteus u.v. endonuclease is enhanced by greater than 2-fold in n-butyrate-treated cells and that there is a corresponding increase in u.v. repair incision rates in intact cells exposed to the drug. Non-toxic levels of n-butyrate induce a block to G1 phase transit and there is a significant growth delay on removal of the drug. Resistance of HT29 cells to u.v.-radiation and adriamycin is enhanced in n-butyrate-treated cells whereas X-ray sensitivity is increased. Although changes in the responses of cells to DNA damaging agents must be considered in relation to the effects of n-butyrate on growth rate and cell-cycle distribution, the results are not inconsistent with the proposal that increased enzymatic-accessibility/repair is biologically favourable for the resistance of cells to u.v.-radiation damage. Overall the results support the suggested operation of a histone acetylation-based chromatin surveillance system in human cells

Live cell imaging combined with high-energy single-ion microbeam

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Guo, Na; Du, Guanghua, E-mail: gh-du@impcas.ac.cn; Liu, Wenjing; Wu, Ruqun; Wei, Junzhe [Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou (China); Guo, Jinlong [Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou (China); Northwest Normal University, Lanzhou (China); Chen, Hao [Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou (China); Institute of Nuclear Science and Technology, University of Lanzhou, Lanzhou (China)

2016-03-15

DNA strand breaks can lead to cell carcinogenesis or cell death if not repaired rapidly and efficiently. An online live cell imaging system was established at the high energy microbeam facility at the Institute of Modern Physics to study early and fast cellular response to DNA damage after high linear energy transfer ion radiation. The HT1080 cells expressing XRCC1-RFP were irradiated with single high energy nickel ions, and time-lapse images of the irradiated cells were obtained online. The live cell imaging analysis shows that strand-break repair protein XRCC1 was recruited to the ion hit position within 20 s in the cells and formed bright foci in the cell nucleus. The fast recruitment of XRCC1 at the ion hits reached a maximum at about 200 s post-irradiation and then was followed by a slower release into the nucleoplasm. The measured dual-exponential kinetics of XRCC1 protein are consistent with the proposed consecutive reaction model, and the measurements obtained that the reaction rate constant of the XRCC1 recruitment to DNA strand break is 1.2 × 10{sup −3} s{sup −1} and the reaction rate constant of the XRCC1 release from the break-XRCC1 complex is 1.2 × 10{sup −2} s{sup −1}.

Live cell imaging combined with high-energy single-ion microbeam

International Nuclear Information System (INIS)

Guo, Na; Du, Guanghua; Liu, Wenjing; Wu, Ruqun; Wei, Junzhe; Guo, Jinlong; Chen, Hao

2016-01-01

DNA strand breaks can lead to cell carcinogenesis or cell death if not repaired rapidly and efficiently. An online live cell imaging system was established at the high energy microbeam facility at the Institute of Modern Physics to study early and fast cellular response to DNA damage after high linear energy transfer ion radiation. The HT1080 cells expressing XRCC1-RFP were irradiated with single high energy nickel ions, and time-lapse images of the irradiated cells were obtained online. The live cell imaging analysis shows that strand-break repair protein XRCC1 was recruited to the ion hit position within 20 s in the cells and formed bright foci in the cell nucleus. The fast recruitment of XRCC1 at the ion hits reached a maximum at about 200 s post-irradiation and then was followed by a slower release into the nucleoplasm. The measured dual-exponential kinetics of XRCC1 protein are consistent with the proposed consecutive reaction model, and the measurements obtained that the reaction rate constant of the XRCC1 recruitment to DNA strand break is 1.2 × 10"−"3 s"−"1 and the reaction rate constant of the XRCC1 release from the break-XRCC1 complex is 1.2 × 10"−"2 s"−"1.

KIN17, XPC, DNA-PKCS and XRCC4 proteins in the cellular response to DNA damages. Relations between nucleotide excision repair and non-homologous end joining in a human syn-genic model

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Despras, Emmanuelle

2006-01-01

The response to genotoxic stress involves many cellular factors in a complex network of mechanisms that aim to preserve the genetic integrity of the organism. These mechanisms enclose the detection and repair of DNA lesions, the regulation of transcription and replication and, eventually, the setting of cell death. Among the nuclear proteins involved in this response, kin17 proteins are zinc-finger proteins conserved through evolution and activated by ultraviolet (UV) or ionizing radiations (IR). We showed that human kin17 protein (HSAkin17) is found in the cell under a soluble form and a form tightly anchored to nuclear structures. A fraction of HSAkin17 protein is directly associated with chromatin. HSAkin17 protein is recruited to nuclear structures 24 hours after treatment with various agents inducing DNA double-strand breaks (DSB) and/or replication forks blockage. Moreover, the reduction of total HSAkin17 protein level sensitizes RKO cells to IR. We also present evidence for the involvement of HSAkin17 protein in DNA replication. This hypothesis was further confirmed by the biochemical demonstration of its belonging to the replication complex. HSAkin17 protein could link DNA replication and DNA repair, a defect in the HSAkin17 pathway leading to an increased radiosensitivity. In a second part, we studied the interactions between two DNA repair mechanisms: nucleotide excision repair (NER) and non-homologous end joining (NHEJ). NER repairs a wide variety of lesions inducing a distortion of the DNA double helix including UV-induced pyrimidine dimers. NHEJ allows the repair of DSB by direct joining of DNA ends. We used a syn-genic model for DNA repair defects based on RNA interference developed in the laboratory. Epstein-Barr virus-derived vectors (pEBV) allow long-term expression of siRNA and specific extinction of the targeted gene. The reduction of the expression of genes involved in NER (XPA and XPC) or NHEJ (DNA-PKcs and XRCC4) leads to the expected

Cdk1 Restrains NHEJ through Phosphorylation of XRCC4-like Factor Xlf1

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Pierre Hentges

2014-12-01

Full Text Available Eukaryotic cells use two principal mechanisms for repairing DNA double-strand breaks (DSBs: homologous recombination (HR and nonhomologous end-joining (NHEJ. DSB repair pathway choice is strongly regulated during the cell cycle. Cyclin-dependent kinase 1 (Cdk1 activates HR by phosphorylation of key recombination factors. However, a mechanism for regulating the NHEJ pathway has not been established. Here, we report that Xlf1, a fission yeast XLF ortholog, is a key regulator of NHEJ activity in the cell cycle. We show that Cdk1 phosphorylates residues in the C terminus of Xlf1 over the course of the cell cycle. Mutation of these residues leads to the loss of Cdk1 phosphorylation, resulting in elevated levels of NHEJ repair in vivo. Together, these data establish that Xlf1 phosphorylation by Cdc2Cdk1 provides a molecular mechanism for downregulation of NHEJ in fission yeast and indicates that XLF is a key regulator of end-joining processes in eukaryotic organisms.

Genetic polymorphisms in 19q13.3 genes associated with alteration of repair capacity to BPDE-DNA adducts in primary cultured lymphocytes.

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Xiao, Mingyang; Xiao, Sha; Straaten, Tahar van der; Xue, Ping; Zhang, Guopei; Zheng, Xiao; Zhang, Qianye; Cai, Yuan; Jin, Cuihong; Yang, Jinghua; Wu, Shengwen; Zhu, Guolian; Lu, Xiaobo

2016-12-01

Benzo[a]pyrene(B[a]P), and its ultimate metabolite Benzo[a]pyrene 7,8-diol 9,10-epoxide (BPDE), are classic DNA damaging carcinogens. DNA damage in cells caused by BPDE is normally repaired by Nucleotide Excision Repair (NER) and Base Excision Repair (BER). Genetic variations in NER and BER can change individual DNA repair capacity to DNA damage induced by BPDE. In the present study we determined the number of in vitro induced BPDE-DNA adducts in lymphocytes, to reflect individual susceptibility to Polycyclic aromatic hydrocarbons (PAHs)-induced carcinogenesis. The BPDE-DNA adduct level in lymphocytes were assessed by high performance liquid chromatography (HPLC) in 281 randomly selected participants. We genotyped for 9 single nucleotide polymorphisms (SNPs) in genes involved in NER (XPB rs4150441, XPC rs2228001, rs2279017 and XPF rs4781560), BER (XRCC1 rs25487, rs25489 and rs1799782) and genes located on chromosome 19q13.2-3 (PPP1R13L rs1005165 and CAST rs967591). We found that 3 polymorphisms in chromosome 19q13.2-3 were associated with lower levels of BPDE-DNA adducts (MinorT allele in XRCC1 rs1799782, minor T allele in PPP1R13L rs1005165 and minor A allele in CAST rs967571). In addition, a modified comet assay was performed to further confirm the above conclusions. We found both minor T allele in PPP1R13L rs1005165 and minor A allele in CAST rs967571 were associated with the lower levels of BPDE-adducts. Our data suggested that the variant genotypes of genes in chromosome 19q13.2-3 are associated with the alteration of repair efficiency to DNA damage caused by Benzo[a]pyrene, and may contribute to enhance predictive value for individual's DNA repair capacity in response to environmental carcinogens. Copyright © 2016 Elsevier B.V. All rights reserved.

Characterization of the linkage disequilibrium structure and identification of tagging-SNPs in five DNA repair genes

International Nuclear Information System (INIS)

Allen-Brady, Kristina; Camp, Nicola J

2005-01-01

Characterization of the linkage disequilibrium (LD) structure of candidate genes is the basis for an effective association study of complex diseases such as cancer. In this study, we report the LD and haplotype architecture and tagging-single nucleotide polymorphisms (tSNPs) for five DNA repair genes: ATM, MRE11A, XRCC4, NBS1 and RAD50. The genes ATM, MRE11A, and XRCC4 were characterized using a panel of 94 unrelated female subjects (47 breast cancer cases, 47 controls) obtained from high-risk breast cancer families. A similar LD structure and tSNP analysis was performed for NBS1 and RAD50, using publicly available genotyping data. We studied a total of 61 SNPs at an average marker density of 10 kb. Using a matrix decomposition algorithm, based on principal component analysis, we captured >90% of the intragenetic variation for each gene. Our results revealed that three of the five genes did not conform to a haplotype block structure (MRE11A, RAD50 and XRCC4). Instead, the data fit a more flexible LD group paradigm, where SNPs in high LD are not required to be contiguous. Traditional haplotype blocks assume recombination is the only dynamic at work. For ATM, MRE11A and XRCC4 we repeated the analysis in cases and controls separately to determine whether LD structure was consistent across breast cancer cases and controls. No substantial difference in LD structures was found. This study suggests that appropriate SNP selection for an association study involving candidate genes should allow for both mutation and recombination, which shape the population-level genomic structure. Furthermore, LD structure characterization in either breast cancer cases or controls appears to be sufficient for future cancer studies utilizing these genes

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

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Katiboina Srinivasa Rao

2014-01-01

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

DNA Repair and Cancer Therapy: Targeting APE1/Ref-1 Using Dietary Agents

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Julian J. Raffoul

2012-01-01

Full Text Available Epidemiological studies have demonstrated the cancer protective effects of dietary agents and other natural compounds isolated from fruits, soybeans, and vegetables on neoplasia. Studies have also revealed the potential for these natural products to be combined with chemotherapy or radiotherapy for the more effective treatment of cancer. In this paper we discuss the potential for targeting the DNA base excision repair enzyme APE1/Ref-1 using dietary agents such as soy isoflavones, resveratrol, curcumin, and the vitamins ascorbate and α-tocopherol. We also discuss the potential role of soy isoflavones in sensitizing cancer cells to the effects of radiotherapy. A comprehensive review of the dual nature of APE1/Ref-1 in DNA repair and redox activation of cellular transcription factors, NF-κB and HIF-1α, is also discussed. Further research efforts dedicated to delineating the role of APE1/Ref-1 DNA repair versus redox activity in sensitizing cancer cells to conventional treatment are warranted.

MD study of pyrimidine base damage on DNA and its recognition by repair enzyme

International Nuclear Information System (INIS)

Pinak, M.

2000-01-01

The molecular dynamics (MD) simulation was used on the study of two specific damages of pyrimidine bases of DNA. Pyrimidine bases are major targets either of free radicals induced by ionizing radiation in DNA surrounding environment or UV radiation. Thymine dimer (TD) is UV induced damage, in which two neighboring thymines in one strand are joined by covalent bonds of C(5)-C(5) and C(6)-C(6) atoms of thymines. Thymine glycol (TG) is ionizing radiation induced damage in which the free water radical adds to unsaturated bond C(5)-C(6) of thymine. Both damages are experimentally suggested to be mutagenetic and carcinogenic unless properly repaired by repair enzymes. In the case of MD of TD, there is detected strong kink around the TD site that is not observed in native DNA. In addition there is observed the different value of electrostatic energy at the TD site - negative '-10 kcal/mol', in contrary to nearly neutral value of native thymine site. Structural changes and specific electrostatic energy - seems to be important for proper recognition of TD damaged site, formation of DNA-enzyme complex and thus for subsequent repair of DNA. In the case of TG damaged DNA there is major structural distortion at the TG site, mainly the increased distance between TG and the C5' of adjacent nucleotide. This enlarged gap between the neighboring nucleotides may prevent the insertion of complementary base during replication causing the replication process to stop. In which extend this structural feature together with energy properties of TG contributes to the proper recognition of TG by repair enzyme Endonuclease III is subject of further computational MD study. (author)

Conserved structural chemistry for incision activity in structurally non-homologous apurinic/apyrimidinic endonuclease APE1 and endonuclease IV DNA repair enzymes.

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Tsutakawa, Susan E.; Shin, David S.; Mol, Clifford D.; Izum, Tadahide; Arvai, Andrew S.; Mantha, Anil K.; Szczesny, Bartosz; Ivanov, Ivaylo N.; Hosfield, David J.; Maiti, Buddhadev; Pique, Mike E.; Frankel, Kenneth A.; Hitomi, Kenichi; Cunningham, Richard P.; Mitra, Sankar; Tainer, John A.

2013-03-22

Non-coding apurinic/apyrimidinic (AP) sites in DNA form spontaneously and as DNA base excision repair intermediates are the most common toxic and mutagenic in vivo DNA lesion. For repair, AP sites must be processed by 5' AP endonucleases in initial stages of base repair. Human APE1 and bacterial Nfo represent the two conserved 5' AP endonuclease families in the biosphere; they both recognize AP sites and incise the phosphodiester backbone 5' to the lesion, yet they lack similar structures and metal ion requirements. Here, we determined and analyzed crystal structures of a 2.4 ? resolution APE1-DNA product complex with Mg(2+) and a 0.92 Nfo with three metal ions. Structural and biochemical comparisons of these two evolutionarily distinct enzymes characterize key APE1 catalytic residues that are potentially functionally similar to Nfo active site components, as further tested and supported by computational analyses. We observe a magnesium-water cluster in the APE1 active site, with only Glu-96 forming the direct protein coordination to the Mg(2+). Despite differences in structure and metal requirements of APE1 and Nfo, comparison of their active site structures surprisingly reveals strong geometric conservation of the catalytic reaction, with APE1 catalytic side chains positioned analogously to Nfo metal positions, suggesting surprising functional equivalence between Nfo metal ions and APE1 residues. The finding that APE1 residues are positioned to substitute for Nfo metal ions is supported by the impact of mutations on activity. Collectively, the results illuminate the activities of residues, metal ions, and active site features for abasic site endonucleases.

Influence of SNP Polymorphisms in DNA Repair Genes on the Level of Persistent Damage in Human Lymphocytes After Exposure to 2 Gy of Ionising Radiation

International Nuclear Information System (INIS)

Milic, M.; Rozgaj, R.; Kasuba, V.; Kubelka, D.; Angelini, S.; Hrelia, P.

2011-01-01

Variation in cell response to ionising radiation could be result of changes in gene expression and/or polymorphisms of DNA repair genes. The aim of the study was to estimate the DNA damage level in human lymphocytes after exposure to 2 Gy of ionising radiation. Medical workers occupationally exposed to low doses of ionising radiation (N = 20) and matched controls (N 20) were genotyped for polymorphic hOGG1, XRCC1, APE1, XPD10, XPD23, XRCC3, PARP1 and MGMT genes. Micronucleus (MN) test was used for the estimation of DNA damage before and after radiation. Incidence of MN in irradiated samples positively correlated with age and negatively with polymorphic variants of XPD23. Significant difference was observed between irradiated homozygotes (HO) and heterozygotes (HE). HO and HE APE1 differed in MN before exposure. HO and polymorphic variants of XPD10 differed in MN after exposure. Gender showed different MN in the exposed group after exposure. Age correlated positively with MN after exposure, working probation and received dose. Multiple regression analysis revealed connection between polymorphic variants of APE1 and XRCC3 with MN before exposure. These results confirm the value of micronucleus assay in DNA damage estimation and suggest possible use of polymorphic genes in monitoring of individuals professionaly exposed to ionising radiation. (author)

Identification of polymorphisms of XRCC1 gene in patients with cancer in a city of northern Brazil

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Artemis Socorro N. Rodrigues

2015-06-01

Full Text Available ABSTRACT Introduction: Cancer is considered a genetic disease. For this reason, identification and characterization of the genes involved in its origin and progression are of fundamental importance in understanding its molecular basis. Objective: Our objective was to determine whether people from Macapá with a diagnosis of cancer have genetic polymorphisms related to the XRCC1 gene. Materials and methods: We analyzed 30 samples of deoxyribonucleic acid (DNA of cases with cancer and 30 control samples. All samples were amplified and analyzed by the polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP method, with the use of restriction enzyme MspI. Results: Regarding the 194T polymorphism, we found that all samples of the cases presented the polymorphic allele Trp (Arg/Trp. In control samples, 96.6% also identify the polymorphic allele Trp and, among these, one was homozygous for the same allele (Trp/Trp. Regarding the 399A polymorphism, 83.3% of the cases and 23.3% of the controls had the Arg/ Gln genotype, respectively. We found that 73.3% of controls and 16.6% of cases had the Arg/Arg genotype. Among the controls, we found only a sample that was homozygous for the polymorphic allele Trp/Trp. Conclusion: Our results demonstrated the allele frequency of 194Trp polymorphism in both sample groups analyzed. We also found a significant number of polymorphic allele 399A in people with cancer. Thus, we can highlight 399Gln polymorphism as a genetic marker of cancer risk in this population.

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

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

2017-03-01

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

DNA repair enzyme APE1 from evolutionarily ancient Hydra reveals redox activity exclusively found in mammalian APE1.

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Pekhale, Komal; Haval, Gauri; Perween, Nusrat; Antoniali, Giulia; Tell, Gianluca; Ghaskadbi, Surendra; Ghaskadbi, Saroj

2017-11-01

Only mammalian apurinic/apyrimidinic endonuclease1 (APE1) has been reported to possess both DNA repair and redox activities. C terminal of the protein is required for base excision repair, while the redox activity resides in the N terminal due to cysteine residues at specific positions. APE1s from other organisms studied so far lack the redox activity in spite of having the N terminal domain. We find that APE1 from the Cnidarian Hydra exhibits both endonuclease and redox activities similar to mammalian APE1. We further show the presence of the three indispensable cysteines in Hydra APE1 for redox activity by site directed mutagenesis. Importance of redox domain but not the repair domain of APE1 in regeneration has been demonstrated by using domain-specific inhibitors. Our findings clearly demonstrate that the redox function of APE1 evolved very early in metazoan evolution and is not a recent acquisition in mammalian APE1 as believed so far. Copyright © 2017 Elsevier B.V. All rights reserved.

The USP1-UAF1 complex interacts with RAD51AP1 to promote homologous recombination repair.

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Cukras, Scott; Lee, Euiho; Palumbo, Emily; Benavidez, Pamela; Moldovan, George-Lucian; Kee, Younghoon

2016-10-01

USP1 deubiquitinating enzyme and its stoichiometric binding partner UAF1 play an essential role in promoting DNA homologous recombination (HR) repair in response to various types of DNA damaging agents. Deubiquitination of FANCD2 may be attributed to the key role of USP1-UAF1 complex in regulating HR repair, however whether USP1-UAF1 promotes HR repair independently of FANCD2 deubiquitination is not known. Here we show evidence that the USP1-UAF1 complex has a FANCD2-independent function in promoting HR repair. Proteomic search of UAF1-interacting proteins revealed that UAF1 associates with RAD51AP1, a RAD51-interacting protein implicated in HR repair. We show that UAF1 mediates the interaction between USP1 and RAD51AP1, and that depletion of USP1 or UAF1 led to a decreased stability of RAD51AP1. Protein interaction mapping analysis identified some key residues within RAD51AP1 required for interacting with the USP1-UAF1 complex. Cells expressing the UAF1 interaction-deficient mutant of RAD51AP1 show increased chromosomal aberrations in response to Mitomycin C treatment. Moreover, similar to the RAD51AP1 depleted cells, the cells expressing UAF1-interaction deficient RAD51AP1 display persistent RAD51 foci following DNA damage exposure, indicating that these factors regulate a later step during the HR repair. These data altogether suggest that the USP1-UAF1 complex promotes HR repair via multiple mechanisms: through FANCD2 deubiquitination, as well as by interacting with RAD51AP1.

Strategies for protection and experiments on repair of irradiated sulfhydryl enzymes

International Nuclear Information System (INIS)

Durchschlag, H.; Zipper, P.

1991-01-01

The investigation of sulfur-containing biomolecules, especially of sulfhydryl proteins, is of particular interest in radiation biology. Sulfhydryl enzymes are useful objects for studying both structural and functional changes caused by radiation. In this context oxidation of enzyme sulfhydryl, inactivation (continuing in the post-irradiation phase), subunit cross-linking, enzyme aggregation, fragmentation, unfolding etc. may be mentioned. For their studies the authors used primarily malate synthase (MS), an enzyme with essential sulfhydryl, which was X-irradiated in aqueous solution in the absence or presence of a variety of additives (thiols, antioxienzymes, typical radical scavengers, inorganic salts, buffer components, substrates, products, substrate and product analogues). Radiation-induced effects were registered during irradiation, after stop of irradiation, and in the post-radiation (p.r.) phase 30 or 60 h p.r. using, e.g., small-angle X-ray scattering (SAXS), polyacrylamide gel electrophoreses (PAGEs), and activity measurements. Repair experiments were initiated by p.r. addition of dithiothreitol (DTT). For comparison, some of the experiments were also carried out with two additional sulfhydryl enzymes (glyceraldehyde-3-phosphate dehydrogenase (GAPDH), lactate dehydrogenase (LDH)) and two disulfide containing proteins (ribonuclease A, serum albumin). 9 refs., 6 figs

The C-Terminal Domain of Cernunnos/XLF Is Dispensable for DNA Repair In Vivo▿ †

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Malivert, Laurent; Callebaut, Isabelle; Rivera-Munoz, Paola; Fischer, Alain; Mornon, Jean-Paul; Revy, Patrick; de Villartay, Jean-Pierre

2009-01-01

The core nonhomologous end-joining DNA repair pathway is composed of seven factors: Ku70, Ku80, DNA-PKcs, Artemis, XRCC4 (X4), DNA ligase IV (L4), and Cernunnos/XLF (Cernunnos). Although Cernunnos and X4 are structurally related and participate in the same complex together with L4, they have distinct functions during DNA repair. L4 relies on X4 but not on Cernunnos for its stability, and L4 is required for optimal interaction of Cernunnos with X4. We demonstrate here, using in vitro-generated Cernunnos mutants and a series of functional assays in vivo, that the C-terminal region of Cernunnos is dispensable for its activity during DNA repair. PMID:19103754

Biochemical Kinetics Model of DSB Repair and GammaH2AX FOCI by Non-homologous End Joining

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Cucinotta, Francis, A.; Pluth, Janice M.; Anderson, Jennifer A.; Harper, Jane V.; O'Neill, Peter

2007-01-01

We developed a biochemical kinetics approach to describe the repair of double strand breaks (DSB) produced by low LET radiation by modeling molecular events associated with the mechanisms of non-homologous end-joining (NHEJ). A system of coupled non-linear ordinary differential equations describes the induction of DSB and activation pathways for major NHEJ components including Ku(sub 70/80), DNA-PK(sub cs), and the Ligase IV-XRCC4 hetero-dimer. The autophosphorylation of DNA-PK(sub cs and subsequent induction of gamma-H2AX foci observed after ionizing radiation exposure were modeled. A two-step model of DNA-PK(sub cs) regulation of repair was developed with the initial step allowing access of other NHEJ components to breaks, and a second step limiting access to Ligase IV-XRCC4. Our model assumes that the transition from the first to second-step depends on DSB complexity, with a much slower-rate for complex DSB. The model faithfully reproduced several experimental data sets, including DSB rejoining as measured by pulsed-field electrophoresis (PFGE), quantification of the induction of gamma-H2AX foci, and live cell imaging of the induction of Ku(sub 70/80). Predictions are made for the behaviors of NHEJ components at low doses and dose-rates, where a steady-state is found at dose-rates of 0.1 Gy/hr or lower.

The cutting edges in DNA repair, licensing, and fidelity: DNA and RNA repair nucleases sculpt DNA to measure twice, cut once.

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Tsutakawa, Susan E; Lafrance-Vanasse, Julien; Tainer, John A

2014-07-01

To avoid genome instability, DNA repair nucleases must precisely target the correct damaged substrate before they are licensed to incise. Damage identification is a challenge for all DNA damage response proteins, but especially for nucleases that cut the DNA and necessarily create a cleaved DNA repair intermediate, likely more toxic than the initial damage. How do these enzymes achieve exquisite specificity without specific sequence recognition or, in some cases, without a non-canonical DNA nucleotide? Combined structural, biochemical, and biological analyses of repair nucleases are revealing their molecular tools for damage verification and safeguarding against inadvertent incision. Surprisingly, these enzymes also often act on RNA, which deserves more attention. Here, we review protein-DNA structures for nucleases involved in replication, base excision repair, mismatch repair, double strand break repair (DSBR), and telomere maintenance: apurinic/apyrimidinic endonuclease 1 (APE1), Endonuclease IV (Nfo), tyrosyl DNA phosphodiesterase (TDP2), UV Damage endonuclease (UVDE), very short patch repair endonuclease (Vsr), Endonuclease V (Nfi), Flap endonuclease 1 (FEN1), exonuclease 1 (Exo1), RNase T and Meiotic recombination 11 (Mre11). DNA and RNA structure-sensing nucleases are essential to life with roles in DNA replication, repair, and transcription. Increasingly these enzymes are employed as advanced tools for synthetic biology and as targets for cancer prognosis and interventions. Currently their structural biology is most fully illuminated for DNA repair, which is also essential to life. How DNA repair enzymes maintain genome fidelity is one of the DNA double helix secrets missed by James Watson and Francis Crick, that is only now being illuminated though structural biology and mutational analyses. Structures reveal motifs for repair nucleases and mechanisms whereby these enzymes follow the old carpenter adage: measure twice, cut once. Furthermore, to measure

Biochemical Characterization of Mycobacterium tuberculosis DNA Repair Enzymes – Nfo, XthA and Nei2

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Sailau Abeldenov

2014-01-01

Full Text Available Introduction: Tuberculosis (TB is a human disease caused by Mycobacterium tuberculosis (Mtb. Treatment of TB requires long-term courses of multi-drug therapies to eliminate subpopulations of bacteria, which sometimes persist against antibiotics. Therefore, understanding of the mechanism of Mtb antibiotic-resistance is extremely important. During infection, Mtb overcomes a variety of body defense mechanisms, including treatment with the reactive species of oxygen and nitrogen. The bases in DNA molecule are susceptible to the damages caused by reactive forms of intermediate compounds of oxygen and nitrogen. Most of this damage is repaired by the base excision repair (BER pathway. In this study, we aimed to biochemically characterize three Mtb DNA repair enzymes of BER pathway. Methods: XthA, nfo, and nei genes were identified in mycobacteria by homology search of genomic sequences available in the GenBank database. We used standard methods of genetic engineering  to clone and sequence Mtb genes, which coded Nfo, XthA and Nei2 repair enzymes. The protein products of Mtb genes were expressed and purified in Escherichia coli using affinity tags. The enzymatic activity of purified Nfo, XthA, and Nei2 proteins were measured using radioactively labeled DNA substrates containing various modified residues. Results: The genes end (Rv0670, xthA (Rv0427c, and nei (Rv3297 were PCR amplified using genomic DNA of Mtb H37Rv with primers that contain specific restriction sites. The amplified products were inserted into pET28c(+ expression vector in such a way that the recombinant proteins contain C-terminal histidine tags. The plasmid constructs were verified by sequencing and then transformed into the Escherichia coli BL21 (DE3 strain. Purification of recombinant proteins was performed using Ni2+ ions immobilized affinity column, coupled with the fast performance liquid chromatography machine AKTA. Identification of the isolated proteins was performed by

Analysis of difference of association between polymorphisms in the XRCC5, RPA3 and RTEL1 genes and glioma, astrocytoma and glioblastoma.

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Jin, Tianbo; Wang, Yuan; Li, Gang; Du, Shuli; Yang, Hua; Geng, Tingting; Hou, Peng; Gong, Yongkuan

2015-01-01

Gliomas are the most common aggressive brain tumors and have many complex pathological types. Previous reports have discovered that genetic mutations are associated with the risk of glioma. However, it is unclear whether uniform genetic mutations exist difference between glioma and its two pathological types in the Han Chinese population. We evaluated 20 SNPs of 703 glioma cases (338 astrocytoma cases, 122 glioblastoma cases) and 635 controls in a Han Chinese population using χ(2) test and genetic model analysis. In three case-control studies, we found rs9288516 in XRCC5 gene showed a decreased risk of glioma (OR, 0.85; 95% CI, 0.73-0.99; P = 0.042) and glioblastoma (OR, 0.70; 95% CI, 0.52-0.92; P = 0.001) in the allele model. We identified rs414805 in RPA3 gene showed an increased risk of glioblastoma in allele model (OR, 1.38; 95% CI, 1.00-1.89; P = 0.047) and dominant model (OR, 1.57; 95% CI, 1.05-2.35; P = 0.027), analysis respectively. Meanwhile, rs2297440 in RTEL1 gene showed an increased risk of glioma (OR, 1.30; 95% CI, 1.10-1.54; P = 0.002) and astrocytoma (OR, 1.26; 95% CI, 1.02-1.54; P = 0.029) in the allele model. In addition, we also observed a haplotype of "GCT" in the RTEL1 gene with an increased risk of astrocytoma (P = 0.005). Polymorphisms in the XRCC5, RPA3 and RTEL1 genes, combinating with previous reaserches, are associated with glioma developing. However, those genes mutations may play different roles in the glioma, astrocytoma and glioblastoma, respectively.

Human longevity and variation in GH/IGF-1/insulin signaling, DNA damage signaling and repair and pro/antioxidant pathway genes: cross sectional and longitudinal studies.

