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

PubMed

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.

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

PubMed

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.

XRCC1 Arg399Gln was associated with repair capacity for DNA damage induced by occupational chromium exposure