Science.gov (United States)

Soerensen, Mette; Dato, Serena; Tan, Qihua; Thinggaard, Mikael; Kleindorp, Rabea; Beekman, Marian; Jacobsen, Rune; Suchiman, H Eka D; de Craen, Anton J M; Westendorp, Rudi G J; Schreiber, Stefan; Stevnsner, Tinna; Bohr, Vilhelm A; Slagboom, P Eline; Nebel, Almut; Vaupel, James W; Christensen, Kaare; McGue, Matt; Christiansen, Lene

2012-05-01

Here we explore association with human longevity of common genetic variation in three major candidate pathways: GH/IGF-1/insulin signaling, DNA damage signaling and repair and pro/antioxidants by investigating 1273 tagging SNPs in 148 genes composing these pathways. In a case-control study of 1089 oldest-old (age 92-93) and 736 middle-aged Danes we found 1 pro/antioxidant SNP (rs1002149 (GSR)), 5 GH/IGF-1/INS SNPs (rs1207362 (KL), rs2267723 (GHRHR), rs3842755 (INS), rs572169 (GHSR), rs9456497 (IGF2R)) and 5 DNA repair SNPs (rs11571461 (RAD52), rs13251813 (WRN), rs1805329 (RAD23B), rs2953983 (POLB), rs3211994 (NTLH1)) to be associated with longevity after correction for multiple testing. In a longitudinal study with 11 years of follow-up on survival in the oldest-old Danes we found 2 pro/antioxidant SNPs (rs10047589 (TNXRD1), rs207444 (XDH)), 1 GH/IGF-1/INS SNP (rs26802 (GHRL)) and 3 DNA repair SNPs (rs13320360 (MLH1), rs2509049 (H2AFX) and rs705649 (XRCC5)) to be associated with mortality in late life after correction for multiple testing. When examining the 11 SNPs from the case-control study in the longitudinal data, rs3842755 (INS), rs13251813 (WRN) and rs3211994 (NTHL1) demonstrated the same directions of effect (ppolymorphisms. Copyright © 2012 Elsevier Inc. All rights reserved.

DNA Damage: Quantum Mechanics/Molecular Mechanics Study on the Oxygen Binding and Substrate Hydroxylation Step in AlkB Repair Enzymes

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Quesne, Matthew G; Latifi, Reza; Gonzalez-Ovalle, Luis E; Kumar, Devesh; de Visser, Sam P

2014-01-01

AlkB repair enzymes are important nonheme iron enzymes that catalyse the demethylation of alkylated DNA bases in humans, which is a vital reaction in the body that heals externally damaged DNA bases. Its mechanism is currently controversial and in order to resolve the catalytic mechanism of these enzymes, a quantum mechanics/molecular mechanics (QM/MM) study was performed on the demethylation of the N1-methyladenine fragment by AlkB repair enzymes. Firstly, the initial modelling identified the oxygen binding site of the enzyme. Secondly, the oxygen activation mechanism was investigated and a novel pathway was found, whereby the catalytically active iron(IV)–oxo intermediate in the catalytic cycle undergoes an initial isomerisation assisted by an Arg residue in the substrate binding pocket, which then brings the oxo group in close contact with the methyl group of the alkylated DNA base. This enables a subsequent rate-determining hydrogen-atom abstraction on competitive σ-and π-pathways on a quintet spin-state surface. These findings give evidence of different locations of the oxygen and substrate binding channels in the enzyme and the origin of the separation of the oxygen-bound intermediates in the catalytic cycle from substrate. Our studies are compared with small model complexes and the effect of protein and environment on the kinetics and mechanism is explained. PMID:24339041

Combustion products of 1,3-butadiene inhibit catalase activity and induce expression of oxidative DNA damage repair enzymes in human bronchial epithelial cells.

Science.gov (United States)

Kennedy, Christopher H; Catallo, W James; Wilson, Vincent L; Mitchell, James B

2009-10-01

1,3-Butadiene, an important petrochemical, is commonly burned off when excess amounts need to be destroyed. This combustion process produces butadiene soot (BDS), which is composed of a complex mixture of polycyclic aromatic hydrocarbons in particulates ranging in size from enzyme inactivation due to protein amino acid oxidation and (2) induce oxidative DNA damage in NHBE cells. Thus, our aims were to determine the effect of butadiene soot ethanol extract (BSEE) on both enzyme activity and the expression of proteins involved in the repair of oxidative DNA damage. Catalase was found to be sensitive to BDS as catalase activity was potently diminished in the presence of BSEE. Using Western analysis, both the alpha isoform of human 8-oxoguanine DNA glycosylase (alpha-hOGG1) and human apurinic/apyrimidinic endonuclease (APE-1) were shown to be significantly overexpressed as compared to untreated controls after exposure of NHBE cells to BSEE. Our results indicate that BSEE is capable of effectively inactivating the antioxidant enzyme catalase, presumably via oxidation of protein amino acids. The presence of oxidized biomolecules may partially explain the extranuclear fluorescence that is detected when NHBE cells are treated with an organic extract of BDS. Overexpression of both alpha-hOGG1 and APE-1 proteins following treatment of NHBE cells with BSEE suggests that this mixture causes oxidative DNA damage.

Association of a XRCC3 polymorphism and rectum mean dose with the risk of acute radio-induced gastrointestinal toxicity in prostate cancer patients

International Nuclear Information System (INIS)

Fachal, Laura; Gómez-Caamaño, Antonio; Peleteiro, Paula; Carballo, Ana; Calvo-Crespo, Patricia; Sánchez-García, Manuel; Lobato-Busto, Ramón; Carracedo, Ángel; Vega, Ana

2012-01-01

Background and purpose: We have performed a case–control study among prostate cancer patients treated with three-dimensional conformational radiotherapy (3D-CRT) in order to investigate the association between single nucleotide polymorphisms (SNPs), treatment and patient features with gastrointestinal and genitourinary acute toxicity. Material and methods: A total of 698 patients were screened for 14 SNPs located in the ATM, ERCC2, LIG4, MLH1 and XRCC3 genes. Gastrointestinal and genitourinary toxicities were recorded prospectively using the Common Terminology Criteria for Adverse Events v3.0. Results: The XRCC3 SNP rs1799794 (G/G OR = 5.65; 95% CI: 1.95–16.38; G/A OR = 2.75; 95% CI: 1.25–6.05; uncorrected p-value = 2.8 × 10 −03 ; corrected p-value = 0.03; FDR q-value = 0.06) as well as the mean dose received by the rectum (OR = 1.06; 95% CI: 1.02–1.1; uncorrected p-value = 2.49 × 10 −03 ; corrected p-value = 0.03; FDR q-value = 0.06) were significantly associated with gastrointestinal toxicity after correction for multiple testing. Those patients who undergone previous prostatectomy were less prone to develop genitourinary toxicity (OR = 0.38; 95% CI: 0.18–0.71; uncorrected p-value = 4.95 × 10 −03 ; corrected p-value = 0.03; FDR q-value = 0.08). Our study excludes the possibility of a >2-fold risk increase in genitourinary acute toxicity being due to rs1801516 ATM SNP, the rs1805386 and rs1805388 LIG4 markers, as well as all the SNPs evaluated in the ERCC2, MLH1 and XRCC3 genes. Conclusions: The XRCC3 rs1799794 SNP and the mean dose received by the rectum are associated with the development of gastrointestinal toxicity after 3D-CRT.

Mitochondrial Targeted Endonuclease III DNA Repair Enzyme Protects against Ventilator Induced Lung Injury in Mice

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Masahiro Hashizume

2014-08-01

Full Text Available The mitochondrial targeted DNA repair enzyme, 8-oxoguanine DNA glycosylase 1, was previously reported to protect against mitochondrial DNA (mtDNA damage and ventilator induced lung injury (VILI. In the present study we determined whether mitochondrial targeted endonuclease III (EndoIII which cleaves oxidized pyrimidines rather than purines from damaged DNA would also protect the lung. Minimal injury from 1 h ventilation at 40 cmH2O peak inflation pressure (PIP was reversed by EndoIII pretreatment. Moderate lung injury due to ventilation for 2 h at 40 cmH2O PIP produced a 25-fold increase in total extravascular albumin space, a 60% increase in W/D weight ratio, and marked increases in MIP-2 and IL-6. Oxidative mtDNA damage and decreases in the total tissue glutathione (GSH and the GSH/GSSH ratio also occurred. All of these indices of injury were attenuated by mitochondrial targeted EndoIII. Massive lung injury caused by 2 h ventilation at 50 cmH2O PIP was not attenuated by EndoIII pretreatment, but all untreated mice died prior to completing the two hour ventilation protocol, whereas all EndoIII-treated mice lived for the duration of ventilation. Thus, mitochondrial targeted DNA repair enzymes were protective against mild and moderate lung damage and they enhanced survival in the most severely injured group.

Individual sensitivity to radiations and DNA repair proficiency: the comet assay contribution; Sensibilite individuelle aux radiations et reparation de l`ADN: apport du test des cometes

Energy Technology Data Exchange (ETDEWEB)

Alapetite, C. [Institut Curie, 75 - Paris (France)

1998-09-01

Some are hereditary syndromes demonstrate high cancer risk and hypersensitivity in response to exposures to agents such as ultraviolet or ionising radiation, and are characterized by a defective processing of DNA damage. They highlight the importance of the individual risk associated to exposures. The comet assay, a simple technique that detects DNA strand breaks, requires few cells and allows examination of DNA repair capacities in established cell lines, in blood samples or biopsies. The assay has been validated on cellular systems with known repair defects such as xeroderma pigmentosum defective in nucleotide excision repair, on mutant rodent cell lines defective in DNA single strand breaks rejoining (XRCC5/Ku80 and XRCC7/DNAPKcs) (neutral conditions). This assay does not allow to distinguish a defective phenotype in ataxia telangiectasia cells. It shows in homozygous mouse embryo fibroblasts Brca2-/- an impaired DNA double strand break rejoining. Simplicity, rapidity and sensitivity of the alkaline comet assay allow to examine the response of lymphocytes. It has been applied to the analysis of the role of DNA repair in the pathogenesis of collagen diseases, and the involvement of individual DNA repair proficiency in the thyroid tumorigenesis induced in some patients after therapeutic irradiation at childhood has been questioned. Preliminary results of these studies suggest that this type of approach could help for adapting treatment modalities and surveillance in subgroups of patients defective in DNA repair process. It could also have some incidence in the radioprotection field. (author)

Effect of specific enzyme inhibitors on replication, total genome DNA repair and on gene-specific DNA repair after UV irradiation in CHO cells

Energy Technology Data Exchange (ETDEWEB)

Jones, J.C.; Stevsner, Tinna; Bohr, Vilhelm A. (National Cancer Institute, NIH, Bethesda, MD (USA). Division of Cancer Treatment, Laboratory of Molecular Pharmacology); Mattern, M.R. (Smith Kline Beecham Pharmaceuticals, King of Prussia, PA (USA). Department of Biomolecular Discovery)

1991-09-01

The effects were studied of some specific enzyme inhibitors on DNA repair and replication after UV damage in Chinese hamster ovary cells. The DNA repair was studied at the level of the average, overall genome and also in the active dihydrofolate reductase gene. Replication was measured in the overall genome. The inhibitors were tested of DNA poly-merase {alpha} and {delta} (aphidicolin), of poly(ADPr) polymerase (3-aminobenzamide), of ribonucleotide reductase (hydroxyurea), of topo-isomerase I (camptothecin), and of topoisomerase II (merbarone, VP-16). In addition, the effects were tested of the potential topoisomerase I activator, {beta}-lapachone. All of these compounds inhibited genome replication and all topoisomerase inhibitors affected the overall genome repair; {beta}-lapachone stimulated it. None of these compounds had any effect on the gene-specific repair. (author). 36 refs.; 3 figs.; 2 tabs.

Polymorphisms in metabolism and repair genes affects DNA damage caused by open-cast coal mining exposure.

Science.gov (United States)

Espitia-Pérez, Lyda; Sosa, Milton Quintana; Salcedo-Arteaga, Shirley; León-Mejía, Grethel; Hoyos-Giraldo, Luz Stella; Brango, Hugo; Kvitko, Katia; da Silva, Juliana; Henriques, João A P

2016-09-15

Increasing evidence suggest that occupational exposure to open-cast coal mining residues like dust particles, heavy metals and Polycyclic Aromatic Hydrocarbons (PAHs) may cause a wide range of DNA damage and genomic instability that could be associated to initial steps in cancer development and other work-related diseases. The aim of our study was to evaluate if key polymorphisms in metabolism genes CYP1A1Msp1, GSTM1Null, GSTT1Null and DNA repair genes XRCC1Arg194Trp and hOGG1Ser326Cys could modify individual susceptibility to adverse coal exposure effects, considering the DNA damage (Comet assay) and micronucleus formation in lymphocytes (CBMN) and buccal mucosa cells (BMNCyt) as endpoints for genotoxicity. The study population is comprised of 200 healthy male subjects, 100 open-cast coal-mining workers from "El Cerrejón" (world's largest open-cast coal mine located in Guajira - Colombia) and 100 non-exposed referents from general population. The data revealed a significant increase of CBMN frequency in peripheral lymphocytes of occupationally exposed workers carrying the wild-type variant of GSTT1 (+) gene. Exposed subjects carrying GSTT1null polymorphism showed a lower micronucleus frequency compared with their positive counterparts (FR: 0.83; P=0.04), while BMNCyt, frequency and Comet assay parameters in lymphocytes: Damage Index (DI) and percentage of DNA in the tail (Tail % DNA) were significantly higher in exposed workers with the GSTM1Null polymorphism. Other exfoliated buccal mucosa abnormalities related to cell death (Karyorrhexis and Karyolysis) were increased in GSTT/M1Null carriers. Nuclear buds were significantly higher in workers carrying the CYP1A1Msp1 (m1/m2, m2/m2) allele. Moreover, BMNCyt frequency and Comet assay parameters were significantly lower in exposed carriers of XRCC1Arg194Trp (Arg/Trp, Trp/Trp) and hOGG1Ser326Cys (Ser/Cys, Cys/Cys), thereby providing new data to the increasing evidence about the protective role of these polymorphisms

Genetic Variability in DNA Repair Proteins in Age-Related Macular Degeneration

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Janusz Blasiak

2012-10-01

Full Text Available The pathogenesis of age-related macular degeneration (AMD is complex and involves interactions between environmental and genetic factors, with oxidative stress playing an important role inducing damage in biomolecules, including DNA. Therefore, genetic variability in the components of DNA repair systems may influence the ability of the cell to cope with oxidative stress and in this way contribute to the pathogenesis of AMD. However, few reports have been published on this subject so far. We demonstrated that the c.977C>G polymorphism (rs1052133 in the hOGG1 gene and the c.972G>C polymorphism (rs3219489 in the MUTYH gene, the products of which play important roles in the repair of oxidatively damaged DNA, might be associated with the risk of AMD. Oxidative stress may promote misincorporation of uracil into DNA, where it is targeted by several DNA glycosylases. We observed that the g.4235T>C (rs2337395 and c.−32A>G (rs3087404 polymorphisms in two genes encoding such glycosylases, UNG and SMUG1, respectively, could be associated with the occurrence of AMD. Polymorphisms in some other DNA repair genes, including XPD (ERCC2, XRCC1 and ERCC6 (CSB have also been reported to be associated with AMD. These data confirm the importance of the cellular reaction to DNA damage, and this may be influenced by variability in DNA repair genes, in AMD pathogenesis.

Environmental Stress Induces Trinucleotide Repeat Mutagenesis in Human Cells by Alt-Nonhomologous End Joining Repair.

Science.gov (United States)

Chatterjee, Nimrat; Lin, Yunfu; Yotnda, Patricia; Wilson, John H

2016-07-31

Multiple pathways modulate the dynamic mutability of trinucleotide repeats (TNRs), which are implicated in neurodegenerative disease and evolution. Recently, we reported that environmental stresses induce TNR mutagenesis via stress responses and rereplication, with more than 50% of mutants carrying deletions or insertions-molecular signatures of DNA double-strand break repair. We now show that knockdown of alt-nonhomologous end joining (alt-NHEJ) components-XRCC1, LIG3, and PARP1-suppresses stress-induced TNR mutagenesis, in contrast to the components of homologous recombination and NHEJ, which have no effect. Thus, alt-NHEJ, which contributes to genetic mutability in cancer cells, also plays a novel role in environmental stress-induced TNR mutagenesis. Published by Elsevier Ltd.

Endogenous DNA Damage and Repair Enzymes

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Arne Klungland

2016-06-01

Full Text Available Tomas Lindahl completed his medical studies at Karolinska Institute in 1970. Yet, his work has always been dedicated to unraveling fundamental mechanisms of DNA decay and DNA repair. His research is characterized with groundbreaking discoveries on the instability of our genome, the identification of novel DNA repair activities, the characterization of DNA repair pathways, and the association to diseases, throughout his 40 years of scientific career.

Reduced cellular DNA repair capacity after environmentally relevant arsenic exposure. Influence of Ogg1 deficiency

International Nuclear Information System (INIS)

Bach, Jordi; Peremartí, Jana; Annangi, Balasubramnayam; Marcos, Ricard; Hernández, Alba

2015-01-01

Highlights: • Repair ability under long-term exposure to arsenic was tested using the comet assay. • Effects were measured under Ogg1 wild-type and deficient backgrounds. • Exposed cells repair less efficiency the DNA damage induced by SA, KBrO 3 , MMA III or UVC radiation. • Oxidative damage and Ogg1 deficient background exacerbate repair deficiencies. • Overexpression of the arsenic metabolizing enzyme As3mt acts as adaptive mechanism. - Abstract: Inorganic arsenic (i-As) is a genotoxic and carcinogenic environmental contaminant known to affect millions of people worldwide. Our previous work demonstrated that chronic sub-toxic i-As concentrations were able to induce biologically significant levels of genotoxic and oxidative DNA damage that were strongly influenced by the Ogg1 genotype. In order to study the nature of the observed levels of damage and the observed differences between MEF Ogg1 +/+ and Ogg1 −/− genetic backgrounds, the genotoxic and oxidative DNA repair kinetics of 18-weeks exposed MEF cells were evaluated by the comet assay. Results indicate that MEF Ogg1 +/+ and Ogg1 −/− cells chronically exposed to i-As repair the DNA damage induced by arsenite, potassium bromide and UVC radiation less efficiently than control cells, being that observation clearly more pronounced in MEF Ogg1 −/− cells. Consequently, exposed cells accumulate a higher percentage of unrepaired DNA damage at the end of the repair period. As an attempt to eliminate i-As associated toxicity, chronically exposed MEF Ogg1 −/− cells overexpress the arsenic metabolizing enzyme As3mt. This adaptive response confers cells a significant resistance to i-As-induced cell death, but at expenses of accumulating high levels of DNA damage due to their repair impairment. Overall, the work presented here evidences that i-As chronic exposure disrupts the normal cellular repair function, and that oxidative DNA damage—and Ogg1 deficiency—exacerbates this phenomenon. The

Reduced cellular DNA repair capacity after environmentally relevant arsenic exposure. Influence of Ogg1 deficiency

Energy Technology Data Exchange (ETDEWEB)

Bach, Jordi; Peremartí, Jana; Annangi, Balasubramnayam [Grup de Mutagènesi, Departament de Genètica i de Microbiologia, Facultat de Biociències, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Barcelona (Spain); Marcos, Ricard, E-mail: ricard.marcos@uab.es [Grup de Mutagènesi, Departament de Genètica i de Microbiologia, Facultat de Biociències, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Barcelona (Spain); CIBER Epidemiología y Salud Pública, ISCIII, Madrid (Spain); Hernández, Alba, E-mail: alba.hernandez@uab.es [Grup de Mutagènesi, Departament de Genètica i de Microbiologia, Facultat de Biociències, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Barcelona (Spain); CIBER Epidemiología y Salud Pública, ISCIII, Madrid (Spain)

2015-09-15

Highlights: • Repair ability under long-term exposure to arsenic was tested using the comet assay. • Effects were measured under Ogg1 wild-type and deficient backgrounds. • Exposed cells repair less efficiency the DNA damage induced by SA, KBrO{sub 3}, MMA{sup III} or UVC radiation. • Oxidative damage and Ogg1 deficient background exacerbate repair deficiencies. • Overexpression of the arsenic metabolizing enzyme As3mt acts as adaptive mechanism. - Abstract: Inorganic arsenic (i-As) is a genotoxic and carcinogenic environmental contaminant known to affect millions of people worldwide. Our previous work demonstrated that chronic sub-toxic i-As concentrations were able to induce biologically significant levels of genotoxic and oxidative DNA damage that were strongly influenced by the Ogg1 genotype. In order to study the nature of the observed levels of damage and the observed differences between MEF Ogg1{sup +/+} and Ogg1{sup −/−} genetic backgrounds, the genotoxic and oxidative DNA repair kinetics of 18-weeks exposed MEF cells were evaluated by the comet assay. Results indicate that MEF Ogg1{sup +/+} and Ogg1{sup −/−} cells chronically exposed to i-As repair the DNA damage induced by arsenite, potassium bromide and UVC radiation less efficiently than control cells, being that observation clearly more pronounced in MEF Ogg1{sup −/−} cells. Consequently, exposed cells accumulate a higher percentage of unrepaired DNA damage at the end of the repair period. As an attempt to eliminate i-As associated toxicity, chronically exposed MEF Ogg1{sup −/−} cells overexpress the arsenic metabolizing enzyme As3mt. This adaptive response confers cells a significant resistance to i-As-induced cell death, but at expenses of accumulating high levels of DNA damage due to their repair impairment. Overall, the work presented here evidences that i-As chronic exposure disrupts the normal cellular repair function, and that oxidative DNA damage—and Ogg1 deficiency�

Split dose recovery studies using homologous recombination deficient gene knockout chicken B lymphocyte cells

International Nuclear Information System (INIS)

Rao, B.S.S.; Tano, Kaori; Utsumi, Hiroshi; Takeda, Shunichi

2007-01-01

To understand the role of proteins involved in double strand breaks (DSB) repair modulating sublethal damage (SLD) recovery, chicken B lymphoma (DT 40) cell lines either proficient or deficient in RAD52, XRCC2, XRCC3, RAD51C and RAD51D were subjected to fractionated irradiation and their survival curves charted. Survival curves of both WT DT40 and RAD52 -/- cells had a big shoulder while all the other cells exhibited small shoulders. However, at the higher doses of radiation, RAD51C -/- cells displayed hypersensitivity comparable to the data obtained for the homologous recombination deficient RAD54 -/- cells. Repair of SLD was measured as an increase in survival after a split dose irradiation with an interval of incubation between the radiation doses. All the cell lines (parental DT40 and genetic knockout cell lines viz., RAD52 -/- , XRCC2 -/- XRCC3 -/- RAD51C -/- and RAD51D -/- ) used in this study demonstrated a typical split-dose recovery capacity with a specific peak, which varied depending on the cell type. The maximum survival of WT DT40 and RAD52 -/- was reached at about 1-2 hours after the first dose of radiation and then decreased to a minimum thereafter (5 h). The increase in the survival peaked once again by about 8 hours. The survival trends observed in XRCC2 -/- , XRCC3 -/- , RAD51C -/- and RAD51D -/- knockout cells were also similar, except for the difference in the initial delay of a peak survival for RAD51D -/- and lower survival ratios. The second phase of increase in the survival in these cell lines was much slower in XRCC2 -/- , XRCC3 -/- , RAD51C -/- nd RAD51D -/- and further delayed when compared with that of RAD52 -/- and parental DT40 cells suggesting a dependence on their cell cycle kinetics. This study demonstrates that the participation of RAD52, XRCC2, XRCC3, RAD51C and RAD51D in the DSB repair via homologous recombination is of less importance in comparison to RAD54, as RAD54 deficient cells demonstrated complete absence of SLD recovery

Polimorfismos em genes de reparo do DNA (XPC, ERCC1, XRCC7) em mulheres com câncer do colo do útero

OpenAIRE

Saffar, Issamir Farias [UNIFESP

2010-01-01

Estudos demonstram que polimorfismos em genes relacionados ao reparo do DNA estão envolvidos na patogênese de diversas doenças neoplásicas, como o câncer ginecológico, particularmente o câncer do colo do útero. O presente estudo, caso-controle, compara os polimorfismos dos genes XPC, ERCC1 e XRCC7 em 77 mulheres com câncer cervical (70 casos de carcinoma espinocelular e 7 casos de adenocarcinoma do colo do útero) e 73 mulheres saudáveis atendidas no Hospital do Câncer Alfredo Abrão, entre Jun...

Yeast redoxyendonuclease, a DNA repair enzyme similar to Escherichia coli endonuclease III

International Nuclear Information System (INIS)

Gossett, J.; Lee, K.; Cunningham, R.P.; Doetsch, P.W.

1988-01-01

A DNA repair endonuclease (redoxyendonuclease) was isolated from bakers' yeast (Saccharomyces cerevisiae). The enzyme has been purified by a series of column chromatography steps and cleaves OsO 4 -damaged, double-stranded DNA at sites of thymine glycol and heavily UV-irradiated DNA at sites of cytosine, thymine, and guanine photoproducts. The base specificity and mechanism of phosphodiester bond cleavage for the yeast redoxyendonuclease appear to be identical with those of Escherichia coli endonuclease III when thymine glycol containing, end-labeled DNA fragments of defined sequence are employed as substrates. Yeast redoxyendonuclease has an apparent molecular size of 38,000-42,000 daltons and is active in the absence of divalent metal cations. The identification of such an enzyme in yeast may be of value in the elucidation of the biochemical basis for radiation sensitivity in certain yeast mutants

Computational study of hydration at the TD damaged site of DNA in complex with repair enzyme T4 endonuclease V

International Nuclear Information System (INIS)

Pinak, Miroslav

2000-02-01

An analysis of the distribution of water around DNA surface focusing on the role of the distribution of water molecules in the proper recognition of damaged site by repair enzyme T4 Endonuclease V was performed. The native DNA dodecamer, dodecamer with the thymine dimer (TD) and complex of DNA and part of repair enzyme T4 Endonuclease V were examined throughout the 500 ps of molecular dynamics simulation. During simulation the number of water molecules close to the DNA atoms and the residence time were calculated. There is an increase in number of water molecules lying in the close vicinity to TD if compared with those lying close to two native thymines (TT). Densely populated area with water molecules around TD is one of the factors detected by enzyme during scanning process. The residence time was found higher for molecule of the complex and the six water molecules were found occupying the stabile positions between the TD and catalytic center close to atoms P, C3' and N3. These molecules originate water mediated hydrogen bond network that contribute to the stability of complex required for the onset of repair process. (author)

Computational study of hydration at the TD damaged site of DNA in complex with repair enzyme T4 endonuclease V

Energy Technology Data Exchange (ETDEWEB)

Pinak, Miroslav [Japan Atomic Energy Research Inst., Tokai, Ibaraki (Japan). Tokai Research Establishment

2000-02-01

An analysis of the distribution of water around DNA surface focusing on the role of the distribution of water molecules in the proper recognition of damaged site by repair enzyme T4 Endonuclease V was performed. The native DNA dodecamer, dodecamer with the thymine dimer (TD) and complex of DNA and part of repair enzyme T4 Endonuclease V were examined throughout the 500 ps of molecular dynamics simulation. During simulation the number of water molecules close to the DNA atoms and the residence time were calculated. There is an increase in number of water molecules lying in the close vicinity to TD if compared with those lying close to two native thymines (TT). Densely populated area with water molecules around TD is one of the factors detected by enzyme during scanning process. The residence time was found higher for molecule of the complex and the six water molecules were found occupying the stabile positions between the TD and catalytic center close to atoms P, C3' and N3. These molecules originate water mediated hydrogen bond network that contribute to the stability of complex required for the onset of repair process. (author)

Non-homologous end joining mediated DNA repair is impaired in the NUP98-HOXD13 mouse model for myelodysplastic syndrome.

Science.gov (United States)

Puthiyaveetil, Abdul Gafoor; Reilly, Christopher M; Pardee, Timothy S; Caudell, David L

2013-01-01

Chromosomal translocations typically impair cell differentiation and often require secondary mutations for malignant transformation. However, the role of a primary translocation in the development of collaborating mutations is debatable. To delineate the role of leukemic translocation NUP98-HOXD13 (NHD13) in secondary mutagenesis, DNA break and repair mechanisms in stimulated mouse B lymphocytes expressing NHD13 were analyzed. Our results showed significantly reduced expression of non-homologous end joining (NHEJ)-mediated DNA repair genes, DNA Pkcs, DNA ligase4, and Xrcc4 leading to cell cycle arrest at G2/M phase. Our results showed that expression of NHD13 fusion gene resulted in impaired NHEJ-mediated DNA break repair. Copyright © 2012 Elsevier Ltd. All rights reserved.

Opposing roles of RNF8/RNF168 and deubiquitinating enzymes in ubiquitination-dependent DNA double-strand break response signaling and DNA-repair pathway choice

International Nuclear Information System (INIS)

Nakada, Shinichiro

2016-01-01

The E3 ubiquitin ligases ring finger protein (RNF) 8 and RNF168 transduce the DNA double-strand break (DSB) response (DDR) signal by ubiquitinating DSB sites. The depletion of RNF8 or RNF168 suppresses the accumulation of DNA-repair regulating factors such as 53BP1 and RAP80 at DSB sites, suggesting roles for RNF8- and RNF168-mediated ubiquitination in DSB repair. This mini-review provides a brief overview of the RNF8- and RNF168-dependent DDR-signaling and DNA-repair pathways. The choice of DNA-repair pathway when RNF8- and RNF168-mediated ubiquitination-dependent DDR signaling is negatively regulated by deubiquitinating enzymes (DUBs) is reviewed to clarify how the opposing roles of RNF8/RNF168 and DUBs regulate ubiquitination-dependent DDR signaling and the choice of DNA-repair pathway

Assay of repair enzyme activity by reactivation of ultraviolet-irradiated infective viral DNA

Energy Technology Data Exchange (ETDEWEB)

Oeda, K; Nakatsu, Y; Sekiguchi, M [Kyushu Univ., Fukuoka (Japan).Faculty of Science

1980-05-01

Treatment of OeX174 replicative form (RF) DNA, pre-exposed to ultraviolet light, with T4 endonuclease V led to a marked increase of infectivity of the RF when the activity was assayed on CaCl/sub 2/-treated cells of Escherichia coli strain defective in uvrA gene. The reaction was specific and the extent of the reactivation was proportional to the concentration of the enzyme. Based on this finding, we developed a procedure to assay endonuclease activities specific for ultraviolet-damaged DNA, that might be involved in the incision step of excision repair of pyrimidine dimers. To find conditions suitable for accurate and rapid assays, we examined conditions affecting transfection with OeX174 RF. The maximum transfection was achieved when more than 2 x 10/sup 8/ CaCl/sub 2/-treated cells, which had been prepared from bacteria harvested during the early or mid-logarithmic phase of growth in L broth, were incubated with the DNA at 0/sup 0/C for 20 min in 50 mM CaCl/sub 2/. Incubation of the cell-DNA mixture at 37/sup 0/C decreased the transfection efficiency to about 30% of the optimal level; thus, heat shock, a step regarded as necessary in the conventional CaCl/sub 2/ methods for transfection and transformation, was eliminated. The CaCl/sub 2/-treated cells remained viable and competent after storage at -20/sup 0/C in a solution containing 15% glycerol. By using the procedure thus established, repair endonuclease activities in crude extracts of T4-infected E. coli and of Micrococcus luteus were determined. The procedure should be of use in assaying and purifying repair enzymes of other organisms.

Heritable susceptibility factors for the development of cancer

International Nuclear Information System (INIS)

Au, William W.

2006-01-01

High frequencies of inherited DNA sequence variations (polymorphisms) are found in the human population. The involvement of polymorphic genes (especially for chemical metabolism and DNA repair) in the development of cancer is under intensive investigation. In our studies, we have irradiated blood lymphocytes from normal non-smokers with γ-rays or UV-light to investigate genotypes and DNA repair functions. We found that XRCC1 399Gln and XRCC3 241Met were deficient in the repair of γ-ray- but not UV-light-induced DNA damage that led to the expression of chromosome aberrations; therefore the variant genotypes are defective in base excision repair. The reverse was found with XPD 312Asn and XPD 751Gln; therefore they are defective in nucleotide excision repair. XRCC1 194Trp, OGG1 326Cys and APE1 148Glu had no DNA repair deficiency based on our experimental conditions. In another study, we investigated the role of some of these genes on the development of lung cancer. We found a significant increase of chromosome aberrations in patients and controls that had the XPD 751Gln and GSTM1 null genotypes, indicating a mechanistic causation of the disease. Therefore, inheritance of susceptibility genes can have significant impact on disease burden in the population. On the other hand, there are many questions that need to be addressed in order to evaluate the impact of susceptibility on cancer. These questions include the understanding of combinations of different polymorphic genes for susceptibility and of specific disease susceptibility for different ethnic populations. (author)

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

International Nuclear Information System (INIS)

Gines, Guillaume; Saint-Pierre, Christine; Gasparutto, Didier

2014-01-01

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

My journey to DNA repair.

Science.gov (United States)

Lindahl, Tomas

2013-02-01

I completed my medical studies at the Karolinska Institute in Stockholm but have always been devoted to basic research. My longstanding interest is to understand fundamental DNA repair mechanisms in the fields of cancer therapy, inherited human genetic disorders and ancient DNA. I initially measured DNA decay, including rates of base loss and cytosine deamination. I have discovered several important DNA repair proteins and determined their mechanisms of action. The discovery of uracil-DNA glycosylase defined a new category of repair enzymes with each specialized for different types of DNA damage. The base excision repair pathway was first reconstituted with human proteins in my group. Cell-free analysis for mammalian nucleotide excision repair of DNA was also developed in my laboratory. I found multiple distinct DNA ligases in mammalian cells, and led the first genetic and biochemical work on DNA ligases I, III and IV. I discovered the mammalian exonucleases DNase III (TREX1) and IV (FEN1). Interestingly, expression of TREX1 was altered in some human autoimmune diseases. I also showed that the mutagenic DNA adduct O(6)-methylguanine (O(6)mG) is repaired without removing the guanine from DNA, identifying a surprising mechanism by which the methyl group is transferred to a residue in the repair protein itself. A further novel process of DNA repair discovered by my research group is the action of AlkB as an iron-dependent enzyme carrying out oxidative demethylation. Copyright © 2013. Production and hosting by Elsevier Ltd.

Dumbbell DNA-templated CuNPs as a nano-fluorescent probe for detection of enzymes involved in ligase-mediated DNA repair.

Science.gov (United States)

Qing, Taiping; He, Xiaoxiao; He, Dinggeng; Ye, Xiaosheng; Shangguan, Jingfang; Liu, Jinquan; Yuan, Baoyin; Wang, Kemin

2017-08-15

DNA repair processes are responsible for maintaining genome stability. Ligase and polynucleotide kinase (PNK) have important roles in ligase-mediated DNA repair. The development of analytical methods to monitor these enzymes involved in DNA repair pathways is of great interest in biochemistry and biotechnology. In this work, we reported a new strategy for label-free monitoring PNK and ligase activity by using dumbbell-shaped DNA templated copper nanoparticles (CuNPs). In the presence of PNK and ligase, the dumbbell-shaped DNA probe (DP) was locked and could resist the digestion of exonucleases and then served as an efficient template for synthesizing fluorescent CuNPs. However, in the absence of ligase or PNK, the nicked DP could be digested by exonucleases and failed to template fluorescent CuNPs. Therefore, the fluorescence changes of CuNPs could be used to evaluate these enzymes activity. Under the optimal conditions, highly sensitive detection of ligase activity of about 1U/mL and PNK activity down to 0.05U/mL is achieved. To challenge the practical application capability of this strategy, the detection of analyte in dilute cells extracts was also investigated and showed similar linear relationships. In addition to ligase and PNK, this sensing strategy was also extended to the detection of phosphatase, which illustrates the versatility of this strategy. Copyright © 2017 Elsevier B.V. All rights reserved.

Long non-coding RNAs as novel expression signatures modulate DNA damage and repair in cadmium toxicology

Science.gov (United States)

Zhou, Zhiheng; Liu, Haibai; Wang, Caixia; Lu, Qian; Huang, Qinhai; Zheng, Chanjiao; Lei, Yixiong

2015-10-01

Increasing evidence suggests that long non-coding RNAs (lncRNAs) are involved in a variety of physiological and pathophysiological processes. Our study was to investigate whether lncRNAs as novel expression signatures are able to modulate DNA damage and repair in cadmium(Cd) toxicity. There were aberrant expression profiles of lncRNAs in 35th Cd-induced cells as compared to untreated 16HBE cells. siRNA-mediated knockdown of ENST00000414355 inhibited the growth of DNA-damaged cells and decreased the expressions of DNA-damage related genes (ATM, ATR and ATRIP), while increased the expressions of DNA-repair related genes (DDB1, DDB2, OGG1, ERCC1, MSH2, RAD50, XRCC1 and BARD1). Cadmium increased ENST00000414355 expression in the lung of Cd-exposed rats in a dose-dependent manner. A significant positive correlation was observed between blood ENST00000414355 expression and urinary/blood Cd concentrations, and there were significant correlations of lncRNA-ENST00000414355 expression with the expressions of target genes in the lung of Cd-exposed rats and the blood of Cd exposed workers. These results indicate that some lncRNAs are aberrantly expressed in Cd-treated 16HBE cells. lncRNA-ENST00000414355 may serve as a signature for DNA damage and repair related to the epigenetic mechanisms underlying the cadmium toxicity and become a novel biomarker of cadmium toxicity.

The involvement of DNA repair genes in the hypoxia-dependent NLCQ-1 (NSC 709257) toxicity and its synergistic interaction with cisplatin or melphalan

International Nuclear Information System (INIS)

Papadopoulou, M.V.; Xue, C.-J.; Bloomer, W.D.

2003-01-01

4-[3-(2-Nitro-1-imidazolyl)-propylamino]-7-chloro-quinoline hydrochloride (NLCQ-1) is a weakly DNA-intercalating hypoxia selective cytotoxin, which synergistically enhances the antitumor effect of several chemotherapeutic agents or radiation against mouse tumors or human xenografts. Synergy with melphalan (L-PAM) or cisplatin (cisPt) requires hypoxic pre-exposure of cells to NLCQ-1 or, in mice, administration of NLCQ-1 about 1 h before L-PAM or cisPt. This suggests that NLCQ-1 may cause DNA lesions upon reductive metabolism. To indirectly identify such lesions, rodent cell lines defective in specific DNA repair genes (EM9 and UV41) and their repair-proficient parental AA8, were exposed to NLCQ-1 alone and in combination with L-PAM or cisPt under hypoxic/aerobic conditions and appropriate routes, and assessed for clonogenicity. Selected comparisons with tirapazamine (TPZ) were also performed. DNA ssbs were identified by using the alkaline comet assay. Synergism was assessed by isobologramic analysis. EM9, which lack the functional XRCC1 gene and are unable to efficiently repair DNA ssbs, were 3.7x and 4.5x more sensitive to NLCQ-1 and TPZ, respectively, than the parental AA8 cells. Similarly, UV41, which are defective in the ERCC4/XPF gene and thus, hypersensitive to DNA cross-linking agents, were 4.1x more sensitive than AA8 cells to NLCQ-1. Equitoxic concentrations of NLCQ-1 and TPZ gave similar numbers of ssbs in AA8 and EM9 cells exposed to each compound for 1 h under hypoxic conditions. In combination with L-PAM or cisPt, synergy was observed in AA8 but not in EM9 or UV41 cells, with either NLCQ-1 or TPZ. These results suggest that NLCQ-1 is involved in the formation of DNA ssbs and interstrand crosslinks, with the latter being most likely responsible for NLCQ-1 hypoxic toxicity. The synergistic interaction of NLCQ-1 with L-PAM or cisPt is probably due to an enhancement in the L-PAM/cisPt-induced DNA interstrand crosslinks, possibly as a result of an inhibited

Modulation of DNA repair capacity and mRNA expression levels of XRCC1, hOGG1 and XPC genes in styrene-exposed workers

Czech Academy of Sciences Publication Activity Database

Hánová, Monika; Štětina, R.; Vodičková, Ludmila; Václavíková, R.; Hlaváč, P.; Šmerhovský, Z.; Naccarati, Alessio; Poláková, Veronika; Souček, P.; Kuricová, M.; Manini, P.; Kumar, R.; Hemminki, K.; Vodička, Pavel

2010-01-01

Roč. 248, č. 3 (2010), s. 194-200 ISSN 0041-008X R&D Projects: GA AV ČR IAA500390806 Grant - others:GA MZd(CZ) NR9423 Program:NR Institutional research plan: CEZ:AV0Z50390512 Keywords : styrene exposure * genotoxicity * DNA repair Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.993, year: 2010

Influence of the complexity of radiation-induced DNA damage on enzyme recognition

International Nuclear Information System (INIS)

Palmer, Philip

2002-01-01

Ionising radiation is unique in inducing DNA clustered damage together with the simple isolated lesions. Understanding how these complex lesions are recognised and repaired by the cell is key to understanding the health risks associated with radiation exposure. This study focuses on whether ionising radiation-induced complex single-strand breaks (SSB) are recognised by DNA-PK and PARP, and whether the complexity of DSB influence their ligation by either DNA ligase lV/XRCC4 (LX) complex or T4 DNA ligase. Plasmid DNA, irradiated in aqueous solution using sparsely ionising γ-rays and densely ionising α-particles produce different yields of complex DNA damages, used as substrates for in vitro DNA-PK and PARP activity assays. The activity of DNA-PK to phosphorylate a peptide was determined using HF19 cell nuclear extracts as a source of DNA-PK. PARP ADP-ribosylation activity was determined using purified PARP enzyme. The activation of DNA-PK and PARP by irradiated DNA is due to SSB and not the low yield of DSB (linear plasmid DNA <10%). A ∼2 fold increase in DNA-PK activation and a ∼3-fold reduction in PARP activity seen on increasing the ionising density of the radiation (proportion of complex damage) are proposed to reflect changes in the complexity of SSB and may relate to damage signalling. Complex DSB synthesised as double-stranded oligonucleotides, with a 2 bp 5'-overhang, and containing modified lesions, 8-oxoguanine and abasic sites, at known positions relative to the termini were used as substrates for in vitro ligation by DNA ligase IV/XRCC4 or T4 ligase. The presence of a modified lesion 2 or 3 bp but not 4 bp from the 3'-termini and 2 or 6 bp from the 5'-termini caused a drastic reduction in the extent of ligation. Therefore, the presence of modified lesions near to the termini of a DSB may compromise their rejoining by non-homologous end-joining (NHEJ) involving the LX complex. (author)

Synthetic lethality between murine DNA repair factors XLF and DNA-PKcs is rescued by inactivation of Ku70

DEFF Research Database (Denmark)

Xing, Mengtan; Bjørås, Magnar; Daniel, Jeremy A

2017-01-01

DNA double-strand breaks (DSBs) are recognized and repaired by the Classical Non-Homologous End-Joining (C-NHEJ) and Homologous Recombination pathways. C-NHEJ includes the core Ku70 and Ku80 (or Ku86) heterodimer that binds DSBs and thus promotes recruitment of accessory downstream NHEJ factors XLF......, PAXX, DNA-PKcs, Artemis and other core subunits, XRCC4 and DNA Ligase 4 (Lig4). In the absence of core C-NHEJ factors, DNA repair can be performed by Alternative End-Joining, which likely depends on DNA Ligase 1 and DNA Ligase 3. Genetic inactivation of C-NHEJ factors, such as Ku70, Ku80, XLF, PAXX...... with severe apoptosis in the central nervous system. Here, we demonstrate that inactivation of the Ku70 gene rescues the synthetic lethality between XLF and DNA-PKcs, resulting in triple knockout mice that are indistinguishable from Ku70-deficient littermates by size or levels of genomic instability. Moreover...

Action of some drugs on enzymes involved in DNA-repair and semiconservative DNA-synthesis

International Nuclear Information System (INIS)

Wawra, E.; Klein, W.; Kocsis, F.; Weniger, P.

1975-07-01

Different antirheumatic and cytostatic drugs had been tested by measurement of the thymidine incorporation into DNA of spleen cells under conditions, under which either DNA-synthesis or repair after gamma- or UV-irradiation takes place. There are substances, which inhibit either only the semiconservative DNA-synthesis (vinblastine, isonicotinic acid hydracide) or only DNA-repair after gamma-irradiation (mixture of penicillin-G and procaine-penicillin-G) or both (cyclophosphamide, phenylbutazone, procarbazine, nalidixic acid). Vincristine shows no effect on the thymidine incorporation in DNA, but by density gradient centrifugation it has been found that it influences the ligase reaction. Two DNA polymerases had been isolated from spleen cells, one of the low molecular and one of the high molecular weight type. The influences of the described drugs on these enzymes and on a deoxyribonuclease I from beef pancreas have been tested in ''in vitro'' systems. In all cases, it has been found that there is no effect or only a very small one, compared with the action of well known inhibitors as e.g. ethidium bromide and p-chloromercuribenzoate, and this cannot be responsible for the suppressions found in DNA-repair and semiconservative DNA-synthesis. (author)

Repair-modification of radiodamaged genes

International Nuclear Information System (INIS)

Volpe, P.; Institute of Experimental Medicine, Rome; Eremenko, T.

1995-01-01

It is proposed that through repair-modification, the modified base 5mC may have facilitated the divergent evolution of coding (hypomethylated exon) and uncoding (hypermethylated promoter and intron) sequences in eukaryotic genes. The radioinduced repair patches appearing in regions lacking 5mC are fully reconstructed by excision-repair, whereas those appearing in regions containing 5mC are incompletely reconstructed by this conventional mechanism. Such a second class of repair patches may, however, become fully reconstructed, in the S phase, by repair-modification. In fact, while DNA polymerase β - which is a key enzyme of excision-repair - is active through the whole interphase. DNA methylase - which is responsible for post-synthetic DNA modification - is essentially active in S. Uncoupling of these two enzyme systems, outside S, might explain why in unsynchronised cells repair patches of non-replicating strands are hypomethylated when compared with specific methylation of replicating strands. In other words, excision-repair would always be able to re-establish the primary ATGC language of both damaged unmethylated and methylated regions, while repair-modification would be able to re-establish the modified ATGC(5mC) language of the damaged methylated regions, only in S, but not in G 1 or G 2 . In these two phases, when DNA methylation is inversely correlated with pre-mRNA transcription (as in the case of many tissue-specific genes), such demethylation might induce a silent transcriptional unit to become active. (Author)

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

Energy Technology Data Exchange (ETDEWEB)

Gines, Guillaume; Saint-Pierre, Christine; Gasparutto, Didier, E-mail: didier.gasparutto@cea.fr

2014-02-17

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{sup −1} and 50 μg mL{sup −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

Preventive Long-Term Effects of a Topical Film-Forming Medical Device with Ultra-High UV Protection Filters and DNA Repair Enzyme in Xeroderma Pigmentosum: A Retrospective Study of Eight Cases

Directory of Open Access Journals (Sweden)

Sandra Giustini

2014-09-01

Full Text Available Skin cancer is common in xeroderma pigmentosum (XP due to a DNA repair mechanisms genetic defect. Ultraviolet (UV exposure is the main cause of increased incidence of actinic keratosis (AK, basal cell carcinoma (BCC and squamous cell carcinoma (SCC observed in XP subjects. Photoprotection is therefore a mandatory strategy in order to reduce skin damage. A topical DNA repair enzyme has been shown to slow down the development of skin lesions in XP. However, there are no data regarding the effects of photoprotection combined with DNA repair strategies in this clinical setting. A film-forming medical device containing the DNA repair enzyme photolyase and very high-protection UV filters (Eryfotona AK-NMSC, Ery is currently available. We report retrospective data regarding the use of Ery in 8 patients (5 women, 3 men with a diagnosis of XP treated for at least 12 consecutive months, comparing the rate of new skin lesions (AK, BCC and SCC during active treatment with Ery and during 12 months just before the use of the product. New AK, BCC and SCC mean lesion numbers during the 1-year Ery treatment were 5, 3 and 0, respectively in comparison with 14, 6.8 and 3 lesions, respectively during the 1-year pre-treatment period. Ery use was associated with a 65% reduction in appearance of new AK lesions and with 56 and 100% reductions in the incidence of new BCC and SCC lesions, respectively. These data suggest that topical use of photoprotection and DNA repair enzyme could help lower skin cancer lesions in XP. Control prospective trials are advisable in this clinical setting.

Altered Gene Expressions and Cytogenetic Repair Efficiency in Cells with Suppressed Expression of XPA after Proton Exposure

Science.gov (United States)

Zhang, Ye; Rohde, Larry H.; Gridley, Daila S.; Mehta, Satish K.; Pierson, Duane L.; Wu, Honglu

2009-01-01

Cellular responses to damages from ionizing radiation (IR) exposure are influenced not only by the genes involved in DNA double strand break (DSB) repair, but also by non- DSB repair genes. We demonstrated previously that suppressed expression of several non-DSB repair genes, such as XPA, elevated IR-induced cytogenetic damages. In the present study, we exposed human fibroblasts that were treated with control or XPA targeting siRNA to 250 MeV protons (0 to 4 Gy), and analyzed chromosome aberrations and expressions of genes involved in DNA repair. As expected, after proton irradiation, cells with suppressed expression of XPA showed a significantly elevated frequency of chromosome aberrations compared with control siRNA treated (CS) cells. Protons caused more severe DNA damages in XPA knock-down cells, as 36% cells contained multiple aberrations compared to 25% in CS cells after 4Gy proton irradiation. Comparison of gene expressions using the real-time PCR array technique revealed that expressions of p53 and its regulated genes in irradiated XPA suppressed cells were altered similarly as in CS cells, suggesting that the impairment of IR induced DNA repair in XPA suppressed cells is p53-independent. Except for XPA, which was more than 2 fold down regulated in XPA suppressed cells, several other DNA damage sensing and repair genes (GTSE1, RBBP8, RAD51, UNG and XRCC2) were shown a more than 1.5 fold difference between XPA knock-down cells and CS cells after proton exposure. The possible involvement of these genes in the impairment of DNA repair in XPA suppressed cells will be further investigated.

Recombinational repair: workshop summary

International Nuclear Information System (INIS)

Howard-Flanders, P.

1983-01-01

Recombinational repair may or may not be synonymous with postreplication repair. Considerable progress has been made in the study of the relevant enzymes, particularly those from bacteria. In this workshop we focus on the recombination enzyme RecA protein. What structural changes take place in the protein and in DNA during repair. How does homologous pairing take place. How is ATP hydrolysis coupled to the stand exchange reaction and the formation of heteroduplx DNA. Turning to another enzyme needed for certain kinds of bacterial recombination, we will ask whether the purified recB protein and recC protein complement each other and are sufficient for exonuclease V activity. In higher cells, we would like to know whether sister exchanges, which occur in bacteria after uv irradiation, are also seen in animal cells

Monitoring of the spatial and temporal dynamics of BER/SSBR pathway proteins, including MYH, UNG2, MPG, NTH1 and NEIL1-3, during DNA replication.

Science.gov (United States)

Bj Rås, Karine Ø; Sousa, Mirta M L; Sharma, Animesh; Fonseca, Davi M; S Gaard, Caroline K; Bj Rås, Magnar; Otterlei, Marit

2017-08-21

Base lesions in DNA can stall the replication machinery or induce mutations if bypassed. Consequently, lesions must be repaired before replication or in a post-replicative process to maintain genomic stability. Base excision repair (BER) is the main pathway for repair of base lesions and is known to be associated with DNA replication, but how BER is organized during replication is unclear. Here we coupled the iPOND (isolation of proteins on nascent DNA) technique with targeted mass-spectrometry analysis, which enabled us to detect all proteins required for BER on nascent DNA and to monitor their spatiotemporal orchestration at replication forks. We demonstrate that XRCC1 and other BER/single-strand break repair (SSBR) proteins are enriched in replisomes in unstressed cells, supporting a cellular capacity of post-replicative BER/SSBR. Importantly, we identify for the first time the DNA glycosylases MYH, UNG2, MPG, NTH1, NEIL1, 2 and 3 on nascent DNA. Our findings suggest that a broad spectrum of DNA base lesions are recognized and repaired by BER in a post-replicative process. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Human longevity and variation in GH/IGF-1/insulin signaling, DNA damage signaling and repair and pro/antioxidant pathway genes

DEFF Research Database (Denmark)

Soerensen, Mette; Dato, Serena; Tan, Qihua

2012-01-01

SNPs (rs13320360 (MLH1), rs2509049 (H2AFX) and rs705649 (XRCC5)) to be associated with mortality in late life after correction for multiple testing. When examining the 11 SNPs from the case-control study in the longitudinal data, rs3842755 (INS), rs13251813 (WRN) and rs3211994 (NTHL1) demonstrated...

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

Directory of Open Access Journals (Sweden)

Petrović Sandra

2015-01-01

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

Effect of Genetic Polymorphisms and Long-Term Tobacco Exposure on the Risk of Breast Cancer

Science.gov (United States)

Verde, Zoraida; Santiago, Catalina; Chicharro, Luis Miguel; Reinoso-Barbero, Luis; Tejerina, Alejandro; Bandrés, Fernando; Gómez-Gallego, Félix

2016-01-01

Introduction: Tobacco smoke contains many potentially harmful compounds that may act differently and at different stages in breast cancer development. The focus of this work was to assess the possible role of cigarette smoking (status, dose, duration or age at initiation) and polymorphisms in genes coding for enzymes involved in tobacco carcinogen metabolism (CYP1A1, CYP2A6) or in DNA repair (XRCC1, APEX1, XRCC3 and XPD) in breast cancer development. Methods: We designed a case control study with 297 patients, 217 histologically verified breast cancers (141 smokers and 76 non-smokers) and 80 healthy smokers in a cohort of Spanish women. Results: We found an association between smoking status and early age at diagnosis of breast cancer. Among smokers, invasive carcinoma subtype incidence increased with intensity and duration of smoking (all Ptrend cancer (OR = 7.12 (1.98–25.59)). Conclusions: Our results support the main effect of CYP1A1 in estrogenic metabolism rather than in tobacco carcinogen activation in breast cancer patients and also confirmed the hypothesis that CYP1A1 Ile462Val, in association with long periods of active smoking, could be a breast cancer risk factor. PMID:27754415

Role of microRNA in Aggressive Prostate Cancer

Science.gov (United States)

2015-09-01

Interleukin enhancer-binding factor 2 OS=Homo sapiens GN=ILF2 PE=1 SV=2 ACACA_HUMAN Acetyl-CoA carboxylase 1 OS=Homo sapiens GN=ACACA PE=1 SV=2...I3L1L3_HUMAN Myb-binding protein 1A (Fragment) OS=Homo sapiens GN=MYBBP1A PE=4 SV=1 XRCC5_HUMAN X-ray repair cross-complementing protein 5 OS=Homo sapiens GN...XRCC5 PE=1 SV=3 SFPQ_HUMAN Splicing factor, proline- and glutamine-rich OS=Homo sapiens GN=SFPQ PE=1 SV=2 ATPA_HUMAN ATP synthase subunit alpha

Radiation damage and repair in cells and cell components. Final report. Part 1

International Nuclear Information System (INIS)

Fluke, D.J.

1984-01-01

An overview of research into the direct action of ionizing radiation, especially the effect of radiation temperature, primarily upon enzymes, into induced repair, and into S.O.S.-related phenomena, is presented

Inhibition of potential lethal damage repair and related gene expression after carbon-ion beam irradiation to human lung cancer grown in nude mice

International Nuclear Information System (INIS)

Yashiro, Tomoyasu; Fujisawa, Takehiko; Koyama-Saegusa, Kumiko; Imai, Takashi; Miyamoto, Tadaaki

2007-01-01

Using cultured and nude mouse tumor cells (IA) derived from a human lung cancer, we previously demonstrated their radiosensitivity by focusing attention on the dynamics of tumor clonogens and the early and rapid survival recovery (potential lethal damage repair: PLD repair) occurring after X-ray irradiation. To the authors' knowledge, this is the first study demonstrating gene expression in association with PLD repair after carbon-ion beam or X-ray irradiation to cancer cells. In this study we tried to detect the mechanism of DNA damage and repair of the clonogens after X-ray or carbon-ion beam irradiation. At first, colony assay method was performed after irradiation of 12 Gy of X-ray or 5 Gy of carbon-ion beam to compare the time dependent cell survival of the IA cells after each irradiation pass. Second, to search the genes causing PLD repair after irradiation of X-ray or carbon-ion beam, we evaluated gene expressions by using semi-quantitative RT-PCR with the selected 34 genes reportedly related to DNA repair. The intervals from the irradiation were 0, 6, 12 and 24 hr for colony assay method, and 0, 3, 18 hr for RT-PCR method. From the result of survival assays, significant PLD repair was not observed in carbon-ion beam as compared to X-ray irradiation. The results of RT-PCR were as follows. The gene showing significantly higher expressions after X-ray irradiation than after carbon-ion beam irradiation was PCNA. The genes showing significantly lower expressions after X-ray irradiation rather than after carbon-ion beam irradiation were RAD50, BRCA1, MRE11A, XRCC3, CHEK1, MLH1, CCNB1, CCNB2 and LIG4. We conclude that PCNA could be a likely candidate gene for PLD repair. (author)

A novel method for monitoring functional lesion-specific recruitment of repair proteins in live cells

Energy Technology Data Exchange (ETDEWEB)

Woodrick, Jordan; Gupta, Suhani; Khatkar, Pooja; Dave, Kalpana; Levashova, Darya; Choudhury, Sujata; Elias, Hadi; Saha, Tapas; Mueller, Susette; Roy, Rabindra, E-mail: rr228@georgetown.edu

2015-05-15

Highlights: • A method of monitoring lesion-specific recruitment of proteins in vivo is described. • Recruitment of repair enzymes to abasic sites is monitored by co-localization. • Repair protein recruitment is consistent with known protein–protein relationships. • Cells demonstrated complete repair of abasic sites by 90 min. - Abstract: DNA–protein relationships have been studied by numerous methods, but a particular gap in methodology lies in the study of DNA adduct-specific interactions with proteins in vivo, which particularly affects the field of DNA repair. Using the repair of a well-characterized and ubiquitous adduct, the abasic (AP) site, as a model, we have developed a comprehensive method of monitoring DNA lesion-specific recruitment of proteins in vivo over time. We utilized a surrogate system in which a Cy3-labeled plasmid containing a single AP-site was transfected into cells, and the interaction of the labeled DNA with BER enzymes, including APE1, Polβ, LIG1, and FEN1, was monitored by immunofluorescent staining of the enzymes by Alexafluor-488-conjugated secondary antibody. The recruitment of enzymes was characterized by quantification of Cy3-Alexafluor-488 co-localization. To validate the microscopy-based method, repair of the transfected AP-site DNA was also quantified at various time points post-transfection using a real time PCR-based method. Notably, the recruitment time kinetics for each enzyme were consistent with AP-site repair time kinetics. This microscopy-based methodology is reliable in detecting the recruitment of proteins to specific DNA substrates and can be extended to study other in vivo DNA–protein relationships in any DNA sequence and in the context of any DNA structure in transfectable proliferating or quiescent cells. The method may be applied to a variety of disciplines of nucleic acid transaction pathways, including repair, replication, transcription, and recombination.

A novel method for monitoring functional lesion-specific recruitment of repair proteins in live cells

International Nuclear Information System (INIS)

Woodrick, Jordan; Gupta, Suhani; Khatkar, Pooja; Dave, Kalpana; Levashova, Darya; Choudhury, Sujata; Elias, Hadi; Saha, Tapas; Mueller, Susette; Roy, Rabindra

2015-01-01

Highlights: • A method of monitoring lesion-specific recruitment of proteins in vivo is described. • Recruitment of repair enzymes to abasic sites is monitored by co-localization. • Repair protein recruitment is consistent with known protein–protein relationships. • Cells demonstrated complete repair of abasic sites by 90 min. - Abstract: DNA–protein relationships have been studied by numerous methods, but a particular gap in methodology lies in the study of DNA adduct-specific interactions with proteins in vivo, which particularly affects the field of DNA repair. Using the repair of a well-characterized and ubiquitous adduct, the abasic (AP) site, as a model, we have developed a comprehensive method of monitoring DNA lesion-specific recruitment of proteins in vivo over time. We utilized a surrogate system in which a Cy3-labeled plasmid containing a single AP-site was transfected into cells, and the interaction of the labeled DNA with BER enzymes, including APE1, Polβ, LIG1, and FEN1, was monitored by immunofluorescent staining of the enzymes by Alexafluor-488-conjugated secondary antibody. The recruitment of enzymes was characterized by quantification of Cy3-Alexafluor-488 co-localization. To validate the microscopy-based method, repair of the transfected AP-site DNA was also quantified at various time points post-transfection using a real time PCR-based method. Notably, the recruitment time kinetics for each enzyme were consistent with AP-site repair time kinetics. This microscopy-based methodology is reliable in detecting the recruitment of proteins to specific DNA substrates and can be extended to study other in vivo DNA–protein relationships in any DNA sequence and in the context of any DNA structure in transfectable proliferating or quiescent cells. The method may be applied to a variety of disciplines of nucleic acid transaction pathways, including repair, replication, transcription, and recombination

The journey of DNA repair.

Science.gov (United States)

Saini, Natalie

2015-12-01

21 years ago, the DNA Repair Enzyme was declared "Molecule of the Year". Today, we are celebrating another "year of repair", with the 2015 Nobel Prize in Chemistry being awarded to Aziz Sancar, Tomas Lindahl and Paul Modrich for their collective work on the different DNA repair pathways.

Inhibition of topoisomerase II activity in repair-proficient CHO K1 cells by 2-[(aminopropyl)amino]ethanethiol (WR-1065)

International Nuclear Information System (INIS)

Grdina, D.J.; Constantinou, A.; Shigematsu, N.

1992-09-01

The aminothiol 2-[(aminopropyl)amino]ethanethiol (WR-1065) is the active thiol of the clinically studied radioprotective agent S-2-(3-aminopropylamino) ethylphosphorothioic acid (WR-2721). WR-1065 is an effective radiation protector under in vitro conditions when it is administered 30 min prior to radiation exposure at a concentration of 4 mM to repair-proficient Chinese hamster ovary Kl cells (i.e., a dose modification factor of 1.4). In contrast, the DNA double-strand break, repair-deficient Chinese hamster ovary xrs-5 cell line is not protected under these conditions (i.e., a dose modification factor of 1.0). Topoisomerase (topo) I and II activities and protein contents were measured in both Kl and xrs-5 cell lines and were found to be similar in magnitude. Neither exposure to radiation, to WR-1065, or to both affected these variables in xrs-5 cells. WR 1065 was effective, however, in reducing topo 11 activity by a factor of 2 in the repair-proficient Kl cell line. Topo II protein content, however, was not affected by these exposure conditions. One of several mechanisms of radiation protection attributed to aminothiol compounds has been their ability to affect enzymatic reactions involved in DNA synthesis, repair, and cell cycle progression. These results demonstrate a modifying effect by 2-[(aminopropyl)amino]ethanethiol on a specific nuclear enzyme (i.e., type H topoisomerase), which is involved in DNA synthesis. These results also suggest that differences do exist between the topo 11 enzymes isolated from the parent repair-proficient Kl and the DNA double-strand break, repair-deficient xrs-5 mutant cell lines

Inhibition of topoisomerase II activity in repair-proficient CHO K1 cells by 2-[(aminopropyl)amino]ethanethiol (WR-1065)

Energy Technology Data Exchange (ETDEWEB)

Grdina, D.J.; Constantinou, A.; Shigematsu, N.

1992-09-01

The aminothiol 2-[(aminopropyl)amino]ethanethiol (WR-1065) is the active thiol of the clinically studied radioprotective agent S-2-(3-aminopropylamino) ethylphosphorothioic acid (WR-2721). WR-1065 is an effective radiation protector under in vitro conditions when it is administered 30 min prior to radiation exposure at a concentration of 4 mM to repair-proficient Chinese hamster ovary Kl cells (i.e., a dose modification factor of 1.4). In contrast, the DNA double-strand break, repair-deficient Chinese hamster ovary xrs-5 cell line is not protected under these conditions (i.e., a dose modification factor of 1.0). Topoisomerase (topo) I and II activities and protein contents were measured in both Kl and xrs-5 cell lines and were found to be similar in magnitude. Neither exposure to radiation, to WR-1065, or to both affected these variables in xrs-5 cells. WR 1065 was effective, however, in reducing topo 11 activity by a factor of 2 in the repair-proficient Kl cell line. Topo II protein content, however, was not affected by these exposure conditions. One of several mechanisms of radiation protection attributed to aminothiol compounds has been their ability to affect enzymatic reactions involved in DNA synthesis, repair, and cell cycle progression. These results demonstrate a modifying effect by 2-[(aminopropyl)amino]ethanethiol on a specific nuclear enzyme (i.e., type H topoisomerase), which is involved in DNA synthesis. These results also suggest that differences do exist between the topo 11 enzymes isolated from the parent repair-proficient Kl and the DNA double-strand break, repair-deficient xrs-5 mutant cell lines.

Inactivation of the HR6B ubiquitin-conjugating DNA repair enzyme in mice causes male sterility associated with chromatin modification.

NARCIS (Netherlands)

J. van Klaveren; J. de Wit (Jan); C.G. van Gurp; M.H.M. Koken (Marcel); M. Vermey; J.H. van Roijen (Jan Herman); J.T.M. Vreeburg (Jan); W.M. Baarends (Willy); D. Bootsma (Dirk); J.A. Grootegoed (Anton); J.H.J. Hoeijmakers (Jan); H.P. Roest (Henk)

1996-01-01

textabstractThe ubiquitin-conjugating yeast enzyme RAD6 and its human homologs hHR6A and hHR6B are implicated in postreplication repair and damage-induced mutagenesis. The yeast protein is also required for sporulation and may modulate chromatin structure via histone ubiquitination. We report the

Wild-type and mutated IDH1/2 enzymes and therapy responses.

Science.gov (United States)

Molenaar, Remco J; Maciejewski, Jaroslaw P; Wilmink, Johanna W; van Noorden, Cornelis J F

2018-04-01

Isocitrate dehydrogenase 1 and 2 (IDH1/2) are key enzymes in cellular metabolism, epigenetic regulation, redox states, and DNA repair. IDH1/2 mutations are causal in the development and/or progression of various types of cancer due to supraphysiological production of D-2-hydroxyglutarate. In various tumor types, IDH1/2-mutated cancers predict for improved responses to treatment with irradiation or chemotherapy. The present review discusses the molecular basis of the sensitivity of IDH1/2-mutated cancers with respect to the function of mutated IDH1/2 in cellular processes and their interactions with novel IDH1/2-mutant inhibitors. Finally, lessons learned from IDH1/2 mutations for future clinical applications in IDH1/2 wild-type cancers are discussed.

Oncometabolite D-2-Hydroxyglutarate Inhibits ALKBH DNA Repair Enzymes and Sensitizes IDH Mutant Cells to Alkylating Agents.

Science.gov (United States)

Wang, Pu; Wu, Jing; Ma, Shenghong; Zhang, Lei; Yao, Jun; Hoadley, Katherine A; Wilkerson, Matthew D; Perou, Charles M; Guan, Kun-Liang; Ye, Dan; Xiong, Yue

2015-12-22

Chemotherapy of a combination of DNA alkylating agents, procarbazine and lomustine (CCNU), and a microtubule poison, vincristine, offers a significant benefit to a subset of glioma patients. The benefit of this regimen, known as PCV, was recently linked to IDH mutation that occurs frequently in glioma and produces D-2-hydroxyglutarate (D-2-HG), a competitive inhibitor of α-ketoglutarate (α-KG). We report here that D-2-HG inhibits the α-KG-dependent alkB homolog (ALKBH) DNA repair enzymes. Cells expressing mutant IDH display reduced repair kinetics, accumulate more DNA damages, and are sensitized to alkylating agents. The observed sensitization to alkylating agents requires the catalytic activity of mutant IDH to produce D-2-HG and can be reversed by the deletion of mutant IDH allele or overexpression of ALKBH2 or AKLBH3. Our results suggest that impairment of DNA repair may contribute to tumorigenesis driven by IDH mutations and that alkylating agents may merit exploration for treating IDH-mutated cancer patients. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Putative Enzymes of UV Photoproduct Repair

Directory of Open Access Journals (Sweden)

Cynthia J. Sakofsky

2011-01-01

Full Text Available In order to determine the biological relevance of two S. acidocaldarius proteins to the repair of UV photoproducts, the corresponding genes (Saci_1227 and Saci_1096 were disrupted, and the phenotypes of the resulting mutants were examined by various genetic assays. The disruption used integration by homologous recombination of a functional but heterologous pyrE gene, promoted by short sequences attached to both ends via PCR. The phenotypic analyses of the disruptants confirmed that ORF Saci_1227 encodes a DNA photolyase which functions in vivo, but they could not implicate ORF Saci_1096 in repair of UV- or other externally induced DNA damage despite its similarity to genes encoding UV damage endonucleases. The success of the gene-disruption strategy, which used 5′ extensions of PCR primers to target cassette integration, suggests potential advantages for routine construction of Sulfolobus strains.

Deficient expression of DNA repair enzymes in early progression to sporadic colon cancer

Science.gov (United States)

2012-01-01

Background Cancers often arise within an area of cells (e.g. an epithelial patch) that is predisposed to the development of cancer, i.e. a "field of cancerization" or "field defect." Sporadic colon cancer is characterized by an elevated mutation rate and genomic instability. If a field defect were deficient in DNA repair, DNA damages would tend to escape repair and give rise to carcinogenic mutations. Purpose To determine whether reduced expression of DNA repair proteins Pms2, Ercc1 and Xpf (pairing partner of Ercc1) are early steps in progression to colon cancer. Results Tissue biopsies were taken during colonoscopies of 77 patients at 4 different risk levels for colon cancer, including 19 patients who had never had colonic neoplasia (who served as controls). In addition, 158 tissue samples were taken from tissues near or within colon cancers removed by resection and 16 tissue samples were taken near tubulovillous adenomas (TVAs) removed by resection. 568 triplicate tissue sections (a total of 1,704 tissue sections) from these tissue samples were evaluated by immunohistochemistry for 4 DNA repair proteins. Substantially reduced protein expression of Pms2, Ercc1 and Xpf occurred in field defects of up to 10 cm longitudinally distant from colon cancers or TVAs and within colon cancers. Expression of another DNA repair protein, Ku86, was infrequently reduced in these areas. When Pms2, Ercc1 or Xpf were reduced in protein expression, then either one or both of the other two proteins most often had reduced protein expression as well. The mean inner colon circumferences, from 32 resections, of the ascending, transverse and descending/sigmoid areas were measured as 6.6 cm, 5.8 cm and 6.3 cm, respectively. When combined with other measurements in the literature, this indicates the approximate mean number of colonic crypts in humans is 10 million. Conclusions The substantial deficiencies in protein expression of DNA repair proteins Pms2, Ercc1 and Xpf in about 1 million

Studies on DNA repair in Bacillus subtilis

International Nuclear Information System (INIS)

Inoue, Tadashi; Kada, Tsuneo

1977-01-01

An enzyme which enhances the priming activity of γ-irradiated DNA for type I DNA polymerase (EC 2.7.7.7) was identified and partially purified from extracts of Bacillus subtilis cells. The enzyme preferentially degraded γ-irradiated DNA into acid-soluble materials. DNA preparations treated with heat, ultraviolet light, pancreatic DNAase (EC 3.1.4.5) or micrococcal DNAase (EC 3.1.4.7) were not susceptible to the enzyme. However, sonication rendered DNA susceptible to the enzyme to some extent. From these results, it is supposed that this enzyme may function by 'cleaning' damaged terminals produced by γ-irradiation to serve as effective primer of sites for repair synthesis by the type I DNA polymerase

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 repair of oxidized modifications both in nuclear and mitochondrial DNA. In order to ascertain whether diminished BER capacity might account for increased levels of oxidative DNA damage in cancer cells, the activities of BER enzymes in three different lung cancer cell lines and their non......-cancerous counterparts were measured using oligonucleotide substrates with single DNA lesions to assess specific BER enzymes. The activities of four BER enzymes, OGG1, NTH1, UDG and APE1, were compared in mitochondrial and nuclear extracts. For each specific lesion, the repair activities were similar among the three...

Radiosensitivity and genes

Energy Technology Data Exchange (ETDEWEB)

Qiyue, Hu; Mingyue, Lun [Suzhou Medical Coll., JS (China)

1995-07-01

Reported effects of some oncogenes, tumour suppressor genes and DNA repair genes on sensitivity of cells to ionizing radiation are reviewed. The role of oncogenes in cellular response to irradiation is discussed, especially the extensively studied oncogenes such as the ras gene family. For tumour suppressor genes, mainly the p53, which is increasingly implicated as a gene affecting radiosensitivity, is reviewed. It is considered that there is a cell cycle checkpoint determinant which is postulated to be able to arrest the irradiated cells in G{sub 1} phase to allow them to repair damage before they undergo DNA synthesis. So far there are six DNA repair genes which have been cloned in mammalian cells, but only one, XRCC1, appears to be involved in repair of human X-ray damage. XRCC1 can correct high sisterchromatid exchange levels when transferred into EM{sub 9} cells, but its expression seems to have no correlation with radiosensitivity of human neck and head tumour cells. Radiosensitivity is an intricate issue which may involve many factors. A scheme of cellular reactions after exposure to irradiation is proposed to indicate a possible sequence of events initiated by ionizing radiation.

Radiosensitivity and genes

International Nuclear Information System (INIS)

Hu Qiyue; Lun Mingyue

1995-07-01

Reported effects of some oncogenes, tumour suppressor genes and DNA repair genes on sensitivity of cells to ionizing radiation are reviewed. The role of oncogenes in cellular response to irradiation is discussed, especially the extensively studied oncogenes such as the ras gene family. For tumour suppressor genes, mainly the p53, which is increasingly implicated as a gene affecting radiosensitivity, is reviewed. It is considered that there is a cell cycle checkpoint determinant which is postulated to be able to arrest the irradiated cells in G 1 phase to allow them to repair damage before they undergo DNA synthesis. So far there are six DNA repair genes which have been cloned in mammalian cells, but only one, XRCC1, appears to be involved in repair of human X-ray damage. XRCC1 can correct high sisterchromatid exchange levels when transferred into EM 9 cells, but its expression seems to have no correlation with radiosensitivity of human neck and head tumour cells. Radiosensitivity is an intricate issue which may involve many factors. A scheme of cellular reactions after exposure to irradiation is proposed to indicate a possible sequence of events initiated by ionizing radiation

Role of isolated and clustered DNA damage and the post-irradiating repair process in the effects of heavy ion beam irradiation

International Nuclear Information System (INIS)

Tokuyama, Yuka; Terato, Hiroaki; Furusawa, Yoshiya; Ide, Hiroshi; Yasui, Akira

2015-01-01

Clustered DNA damage is a specific type of DNA damage induced by ionizing radiation. Any type of ionizing radiation traverses the target DNA molecule as a beam, inducing damage along its track. Our previous study showed that clustered DNA damage yields decreased with increased linear energy transfer (LET), leading us to investigate the importance of clustered DNA damage in the biological effects of heavy ion beam radiation. In this study, we analyzed the yield of clustered base damage (comprising multiple base lesions) in cultured cells irradiated with various heavy ion beams, and investigated isolated base damage and the repair process in post-irradiation cultured cells. Chinese hamster ovary (CHO) cells were irradiated by carbon, silicon, argon and iron ion beams with LETs of 13, 55, 90 and 200 keV µm -1 , respectively. Agarose gel electrophoresis of the cells with enzymatic treatments indicated that clustered base damage yields decreased as the LET increased. The aldehyde reactive probe procedure showed that isolated base damage yields in the irradiated cells followed the same pattern. To analyze the cellular base damage process, clustered DNA damage repair was investigated using DNA repair mutant cells. DNA double-strand breaks accumulated in CHO mutant cells lacking Xrcc1 after irradiation, and the cell viability decreased. On the other hand, mouse embryonic fibroblast (Mef) cells lacking both Nth1 and Ogg1 became more resistant than the wild type Mef. Thus, clustered base damage seems to be involved in the expression of heavy ion beam biological effects via the repair process. (author)

Metabolite damage and repair in metabolic engineering design

Energy Technology Data Exchange (ETDEWEB)

Sun, Jiayi; Jeffryes, James G.; Henry, Christopher S.; Bruner, Steven D.; Hanson, Andrew D.

2017-11-01

The necessarily sharp focus of metabolic engineering and metabolic synthetic biology on pathways and their fluxes has tended to divert attention from the damaging enzymatic and chemical side-reactions that pathway metabolites can undergo. Although historically overlooked and underappreciated, such metabolite damage reactions are now known to occur throughout metabolism and to generate (formerly enigmatic) peaks detected in metabolomics datasets. It is also now known that metabolite damage is often countered by dedicated repair enzymes that undo or prevent it. Metabolite damage and repair are highly relevant to engineered pathway design: metabolite damage reactions can reduce flux rates and product yields, and repair enzymes can provide robust, host-independent solutions. Herein, after introducing the core principles of metabolite damage and repair, we use case histories to document how damage and repair processes affect efficient operation of engineered pathways - particularly those that are heterologous, non-natural, or cell-free. We then review how metabolite damage reactions can be predicted, how repair reactions can be prospected, and how metabolite damage and repair can be built into genome-scale metabolic models. Lastly, we propose a versatile 'plug and play' set of well-characterized metabolite repair enzymes to solve metabolite damage problems known or likely to occur in metabolic engineering and synthetic biology projects.

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

International Nuclear Information System (INIS)

Kim, Hyun-Suk; Guo, Chunlu; Thompson, Eric L.; Jiang, Yanlin; Kelley, Mark R.; Vasko, Michael R.; Lee, Suk-Hee

2015-01-01

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

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.

A hypothesis: factor VII governs clot formation, tissue repair and apoptosis.

Science.gov (United States)

Coleman, Lewis S

2007-01-01

A hypothesis: thrombin is a "Universal Enzyme of Energy Transduction" that employs ATP energy in flowing blood to activate biochemical reactions and cell effects in both hemostasis and tissue repair. All cells possess PAR-1 (thrombin) receptors and are affected by thrombin elevations, and thrombin effects on individual cell types are determined by their unique complement of PAR-1 receptors. Disruption of the vascular endothelium (VE) activates a tissue repair mechanism (TRM) consisting of the VE, tissue factor (TF), and circulating Factors VII, IX and X that governs localized thrombin elevations to activate clot formation and cellular effects that repair tissue damage. The culmination of the repair process occurs with the restoration of the VE followed by declines in thrombin production that causes Apoptosis ("programmed cell death") in wound-healing fibroblasts, which functions as a mechanism to draw wound edges together. The location and magnitude of TRM activity governs the location and magnitude of Factor VIII activity and clot formation, but the large size of Factor VIII prevents it from penetrating the clot formed by its activity, so that its effects are self-limiting. Factors VII, IX and X function primarily as tissue repair enzymes, while Factor VIII and Factor XIII are the only serine protease enzymes in the "Coagulation Cascade" that are exclusively associated with hemostasis.

Single nucleotide polymorphisms and unacceptable late toxicity in breast cancer adjuvant radiotherapy: a case report

Directory of Open Access Journals (Sweden)

Lazzari G

2017-05-01

Full Text Available Grazia Lazzari,1 Maria Iole Natalicchio,2 Angela Terlizzi,3 Francesco Perri,4 Giovanni Silvano1 1Radiation Oncology Unit, San Giuseppe Moscati Hospital, Taranto, 2Molecular Biology Laboratory, Pathological Anatomy Department, Ospedali Riuniti, Foggia, 3Medical Physic Unit, San Giuseppe Moscati Hospital, 4Medical Oncology Unit, Presidio Ospedaliero Centrale - Santissima Annunziata, Taranto, Italy Background: There has recently been a strong interest in the inter-individual variation in normal tissue and tumor response to radiotherapy (RT, because tissue radiosensitivity seems to be under genetic control. Evidence is accumulating on the role of polymorphic genetic variants, such as single nucleotide polymorphisms (SNPs that could influence normal tissue response after radiation. The most studied SNPs include those in genes involved in DNA repair (single- and double-strand breaks, and base excision and those active in the response to oxidative stress.Case report: We present the case report of a 60-year-old woman with early breast cancer who underwent adjuvant hormone therapy and conventional radiotherapy, and subsequently developed unacceptable cosmetic toxicities of the irradiated breast requiring a genetic test of genes involved in DNA repair mechanisms. The patient was found to be heterozygous for G28152A (T/C and C18067T (A/G mutations in X-ray repair cross-complementing group 1 (XRCC1 and 3 (XRCC3, respectively, homozygous for A313G (G/G mutation in glutathione S transferase Pi 1 (GSTP1, and wild-type for A4541G (A/A in XRCC3 and G135C (G/G in RAD51 recombinase.Conclusion: The role of SNPs should be taken into account when a severe phenomenon appears in normal tissues after radiation treatment, because understanding the molecular basis of individual radiosensitivity may be useful for identifying moderately or extremely radiosensitive patients who may need tailored therapeutic strategies. Keywords: radiosensitivity, SNPs, fibrosis, DNA repair

Influence of the environment and phototoxicity of the live cell imaging system at IMP microbeam facility

Science.gov (United States)

Liu, Wenjing; Du, Guanghua; Guo, Jinlong; Wu, Ruqun; Wei, Junzhe; Chen, Hao; Li, Yaning; Zhao, Jing; Li, Xiaoyue

2017-08-01

To investigate the spatiotemporal dynamics of DNA damage and repair after the ion irradiation, an online live cell imaging system has been established based on the microbeam facility at Institute of Modern Physics (IMP). The system could provide a sterile and physiological environment by making use of heating plate and live cell imaging solution. The phototoxicity was investigated through the evaluation of DNA repair protein XRCC1 foci formed in HT1080-RFP cells during the imaging exposure. The intensity of the foci induced by phototoxicity was much lower compared with that of the foci induced by heavy ion hits. The results showed that although spontaneous foci were formed due to RFP exposure during live cell imaging, they had little impact on the analysis of the recruitment kinetics of XRCC1 in the foci induced by the ion irradiation.

Radiobiological significance of DNA repair

International Nuclear Information System (INIS)

Kuzin, A.M.

1978-01-01

A short outline is given on the history of the problem relating to the repair of radiation injuries, specifically its molecular mechanisms. The most urgent problems which currently confront the researchers are noted. This is a further study on the role of DNA repair in post-radiation recovery, search for ways to activate and suppress DNA repair, investigations into the activity balance of various repair enzymes as well as the problem of errors in the structure of repairing DNA. An important role is attached to the investigations of DNA repair in solving a number of practical problems

Metabolite damage and repair in metabolic engineering design.

Science.gov (United States)

Sun, Jiayi; Jeffryes, James G; Henry, Christopher S; Bruner, Steven D; Hanson, Andrew D

2017-11-01

The necessarily sharp focus of metabolic engineering and metabolic synthetic biology on pathways and their fluxes has tended to divert attention from the damaging enzymatic and chemical side-reactions that pathway metabolites can undergo. Although historically overlooked and underappreciated, such metabolite damage reactions are now known to occur throughout metabolism and to generate (formerly enigmatic) peaks detected in metabolomics datasets. It is also now known that metabolite damage is often countered by dedicated repair enzymes that undo or prevent it. Metabolite damage and repair are highly relevant to engineered pathway design: metabolite damage reactions can reduce flux rates and product yields, and repair enzymes can provide robust, host-independent solutions. Herein, after introducing the core principles of metabolite damage and repair, we use case histories to document how damage and repair processes affect efficient operation of engineered pathways - particularly those that are heterologous, non-natural, or cell-free. We then review how metabolite damage reactions can be predicted, how repair reactions can be prospected, and how metabolite damage and repair can be built into genome-scale metabolic models. Lastly, we propose a versatile 'plug and play' set of well-characterized metabolite repair enzymes to solve metabolite damage problems known or likely to occur in metabolic engineering and synthetic biology projects. Copyright © 2017 International Metabolic Engineering Society. All rights reserved.

Feedback-regulated poly(ADP-ribosyl)ation by PARP-1 is required for rapid response to DNA damage in living cells

Science.gov (United States)

Mortusewicz, Oliver; Amé, Jean-Christophe; Leonhardt, Heinrich

2007-01-01

Genome integrity is constantly threatened by DNA lesions arising from numerous exogenous and endogenous sources. Survival depends on immediate recognition of these lesions and rapid recruitment of repair factors. Using laser microirradiation and live cell microscopy we found that the DNA-damage dependent poly(ADP-ribose) polymerases (PARP) PARP-1 and PARP-2 are recruited to DNA damage sites, however, with different kinetics and roles. With specific PARP inhibitors and mutations, we could show that the initial recruitment of PARP-1 is mediated by the DNA-binding domain. PARP-1 activation and localized poly(ADP-ribose) synthesis then generates binding sites for a second wave of PARP-1 recruitment and for the rapid accumulation of the loading platform XRCC1 at repair sites. Further PARP-1 poly(ADP-ribosyl)ation eventually initiates the release of PARP-1. We conclude that feedback regulated recruitment of PARP-1 and concomitant local poly(ADP-ribosyl)ation at DNA lesions amplifies a signal for rapid recruitment of repair factors enabling efficient restoration of genome integrity. PMID:17982172

DNA damage and repair activity after broccoli intake in young healthy smokers

DEFF Research Database (Denmark)

Riso, Patrizia; Martini, Daniela; Møller, Peter

2010-01-01

compounds, including smokers. The aim of the study was to evaluate the effect of broccoli intake on biomarkers of DNA damage and repair. Twenty-seven young healthy smokers consumed a portion of steamed broccoli (250 g/day) or a control diet for 10 days each within a crossover design with a washout period...... mRNA expression levels of repair and defence enzymes: 8-oxoguanine DNA glycosylase (OGG1), nucleoside diphosphate linked moiety X-type motif 1 (NUDT1) and heme oxygenase 1 (HO-1). After broccoli consumption, the level of oxidised DNA lesions decreased by 41% (95% confidence interval: 10%, 72......%) and the resistance to H(2)O(2)-induced DNA strand breaks increased by 23% (95% CI: 13%, 34%). Following broccoli intake, a higher protection was observed in subjects with glutathione S-transferase (GST) M1-null genotype. The expression level and activity of repair enzymes was unaltered. In conclusion, broccoli...

Conformational Analysis of DNA Repair Intermediates by Time-Resolved Fluorescence Spectroscopy

OpenAIRE

Lin, Su; Horning, David P.; Szostak, Jack W.; Chaput, John C.

2009-01-01

DNA repair enzymes are essential for maintaining the integrity of the DNA sequence. Unfortunately, very little is known about how these enzymes recognize damaged regions along the helix. Structural analysis of cellular repair enzymes bound to DNA reveals that these enzymes are able to recognize DNA in a variety of conformations. However, the prevalence of these deformations in the absence of enzymes remains unclear, as small populations of DNA conformations are often difficult to detect by NM...

Co-expression of antioxidant enzymes with expression of p53, DNA repair, and heat shock protein genes in the gamma ray-irradiated hermaphroditic fish Kryptolebias marmoratus larvae

Energy Technology Data Exchange (ETDEWEB)

Rhee, Jae-Sung [Research Institute for Natural Sciences, Hanyang University, Seoul 133-791 (Korea, Republic of); Kim, Bo-Mi; Kim, Ryeo-Ok [Department of Chemistry, College of Natural Sciences, Hanyang University, Seoul 133-791 (Korea, Republic of); Seo, Jung Soo [Pathology Team, National Fisheries Research and Development Institute, Busan 619-902 (Korea, Republic of); Kim, Il-Chan [Division of Life Sciences, Korea Polar Research Institute, Korea Institute of Ocean Science and Technology, Incheon 406-840 (Korea, Republic of); Lee, Young-Mi, E-mail: ymlee70@smu.ac.kr [Department of Green Life Science, College of Convergence, Sangmyung University, Seoul 110-743 (Korea, Republic of); Lee, Jae-Seong, E-mail: jslee2@hanyang.ac.kr [Research Institute for Natural Sciences, Hanyang University, Seoul 133-791 (Korea, Republic of); Department of Chemistry, College of Natural Sciences, Hanyang University, Seoul 133-791 (Korea, Republic of)

2013-09-15

Highlights: •Novel identification of DNA repair-related genes in fish. •Investigation of whole expression profiling of DNA repair genes upon gamma radiation. •Analysis of effects of gamma radiation on antioxidant system and cell stress proteins. •Usefulness of verification of pathway-based profiling for mechanistic understanding. -- Abstract: To investigate effects of gamma ray irradiation in the hermaphroditic fish, Kryptolebias marmoratus larvae, we checked expression of p53, DNA repair, and heat shock protein genes with several antioxidant enzyme activities by quantitative real-time RT-PCR and biochemical methods in response to different doses of gamma radiation. As a result, the level of gamma radiation-induced DNA damage was initiated after 4 Gy of radiation, and biochemical and molecular damage became substantial from 8 Gy. In particular, several DNA repair mechanism-related genes were significantly modulated in the 6 Gy gamma radiation-exposed fish larvae, suggesting that upregulation of such DNA repair genes was closely associated with cell survival after gamma irradiation. The mRNA expression of p53 and most hsps was also significantly upregulated at high doses of gamma radiation related to cellular damage. This finding indicates that gamma radiation can induce oxidative stress with associated antioxidant enzyme activities, and linked to modulation of the expression of DNA repair-related genes as one of the defense mechanisms against radiation damage. This study provides a better understanding of the molecular mode of action of defense mechanisms upon gamma radiation in fish larvae.

A data mining approach for classifying DNA repair genes into ageing-related or non-ageing-related

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Vasieva Olga

2011-01-01

Full Text Available Abstract Background The ageing of the worldwide population means there is a growing need for research on the biology of ageing. DNA damage is likely a key contributor to the ageing process and elucidating the role of different DNA repair systems in ageing is of great interest. In this paper we propose a data mining approach, based on classification methods (decision trees and Naive Bayes, for analysing data about human DNA repair genes. The goal is to build classification models that allow us to discriminate between ageing-related and non-ageing-related DNA repair genes, in order to better understand their different properties. Results The main patterns discovered by the classification methods are as follows: (a the number of protein-protein interactions was a predictor of DNA repair proteins being ageing-related; (b the use of predictor attributes based on protein-protein interactions considerably increased predictive accuracy of attributes based on Gene Ontology (GO annotations; (c GO terms related to "response to stimulus" seem reasonably good predictors of ageing-relatedness for DNA repair genes; (d interaction with the XRCC5 (Ku80 protein is a strong predictor of ageing-relatedness for DNA repair genes; and (e DNA repair genes with a high expression in T lymphocytes are more likely to be ageing-related. Conclusions The above patterns are broadly integrated in an analysis discussing relations between Ku, the non-homologous end joining DNA repair pathway, ageing and lymphocyte development. These patterns and their analysis support non-homologous end joining double strand break repair as central to the ageing-relatedness of DNA repair genes. Our work also showcases the use of protein interaction partners to improve accuracy in data mining methods and our approach could be applied to other ageing-related pathways.

A history of the DNA repair and mutagenesis field: The discovery of base excision repair.

Science.gov (United States)

Friedberg, Errol C

2016-01-01

This article reviews the early history of the discovery of an DNA repair pathway designated as base excision repair (BER), since in contrast to the enzyme-catalyzed removal of damaged bases from DNA as nucleotides [called nucleotide excision repair (NER)], BER involves the removal of damaged or inappropriate bases, such as the presence of uracil instead of thymine, from DNA as free bases. Copyright © 2015. Published by Elsevier B.V.

The journey of DNA repair

OpenAIRE

Saini, Natalie

2015-01-01

21 years ago, the DNA Repair Enzyme was declared “Molecule of the Year”. Today, we are celebrating another “year of repair”, with the 2015 Nobel Prize in Chemistry being awarded to Aziz Sancar, Tomas Lindahl and Paul Modrich for their collective work on the different DNA repair pathways.

The CNA-1 (Nuclear Power Plant Atucha-1) QK-01 repairing project

International Nuclear Information System (INIS)

Pizzaferri, J.C.; Cabot, P.

1997-01-01

The repair/maintenance of the CNA-1 QK-01 Moderator Cooler will be a leading case of the repair of a class 1 nuclear component in a high radiation environment; utilizing for the work, sophisticated remotely operated equipment. This paper describes the component, the repair-maintenance objective, and the equipment-procedures developed for the intervention. (author) [es

Polymorphic trial in oxidative damage of arsenic exposed Vietnamese

International Nuclear Information System (INIS)

Fujihara, Junko; Soejima, Mikiko; Yasuda, Toshihiro; Koda, Yoshiro; Kunito, Takashi; Iwata, Hisato; Tanabe, Shinsuke; Takeshita, Haruo

2011-01-01

Arsenic causes DNA damage and changes the cellular capacity for DNA repair. Genes in the base excision repair (BER) pathway influence the generation and repair of oxidative lesions. Single nucleotide polymorphisms (SNPs) in human 8-oxoguanine DNA glycosylase (hOGG1) Ser326Cys; apurinic/apyrimidinic endonuclease (APE1) Asp148Glu; X-ray and repair and cross-complementing group 1 (XRCC1) Arg280His and Arg399Gln in the BER genes were analyzed, and the relationship between these 4 SNPs and the urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) concentrations of 100 Vietnamese population exposed to arsenic was investigated. Individuals with hOGG1 326Cys/Cys showed significantly higher urinary 8-OHdG concentrations than did those with 326 Ser/Cys and Ser/Ser. As for APE1 Asp148Glu, heterozygous subjects showed significantly higher urinary 8-OHdG concentrations than did those homozygous for Asp/Asp. Moreover, global ethnic comparison of the allelic frequencies of the 4SNPs was performed in 10 population and previous reported data. The mutant allele frequencies of hOGG1 Ser326Cys in the Asian populations were higher than those in the African and Caucasian populations. As for APE1 Asp148Glu, Caucasians showed higher mutant frequencies than those shown by African and Asian populations. Among Asian populations, the Bangladeshi population showed relatively higher mutant allele frequencies of the APE1 Asp148Glu polymorphism. This study is the first to demonstrate the existence of genetic heterogeneity in a worldwide distribution of SNPs (hOGG1 Ser326Cys, APE1 Asp148Glu, XRCC1 Arg280His, and XRCC1 Arg399Gln) in the BER genes. - Highlights: → We showed that hOGG1 and APE1 are associated with urinary 8-OHdG concentrations. → We showed the existence of inter-ethnic differences in hOGG1 and APE1 polymorphism. → These polymorphisms is a genetic marker of susceptibility to oxidative stress.

Aspects of DNA repair and nucleotide pool imbalance

Energy Technology Data Exchange (ETDEWEB)

Holliday, R.

1985-01-01

Evidence that optimum repair depends on adequate pools of deoxynucleotide triphosphates (dNTPs) comes from the study of pyrimidine auxotrophs of Ustilago maydis. These strains are sensitive to UV light and X-rays, and for pyr1-1 it has been shown that the intracellular concentration of dTTP is reduced about 7-fold. The survival curve of pyr1-1 after UV-treatment, and split dose experiments with wild-type cells, provide evidence for an inducible repair mechanism, which probably depends on genetic recombination. Although inducible repair saves cellular resources, it has the disadvantage of becoming ineffective at doses which are high enough to inactivate the repressed structural gene(s) for repair enzymes. It is clear that a wide variety of repair mechanisms have evolved to remove lesions which arise either spontaneously or as a result of damage from external agents. Nevertheless, it would be incorrect to assume that all species require all possible pathways of repair. It is now well established that the accuracy of DNA and protein synthesis depends on proof-reading or editing mechanisms. Optimum accuracy levels will evolve from the balance between error avoidance in macromolecular synthesis and physiological efficiency in growth and propagation.

Differential repair of etheno-DNA adducts by bacterial and human AlkB proteins.

Science.gov (United States)

Zdżalik, Daria; Domańska, Anna; Prorok, Paulina; Kosicki, Konrad; van den Born, Erwin; Falnes, Pål Ø; Rizzo, Carmelo J; Guengerich, F Peter; Tudek, Barbara

2015-06-01

AlkB proteins are evolutionary conserved Fe(II)/2-oxoglutarate-dependent dioxygenases, which remove alkyl and highly promutagenic etheno(ɛ)-DNA adducts, but their substrate specificity has not been fully determined. We developed a novel assay for the repair of ɛ-adducts by AlkB enzymes using oligodeoxynucleotides with a single lesion and specific DNA glycosylases and AP-endonuclease for identification of the repair products. We compared the repair of three ɛ-adducts, 1,N(6)-ethenoadenine (ɛA), 3,N(4)-ethenocytosine (ɛC) and 1,N(2)-ethenoguanine (1,N(2)-ɛG) by nine bacterial and two human AlkBs, representing four different structural groups defined on the basis of conserved amino acids in the nucleotide recognition lid, engaged in the enzyme binding to the substrate. Two bacterial AlkB proteins, MT-2B (from Mycobacterium tuberculosis) and SC-2B (Streptomyces coelicolor) did not repair these lesions in either double-stranded (ds) or single-stranded (ss) DNA. Three proteins, RE-2A (Rhizobium etli), SA-2B (Streptomyces avermitilis), and XC-2B (Xanthomonas campestris) efficiently removed all three lesions from the DNA substrates. Interestingly, XC-2B and RE-2A are the first AlkB proteins shown to be specialized for ɛ-adducts, since they do not repair methylated bases. Three other proteins, EcAlkB (Escherichia coli), SA-1A, and XC-1B removed ɛA and ɛC from ds and ssDNA but were inactive toward 1,N(2)-ɛG. SC-1A repaired only ɛA with the preference for dsDNA. The human enzyme ALKBH2 repaired all three ɛ-adducts in dsDNA, while only ɛA and ɛC in ssDNA and repair was less efficient in ssDNA. ALKBH3 repaired only ɛC in ssDNA. Altogether, we have shown for the first time that some AlkB proteins, namely ALKBH2, RE-2A, SA-2B and XC-2B can repair 1,N(2)-ɛG and that ALKBH3 removes only ɛC from ssDNA. Our results also suggest that the nucleotide recognition lid is not the sole determinant of the substrate specificity of AlkB proteins. Copyright © 2015 Elsevier B

Acute Normal Tissue Reactions in Head-and-Neck Cancer Patients Treated With IMRT: Influence of Dose and Association With Genetic Polymorphisms in DNA DSB Repair Genes

International Nuclear Information System (INIS)

Werbrouck, Joke; Ruyck, Kim de; Duprez, Frederic; Veldeman, Liv; Claes, Kathleen; Eijkeren, Marc van; Boterberg, Tom; Willems, Petra; Vral, Anne; Neve, Wilfried de; Thierens, Hubert

2009-01-01

Purpose: To investigate the association between dose-related parameters and polymorphisms in DNA DSB repair genes XRCC3 (c.-1843A>G, c.562-14A>G, c.722C>T), Rad51 (c.-3429G>C, c.-3392G>T), Lig4 (c.26C>T, c.1704T>C), Ku70 (c.-1310C>G), and Ku80 (c.2110-2408G>A) and the occurrence of acute reactions after radiotherapy. Materials and Methods: The study population consisted of 88 intensity-modulated radiation therapy (IMRT)-treated head-and-neck cancer patients. Mucositis, dermatitis, and dysphagia were scored using the Common Terminology Criteria (CTC) for Adverse Events v.3.0 scale. The population was divided into a CTC0-2 and CTC3+ group for the analysis of each acute effect. The influence of the dose on critical structures was analyzed using dose-volume histograms. Genotypes were determined by polymerase chain reaction (PCR) combined with restriction fragment length polymorphism or PCR-single base extension assays. Results: The mean dose (D mean ) to the oral cavity and constrictor pharyngeus (PC) muscles was significantly associated with the development of mucositis and dysphagia, respectively. These parameters were considered confounding factors in the radiogenomics analyses. The XRCC3c.722CT/TT and Ku70c.-1310CG/GG genotypes were significantly associated with the development of severe dysphagia (CTC3+). No association was found between the investigated polymorphisms and the development of mucositis or dermatitis. A risk analysis model for severe dysphagia, which was developed based on the XRCC3c.722CT/TT and Ku70c.-1310CG/GG genotypes and the PC dose, showed a sensitivity of 78.6% and a specificity of 77.6%. Conclusions: The XRCC3c.722C>T and Ku70c.-1310C>G polymorphisms as well as the D mean to the PC muscles were highly associated with the development of severe dysphagia after IMRT. The prediction model developed using these parameters showed a high sensitivity and specificity

Protozoan ALKBH8 Oxygenases Display both DNA Repair and tRNA Modification Activities

DEFF Research Database (Denmark)

Zdżalik, Daria; Vågbø, Cathrine B; Kirpekar, Finn

2014-01-01

The ALKBH family of Fe(II) and 2-oxoglutarate dependent oxygenases comprises enzymes that display sequence homology to AlkB from E. coli, a DNA repair enzyme that uses an oxidative mechanism to dealkylate methyl and etheno adducts on the nucleobases. Humans have nine different ALKBH proteins, ALKBH......1-8 and FTO. Mammalian and plant ALKBH8 are tRNA hydroxylases targeting 5-methoxycarbonylmethyl-modified uridine (mcm5U) at the wobble position of tRNAGly(UCC). In contrast, the genomes of some bacteria encode a protein with strong sequence homology to ALKBH8, and robust DNA repair activity...... was previously demonstrated for one such protein. To further explore this apparent functional duality of the ALKBH8 proteins, we have here enzymatically characterized a panel of such proteins, originating from bacteria, protozoa and mimivirus. All the enzymes showed DNA repair activity in vitro, but...

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

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David G. Johnson

2012-10-01

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

New paradigms in the repair of oxidative damage in human genome: mechanisms ensuring repair of mutagenic base lesions during replication and involvement of accessory proteins.

Science.gov (United States)

Dutta, Arijit; Yang, Chunying; Sengupta, Shiladitya; Mitra, Sankar; Hegde, Muralidhar L

2015-05-01

Oxidized bases in the mammalian genome, which are invariably mutagenic due to their mispairing property, are continuously induced by endogenous reactive oxygen species and more abundantly after oxidative stress. Unlike bulky base adducts induced by UV and other environmental mutagens in the genome that block replicative DNA polymerases, oxidatively damaged bases such as 5-hydroxyuracil, produced by oxidative deamination of cytosine in the template strand, do not block replicative polymerases and thus need to be repaired prior to replication to prevent mutation. Following up our earlier studies, which showed that the Nei endonuclease VIII like 1 (NEIL1) DNA glycosylase, one of the five base excision repair (BER)-initiating enzymes in mammalian cells, has enhanced expression during the S-phase and higher affinity for replication fork-mimicking single-stranded (ss) DNA substrates, we recently provided direct experimental evidence for NEIL1's role in replicating template strand repair. The key requirement for this event, which we named as the 'cow-catcher' mechanism of pre-replicative BER, is NEIL1's non-productive binding (substrate binding without product formation) to the lesion base in ss DNA template to stall DNA synthesis, causing fork regression. Repair of the lesion in reannealed duplex is then carried out by NEIL1 in association with the DNA replication proteins. NEIL1 (and other BER-initiating enzymes) also interact with several accessory and non-canonical proteins including the heterogeneous nuclear ribonucleoprotein U and Y-box-binding protein 1 as well as high mobility group box 1 protein, whose precise roles in BER are still obscure. In this review, we have discussed the recent advances in our understanding of oxidative genome damage repair pathways with particular focus on the pre-replicative template strand repair and the role of scaffold factors like X-ray repairs cross-complementing protein 1 and poly (ADP-ribose) polymerase 1 and other accessory

In normal human fibroblasts variation in DSB repair capacity cannot be ascribed to radiation-induced changes in the localisation, expression or activity of major NHEJ proteins

DEFF Research Database (Denmark)

Kasten-Pisula, Ulla; Vronskaja, Svetlana; Overgaard, Jens

2008-01-01

in the activity of the DNA-PK complex induced upon irradiation. CONCLUSIONS: For normal human fibroblasts, the level or activity of NHEJ proteins measured prior to or after irradiation cannot be used to predict the DSB repair capacity or cellular radiosensitivity. Udgivelsesdato: 2008-Mar......BACKGROUND AND PURPOSE: The aim of the present study was to test whether for normal human fibroblasts the variation in double-strand break (DSB) repair capacity results from radiation-induced differences in localisation, expression or activity of major non-homologous end-joining (NHEJ) proteins....... MATERIALS AND METHODS: Experiments were performed with 11 normal human fibroblast strains AF01-11. NHEJ proteins were determined by Western blot and DNA-PK activity by pulldown-assay. RESULTS: The four NHEJ proteins tested (Ku70, Ku80, XRCC4 and DNA-PKcs) were found to be localised almost exclusively...

The Seed Repair Response during Germination: Disclosing Correlations between DNA Repair, Antioxidant Response, and Chromatin Remodeling in Medicago truncatula

Directory of Open Access Journals (Sweden)

Andrea Pagano

2017-11-01

Full Text Available This work provides novel insights into the effects caused by the histone deacetylase inhibitor trichostatin A (TSA during Medicago truncatula seed germination, with emphasis on the seed repair response. Seeds treated with H2O and TSA (10 and 20 μM were collected during imbibition (8 h and at the radicle protrusion phase. Biometric data showed delayed germination and impaired seedling growth in TSA-treated samples. Comet assay, performed on radicles at the protrusion phase and 4-days old M. truncatula seedlings, revealed accumulation of DNA strand breaks upon exposure to TSA. Activation of DNA repair toward TSA-mediated genotoxic damage was evidenced by the up-regulation of MtOGG1(8-OXOGUANINE GLYCOSYLASE/LYASE gene involved in the removal of oxidative DNA lesions, MtLIGIV(LIGASE IV gene, a key determinant of seed quality, required for the rejoining of DNA double strand breaks and TDP(TYROSYL-DNA PHOSPHODIESTERASE genes encoding the multipurpose DNA repair enzymes tyrosyl-DNA phosphodiesterases. Since radical scavenging can prevent DNA damage, the specific antioxidant activity (SAA was measured by DPPH (1,1-diphenyl-2-picrylhydrazyl and Folin-Ciocalteu reagent assays. Fluctuations of SAA were observed in TSA-treated seeds/seedlings concomitant with the up-regulation of antioxidant genes MtSOD(SUPEROXIDE DISMUTASE, MtAPX(ASCORBATE PEROXIDASE and MtMT2(TYPE 2 METALLOTHIONEIN. Chromatin remodeling, required to facilitate the access of DNA repair enzymes at the damaged sites, is also part of the multifaceted seed repair response. To address this aspect, still poorly explored in plants, the MtTRRAP(TRANSFORMATION/TRANSACTIVATION DOMAIN-ASSOCIATED PROTEIN gene was analyzed. TRRAP is a transcriptional adaptor, so far characterized only in human cells where it is needed for the recruitment of histone acetyltransferase complexes to chromatin during DNA repair. The MtTRRAP gene and the predicted interacting partners MtHAM2 (HISTONE ACETYLTRANSFERASE OF

Repair of DNA treated with γ-irradiation and chemical carcinogens. Final report, June 1, 1981-May 31, 1984

International Nuclear Information System (INIS)

Goldthwait, D.A.

1984-01-01

Work done in the past three years has been on DNA repair, on genetic transposition and on the effect of carcinogens on alu sequence transcription. DNA repair work was completed on β-propiolactone DNA adducts, on procaryotic and eucaryotic enzymes capable of removal of 3-methyladenine from DNA, and on in vitro repair of neucleosomal core particle DNA and chromatin DNA. Attempts were made to isolate a human transposable element through the isolation of double stranded RNA and probing of a human library. Experiments were also done to determine whether carcinogens altered the expression of alu sequences in human DNA

Structure/function analysis of PARP-1 in oxidative and nitrosative stress-induced monomeric ADPR formation.

Directory of Open Access Journals (Sweden)

Ben Buelow

2009-07-01

Full Text Available Poly adenosine diphosphate-ribose polymerase-1 (PARP-1 is a multifunctional enzyme that is involved in two major cellular responses to oxidative and nitrosative (O/N stress: detection and response to DNA damage via formation of protein-bound poly adenosine diphosphate-ribose (PAR, and formation of the soluble 2(nd messenger monomeric adenosine diphosphate-ribose (mADPR. Previous studies have delineated specific roles for several of PARP-1's structural domains in the context of its involvement in a DNA damage response. However, little is known about the relationship between the mechanisms through which PARP-1 participates in DNA damage detection/response and those involved in the generation of monomeric ADPR. To better understand the relationship between these events, we undertook a structure/function analysis of PARP-1 via reconstitution of PARP-1 deficient DT40 cells with PARP-1 variants deficient in catalysis, DNA binding, auto-PARylation, and PARP-1's BRCT protein interaction domain. Analysis of responses of the respective reconstituted cells to a model O/N stressor indicated that PARP-1 catalytic activity, DNA binding, and auto-PARylation are required for PARP-dependent mADPR formation, but that BRCT-mediated interactions are dispensable. As the BRCT domain is required for PARP-dependent recruitment of XRCC1 to sites of DNA damage, these results suggest that DNA repair and monomeric ADPR 2(nd messenger generation are parallel mechanisms through which PARP-1 modulates cellular responses to O/N stress.

Effects of radiations on DNA and repair of the damage. Progress report, May 1, 1976--March 31, 1977

International Nuclear Information System (INIS)

Hutchinson, F.

1977-01-01

Last year's report that repair of DNA double-strand breaks from gamma rays occurs in E. coli was verified by additional experiments. Such repair requires recA function and the presence of another DNA molecule of the same base sequence, so it may involve a recombination-like event. Ultraviolet light acting on DNA containing bromouracil produces double-strand breaks by single photochemical events, and a single model can explain this as well as other results. Strains of E. coli which are unusually mutable by bromouracil--uvrE, mutL, mutR, mutS, are defective in mismatch repair. This strengthens the suggestion in last year's report that such mutagenesis occurs when enzymes responsible for the removal of mismatched bases are unable to remove all the mismatches. Ultraviolet mutagenesis of lambda phage may be a useful model for the study of mutagenesis in cells, because the effects of lesions in the gene mutated (i.e., in the phage) and changes in enzyme systems (by treating the host cells) can be examined separately. Quantitative data support this approach

Effects of radiations on DNA and repair of the damage. Progress report, May 1, 1974--June 30, 1977

International Nuclear Information System (INIS)

Hutchinson, F.

1977-01-01

Repair of DNA double-strand breaks produced by gamma rays takes place in E. coli. Such repair requires recA function and the presence of another DNA molecule of the same base sequence, so it may involve a recombination-like event. Ultraviolet light acting on DNA containing bromouracil produces doublestrand breaks by single photochemical events, and a simple model can explain this, as well as other results. Bromouracil mutagenesis of either E. coli or lambda phage does not involve the recA or red functions. Bromouracil mutagenesis is greatly increased in E. coli mutants such as uvrE, mutL, mutR and mutS, which are defective in mismatch repair. This, and other results, suggest that bromouracil mutagenesis occurs when cell enzymes fail to remove mismatched bases. Ultraviolet mutagenesis of lambda phage may be a useful model for the study of mutagenesis in cells, because the effects of lesions in the gene mutated (i.e. in the phage) and changes in enzyme systems (by treating the host cells) can be examined separately. Quantitative data support this approach

46 CFR 97.30-1 - Repairs to boilers and pressure vessels.

Science.gov (United States)

2010-10-01

... 46 Shipping 4 2010-10-01 2010-10-01 false Repairs to boilers and pressure vessels. 97.30-1 Section... VESSELS OPERATIONS Reports of Accidents, Repairs, and Unsafe Equipment § 97.30-1 Repairs to boilers and pressure vessels. (a) Before making any repairs to boilers or unfired pressure vessels, the chief engineer...

46 CFR 196.30-1 - Repairs to boilers and pressure vessels.

Science.gov (United States)

2010-10-01

... 46 Shipping 7 2010-10-01 2010-10-01 false Repairs to boilers and pressure vessels. 196.30-1... VESSELS OPERATIONS Reports of Accidents, Repairs, and Unsafe Equipment § 196.30-1 Repairs to boilers and pressure vessels. (a) Before making any repairs to boilers or unfired pressure vessels, the Chief Engineer...

46 CFR 78.33-1 - Repairs of boiler and pressure vessels.

Science.gov (United States)

2010-10-01

... 46 Shipping 3 2010-10-01 2010-10-01 false Repairs of boiler and pressure vessels. 78.33-1 Section... OPERATIONS Reports of Accidents, Repairs, and Unsafe Equipment § 78.33-1 Repairs of boiler and pressure vessels. (a) Before making any repairs to boilers or unfired pressure vessels, the chief engineer shall...

CEBPG transcription factor correlates with antioxidant and DNA repair genes in normal bronchial epithelial cells but not in individuals with bronchogenic carcinoma

International Nuclear Information System (INIS)

Mullins, D'Anna N; Crawford, Erin L; Khuder, Sadik A; Hernandez, Dawn-Alita; Yoon, Youngsook; Willey, James C

2005-01-01

Cigarette smoking is the primary cause of bronchogenic carcinoma (BC), yet only 10–15% of heavy smokers develop BC and it is likely that this variation in risk is, in part, genetically determined. We previously reported a set of antioxidant genes for which transcript abundance was lower in normal bronchial epithelial cells (NBEC) of BC individuals compared to non-BC individuals. In unpublished studies of the same NBEC samples, transcript abundance values for several DNA repair genes were correlated with these antioxidant genes. From these data, we hypothesized that antioxidant and DNA repair genes are co-regulated by one or more transcription factors and that inter-individual variation in expression and/or function of one or more of these transcription factors is responsible for inter-individual variation in risk for BC. The putative transcription factor recognition sites common to six of the antioxidant genes were identified through in silico DNA sequence analysis. The transcript abundance values of these transcription factors (n = 6) and an expanded group of antioxidant and DNA repair genes (n = 16) were measured simultaneously by quantitative PCR in NBEC of 24 non-BC and 25 BC individuals. CEBPG transcription factor was significantly (p < 0.01) correlated with eight of the antioxidant or DNA repair genes in non-BC individuals but not in BC individuals. In BC individuals the correlation with CEBPG was significantly (p < 0.01) lower than that of non-BC individuals for four of the genes (XRCC1, ERCC5, GSTP1, and SOD1) and the difference was nearly significant for GPX1. The only other transcription factor correlated with any of these five target genes in non-BC individuals was E2F1. E2F1 was correlated with GSTP1 among non-BC individuals, but in contrast to CEBPG, there was no significant difference in this correlation in non-BC individuals compared to BC individuals. We conclude that CEBPG is the transcription factor primarily responsible for regulating

Enhanced DNA repair of cyclobutane pyrimidine dimers changes the biological response to UV-B radiation

Energy Technology Data Exchange (ETDEWEB)

Yarosh, Daniel B

2002-11-30

The goal of DNA repair enzyme therapy is the same as that for gene therapy: to rescue a defective proteome/genome by introducing a substitute protein/DNA. The danger of inadequate DNA repair is highlighted in the genetic disease xeroderma pigmentosum. These patients are hypersensitive to sunlight and develop multiple cutaneous neoplasms very early in life. The bacterial DNA repair enzyme T4 endonuclease V was shown over 25 years ago to be capable of reversing the defective repair in xeroderma pigmentosum cells. This enzyme, packaged in an engineered delivery vehicle, has been shown to traverse the stratum corneum, reach the nuclei of living cells of the skin, and enhance the repair of UV-induced cyclobutane pyrimidine dimers (CPD). In such a system, changes in DNA repair, mutagenesis, and cell signaling can be studied without manipulation of the genome.

Evaluation of the XRCC1 gene as a phenotypic modifier in BRCA1/2 mutation carriers. Results from the consortium of investigators of modifiers of BRCA1/BRCA2

NARCIS (Netherlands)

Osorio, A.; Milne, R. L.; Alonso, R.; Pita, G.; Peterlongo, P.; Teulé, A.; Nathanson, K. L.; Domchek, S. M.; Rebbeck, T.; Lasa, A.; Konstantopoulou, I.; Hogervorst, F. B.; Verhoef, S.; van Dooren, M. F.; Jager, A.; Ausems, M. G. E. M.; Aalfs, C. M.; van Asperen, C. J.; Vreeswijk, M.; Waisfisz, Q.; van Roozendaal, C. E.; Ligtenberg, M. J.; Easton, D. F.; Peock, S.; Cook, M.; Oliver, C. T.; Frost, D.; Curzon, B.; Evans, D. G.; Lalloo, F.; Eeles, R.; Izatt, L.; Davidson, R.; Adlard, J.; Eccles, D.; Ong, K.-r; Douglas, F.; Downing, S.; Brewer, C.; Walker, L.; Nevanlinna, H.; Aittomäki, K.; Couch, F. J.; Fredericksen, Z.; Lindor, N. M.; Godwin, A.; Isaacs, C.; Caligo, M. A.; Loman, N.; Jernström, H.; Barbany-Bustinza, G.; Liljegren, A.; Ehrencrona, H.; Stenmark-Askmalm, M.; Feliubadaló, L.; Manoukian, S.; Peissel, B.; Zaffaroni, D.; Bonanni, B.; Fortuzzi, S.; Johannsson, O. T.; Chenevix-Trench, G.; Chen, X.-C.; Beesley, J.; Spurdle, A. B.; Sinilnikova, O. M.; Healey, S.; McGuffog, L.; Antoniou, A. C.; Brunet, J.; Radice, P.; Benítez, J.; Hogervorst, F. B. L.; Verheus, M.; van 't Veer, L. J.; van Leeuwen, F. E.; Rookus, M. A.; Collée, M.; van den Ouweland, A. M. W.; Hooning, M. J.; Tilanus-Linthorst, M. M. A.; Seynaeve, C.; Wijnen, J. T.; Vreeswijk, M. P.; Tollenaar, R. A.; Devilee, P.; Hoogerbrugge, N.; Ausems, M. G.; van der Luijt, R. B.; van Os, T. A.; Gille, J. J. P.; Meijers-Heijboer, H. E. J.; Gomez-Garcia, E. B.; Blok, Marinus J.; Caanen, B.; Oosterwijk, J. C.; van der Hout, A. H.; Mourits, M. J.; Vasen, H. F.; Peock, Susan; Cook, Margaret; Oliver, Clare; Frost, Debra; Miedzybrodzka, Zosia; Gregory, Helen; Morrison, Patrick; Jeffers, Lisa; Cole, Trevor; McKeown, Carole; Ong, Kai-Ren; Hoffman, Jonathan; Donaldson, Alan; Paterson, Joan; Downing, Sarah; Taylor, Amy; Murray, Alexandra; Rogers, Mark T.; McCann, Emma; Kennedy, M. John; Barton, David; East, South; Porteous, Mary; Drummond, Sarah; Brewer, Carole; Kivuva, Emma; Searle, Anne; Goodman, Selina; Hill, Kathryn; Davidson, Rosemarie; Bradshaw, Nicola; Snadden, Lesley; Longmuir, Mark; Watt, Catherine; Gibson, Sarah; Izatt, Louise; Jacobs, Chris; Langman, Caroline; Whaite, Anna; Dorkins, Huw; Barwell, Julian; Adlard, Julian; Chu, Carol; Miller, Julie; Ellis, Ian; Evans, D. Gareth; Lalloo, Fiona; Taylor, Jane; Side, Lucy; Male, Alison; Berlin, Cheryl; Eason, Jacqueline; Collier, Rebecca; Douglas, Fiona; Claber, Oonagh; Walker, Lisa; McLeod, Diane; Halliday, Dorothy; Durell, Sarah; Stayner, Barbara; Eeles, Ros; Shanley, Susan; Rahman, Nazneen; Houlston, Richard; Bancroft, Elizabeth; D'Mello, Lucia; Page, Elizabeth; Ardern-Jones, Audrey; Kohut, Kelly; Wiggins, Jennifer; Castro, Elena; Mitra, Anitra; Robertson, Lisa; Cook, Jackie; Quarrell, Oliver; Bardsley, Cathryn; Hodgson, Shirley; Goff, Sheila; Brice, Glen; Winchester, Lizzie; Eddy, Charlotte; Tripathi, Vishakha; Attard, Virginia; Eccles, Diana; Lucassen, Anneke; Crawford, Gillian; McBride, Donna; Smalley, Sarah; Godwin, A. K.; Karlsson, Per; Nordling, Margareta; Bergman, Annika; Einbeigi, Zakaria; Stenmark- Askmalm, Marie; Liedgren, Sigrun; Borg, Ake; Loman, Niklas; Olsson, Håkan; Kristoffersson, Ulf; Jernström, Helena; Harbst, Katja; Henriksson, Karin; Lindblom, Annika; Arver, Brita; Wachenfeldt, Anna von; Liljegren, Annelie; Barbany-Bustinza, Gisela; Rantala, Johanna; Melin, Beatrice; Grönberg, Henrik; Stattin, Eva-Lena; Emanuelsson, Monica; Ehrencrona, Hans; Rosenquist Brandell, Richard; Dahl, Niklas

2011-01-01

Single-nucleotide polymorphisms (SNPs) in genes involved in DNA repair are good candidates to be tested as phenotypic modifiers for carriers of mutations in the high-risk susceptibility genes BRCA1 and BRCA2. The base excision repair (BER) pathway could be particularly interesting given the relation

The effects of gamma irradiation on growth and expression of genes encoding DNA repair-related proteins in Lombardy poplar (Populus nigra var. italica).

Science.gov (United States)

Nishiguchi, Mitsuru; Nanjo, Tokihiko; Yoshida, Kazumasa

2012-07-01

In this study, to elucidate the mechanisms of adaptation and tolerance to ionizing radiation in woody plants, we investigated the various biological effects of γ-rays on the Lombardy poplar (Populus nigra L. var. italica Du Roi). We detected abnormal leaf shape and color, fusion, distorted venation, shortened internode, fasciation and increased axillary shoots in γ-irradiated poplar plants. Acute γ-irradiation with a dose of 100Gy greatly reduced the height, stem diameter and biomass of poplar plantlets. After receiving doses of 200 and 300Gy, all the plantlets stopped growing, and then most of them withered after 4-10 weeks of γ-irradiation. Comet assays showed that nuclear DNA in suspension-cultured poplar cells had been damaged by γ-rays. To determine whether DNA repair-related proteins are involved in the response to γ-rays in Lombardy poplars, we cloned the PnRAD51, PnLIG4, PnKU70, PnXRCC4, PnPCNA and PnOGG1 cDNAs and investigated their mRNA expression. The PnRAD51, PnLIG4, PnKU70, PnXRCC4 and PnPCNA mRNAs were increased by γ-rays, but the PnOGG1 mRNA was decreased. Moreover, the expression of PnLIG4, PnKU70 and PnRAD51 was also up-regulated by Zeocin known as a DNA cleavage agent. These observations suggest that the morphogenesis, growth and protective gene expression in Lombardy poplars are severely affected by the DNA damage and unknown cellular events caused by γ-irradiation. Copyright © 2012 Elsevier Ltd. All rights reserved.

Repair of DNA damage in Deinococcus radiodurans

International Nuclear Information System (INIS)

Evans, D.M.

1984-01-01

The repair of DNA lesions in Deinococcus radiodurans was examined with particular reference to DNA excision repair of ultraviolet light (UV) induced pyrimidine dimers. The characteristics of excision repair via UV endonucleases α and β in vivo varied with respect to (a) the substrate range of the enzymes, (b) the rate of repair of DNA damage (c) the requirement for a protein synthesised in response to DNA damage to attenuate exonuclease action at repairing regions. UV endonuclease α is postulated to incise DNA in a different manner from UV endonuclease β thus defining the method of subsequent repair. Several DNA damage specific endonuclease activities independent of α and β are described. Mutations of the uvsA, uvsF and uvsG genes resulted in an increase in single-strand breaks in response to DNA damage producing uncontrolled DNA degradation. Evidence is presented that these genes have a role in limiting the access of UV endonuclease β to DNA lesions. uvsF and uvsG are also shown to be linked to the mtoA gene. Mutation of uvsH and reo-1 produces further distinct phenotypes which are discussed. An overall model of excision repair of DNA damage in Deinococcus radiodurans is presented. (author)

DNA repair is indispensable for survival after acute inflammation

Science.gov (United States)

Calvo, Jennifer A.; Meira, Lisiane B.; Lee, Chun-Yue I.; Moroski-Erkul, Catherine A.; Abolhassani, Nona; Taghizadeh, Koli; Eichinger, Lindsey W.; Muthupalani, Sureshkumar; Nordstrand, Line M.; Klungland, Arne; Samson, Leona D.

2012-01-01

More than 15% of cancer deaths worldwide are associated with underlying infections or inflammatory conditions, therefore understanding how inflammation contributes to cancer etiology is important for both cancer prevention and treatment. Inflamed tissues are known to harbor elevated etheno-base (ε-base) DNA lesions induced by the lipid peroxidation that is stimulated by reactive oxygen and nitrogen species (RONS) released from activated neutrophils and macrophages. Inflammation contributes to carcinogenesis in part via RONS-induced cytotoxic and mutagenic DNA lesions, including ε-base lesions. The mouse alkyl adenine DNA glycosylase (AAG, also known as MPG) recognizes such base lesions, thus protecting against inflammation-associated colon cancer. Two other DNA repair enzymes are known to repair ε-base lesions, namely ALKBH2 and ALKBH3; thus, we sought to determine whether these DNA dioxygenase enzymes could protect against chronic inflammation-mediated colon carcinogenesis. Using established chemically induced colitis and colon cancer models in mice, we show here that ALKBH2 and ALKBH3 provide cancer protection similar to that of the DNA glycosylase AAG. Moreover, Alkbh2 and Alkbh3 each display apparent epistasis with Aag. Surprisingly, deficiency in all 3 DNA repair enzymes confers a massively synergistic phenotype, such that animals lacking all 3 DNA repair enzymes cannot survive even a single bout of chemically induced colitis. PMID:22684101

Archaeal DNA Polymerase-B as a DNA Template Guardian: Links between Polymerases and Base/Alternative Excision Repair Enzymes in Handling the Deaminated Bases Uracil and Hypoxanthine

Directory of Open Access Journals (Sweden)

Javier Abellón-Ruiz

2016-01-01

Full Text Available In Archaea repair of uracil and hypoxanthine, which arise by deamination of cytosine and adenine, respectively, is initiated by three enzymes: Uracil-DNA-glycosylase (UDG, which recognises uracil; Endonuclease V (EndoV, which recognises hypoxanthine; and Endonuclease Q (EndoQ, (which recognises both uracil and hypoxanthine. Two archaeal DNA polymerases, Pol-B and Pol-D, are inhibited by deaminated bases in template strands, a feature unique to this domain. Thus the three repair enzymes and the two polymerases show overlapping specificity for uracil and hypoxanthine. Here it is demonstrated that binding of Pol-D to primer-templates containing deaminated bases inhibits the activity of UDG, EndoV, and EndoQ. Similarly Pol-B almost completely turns off EndoQ, extending earlier work that demonstrated that Pol-B reduces catalysis by UDG and EndoV. Pol-B was observed to be a more potent inhibitor of the enzymes compared to Pol-D. Although Pol-D is directly inhibited by template strand uracil, the presence of Pol-B further suppresses any residual activity of Pol-D, to near-zero levels. The results are compatible with Pol-D acting as the replicative polymerase and Pol-B functioning primarily as a guardian preventing deaminated base-induced DNA mutations.

The time course of repair of ultraviolet-induced DNA damage; implications for the structural organization of repair

International Nuclear Information System (INIS)

Collins, A.; Squires, S.

1986-01-01

Alternative molecular mechanisms can be envisaged for the cellular repair of UV-damaged DNA. In the 'random collision' model, DNA damage distributed throughout the genome is recognised and repaired by a process of random collision between DNA damage and repair enzymes. The other model assumes a 'processive' mechanism, whereby DNA is scanned for damage by a repair complex moving steadily along its length. Random collision should result in a declining rate of repair with time as the concentration of lesions in the DNA falls; but the processive model predicts a constant rate until scanning is complete. The authors have examined the time course of DNA repair in human fibroblasts given low doses of UV light. Using 3 distinct assays, the authors find no sign of a constant repair rate after 4 J/m 2 or less, even when the first few hours after irradiation are examined. Thus DNA repair is likely to depend on random collision. (Auth.)

DNA repair in human cells: Methods for the determination of calmodulin involvement

International Nuclear Information System (INIS)

Charp, P.A.

1987-01-01

Exposure of DNA to either physical or chemical agents can result in the formation of a number of different lesions which must be repaired enzymatically in order for DNA to carry on normal replication and transcription. In most cases, the enzymes involved in this repair of damaged DNA include endonucleases, exonucleases, glycosylases, polymerases, and ligases. Each group of enzymes is involved in precise steps in DNA repair. Exposure to physical agents such as ultraviolet light (UV) at a wavelength of 254 nm is repaired by two distinct and different mechanisms. One mode of enzymatic repair of pyrimidine dimers is accomplished in situ by photoreactivation of UV-induced pyrimidine dimers by photoreactivating light. The second mode of enzymatic repair is the excision repair of pyrimidine dimers involving several different enzymes including endonuclease, exonuclease, and DNA ligase. A summary of the sequence of enzymatic steps involved is shown. It has been observed that specific drugs which bind to and alter the action of calmodulin in cells block DNA synthesis. This suggests that calmodulin may play a role both in normal DNA replication and repair. Others using an indirect method measuring the degree of DNA nucleoid sedimentation, showed that the specific anti-calmodulin agent W-13 slowed the rate of DNA repair. Others showed that DNA synthesis in T51B rat liver cells could be blocked with the addition of either chlorpromazine or trifluoperazine

High fat diet accelerates cartilage repair in DBA/1 mice.

Science.gov (United States)

Wei, Wu; Bastiaansen-Jenniskens, Yvonne M; Suijkerbuijk, Mathijs; Kops, Nicole; Bos, Pieter K; Verhaar, Jan A N; Zuurmond, Anne-Marie; Dell'Accio, Francesco; van Osch, Gerjo J V M

2017-06-01

Obesity is a well-known risk factor for osteoarthritis, but it is unknown what it does on cartilage repair. Here we investigated whether a high fat diet (HFD) influences cartilage repair in a mouse model of cartilage repair. We fed DBA/1 mice control or HFD (60% energy from fat). After 2 weeks, a full thickness cartilage defect was made in the trochlear groove. Mice were sacrificed, 1, 8, and 24 weeks after operation. Cartilage repair was evaluated on histology. Serum glucose, insulin and amyloid A were measured 24 h before operation and at endpoints. Immunohistochemical staining was performed on synovium and adipose tissue to evaluate macrophage infiltration and phenotype. One week after operation, mice on HFD had defect filling with fibroblast-like cells and more cartilage repair as indicated by a lower Pineda score. After 8 weeks, mice on a HFD still had a lower Pineda score. After 24 weeks, no mice had complete cartilage repair and we did not detect a significant difference in cartilage repair between diets. Bodyweight was increased by HFD, whereas serum glucose, amyloid A and insulin were not influenced. Macrophage infiltration and phenotype in adipose tissue and synovium were not influenced by HFD. In contrast to common wisdom, HFD accelerated intrinsic cartilage repair in DBA/1 mice on the short term. Resistance to HFD induced inflammatory and metabolic changes could be associated with accelerated cartilage repair. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1258-1264, 2017. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Radio-sensitization of esophageal cancer EC9706 cells to X ray by histone deacetylase inhibiter Trichostatin A

International Nuclear Information System (INIS)

Lin Xiaoli; Qin Guangyong; Fang Huasheng; Zhang Fengqiu; Ya Huiyuan

2009-01-01

Cell apoptosis and the expression of DNA damage repair-related genes XRCC2, ATM and Lig4 were detected separately by flow cytometry analysis and real-time fluorescence quantitative (RT-PCR), in order to investigate the effect of Trichostatin A (TSA) pre-treatment on the radiosensitivity of esophageal cancer EC9706 cells to X ray in vitro. The results indicate that pre-irradiation exposure to TSA enhances cell apoptotic rate by X ray markedly (P<0.05) and the mRNA levels of XRCC2, ATM and Lig4 (except 8Gy) are up-regulated by X ray individually (P<0.05). TSA alone and TSA in combination with X ray all reduce mRNA levels of XRCC2, ATM and Lig4. In conclusions, TSA may radio-sensitize EC9706 cells to X ray by reducing mRNA levels of XRCC2, ATM and Lig4. (authors)

Determination of the frequency of polymorphisms in genes related to the genome stability maintenance of the population residing at Monte Alegre, PA (Brazil) municipality; Determinacao da frequencia de polimorfismos em genes relacionados a manutencao da estabilidade do genoma na populacao residente no municipio de Monte Alegre, PA

Energy Technology Data Exchange (ETDEWEB)

Hozumi, Cristiny Gomes

2010-07-01

The human exposure to ionizing radiation coming from natural sources is an inherent feature of human life on earth, for man and all living things have always been exposed to these sources. Ionizing radiation is a known genotoxic agent which can affect the genomic stability and genes related to DNA repair may play a role when they have committed certain polymorphism. This study aimed to analyze the frequency of polymorphisms (SNPs) in genes of DNA repair and cell cycle control: hOGG1 (Ser326Cys), XRCC3 (Thr241 Met) and p53 (Arg72Pro) in saliva samples from a population located Monte Alegre, state of Para were collected in August 2008 and 40 samples of men and 46 samples of women, adding a total of 86 samples. By RFLP was determined the frequency of homozygous genotypes and / or heterozygous for polymorphic genes. The I)OGG1 gene was 5% of the allele 326Cys, XRCC3 gene found about 21 % of the allele 241 Met and p53 gene showed 40.8% of the 72Pro allele. And the genotype frequencies of individuals for the three genes were 91.04%, 88.06% and 59.7% for homozygous wild genotype, 5.97%, 11.94% and 22.39% for heterozygote genotype and 2,99%, zero and 17:91% for homozygous polymorphic hOGG1 genes respectively, XRCC3, p53. These values are similar to those found in previous studies. The influence of these polymorphisms, which are involved in DNA repair and consequent genotoxicity induced by radiation depends on dose and exposure factors such as smoking, which is statistically a factor in public health surveillance in the region. This study gathered information and molecular epidemiology in Monte Alegre, that help to characterization of local population. (author)

Determination of the frequency of polymorphisms in genes related to the genome stability maintenance of the population residing at Monte Alegre, PA (Brazil) municipality

International Nuclear Information System (INIS)

Hozumi, Cristiny Gomes

2010-01-01

The human exposure to ionizing radiation coming from natural sources is an inherent feature of human life on earth, for man and all living things have always been exposed to these sources. Ionizing radiation is a known genotoxic agent which can affect the genomic stability and genes related to DNA repair may play a role when they have committed certain polymorphism. This study aimed to analyze the frequency of polymorphisms (SNPs) in genes of DNA repair and cell cycle control: hOGG1 (Ser326Cys), XRCC3 (Thr241 Met) and p53 (Arg72Pro) in saliva samples from a population located Monte Alegre, state of Para were collected in August 2008 and 40 samples of men and 46 samples of women, adding a total of 86 samples. By RFLP was determined the frequency of homozygous genotypes and / or heterozygous for polymorphic genes. The I)OGG1 gene was 5% of the allele 326Cys, XRCC3 gene found about 21 % of the allele 241 Met and p53 gene showed 40.8% of the 72Pro allele. And the genotype frequencies of individuals for the three genes were 91.04%, 88.06% and 59.7% for homozygous wild genotype, 5.97%, 11.94% and 22.39% for heterozygote genotype and 2,99%, zero and 17:91% for homozygous polymorphic hOGG1 genes respectively, XRCC3, p53. These values are similar to those found in previous studies. The influence of these polymorphisms, which are involved in DNA repair and consequent genotoxicity induced by radiation depends on dose and exposure factors such as smoking, which is statistically a factor in public health surveillance in the region. This study gathered information and molecular epidemiology in Monte Alegre, that help to characterization of local population. (author)

ErbB2 regulates NHEJ repair pathway by affecting erbB1-triggered IR-induced Akt activity

International Nuclear Information System (INIS)

Toulany, Mahmoud; Peter Rodemann, H.

2009-01-01

We have already reported that erbBl-PI3K-AKT signaling is an important pathway in regulating radiation sensitivity and DNA double strand break repair of human tumor cells. In the present study using small interfering RNA and pharmacological inhibitors in non-small cell lung cancer cell lines we investigated the role of Aktl on radiation-induced DNA-PKcs activity and DNA-double strand break (DNA-DSB) repair. Likewise, the function of erbB2 as hetrodimerization partner of erbBl in radiation-induced Akt activity and regulation of DNA-dsb repair through DNA-PKcs was evaluated. In A549 and H460 transfected with AKTl-siRNA radiation-induced phosphorylation of DNA-PKcs the key enzyme regulating NHEJ repair pathway was markedly inhibited. In both cell lines downregulation of Aktl led to a significant enhancement of residual DNA-DSB, i.e. impaired DNA-DSB repair. Interestingly, in cells transfected with DNA-PKcs-siRNA a lack of effect of AKTl-siRNA on enhancement of residual DNA-DSBs was observed. This results indicate that Aktl regulates NHEJ repair in a DNA-PKcs dependent manner

Retraction RETRACTION of "Association between polymorphisms in the XRCC1 gene and the risk of non-small cell lung cancer", by Han JC, Zhang YJ and Li XD - Genet. Mol. Res. 14 (4): 12888-12893 (2015).

Science.gov (United States)

Han, J C; Zhang, Y J; Li, X D

2016-10-07

The retracted article is: Han JC, Zhang YJ and Li XD (2015). Association between polymorphisms in the XRCC1 gene and the risk of non-small cell lung cancer. Genet. Mol. Res. 14: 12888-12893. The GMR editorial staff was alerted about this article (received on May 3, 2015; accepted on August 18, 2015) published on October 21, 2015 (DOI: 10.4238/2015.October.21.9) that was found to be substantially similar to the publication of "Association of XRCC1 gene polymorphisms with risk of non-small cell lung cancer" (received on January 25, 2015; accepted on March 23, 2015; e-published on April 1, 2015) by Kang et al., published in the International Journal of Clinical Experimental Pathology 8 (4): 4171-4176. The authors were aware of the Kang et al.'s paper, since they cite it several times in the manuscript published in GMR. Some of the language is similar between the two manuscripts, but what is the most concerning is that several of the tables in the papers are nearly identical. Tables 2 and 3 are exactly identical between the two articles, suggesting that the publication in GMR was plagiarized from the publication in the International Journal of Clinical Experimental Pathology. The Publisher and Editor decided to retract these articles in accordance with the recommendations of the Committee on Publication Ethics (COPE). After a thorough investigation, we have strong reason to believe that the peer review process was failure and, after review and contacting the authors, the editors of Genetics and Molecular Research decided to retract the article. The authors and their institutions were advised of this serious breach of ethics.

N-acetylcysteine normalizes the urea cycle and DNA repair in cells from patients with Batten disease.

Science.gov (United States)

Kim, June-Bum; Lim, Nary; Kim, Sung-Jo; Heo, Tae-Hwe

2012-12-01

Batten disease is an inherited disorder characterized by early onset neurodegeneration due to the mutation of the CLN3 gene. The function of the CLN3 protein is not clear, but an association with oxidative stress has been proposed. Oxidative stress and DNA damage play critical roles in the pathogenesis of neurodegenerative diseases. Antioxidants are of interest because of their therapeutic potential for treating neurodegenerative diseases. We tested whether N-acetylcysteine (NAC), a well-known antioxidant, improves the pathology of cells from patients with Batten disease. At first, the expression levels of urea cycle components and DNA repair enzymes were compared between Batten disease cells and normal cells. We used both mRNA expression levels and Western blot analysis. We found that carbamoyl phosphate synthetase 1, an enzyme involved in the urea cycle, 8-oxoguanine DNA glycosylase 1 and DNA polymerase beta, enzymes involved in DNA repair, were expressed at higher levels in Batten disease cells than in normal cells. The treatment of Batten disease cells with NAC for 48 h attenuated activities of the urea cycle and of DNA repair, as indicated by the substantially decreased expression levels of carbamoyl phosphate synthetase 1, 8-oxoguanine DNA glycosylase 1 and DNA polymerase beta proteins compared with untreated Batten cells. NAC may serve in alleviating the burden of urea cycle and DNA repair processes in Batten disease cells. We propose that NAC may have beneficial effects in patients with Batten disease. Copyright © 2012 John Wiley & Sons, Ltd.

Relationship of DNA repair processes to mutagenesis and carcinogenesis in mammalian cells. Progress report, November 1, 1979-October 31, 1980

International Nuclear Information System (INIS)

Evans, H.H.

1980-10-01

The objective of this research is to determine the role of DNA repair in mutagenesis and carcinogenesis in mammalian cells. Use of the host-cell reactivation viral suicide enrichment procedure was initiated in the isolation of repair-deficient mutants. Lightly mutagenized BHK cells were infected with irradiated Herpes simplex virus (HSV); several radiation-sensitive strains were isolated among the survivors of the infection. The characterization of these strains is progressing and the enrichments are continuing. That alterations in the frequency of mutation of C3H/10T 1/2 cells, occurring as a result of holding the cells in a confluent state following treatment with ethylmethane sulfonate, parallel the alterations in the frequency of neoplastic transformation was found. The repair capabilities of BHK cells were found to be intermediate in comparison to repair-proficient and -deficient human cells with regard to the reactivation of HSV treated with various inactivating agents. The effect of confluency and of low serum levels on DNA synthesis, as well as the response to the cytotoxic effects of MNNG and acriflavin were determined in BHK cells in preparation for the investigation of the role of DNA repair in mutagenesis and transformation. It was also found that C3H/10T 1/2 cells partially recover from the toxic effects of 4-nitroquinoline-1-oxide if they are held in a confluent state for 6 to 22 hrs following treatment. Addition of catalase did not alleviate the toxic effects of 4-NQO. The cells contain a relatively high endogenous level of this enzyme

Relationship of DNA repair processes to mutagenesis and carcinogenesis in mammalian cells. Progress report, November 1, 1979-October 31, 1980

Energy Technology Data Exchange (ETDEWEB)

Evans, H.H.

1980-10-01

The objective of this research is to determine the role of DNA repair in mutagenesis and carcinogenesis in mammalian cells. Use of the host-cell reactivation viral suicide enrichment procedure was initiated in the isolation of repair-deficient mutants. Lightly mutagenized BHK cells were infected with irradiated Herpes simplex virus (HSV); several radiation-sensitive strains were isolated among the survivors of the infection. The characterization of these strains is progressing and the enrichments are continuing. That alterations in the frequency of mutation of C3H/10T 1/2 cells, occurring as a result of holding the cells in a confluent state following treatment with ethylmethane sulfonate, parallel the alterations in the frequency of neoplastic transformation was found. The repair capabilities of BHK cells were found to be intermediate in comparison to repair-proficient and -deficient human cells with regard to the reactivation of HSV treated with various inactivating agents. The effect of confluency and of low serum levels on DNA synthesis, as well as the response to the cytotoxic effects of MNNG and acriflavin were determined in BHK cells in preparation for the investigation of the role of DNA repair in mutagenesis and transformation. It was also found that C3H/10T 1/2 cells partially recover from the toxic effects of 4-nitroquinoline-1-oxide if they are held in a confluent state for 6 to 22 hrs following treatment. Addition of catalase did not alleviate the toxic effects of 4-NQO. The cells contain a relatively high endogenous level of this enzyme. (ERB)

Phosphorylation states of cell cycle and DNA repair proteins can be altered by the nsSNPs

International Nuclear Information System (INIS)

Savas, Sevtap; Ozcelik, Hilmi

2005-01-01

Phosphorylation is a reversible post-translational modification that affects the intrinsic properties of proteins, such as structure and function. Non-synonymous single nucleotide polymorphisms (nsSNPs) result in the substitution of the encoded amino acids and thus are likely to alter the phosphorylation motifs in the proteins. In this study, we used the web-based NetPhos tool to predict candidate nsSNPs that either introduce or remove putative phosphorylation sites in proteins that act in DNA repair and cell cycle pathways. Our results demonstrated that a total of 15 nsSNPs (16.9%) were likely to alter the putative phosphorylation patterns of 14 proteins. Three of these SNPs (CDKN1A-S31R, OGG1-S326C, and XRCC3-T241M) have already found to be associated with altered cancer risk. We believe that this set of nsSNPs constitutes an excellent resource for further molecular and genetic analyses. The novel systematic approach used in this study will accelerate the understanding of how naturally occurring human SNPs may alter protein function through the modification of phosphorylation mechanisms and contribute to disease susceptibility

DNA repair in ultraviolet-irradiated spores of Bacillus subtilis

International Nuclear Information System (INIS)

Wang, T.C.V.

1976-01-01

It has been shown previously by others that at least two independent repair mechanisms are present in Bacillus subtilis for removing ''spore photoproduct'' from DNA of ultraviolet (254 nm)-irradiated spores after germination. One of these, designated as ''spore repair,'' is shown in this study to restore ''spore photoproduct'' to two thymine residues, leaving the DNA backbone intact at the end of the process in vivo. The circumstances under which this repair can occur and some characteristics of its energy requirements have been clarified. The second repair process is identified as excision repair, which can excise both ''spore photoproduct'' from DNA of irradiated spores and cyclobutane-type pyrimidine dimers from DNA of irradiated vegetative cells. In this study it is shown that the gene hcr 1 affects an enzyme activity for the incision step initiating this repair, while the gene hcr 42 affects a step subsequent to incision in the mechanism. In addition a third, independent repair system, termed ''germinative excision repair,'' is discovered and shown to be specific for excising only cyclobutane-type pyrimidine dimers but not ''spore photoproduct.'' This repair system is responsible for the observed high ultraviolet-resistance and temporary capacity for host cell reactivation on recently germinated spores of Bacillus subtilis HCR - strains

Repair of DNA-polypeptide crosslinks by human excision nuclease

Science.gov (United States)

Reardon, Joyce T.; Sancar, Aziz

2006-03-01

DNA-protein crosslinks are relatively common DNA lesions that form during the physiological processing of DNA by replication and recombination proteins, by side reactions of base excision repair enzymes, and by cellular exposure to bifunctional DNA-damaging agents such as platinum compounds. The mechanism by which pathological DNA-protein crosslinks are repaired in humans is not known. In this study, we investigated the mechanism of recognition and repair of protein-DNA and oligopeptide-DNA crosslinks by the human excision nuclease. Under our assay conditions, the human nucleotide excision repair system did not remove a 16-kDa protein crosslinked to DNA at a detectable level. However, 4- and 12-aa-long oligopeptides crosslinked to the DNA backbone were recognized by some of the damage recognition factors of the human excision nuclease with moderate selectivity and were excised from DNA at relatively efficient rates. Our data suggest that, if coupled with proteolytic degradation of the crosslinked protein, the human excision nuclease may be the major enzyme system for eliminating protein-DNA crosslinks from the genome. damage recognition | nucleotide excision repair

Enhanced susceptibility of ovaries from obese mice to 7,12-dimethylbenz[a]anthracene-induced DNA damage

International Nuclear Information System (INIS)

Ganesan, Shanthi; Nteeba, Jackson; Keating, Aileen F.

2014-01-01

7,12-Dimethylbenz[a]anthracene (DMBA) depletes ovarian follicles and induces DNA damage in extra-ovarian tissues, thus, we investigated ovarian DMBA-induced DNA damage. Additionally, since obesity is associated with increased offspring birth defect incidence, we hypothesized that a DMBA-induced DNA damage response (DDR) is compromised in ovaries from obese females. Wild type (lean) non agouti (a/a) and KK.Cg-Ay/J heterozygote (obese) mice were dosed with sesame oil or DMBA (1 mg/kg; intraperitoneal injection) at 18 weeks of age, for 14 days. Total ovarian RNA and protein were isolated and abundance of Ataxia telangiectasia mutated (Atm), X-ray repair complementing defective repair in Chinese hamster cells 6 (Xrcc6), breast cancer type 1 (Brca1), Rad 51 homolog (Rad51), poly [ADP-ribose] polymerase 1 (Parp1) and protein kinase, DNA-activated, catalytic polypeptide (Prkdc) were quantified by RT-PCR or Western blot. Phosphorylated histone H2AX (γH2AX) level was determined by Western blotting. Obesity decreased (P < 0.05) basal protein abundance of PRKDC and BRCA1 proteins but increased (P < 0.05) γH2AX and PARP1 proteins. Ovarian ATM, XRCC6, PRKDC, RAD51 and PARP1 proteins were increased (P < 0.05) by DMBA exposure in lean mice. A blunted DMBA-induced increase (P < 0.05) in XRCC6, PRKDC, RAD51 and BRCA1 was observed in ovaries from obese mice, relative to lean counterparts. Taken together, DMBA exposure induced γH2AX as well as the ovarian DDR, supporting that DMBA causes ovarian DNA damage. Additionally, ovarian DDR was partially attenuated in obese females raising concern that obesity may be an additive factor during chemical-induced ovotoxicity. - Highlights: • DMBA induces markers of ovarian DNA damage. • Obesity induces low level ovarian DNA damage. • DMBA-induced DNA repair response is altered by obesity

Enhanced susceptibility of ovaries from obese mice to 7,12-dimethylbenz[a]anthracene-induced DNA damage

Energy Technology Data Exchange (ETDEWEB)

Ganesan, Shanthi, E-mail: shanthig@iastate.edu; Nteeba, Jackson, E-mail: nteeba@iastate.edu; Keating, Aileen F., E-mail: akeating@iastate.edu

2014-12-01

7,12-Dimethylbenz[a]anthracene (DMBA) depletes ovarian follicles and induces DNA damage in extra-ovarian tissues, thus, we investigated ovarian DMBA-induced DNA damage. Additionally, since obesity is associated with increased offspring birth defect incidence, we hypothesized that a DMBA-induced DNA damage response (DDR) is compromised in ovaries from obese females. Wild type (lean) non agouti (a/a) and KK.Cg-Ay/J heterozygote (obese) mice were dosed with sesame oil or DMBA (1 mg/kg; intraperitoneal injection) at 18 weeks of age, for 14 days. Total ovarian RNA and protein were isolated and abundance of Ataxia telangiectasia mutated (Atm), X-ray repair complementing defective repair in Chinese hamster cells 6 (Xrcc6), breast cancer type 1 (Brca1), Rad 51 homolog (Rad51), poly [ADP-ribose] polymerase 1 (Parp1) and protein kinase, DNA-activated, catalytic polypeptide (Prkdc) were quantified by RT-PCR or Western blot. Phosphorylated histone H2AX (γH2AX) level was determined by Western blotting. Obesity decreased (P < 0.05) basal protein abundance of PRKDC and BRCA1 proteins but increased (P < 0.05) γH2AX and PARP1 proteins. Ovarian ATM, XRCC6, PRKDC, RAD51 and PARP1 proteins were increased (P < 0.05) by DMBA exposure in lean mice. A blunted DMBA-induced increase (P < 0.05) in XRCC6, PRKDC, RAD51 and BRCA1 was observed in ovaries from obese mice, relative to lean counterparts. Taken together, DMBA exposure induced γH2AX as well as the ovarian DDR, supporting that DMBA causes ovarian DNA damage. Additionally, ovarian DDR was partially attenuated in obese females raising concern that obesity may be an additive factor during chemical-induced ovotoxicity. - Highlights: • DMBA induces markers of ovarian DNA damage. • Obesity induces low level ovarian DNA damage. • DMBA-induced DNA repair response is altered by obesity.

Introduction of the yeast DNA repair gene PHR1 into normal and xeroderma pigmentosum human cells

International Nuclear Information System (INIS)

Whyte, D.B.

1988-01-01

The goal of the work described herein is to determine how UV light kills and mutates human cells. Specifically, the hypothesis to be tested states that the major cause of cell death is the cyclobutane dimer. The yeast (S. cerevisiae) enzyme photolyase provides an elegant means of dissecting the biological effects of the two lesions. Photolyase, the product of the PHR1 gene, catalyzes the visible light-dependent reversal of cyclobutane pyrimidine dimers. Introducing the gene for photolyase into human cells, which do not have a functional photoreactivation mechanism, should allow specific repair of cyclobutane pyrimidine dimers. To express the yeast DNA repair gene in human cells, the yeast PHR1 coding sequence was cloned into the mammalian expression vector pRSV4NEO-I. The resulting plasmid, pRSVPHR1, contains the coding sequence of the yeast gene, under control of transcription signals recognized by mammalian cells, and the dominant selectable gene neo. pRSVPHR1 was introduced into normal and XP SV40-transformed fibroblasts by the calcium phosphate coprecipitation technique, and G418-resistant clones were isolated. The level of PHR1 expression was determined by cytoplasmic RNA dot blots. Two clones, XP-3B and GM-20A, had high levels of expression

Protein damage and repair controlling seed vigor and longevity.

Science.gov (United States)

Ogé, Laurent; Broyart, Caroline; Collet, Boris; Godin, Béatrice; Jallet, Denis; Bourdais, Gildas; Job, Dominique; Grappin, Philippe

2011-01-01

The formation of abnormal isoaspartyl residues derived from aspartyl or asparaginyl residues is a major source of spontaneous protein misfolding in cells. The repair enzyme protein L: -isoaspartyl methyltransferase (PIMT) counteracts such damage by catalyzing the conversion of abnormal isoaspartyl residues to their normal aspartyl forms. Thus, this enzyme contributes to the survival of many organisms, including plants. Analysis of the accumulation of isoaspartyl-containing proteins and its modulation by the PIMT repair pathway, using germination tests, immunodetection, enzymatic assays, and HPLC analysis, gives new insights in understanding controlling mechanisms of seed longevity and vigor.

RTEL1 contributes to DNA replication and repair and telomere maintenance.

OpenAIRE

Uringa, E.-J.; Lisaingo, K.; Pickett, H. A.; Brind'Amour, J.; Rohde, J.-H.; Zelensky, A.; Essers, J.; Lansdorp, P. M.

2012-01-01

textabstractTelomere maintenance and DNA repair are important processes that protect the genome against instability. mRtel1, an essential helicase, is a dominant factor setting telomere length in mice. In addition, mRtel1 is involved in DNA double-strand break repair. The role of mRtel1 in telomere maintenance and genome stability is poorly understood. Therefore we used mRtel1-deficient mouse embryonic stem cells to examine the function of mRtel1 in replication, DNA repair, recombination, and...

PCAF/GCN5-Mediated Acetylation of RPA1 Promotes Nucleotide Excision Repair

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Meimei Zhao

2017-08-01

Full Text Available The RPA complex can integrate multiple stress signals into diverse responses by activating distinct DNA repair pathways. However, it remains unclear how RPA1 elects to activate a specific repair pathway during different types of DNA damage. Here, we report that PCAF/GCN5-mediated K163 acetylation of RPA1 is crucial for nucleotide excision repair (NER but is dispensable for other DNA repair pathways. Mechanistically, we demonstrate that the acetylation of RPA1 is critical for the steady accumulation of XPA at damaged DNA sites and preferentially activates the NER pathway. DNA-PK phosphorylates and activates PCAF upon UV damage and consequently promotes the acetylation of RPA1. Moreover, the acetylation of RPA1 is tightly regulated by HDAC6 and SIRT1. Together, our results demonstrate that the K163 acetylation of RPA1 plays a key role in the repair of UV-induced DNA damage and reveal how the specific RPA1 modification modulates the choice of distinct DNA repair pathways.

Ku recruits XLF to DNA double-strand breaks.

Science.gov (United States)

Yano, Ken-ichi; Morotomi-Yano, Keiko; Wang, Shih-Ya; Uematsu, Naoya; Lee, Kyung-Jong; Asaithamby, Aroumougame; Weterings, Eric; Chen, David J

2008-01-01

XRCC4-like factor (XLF)--also known as Cernunnos--has recently been shown to be involved in non-homologous end-joining (NHEJ), which is the main pathway for the repair of DNA double-strand breaks (DSBs) in mammalian cells. XLF is likely to enhance NHEJ by stimulating XRCC4-ligase IV-mediated joining of DSBs. Here, we report mechanistic details of XLF recruitment to DSBs. Live cell imaging combined with laser micro-irradiation showed that XLF is an early responder to DSBs and that Ku is essential for XLF recruitment to DSBs. Biochemical analysis showed that Ku-XLF interaction occurs on DNA and that Ku stimulates XLF binding to DNA. Unexpectedly, XRCC4 is dispensable for XLF recruitment to DSBs, although photobleaching analysis showed that XRCC4 stabilizes the binding of XLF to DSBs. Our observations showed the direct involvement of XLF in the dynamic assembly of the NHEJ machinery and provide mechanistic insights into DSB recognition.

DNA Polymerases λ and β: The Double-Edged Swords of DNA Repair

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Elisa Mentegari

2016-08-01

Full Text Available DNA is constantly exposed to both endogenous and exogenous damages. More than 10,000 DNA modifications are induced every day in each cell’s genome. Maintenance of the integrity of the genome is accomplished by several DNA repair systems. The core enzymes for these pathways are the DNA polymerases. Out of 17 DNA polymerases present in a mammalian cell, at least 13 are specifically devoted to DNA repair and are often acting in different pathways. DNA polymerases β and λ are involved in base excision repair of modified DNA bases and translesion synthesis past DNA lesions. Polymerase λ also participates in non-homologous end joining of DNA double-strand breaks. However, recent data have revealed that, depending on their relative levels, the cell cycle phase, the ratio between deoxy- and ribo-nucleotide pools and the interaction with particular auxiliary proteins, the repair reactions carried out by these enzymes can be an important source of genetic instability, owing to repair mistakes. This review summarizes the most recent results on the ambivalent properties of these enzymes in limiting or promoting genetic instability in mammalian cells, as well as their potential use as targets for anticancer chemotherapy.

DNA Polymerases λ and β: The Double-Edged Swords of DNA Repair.

Science.gov (United States)

Mentegari, Elisa; Kissova, Miroslava; Bavagnoli, Laura; Maga, Giovanni; Crespan, Emmanuele

2016-08-31

DNA is constantly exposed to both endogenous and exogenous damages. More than 10,000 DNA modifications are induced every day in each cell's genome. Maintenance of the integrity of the genome is accomplished by several DNA repair systems. The core enzymes for these pathways are the DNA polymerases. Out of 17 DNA polymerases present in a mammalian cell, at least 13 are specifically devoted to DNA repair and are often acting in different pathways. DNA polymerases β and λ are involved in base excision repair of modified DNA bases and translesion synthesis past DNA lesions. Polymerase λ also participates in non-homologous end joining of DNA double-strand breaks. However, recent data have revealed that, depending on their relative levels, the cell cycle phase, the ratio between deoxy- and ribo-nucleotide pools and the interaction with particular auxiliary proteins, the repair reactions carried out by these enzymes can be an important source of genetic instability, owing to repair mistakes. This review summarizes the most recent results on the ambivalent properties of these enzymes in limiting or promoting genetic instability in mammalian cells, as well as their potential use as targets for anticancer chemotherapy.

[Studies of the repair of radiation-induced genetic damage in Drosophila]. Annual progress report, June 1, 1992 - June 30, 1993

International Nuclear Information System (INIS)

1998-01-01

The most interesting discovery made over the past year derives from sequence analysis of cDNAs from the putative mus308 gene. The theoretical translation product of this gene contains a DNA polymerase domain near the carboxy terminus and DNA/RNA helicase motifs near the amino terminus. There is currently no precedent in the literature for a single polypeptide containing both of these domains. The protein appears to be a novel DNA repair enzyme which should be fruitful ground for future enzymological analysis. The authors have identified two ORFs by sequence analysis of the transforming fragment containing the mei-41 gene and of corresponding cDNAs. ORF 1 includes the P element insertion sites and encodes a peptide of 757 amino acids. ORF 2 starts 900 base pairs downstream of ORF 1 and encodes a peptide of 1,037 amino acids. This putative peptide shows homology to the yeast DNA repair genes, rad50 of S. cerevisiae and rad3 of S. pombe

UV-B induction of NADP-malic enzyme in etiolated and green maize seedlings

International Nuclear Information System (INIS)

Drincovich, M.F.; Casati, P.; Andreo, C.S.; Donahue, R.; Edwards, G.E.

1998-01-01

The effect of treatment of etiolated maize seedlings with UV-B and UV-A radiation, and different levels of photosynthetically active radiation (PAR, 400–700 nm), on the activity and quantity of NADP-malic enzyme (NADPME) and on RNA levels was determined. Under low levels of PAR (14 µmol m –2 s –1 ), exposure to UV-B radiation (9 µmol m –2 s –1 ) but not UV-A radiation (11 µmol m –2 s –1 ) for 6–24 h caused a marked increase in the activity of the enzyme similar to that observed under high PAR (300 µmol m –2 s –1 ) in the absence of UV-B. Western blot analysis indicated there was a specific increase of the photosynthetically active isoform of the enzyme. This increase was also measured at the RNA level by dot blot analysis, indicating that the induction is displayed at the level of NADP-ME transcription. UV-B treatment of green leaves after a 12 h dark period also caused an increase in the activity and level of NADP-ME. The UV-B induction of NADP-ME synthesis may reflect a mechanism for induction of photosynthetic processes in C4 photosynthesis. Alternatively, the relatively low intensity of UV-B radiation present under full sunlight might provide a signal that facilitates repair of UV-B-induced damage through the increased activity of different enzymes such as NADP-ME. It is speculated that the reducing power and pyruvate generated by activity of NADP-ME may be used for respiration in cellular repair processes and as substrates for the fatty acid synthesis required for membrane repair. (author)

A damage-responsive DNA binding protein regulates transcription of the yeast DNA repair gene PHR1

International Nuclear Information System (INIS)

Sebastian, J.; Sancar, G.B.

1991-01-01

The PHR1 gene of Saccharomyces cerevisiae encodes the DNA repair enzyme photolyase. Transcription of PHR1 increases in response to treatment of cells with 254-nm radiation and chemical agents that damage DNA. The authors here the identification of a damage-responsive DNA binding protein, termed photolyase regulatory protein (PRP), and its cognate binding site, termed the PHR1 transcription after DNA damage. PRP activity, monitored by electrophoretic-mobility-shift assay, was detected in cells during normal growth but disappeared within 30 min after irradiation. Copper-phenanthroline footprinting of PRP-DNA complexes revealed that PRP protects a 39-base-pair region of PHR1 5' flanking sequence beginning 40 base pairs upstream from the coding sequence. Thus these observations establish that PRP is a damage-responsive repressor of PHR1 transcription

Ability of Bacillus subtilis protoplasts to repair irradiated bacteriophage deoxyribonucleic acid via acquired and natural enzymatic systems

International Nuclear Information System (INIS)

Yasbin, R.E.; Andersen, B.J.; Sutherland, B.M.

1981-01-01

A novel form of enzyme therapy was achieved by utilizing protoplasts of Bacillus subtilis. Photoreactivating enzyme of Escherichia coli was successfully inserted into the protoplasts of B. subtilis treated with polyethylene glycol. This enzyme was used to photoreactivate ultraviolet-damaged bacteriophage deoxyribonucleic acid (DNA). Furthermore, in polyethylene glycol-treated protoplasts, ultraviolet-irradiated transfecting bacteriophage DNA was shown to be a functional substrate for the host DNA excision repair system. Previous results (R.E. Yasbin, J.D. Fernwalt, and P.I. Fields, J. Bacteriol.; 137: 391-396) showed that ultraviolet-irradiated bacteriophage DNA could not be repaired via the excision repair system of competent cells. Therefore, the processing of bacteriophage DNA by protoplasts and by competent cells must be different. This sensitive protoplast assay can be used to identify and to isolate various types of DNA repair enzymes

Apolipoprotein A-1 mimetic peptide 4F promotes endothelial repairing and compromises reendothelialization impaired by oxidized HDL through SR-B1

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Dan He

2018-05-01

Full Text Available Disruption of endothelial monolayer integrity is the primary instigating factor for many cardiovascular diseases. High density lipoprotein (HDL oxidized by heme enzyme myeloperoxidase (MPO is dysfunctional in promoting endothelial repair. Apolipoprotein A-1 mimetic 4F with its pleiotropic benefits has been proven effective in many in vivo models. In this study we investigated whether 4F promotes endothelial repair and restores the impaired function of oxidized HDL (Cl/NO2-HDL in promoting re-endothelialization. We demonstrate that 4F and Cl/NO2-HDL act on scavenger receptor type I (SR-B1 using human aorta endothelial cells (HAEC and SR-B1 (-/- mouse aortic endothelial cells. Wound healing, transwell migration, lamellipodia formation and single cell migration assay experiments show that 4F treatment is associated with a recovery of endothelial cell migration and associated with significantly increased endothelial nitric oxide synthase (eNOS activity, Akt phosphorylation and SR-B1 expression. 4F increases NO generation and diminishes oxidative stress. In vivo, 4F can stimulate cell proliferation and re-endothelialization in the carotid artery after treatment with Cl/NO2-HDL in a carotid artery electric injury model but fails to do so in SR-B1(-/- mice. These findings demonstrate that 4F promotes endothelial cell migration and has a potential therapeutic benefit against early endothelial injury in cardiovascular diseases.

Genetic polymorphisms and expression of minisatellite mutations in a 3-generation population around the Semipalatinsk nuclear explosion test-site, Kazakhstan.

Science.gov (United States)

Bolegenova, N K; Bekmanov, B O; Djansugurova, L B; Bersimbaev, R I; Salama, S A; Au, W W

2009-11-01

We have reported previously that a population near the Semipalatinsk nuclear explosion test site had significantly increased minisatellite mutations (MM), suggesting increased germ-line mutation rates from the exposure in 3 generations. We hypothesize that the MM can be used as a surrogate biomarker for functional genetic alterations, e.g. gene mutations and chromosome aberrations. Therefore, we have investigated the influence of polymorphisms in genes on the expression of MM in the same two populations (247 and 172 individuals, for exposed and control, respectively, in 3 generations), and their relationships with radiation exposure. We have chosen the analyses of three polymorphic DNA - repair genes (XRCC1, XRCC1 and XRCC3) and two xenobiotic detoxification genes (GSTT1 and GSTM1). Among the exposed and in comparison with the wild-type gene, the functionally active XRCC1 Arg194Trp was significantly associated with low MM and over-represented in the exposed compared with the control populations. In a similar analysis, the functionally deficient XRCC1 Arg399Glu and XRCC3 Trp241Met were associated with increased and significantly reduced MM, respectively, but these variant genes were under-represented in the exposed population. Both GSTT1 and GSTM1 nulls were significantly associated with increased MM. The former was under-represented but the latter was significantly over-represented in the exposed compared with the control populations. In summary, the data indicate that the expected enzymatic functions of the polymorphic genes are consistent with the MM expression, except the XRCC1 Arg399Glu variant gene. In addition, the variant genes were retained in the three generations in association with their useful function, except for the GSTM1 null. However, the MM frequencies in the exposed were not consistently and significantly higher than those in the control populations, radiation exposure may therefore not have been the only cause for the high MM frequency among the

Resistance of hypoxic cells to ionizing radiation is influenced by homologous recombination status

International Nuclear Information System (INIS)

Sprong, Debbie; Janssen, Hilde L.; Vens, Conchita; Begg, Adrian C.

2006-01-01

Purpose: To determine the role of DNA repair in hypoxic radioresistance. Methods and Materials: Chinese hamster cell lines with mutations in homologous recombination (XRCC2, XRCC3, BRAC2, RAD51C) or nonhomologous end-joining (DNA-PKcs) genes were irradiated under normoxic (20% oxygen) and hypoxic (<0.1% oxygen) conditions, and the oxygen enhancement ratio (OER) was calculated. In addition, Fanconi anemia fibroblasts (complementation groups C and G) were compared with fibroblasts from nonsyndrome patients. RAD51 foci were studied using immunofluorescence. Results: All hamster cell lines deficient in homologous recombination showed a decrease in OER (1.5-2.0 vs. 2.6-3.0 for wild-types). In contrast, the OER for the DNA-PKcs-deficient line was comparable to wild-type controls. The two Fanconi anemia cell strains also showed a significant reduction in OER. The OER for RAD51 foci formation at late times after irradiation was considerably lower than that for survival in wild-type cells. Conclusion: Homologous recombination plays an important role in determining hypoxic cell radiosensitivity. Lower OERs have also been reported in cells deficient in XPF and ERCC1, which, similar to homologous recombination genes, are known to play a role in cross-link repair. Because Fanconi anemia cells are also sensitive to cross-linking agents, this strengthens the notion that the capacity to repair cross-links determines hypoxic radiosensitivity

Exonuclease 1 and its versatile roles in DNA repair

DEFF Research Database (Denmark)

Keijzers, Guido; Liu, Dekang; Rasmussen, Lene Juel

2016-01-01

Exonuclease 1 (EXO1) is a multifunctional 5' → 3' exonuclease and a DNA structure-specific DNA endonuclease. EXO1 plays roles in DNA replication, DNA mismatch repair (MMR) and DNA double-stranded break repair (DSBR) in lower and higher eukaryotes and contributes to meiosis, immunoglobulin...... maturation, and micro-mediated end-joining in higher eukaryotes. In human cells, EXO1 is also thought to play a role in telomere maintenance. Mutations in the human EXO1 gene correlate with increased susceptibility to some cancers. This review summarizes recent studies on the enzymatic functions...

Similarities between long interspersed element-1 (LINE-1) reverse transcriptase and telomerase.

Science.gov (United States)

Kopera, Huira C; Moldovan, John B; Morrish, Tammy A; Garcia-Perez, Jose Luis; Moran, John V

2011-12-20

Long interspersed element-1 (LINE-1 or L1) retrotransposons encode two proteins (ORF1p and ORF2p) that contain activities required for conventional retrotransposition by a mechanism termed target-site primed reverse transcription. Previous experiments in XRCC4 or DNA protein kinase catalytic subunit-deficient CHO cell lines, which are defective for the nonhomologous end-joining DNA repair pathway, revealed an alternative endonuclease-independent (ENi) pathway for L1 retrotransposition. Interestingly, some ENi retrotransposition events in DNA protein kinase catalytic subunit-deficient cells are targeted to dysfunctional telomeres. Here we used an in vitro assay to detect L1 reverse transcriptase activity to demonstrate that wild-type or endonuclease-defective L1 ribonucleoprotein particles can use oligonucleotide adapters that mimic telomeric ends as primers to initiate the reverse transcription of L1 mRNA. Importantly, these ribonucleoprotein particles also contain a nuclease activity that can process the oligonucleotide adapters before the initiation of reverse transcription. Finally, we demonstrate that ORF1p is not strictly required for ENi retrotransposition at dysfunctional telomeres. Thus, these data further highlight similarities between the mechanism of ENi L1 retrotransposition and telomerase.

ERCC2 2251A>C genetic polymorphism was highly correlated with early relapse in high-risk stage II and stage III colorectal cancer patients: A preliminary study

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Lee Su-Chen

2008-02-01

Full Text Available Abstract Background Early relapse in colorectal cancer (CRC patients is attributed mainly to the higher malignant entity (such as an unfavorable genotype, deeper tumor invasion, lymph node metastasis and advance cancer stage and poor response to chemotherapy. Several investigations have demonstrated that genetic polymorphisms in drug-targeted genes, metabolizing enzymes, and DNA-repairing enzymes are all strongly correlated with inter-individual differences in the efficacy and toxicity of many treatment regimens. This preliminary study attempts to identify the correlation between genetic polymorphisms and clinicopathological features of CRC, and evaluates the relationship between genetic polymorphisms and chemotherapeutic susceptibility of Taiwanese CRC patients. To our knowledge, this study discusses, for the first time, early cancer relapse and its indication by multiple genes. Methods Six gene polymorphisms functional in drug-metabolism – GSTP1 Ile105Val, ABCB1 Ile1145Ile, MTHFR Ala222Val, TYMS double (2R or triple (3R tandem repeat – and DNA-repair genes – ERCC2 Lys751Gln and XRCC1 Arg399Gln – were assessed in 201 CRC patients using a polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP technique and DNA sequencing. Patients were diagnosed as either high-risk stage II (T2 and 3 N0 M0 or III (any T N1 and 2 M0 and were administered adjuvant chemotherapy regimens that included 5-fluorouracil (5FU and leucovorin (LV. The correlations between genetic polymorphisms and patient clinicopathological features and relapses were investigated. Results In this study, the distributions of GSTP1 (P = 0.003, ABCB1 (P = 0.001, TYMS (P ERCC2 (P XRCC1 (P = 0.006 genotypes in the Asian population, with the exception of MTHFR (P = 0.081, differed significantly from their distributions in a Caucasian population. However, the unfavorable genotype ERCC2 2251A>C (P = 0.006, tumor invasion depth (P = 0.025, lymph node metastasis (P = 0

Oxidatively-induced DNA damage and base excision repair in euthymic patients with bipolar disorder.

Science.gov (United States)

Ceylan, Deniz; Tuna, Gamze; Kirkali, Güldal; Tunca, Zeliha; Can, Güneş; Arat, Hidayet Ece; Kant, Melis; Dizdaroglu, Miral; Özerdem, Ayşegül

2018-05-01

Oxidatively-induced DNA damage has previously been associated with bipolar disorder. More recently, impairments in DNA repair mechanisms have also been reported. We aimed to investigate oxidatively-induced DNA lesions and expression of DNA glycosylases involved in base excision repair in euthymic patients with bipolar disorder compared to healthy individuals. DNA base lesions including both base and nucleoside modifications were measured using gas chromatography-tandem mass spectrometry and liquid chromatography-tandem mass spectrometry with isotope-dilution in DNA samples isolated from leukocytes of euthymic patients with bipolar disorder (n = 32) and healthy individuals (n = 51). The expression of DNA repair enzymes OGG1 and NEIL1 were measured using quantitative real-time polymerase chain reaction. The levels of malondialdehyde were measured using high performance liquid chromatography. Seven DNA base lesions in DNA of leukocytes of patients and healthy individuals were identified and quantified. Three of them had significantly elevated levels in bipolar patients when compared to healthy individuals. No elevation of lipid peroxidation marker malondialdehyde was observed. The level of OGG1 expression was significantly reduced in bipolar patients compared to healthy individuals, whereas the two groups exhibited similar levels of NEIL1 expression. Our results suggest that oxidatively-induced DNA damage occurs and base excision repair capacity may be decreased in bipolar patients when compared to healthy individuals. Measurement of oxidatively-induced DNA base lesions and the expression of DNA repair enzymes may be of great importance for large scale basic research and clinical studies of bipolar disorder. Copyright © 2018 Elsevier B.V. All rights reserved.

Clinicopathologic Significance of Excision Repair Cross-Complementation 1 Expression in Patients Treated With Breast-Conserving Surgery and Radiation Therapy

International Nuclear Information System (INIS)

Goyal, Sharad; Parikh, Rahul R.; Green, Camille; Schiff, Devora B.S.; Moran, Meena S.; Yang Qifeng; Haffty, Bruce G.

2010-01-01

Purpose: The excision repair cross-complementation 1 (ERCC1) enzyme plays a rate-limiting role in the nucleotide excision repair pathway and is associated with resistance to platinum-based chemotherapy in cancers of the head and neck and the lung. The purpose of this study was to evaluate the clinicopathologic and prognostic significance of ERCC1 expression in a cohort of early-stage breast cancer patients treated with breast conservation therapy. Methods and Materials: Paraffin specimens from 504 women with early-stage breast cancer treated with breast conservation therapy were constructed into tissue microarrays. The array was stained for estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) and ERCC1. This was then correlated with clinicopathologic factors and outcomes data. Results: ERCC-1 expression was evaluable in 366 cases (72%). In this group, 32% and 38% of patients received adjuvant chemotherapy and hormonal therapy, respectively. Increased ERCC-1 expression was found to be correlated with ER positivity (p 50 (p 50. To our knowledge, this is the first study investigating ERCC1 expression in patients receiving adjuvant radiation therapy for breast cancer.

A small interfering RNA screen of genes involved in DNA repair identifies tumor-specific radiosensitization by POLQ knockdown

DEFF Research Database (Denmark)

Higgins, Geoff S; Prevo, Remko; Lee, Yin-Fai

2010-01-01

The effectiveness of radiotherapy treatment could be significantly improved if tumor cells could be rendered more sensitive to ionizing radiation (IR) without altering the sensitivity of normal tissues. However, many of the key therapeutically exploitable mechanisms that determine intrinsic tumor...... radiosensitivity are largely unknown. We have conducted a small interfering RNA (siRNA) screen of 200 genes involved in DNA damage repair aimed at identifying genes whose knockdown increased tumor radiosensitivity. Parallel siRNA screens were conducted in irradiated and unirradiated tumor cells (SQ20B......) and irradiated normal tissue cells (MRC5). Using gammaH2AX foci at 24 hours after IR, we identified several genes, such as BRCA2, Lig IV, and XRCC5, whose knockdown is known to cause increased cell radiosensitivity, thereby validating the primary screening end point. In addition, we identified POLQ (DNA...

PAH-DNA adducts in environmentally exposed population in relation to metabolic and DNA repair gene polymorphisms

International Nuclear Information System (INIS)

Binkova, Blanka; Chvatalova, Irena; Lnenickova, Zdena; Milcova, Alena; Tulupova, Elena; Farmer, Peter B.; Sram, Radim J.

2007-01-01

Epidemiologic studies indicate that prolonged exposure to particulate air pollution may be associated with increased risk of cardiovascular diseases and cancer in general population. These effects may be attributable to polycyclic aromatic hydrocarbons (PAHs) adsorbed to respirable air particles. It is expected that metabolic and DNA repair gene polymorphisms may modulate individual susceptibility to PAH exposure. This study investigates relationships between exposure to PAHs, polymorphisms of these genes and DNA adducts in group of occupationally exposed policemen (EXP, N = 53, males, aged 22-50 years) working outdoors in the downtown area of Prague and in matched 'unexposed' controls (CON, N = 52). Personal exposure to eight carcinogenic PAHs (c-PAHs) was evaluated by personal samplers during working shift prior to collection of biological samples. Bulky-aromatic DNA adducts were analyzed in lymphocytes by 32 P-postlabeling assay. Polymorphisms of metabolizing (GSTM1, GSTP1, GSTT1, EPHX1, CYP1A1-MspI) and DNA repair (XRCC1, XPD) genes were determined by PCR-based RFLP assays. As potential modifiers and/or cofounders, urinary cotinine levels were analyzed by radioimmunoassay, plasma levels of vitamins A, C, E and folates by HPLC, cholesterol and triglycerides using commercial kits. During the sampling period ambient particulate air pollution was as follows: PM10 32-55 μg/m 3 , PM2.5 27-38 μg/m 3 , c-PAHs 18-22 ng/m 3 ; personal exposure to c-PAHs: 9.7 ng/m 3 versus 5.8 ng/m 3 (P 8 nucleotides versus 0.82 ± 0.23 adducts/10 8 nucleotides, P = 0.065), whereas the level of the B[a]P-'like' adduct was significantly higher in exposed group (0.122 ± 0.036 adducts/10 8 nucleotides versus 0.099 ± 0.035 adducts/10 8 nucleotides, P = 0.003). A significant difference in both the total (P < 0.05) and the B[a]P-'like' DNA adducts (P < 0.01) between smokers and nonsmokers within both groups was observed. A significant positive association between DNA adduct and cotinine

Divergent Requirement for a DNA Repair Enzyme during Enterovirus Infections

Directory of Open Access Journals (Sweden)

Sonia Maciejewski

2015-12-01

Full Text Available Viruses of the Enterovirus genus of picornaviruses, including poliovirus, coxsackievirus B3 (CVB3, and human rhinovirus, commandeer the functions of host cell proteins to aid in the replication of their small viral genomic RNAs during infection. One of these host proteins is a cellular DNA repair enzyme known as 5′ tyrosyl-DNA phosphodiesterase 2 (TDP2. TDP2 was previously demonstrated to mediate the cleavage of a unique covalent linkage between a viral protein (VPg and the 5′ end of picornavirus RNAs. Although VPg is absent from actively translating poliovirus mRNAs, the removal of VPg is not required for the in vitro translation and replication of the RNA. However, TDP2 appears to be excluded from replication and encapsidation sites during peak times of poliovirus infection of HeLa cells, suggesting a role for TDP2 during the viral replication cycle. Using a mouse embryonic fibroblast cell line lacking TDP2, we found that TDP2 is differentially required among enteroviruses. Our single-cycle viral growth analysis shows that CVB3 replication has a greater dependency on TDP2 than does poliovirus or human rhinovirus replication. During infection, CVB3 protein accumulation is undetectable (by Western blot analysis in the absence of TDP2, whereas poliovirus protein accumulation is reduced but still detectable. Using an infectious CVB3 RNA with a reporter, CVB3 RNA could still be replicated in the absence of TDP2 following transfection, albeit at reduced levels. Overall, these results indicate that TDP2 potentiates viral replication during enterovirus infections of cultured cells, making TDP2 a potential target for antiviral development for picornavirus infections.

Inactivation of ultraviolet repair in normal and xeroderma pigmentosum cells by methyl methanesulfonate

International Nuclear Information System (INIS)

Cleaver, J.E.

1982-01-01

Excision repair of ultraviolet damage in the DNA of normal and xeroderma pigmentosum (Groups C, D, and variant) cells was inactivated by exposure of cells to methyl methanesulfonate immediately before irradiation independent of the presence of 0 to 10% fetal calf serum. The inactivation could be represented by a semilog relationship between the amount of repair and methyl methanesulfonate concentration up to approximately 5 mM. The inactivation can be considered to occur as the result of alkylation of a large (about 10(6) daltons) repair enzyme complex, and the dose required to reduce repair to 37% for most cells types was between 4 and 7 mM. No consistent, large difference in sensitivity to methyl methanesulfonate was found in any xeroderma pigmentosum complementation group compared to normal cells, implying that reduced repair in these groups may be caused by small inherited changes in the amino acid composition (i.e., point mutations or small deletions) rather than by losses of major components of the repair enzyme complex

Inhibition of poly(ADP-ribose)polymerase-1 and DNA repair by uranium

Energy Technology Data Exchange (ETDEWEB)

Cooper, Karen L.; Dashner, Erica J. [Department of Pharmaceutical Sciences, College of Pharmacy, University of New Mexico Health Sciences Center, Albuquerque, NM 87131 (United States); Tsosie, Ranalda [Department of Chemistry and Biochemistry, University of Montana, Missoula, MT 59812 (United States); Cho, Young Mi [Department of Food and Nutrition, College of Human Ecology, Hanyang University, Seoul 133-791 (Korea, Republic of); Lewis, Johnnye [Department of Pharmaceutical Sciences, College of Pharmacy, University of New Mexico Health Sciences Center, Albuquerque, NM 87131 (United States); Community Environmental Health Program, University of New Mexico Health Sciences Center College of Pharmacy, Albuquerque, NM 87131 (United States); Hudson, Laurie G., E-mail: lhudson@salud.unm.edu [Department of Pharmaceutical Sciences, College of Pharmacy, University of New Mexico Health Sciences Center, Albuquerque, NM 87131 (United States)

2016-01-15

Uranium has radiological and non-radiological effects within biological systems and there is increasing evidence for genotoxic and carcinogenic properties attributable to uranium through its heavy metal properties. In this study, we report that low concentrations of uranium (as uranyl acetate; < 10 μM) is not cytotoxic to human embryonic kidney cells or normal human keratinocytes; however, uranium exacerbates DNA damage and cytotoxicity induced by hydrogen peroxide, suggesting that uranium may inhibit DNA repair processes. Concentrations of uranyl acetate in the low micromolar range inhibited the zinc finger DNA repair protein poly(ADP-ribose) polymerase (PARP)-1 and caused zinc loss from PARP-1 protein. Uranyl acetate exposure also led to zinc loss from the zinc finger DNA repair proteins Xeroderma Pigmentosum, Complementation Group A (XPA) and aprataxin (APTX). In keeping with the observed inhibition of zinc finger function of DNA repair proteins, exposure to uranyl acetate enhanced retention of induced DNA damage. Co-incubation of uranyl acetate with zinc largely overcame the impact of uranium on PARP-1 activity and DNA damage. These findings present evidence that low concentrations of uranium can inhibit DNA repair through disruption of zinc finger domains of specific target DNA repair proteins. This may provide a mechanistic basis to account for the published observations that uranium exposure is associated with DNA repair deficiency in exposed human populations. - Highlights: • Low micromolar concentration of uranium inhibits polymerase-1 (PARP-1) activity. • Uranium causes zinc loss from multiple DNA repair proteins. • Uranium enhances retention of DNA damage caused by ultraviolet radiation. • Zinc reverses the effects of uranium on PARP activity and DNA damage repair.

Effects of low dose radiation on repair processes in human lymphocytes

International Nuclear Information System (INIS)

Tuschl, H.; Altmann, H.; Kovac, R.; Topaloglou, A.; Egg, D.; Guenther, R.

1978-10-01

DNA excision repair was investigated in lymphocytes of persons occupationally exposed to low dose radiation of 222 Rn. Autoradiographic studies of unscheduled DNA synthesis and measurement of 3 H-thymidine incorporation by repair replication into double stranded and single-strand containing DNA fractions obtained by BND cellulose chromatography seem to indicate a stimulatory effect of repeated low dose radiation on repair enzymes. (author)

Attenuated DNA damage repair by trichostatin A through BRCA1 suppression.

Science.gov (United States)

Zhang, Yin; Carr, Theresa; Dimtchev, Alexandre; Zaer, Naghmeh; Dritschilo, Anatoly; Jung, Mira

2007-07-01

Recent studies have demonstrated that some histone deacetylase (HDAC) inhibitors enhance cellular radiation sensitivity. However, the underlying mechanism for such a radiosensitizing effect remains unexplored. Here we show evidence that treatment with the HDAC inhibitor trichostatin A (TSA) impairs radiation-induced repair of DNA damage. The effect of TSA on the kinetics of DNA damage repair was measured by performing the comet assay and gamma-H2AX focus analysis in radioresistant human squamous carcinoma cells (SQ-20B). TSA exposure increased the amount of radiation-induced DNA damage and slowed the repair kinetics. Gene expression profiling also revealed that a majority of the genes that control cell cycle, DNA replication and damage repair processes were down-regulated after TSA exposure, including BRCA1. The involvement of BRCA1 was further demonstrated by expressing ectopic wild-type BRCA1 in a BRCA1 null cell line (HCC-1937). TSA treatment enhanced radiation sensitivity of HCC-1937/wtBRCA1 clonal cells, which restored cellular radiosensitivity (D(0) = 1.63 Gy), to the control level (D(0) = 1.03 Gy). However, TSA had no effect on the level of radiosensitivity of BRCA1 null cells. Our data demonstrate for the first time that TSA treatment modulates the radiation-induced DNA damage repair process, in part by suppressing BRCA1 gene expression, suggesting that BRCA1 is one of molecular targets of TSA.

A plant gene for photolyase: an enzyme catalyzing the repair of UV-light-induced DNA damage

International Nuclear Information System (INIS)

Batschauer, A.

1993-01-01

Photolyases are thought to be critical components of the defense of plants against damage to DNA by solar ultraviolet light, but nothing is known about their molecular or enzymatic nature. The molecular cloning of a photolyase from mustard (Sinapis alba) described here is intended to increase the knowledge about this important repair mechanism in plant species at a molecular level. The gene encodes a polypeptide of 501 amino acids with a predicted molecular mass of 57 kDa. There is a strong sequence similarity to bacterial and yeast photolyases, with a close relationship to enzymes with a deazaflavin chromophor. The plant photolyase is shown to be functional in Escherichia coli which also indicates conservation of photolyases during evolution. It is demonstrated that photolyase expression in plants is light induced, thus providing good evidence for the adaptation of plants to their environment in order to diminish the harmful effects of sunlight. (author)

Polymorphisms of homologous recombination genes and clinical outcomes of non-small cell lung cancer patients treated with definitive radiotherapy.

Directory of Open Access Journals (Sweden)

Ming Yin

Full Text Available The repair of DNA double-strand breaks (DSBs is the major mechanism to maintain genomic stability in response to irradiation. We hypothesized that genetic polymorphisms in DSB repair genes may affect clinical outcomes among non-small cell lung cancer (NSCLC patients treated with definitive radio(chemotherapy. We genotyped six potentially functional single nucleotide polymorphisms (SNPs (i.e., RAD51 -135G>C/rs1801320 and -172G>T/rs1801321, XRCC2 4234G>C/rs3218384 and R188H/rs3218536 G>A, XRCC3 T241M/rs861539 and NBN E185Q/rs1805794 and estimated their associations with overall survival (OS and radiation pneumonitis (RP in 228 NSCLC patients. We found a predictive role of RAD51 -135G>C SNP in RP development (adjusted hazard ratio [HR] = 0.52, 95% confidence interval [CI], 0.31-0.86, P = 0.010 for CG/CC vs. GG. We also found that RAD51 -135G>C and XRCC2 R188H SNPs were independent prognostic factors for overall survival (adjusted HR = 1.70, 95% CI, 1.14-2.62, P = 0.009 for CG/CC vs. GG; and adjusted HR = 1.70; 95% CI, 1.02-2.85, P = 0.043 for AG vs. GG, respectively and that the SNP-survival association was most pronounced in the presence of RP. Our study suggests that HR genetic polymorphisms, particularly RAD51 -135G>C, may influence overall survival and radiation pneumonitis in NSCLC patients treated with definitive radio(chemotherapy. Large studies are needed to confirm our findings.

Challenges in biotechnology at LLNL: from genes to proteins; TOPICAL

International Nuclear Information System (INIS)

Albala, J S

1999-01-01

This effort has undertaken the task of developing a link between the genomics, DNA repair and structural biology efforts within the Biology and Biotechnology Research Program at LLNL. Through the advent of the I.M.A.G.E. (Integrated Molecular Analysis of Genomes and their Expression) Consortium, a world-wide effort to catalog the largest public collection of genes, accepted and maintained within BBRP, it is now possible to systematically express the protein complement of these to further elucidate novel gene function and structure. The work has ensued in four phases, outlined as follows: (1) Gene and System selection; (2) Protein expression and purification; (3) Structural analysis; and (4) biological integration. Proteins to be expressed have been those of high programmatic interest. This includes, in particular, proteins involved in the maintenance of genome integrity, particularly those involved in the repair of DNA damage, including ERCC1, ERCC4, XRCC2, XRCC3, XRCC9, HEX1, APN1, p53, RAD51B, RAD51C, and RAD51. Full-length cDNA cognates of selected genes were isolated, and cloned into baculovirus-based expression vectors. The baculoviral expression system for protein over-expression is now well-established in the Albala laboratory. Procedures have been successfully optimized for full-length cDNA clining into expression vectors for protein expression from recombinant constructs. This includes the reagents, cell lines, techniques necessary for expression of recombinant baculoviral constructs in Spodoptera frugiperda (Sf9) cells. The laboratory has also generated a high-throughput baculoviral expression paradigm for large scale expression and purification of human recombinant proteins amenable to automation

Uracil excision repair in Mycobacterium tuberculosis cell-free extracts.

Science.gov (United States)

Kumar, Pradeep; Bharti, Sanjay Kumar; Varshney, Umesh

2011-05-01

Uracil excision repair is ubiquitous in all domains of life and initiated by uracil DNA glycosylases (UDGs) which excise the promutagenic base, uracil, from DNA to leave behind an abasic site (AP-site). Repair of the resulting AP-sites requires an AP-endonuclease, a DNA polymerase, and a DNA ligase whose combined activities result in either short-patch or long-patch repair. Mycobacterium tuberculosis, the causative agent of tuberculosis, has an increased risk of accumulating uracils because of its G + C-rich genome, and its niche inside host macrophages where it is exposed to reactive nitrogen and oxygen species, two major causes of cytosine deamination (to uracil) in DNA. In vitro assays to study DNA repair in this important human pathogen are limited. To study uracil excision repair in mycobacteria, we have established assay conditions using cell-free extracts of M. tuberculosis and M. smegmatis (a fast-growing mycobacterium) and oligomer or plasmid DNA substrates. We show that in mycobacteria, uracil excision repair is completed primarily via long-patch repair. In addition, we show that M. tuberculosis UdgB, a newly characterized family 5 UDG, substitutes for the highly conserved family 1 UDG, Ung, thereby suggesting that UdgB might function as backup enzyme for uracil excision repair in mycobacteria. Copyright © 2011 Elsevier Ltd. All rights reserved.

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.

Oxidative DNA damage & repair: An introduction.

Science.gov (United States)

Cadet, Jean; Davies, Kelvin J A

2017-06-01

This introductory article should be viewed as a prologue to the Free Radical Biology & Medicine Special Issue devoted to the important topic of Oxidatively Damaged DNA and its Repair. This special issue is dedicated to Professor Tomas Lindahl, co-winner of the 2015 Nobel Prize in Chemistry for his seminal discoveries in the area repair of oxidatively damaged DNA. In the past several years it has become abundantly clear that DNA oxidation is a major consequence of life in an oxygen-rich environment. Concomitantly, survival in the presence of oxygen, with the constant threat of deleterious DNA mutations and deletions, has largely been made possible through the evolution of a vast array of DNA repair enzymes. The articles in this Oxidatively Damaged DNA & Repair special issue detail the reactions by which intracellular DNA is oxidatively damaged, and the enzymatic reactions and pathways by which living organisms survive such assaults by repair processes. Copyright © 2017 Elsevier Inc. All rights reserved.

RTEL1 contributes to DNA replication and repair and telomere maintenance

NARCIS (Netherlands)

Uringa, Evert-Jan; Lisaingo, Kathleen; Pickett, Hilda A.; Brind'Amour, Julie; Rohde, Jan-Hendrik; Zelensky, Alex; Essers, Jeroen; Lansdorp, Peter M.

2012-01-01

Telomere maintenance and DNA repair are important processes that protect the genome against instability. mRtel1, an essential helicase, is a dominant factor setting telomere length in mice. In addition, mRtel1 is involved in DNA double-strand break repair. The role of mRtel1 in telomere maintenance

RTEL1 contributes to DNA replication and repair and telomere maintenance

NARCIS (Netherlands)

E.J. Uringa; K. Lisaingo (Kathleen); H.A. Pickett (Hilda); J. Brind'Amour (Julie); J.-H. Rohde (Jan-Hendrik); A. Zelensky (Alexander); J. Essers (Jeroen); P.M. Lansdorp (Peter)

2012-01-01

textabstractTelomere maintenance and DNA repair are important processes that protect the genome against instability. mRtel1, an essential helicase, is a dominant factor setting telomere length in mice. In addition, mRtel1 is involved in DNA double-strand break repair. The role of mRtel1 in telomere

Association of single nucleotide polymorphisms in the genes ATM, GSTP1, SOD2, TGFB1, XPD and XRCC1 with risk of severe erythema after breast conserving radiotherapy

International Nuclear Information System (INIS)

Raabe, Annette; Derda, Katharina; Reuther, Sebastian; Szymczak, Silke; Borgmann, Kerstin; Hoeller, Ulrike; Ziegler, Andreas; Petersen, Cordula; Dikomey, Ekkehard

2012-01-01

To examine the association of polymorphisms in ATM (codon 158), GSTP1 (codon 105), SOD2 (codon 16), TGFB1 (position −509), XPD (codon 751), and XRCC1 (codon 399) with the risk of severe erythema after breast conserving radiotherapy. Retrospective analysis of 83 breast cancer patients treated with breast conserving radiotherapy. A total dose of 50.4 Gy was administered, applying 1.8 Gy/fraction within 42 days. Erythema was evaluated according to the Radiation Therapy Oncology Group (RTOG) score. DNA was extracted from blood samples and polymorphisms were determined using either the Polymerase Chain Reaction based Restriction-Fragment-Length-Polymorphism (PCR-RFL) technique or Matrix-Assisted-Laser-Desorption/Ionization –Time-Of-Flight-Mass-Spectrometry (MALDI-TOF). Relative excess heterozygosity (REH) was investigated to check compatibility of genotype frequencies with Hardy-Weinberg equilibrium (HWE). In addition, p-values from the standard exact HWE lack of fit test were calculated using 100,000 permutations. HWE analyses were performed using R. Fifty-six percent (46/83) of all patients developed erythema of grade 2 or 3, with this risk being higher for patients with large breast volume (odds ratio, OR = 2.55, 95% confidence interval, CI: 1.03–6.31, p = 0.041). No significant association between SNPs and risk of erythema was found when all patients were considered. However, in patients with small breast volume the TGFB1 SNP was associated with erythema (p = 0.028), whereas the SNP in XPD showed an association in patients with large breast volume (p = 0.046). A risk score based on all risk alleles was neither significant in all patients nor in patients with small or large breast volume. Risk alleles of most SNPs were different compared to a previously identified risk profile for fibrosis. The genetic risk profile for erythema appears to be different for patients with small and larger breast volume. This risk profile seems to be specific for erythema as

46 CFR 167.30-1 - Notice of repairs or alterations required.

Science.gov (United States)

2010-10-01

... Inspection, whenever repairs or alterations are required, or will be made on a nautical school ship. (b) Whenever a nautical school ship is placed upon the dock, it shall be the duty of the master, owner or agent... NAUTICAL SCHOOL SHIPS Repairs or Alterations § 167.30-1 Notice of repairs or alterations required. (a) It...

The AlkB Family of Fe(II)/α-Ketoglutarate-dependent Dioxygenases: Repairing Nucleic Acid Alkylation Damage and Beyond.

Science.gov (United States)

Fedeles, Bogdan I; Singh, Vipender; Delaney, James C; Li, Deyu; Essigmann, John M

2015-08-21

The AlkB family of Fe(II)- and α-ketoglutarate-dependent dioxygenases is a class of ubiquitous direct reversal DNA repair enzymes that remove alkyl adducts from nucleobases by oxidative dealkylation. The prototypical and homonymous family member is an Escherichia coli "adaptive response" protein that protects the bacterial genome against alkylation damage. AlkB has a wide variety of substrates, including monoalkyl and exocyclic bridged adducts. Nine mammalian AlkB homologs exist (ALKBH1-8, FTO), but only a subset functions as DNA/RNA repair enzymes. This minireview presents an overview of the AlkB proteins including recent data on homologs, structural features, substrate specificities, and experimental strategies for studying DNA repair by AlkB family proteins. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Simultaneous demonstration of UV-type and ionizing radiation-type DNA repair by the nucleoid sedimentation technique

International Nuclear Information System (INIS)

Aldenhoff, P.; Sperling, K.

1984-01-01

The nucleoid sedimentation technique is one of the most sensitive methods for measuring DNA excision repair. With this technique, it is shown that both UV- and ionizing radiation-type repair (the latter induced by bleomycin) can be discriminated in HeLa and normal diploid cells using 1-β-D-arabinofuranosylcytosine. The latter compound inhibits UV-type repair synthesis, and thus causes DNA breaks due to enzymic incision to persist, but has no effect on rejoining DNA after ionizing radiation-type damage. It was then possible to prove that 4-nitroquinoline-1-oxide induces both types of lesions which are repaired simultaneously. This effect could be demonstrated in HeLa and normal human diploid cells in a single experimental set-up. (Auth.)

Repair of DNA treated with γ-irradiation and chemical carcinogens. Progress report, June 1, 1975--January 31, 1976

International Nuclear Information System (INIS)

Goldthwait, D.A.

1976-02-01

Enzymatic mechanisms of DNA repair are under investigation. From E. coli an endonuclease active on apurinic acid has been separated from endonuclease II active on DNA treated with methylmethane sulfonate, methylnitrosourea, 7-bromomethyl-12-methylbenz[a]anthracene and γ-irradiation. Mutants have been identified for each enzyme. The purification of both enzymes is proceeding and the mechanism of action of endonuclease II which has both an N-glycosidase and a phosphodiester hydrolase activity is under investigation. Evidence demonstrating exonuclease III is an enzyme separate from the apurinic acid endonuclease and endonuclease II has been accumulated but this is still under investigation. Work has been completed on 7-bromomethyl-12-methylbenz[a]anthracene treated DNA as a substrate and is being continued on the inhibitory effects of phorbol ester on endonuclease II. Finally, the properties of an enzyme from calf liver active on depurinated DNA have been compared with those of a comparable enzyme from calf thymus. Evidence that they are isozymes has been found

S1 nuclease from Aspergillus oryzae for the detection of DNA damage and repair in the gamma-irradiated intracerebral rat gliosarcoma 9L

International Nuclear Information System (INIS)

Gutin, P.H.; Hilton, J.; Fein, V.J.; Allen, A.E.; Walker, M.D.

1977-01-01

DNA damage and repair in a rat brain tumor following irradiation in vivo were measured by analysis of the rate of strand separation of the tumor DNA in alkali. Tumors were removed after irradiation and mechanically dissociated to a cellular suspension. Tumor cells were injected into alkali (pH 12) for 20 min at 22 0 C. The fraction of tumor DNA remaining double-stranded after this exposure to alkali was determined by its resistance to S 1 nuclease from Aspergillus oryzae. Double-stranded DNA remains (after enzyme exposure) acid-precipitable for fluorescent assay. The double-stranded fraction after exposure to alkali decreases with increasing radiation dose following first-order kinetics. DNA from tumors excised at intervals after irradiation showed a greater double-stranded fraction in alkali than that from tumors excised immediately, indicating repair of single-strand breaks. Repair of damage produced by 600 rad proceeded with a half-time of approximately 15 min

Divergent Requirement for a DNA Repair Enzyme during Enterovirus Infections.

Science.gov (United States)

Maciejewski, Sonia; Nguyen, Joseph H C; Gómez-Herreros, Fernando; Cortés-Ledesma, Felipe; Caldecott, Keith W; Semler, Bert L

2015-12-29

Viruses of the Enterovirus genus of picornaviruses, including poliovirus, coxsackievirus B3 (CVB3), and human rhinovirus, commandeer the functions of host cell proteins to aid in the replication of their small viral genomic RNAs during infection. One of these host proteins is a cellular DNA repair enzyme known as 5' tyrosyl-DNA phosphodiesterase 2 (TDP2). TDP2 was previously demonstrated to mediate the cleavage of a unique covalent linkage between a viral protein (VPg) and the 5' end of picornavirus RNAs. Although VPg is absent from actively translating poliovirus mRNAs, the removal of VPg is not required for the in vitro translation and replication of the RNA. However, TDP2 appears to be excluded from replication and encapsidation sites during peak times of poliovirus infection of HeLa cells, suggesting a role for TDP2 during the viral replication cycle. Using a mouse embryonic fibroblast cell line lacking TDP2, we found that TDP2 is differentially required among enteroviruses. Our single-cycle viral growth analysis shows that CVB3 replication has a greater dependency on TDP2 than does poliovirus or human rhinovirus replication. During infection, CVB3 protein accumulation is undetectable (by Western blot analysis) in the absence of TDP2, whereas poliovirus protein accumulation is reduced but still detectable. Using an infectious CVB3 RNA with a reporter, CVB3 RNA could still be replicated in the absence of TDP2 following transfection, albeit at reduced levels. Overall, these results indicate that TDP2 potentiates viral replication during enterovirus infections of cultured cells, making TDP2 a potential target for antiviral development for picornavirus infections. Picornaviruses are one of the most prevalent groups of viruses that infect humans and livestock worldwide. These viruses include the human pathogens belonging to the Enterovirus genus, such as poliovirus, coxsackievirus B3 (CVB3), and human rhinovirus. Diseases caused by enteroviruses pose a major problem

Measurement of enzyme-sensitive sites in uv- or. gamma. -irradiated human cells using Micrococcus luteus extracts

Energy Technology Data Exchange (ETDEWEB)

Paterson, M C; Smith, B P; Smith, P J

1979-01-01

The study of DNA damage and its enzymatic repair has undergone rapid expansion in recent years. Laboratory observations at the molecular level have been facilitated greatly by the availability of a battery of physicochemical techniques capable of monitoring hallmarks of different repair mechanisms. One technique exploits the unique ability of certain putative repair enzymes (endonucleases and DNA glycosylases of prokaryotic origin) to selectively attack DNA at sites containing altered base or sugar residues; the sites are subsequently observed as single-strand break, by velocity sedimentatn of the DNA in an alkaline sucrose gradient. Incubation of carcinogen-treated cell cultures for varying times, followed by enzymatic analysis of their radionuclide-labeled DNA, yields the time course of disappearace of such sites; this is taken as an indirect expression of the kinetics of lesion repair. Although there are several variations of the enzymatic assay two basic protocols are in current use. The only major difference is the way in which the damaged DNA is treated with the lesion-detecting enzyme(s). In one protocol this is achieved by rendering the cells porous to extracellular proteins prior to incubation with the test enzyme(s). In the second protocol the damaged DNA is extracted from the cells and is then exposed to the lesion-recognizing enzyme(s) in vitro. The enzymatic assay developed in our laboratory follows this second protocol, and the procedure is described.

Detection of individual radiosensitivity by radiation–induced micronuclei in human peripheral blood lymphocytes and polymorphisms in DNA repair genes

Energy Technology Data Exchange (ETDEWEB)

Staynova, A.; Hadjidekova, V.; Popova, L.; Hristova, R. [Radiation Genetics Laboratory, National Centre of Radiobology and Radiation Protection, Sofia (Bulgaria); Savov, A. [National Genetic Laboratory, University Hospital of Obstetrics and Gynecology, Sofia (Bulgaria)

2013-07-01

Aim: To investigate the association of two polymorphisms – in XRCC1 gene (Arg399Gln) and in APE1 gene (Asp148Glu) and the radiation induced frequency of micronuclei in human peripheral blood lymphocytes. Material and methods: Genomic DNA from 34 cancer patients and 52 controls were genotyped using PCR–RFLP technique. Micronucleus test (MNT) was performed on 15 cancer patients and 15 controls, before and after in vitro irradiation with 2Gy gamma rays. Results: The data showed that cancer patients had a significantly higher spontaneous frequency of cells with micronuclei than controls (P=0.009). No statistical difference was registered when comparing the mean frequency of cells with micronuclei after in vitro irradiation between these groups. Four subjects were selected as radiosensitive after applying cut–off of the mean frequency of radiation induced micronuclei. Three of them are carriers of the XRCC1 399Gln allele and two of them are carriers of the APE1 148Glu allele. (author)

Inhibition of poly(ADP-ribose)polymerase-1 and DNA repair by uranium.

Science.gov (United States)

Cooper, Karen L; Dashner, Erica J; Tsosie, Ranalda; Cho, Young Mi; Lewis, Johnnye; Hudson, Laurie G

2016-01-15

Uranium has radiological and non-radiological effects within biological systems and there is increasing evidence for genotoxic and carcinogenic properties attributable to uranium through its heavy metal properties. In this study, we report that low concentrations of uranium (as uranyl acetate; uranium exacerbates DNA damage and cytotoxicity induced by hydrogen peroxide, suggesting that uranium may inhibit DNA repair processes. Concentrations of uranyl acetate in the low micromolar range inhibited the zinc finger DNA repair protein poly(ADP-ribose) polymerase (PARP)-1 and caused zinc loss from PARP-1 protein. Uranyl acetate exposure also led to zinc loss from the zinc finger DNA repair proteins Xeroderma Pigmentosum, Complementation Group A (XPA) and aprataxin (APTX). In keeping with the observed inhibition of zinc finger function of DNA repair proteins, exposure to uranyl acetate enhanced retention of induced DNA damage. Co-incubation of uranyl acetate with zinc largely overcame the impact of uranium on PARP-1 activity and DNA damage. These findings present evidence that low concentrations of uranium can inhibit DNA repair through disruption of zinc finger domains of specific target DNA repair proteins. This may provide a mechanistic basis to account for the published observations that uranium exposure is associated with DNA repair deficiency in exposed human populations. Copyright © 2015 Elsevier Inc. All rights reserved.

EGR1 induces tenogenic differentiation of tendon stem cells and promotes rabbit rotator cuff repair.

Science.gov (United States)

Tao, Xu; Liu, Junpeng; Chen, Lei; Zhou, You; Tang, Kanglai

2015-01-01

The rate of healing failure after surgical repair of chronic rotator cuff tears is considerably high. The aim of this study was to investigate the function of the zinc finger transcription factor early growth response 1 (EGR1) in the differentiation of tendon stem cells (TSCs) and in tendon formation, healing, and tendon tear repair using an animal model of rotator cuff repair. Tenocyte, adipocyte, osteocyte, and chondrocyte differentiation as well as the expression of related genes were determined in EGR1-overexpressing TSCs (EGR1-TSCs) using tissue-specific staining, immunofluorescence staining, quantitative PCR, and western blotting. A rabbit rotator cuff repair model was established, and TSCs and EGR1-TSCs in a fibrin glue carrier were applied onto repair sites. The rabbits were sacrificed 8 weeks after repair operation, and tissues were histologically evaluated and tenocyte-related gene expression was determined. EGR1 induced tenogenic differentiation of TSCs and inhibited non-tenocyte differentiation of TSCs. Furthermore, EGR1 promoted tendon repair in a rabbit model of rotator cuff injury. The BMP12/Smad1/5/8 signaling pathway was involved in EGR1-induced tenogenic differentiation and rotator cuff tendon repair. EGR1 plays a key role in tendon formation, healing, and repair through BMP12/Smad1/5/8 pathway. EGR1-TSCs is a promising treatment for rotator cuff tendon repair surgeries. © 2015 S. Karger AG, Basel.

EGR1 Induces Tenogenic Differentiation of Tendon Stem Cells and Promotes Rabbit Rotator Cuff Repair

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Xu Tao

2015-01-01

Full Text Available Background/Aims: The rate of healing failure after surgical repair of chronic rotator cuff tears is considerably high. The aim of this study was to investigate the function of the zinc finger transcription factor early growth response 1 (EGR1 in the differentiation of tendon stem cells (TSCs and in tendon formation, healing, and tendon tear repair using an animal model of rotator cuff repair. Methods: Tenocyte, adipocyte, osteocyte, and chondrocyte differentiation as well as the expression of related genes were determined in EGR1-overexpressing TSCs (EGR1-TSCs using tissue-specific staining, immunofluorescence staining, quantitative PCR, and western blotting. A rabbit rotator cuff repair model was established, and TSCs and EGR1-TSCs in a fibrin glue carrier were applied onto repair sites. The rabbits were sacrificed 8 weeks after repair operation, and tissues were histologically evaluated and tenocyte-related gene expression was determined. Results: EGR1 induced tenogenic differentiation of TSCs and inhibited non-tenocyte differentiation of TSCs. Furthermore, EGR1 promoted tendon repair in a rabbit model of rotator cuff injury. The BMP12/Smad1/5/8 signaling pathway was involved in EGR1-induced tenogenic differentiation and rotator cuff tendon repair. Conclusion: EGR1 plays a key role in tendon formation, healing, and repair through BMP12/Smad1/5/8 pathway. EGR1-TSCs is a promising treatment for rotator cuff tendon repair surgeries.

Studies on the enzymes produced by Basidiomycetes. Part 1. The production of crude enzymes

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Hong, J. S.; Kim, D.H.

1981-01-01

Cellulase, protease, and xylanase, formation by the basidiomycetes, Pleurotus ostreatus 301 and Lentinus edodes 3-1 in growth on rice straw medium were studied. Cultural conditions adequate for enzyme production and effects of various materials and inorganic salts added to the rice straw media were investigated. Lentinus edodes 3-1 was an excellent producer of cellulase and xylanase, and Pleurotus ostreatus 301 of protease. The optimum conditions for enzyme production were 30 degrees for cellulase production and at 25 degrees for xylanase and protease production, with 75% moisture content and initial pH of 5.0-6.0. The appropriate incubation times for enzyme production were 30 days and 35 days for Pleurotus ostreatus 301 and Lentinus edodes 3-1, respectively. Among the various materials added, defatted soybean, defatted rape seed, or defatted sesame were all effective in enzyme production but reduced mycelial growth. Rice bran was also effective, particularly at a 30% concentration. The addition of inorganic salts enhanced enzyme production. Among inorganic salts, the optimum concentration of CaCO3 was 5%, and that of CaSO4 was 2%.

RTEL1 contributes to DNA replication and repair and telomere maintenance.

Science.gov (United States)

Uringa, Evert-Jan; Lisaingo, Kathleen; Pickett, Hilda A; Brind'Amour, Julie; Rohde, Jan-Hendrik; Zelensky, Alex; Essers, Jeroen; Lansdorp, Peter M

2012-07-01

Telomere maintenance and DNA repair are important processes that protect the genome against instability. mRtel1, an essential helicase, is a dominant factor setting telomere length in mice. In addition, mRtel1 is involved in DNA double-strand break repair. The role of mRtel1 in telomere maintenance and genome stability is poorly understood. Therefore we used mRtel1-deficient mouse embryonic stem cells to examine the function of mRtel1 in replication, DNA repair, recombination, and telomere maintenance. mRtel1-deficient mouse embryonic stem cells showed sensitivity to a range of DNA-damaging agents, highlighting its role in replication and genome maintenance. Deletion of mRtel1 increased the frequency of sister chromatid exchange events and suppressed gene replacement, demonstrating the involvement of the protein in homologous recombination. mRtel1 localized transiently at telomeres and is needed for efficient telomere replication. Of interest, in the absence of mRtel1, telomeres in embryonic stem cells appeared relatively stable in length, suggesting that mRtel1 is required to allow extension by telomerase. We propose that mRtel1 is a key protein for DNA replication, recombination, and repair and efficient elongation of telomeres by telomerase.

Complementary roles of KCa3.1 channels and β1-integrin during alveolar epithelial repair.

Science.gov (United States)

Girault, Alban; Chebli, Jasmine; Privé, Anik; Trinh, Nguyen Thu Ngan; Maillé, Emilie; Grygorczyk, Ryszard; Brochiero, Emmanuelle

2015-09-04

Extensive alveolar epithelial injury and remodelling is a common feature of acute lung injury and acute respiratory distress syndrome (ARDS) and it has been established that epithelial regeneration, and secondary lung oedema resorption, is crucial for ARDS resolution. Much evidence indicates that K(+) channels are regulating epithelial repair processes; however, involvement of the KCa3.1 channels in alveolar repair has never been investigated before. Wound-healing assays demonstrated that the repair rates were increased in primary rat alveolar cell monolayers grown on a fibronectin matrix compared to non-coated supports, whereas an anti-β1-integrin antibody reduced it. KCa3.1 inhibition/silencing impaired the fibronectin-stimulated wound-healing rates, as well as cell migration and proliferation, but had no effect in the absence of coating. We then evaluated a putative relationship between KCa3.1 channel and the migratory machinery protein β1-integrin, which is activated by fibronectin. Co-immunoprecipitation and immunofluorescence experiments indicated a link between the two proteins and revealed their cellular co-distribution. In addition, we demonstrated that KCa3.1 channel and β1-integrin membrane expressions were increased on a fibronectin matrix. We also showed increased intracellular calcium concentrations as well as enhanced expression of TRPC4, a voltage-independent calcium channel belonging to the large TRP channel family, on a fibronectin matrix. Finally, wound-healing assays showed additive effects of KCa3.1 and TRPC4 inhibitors on alveolar epithelial repair. Taken together, our data demonstrate for the first time complementary roles of KCa3.1 and TRPC4 channels with extracellular matrix and β1-integrin in the regulation of alveolar repair processes.

The repair of damage to DNA in different cell types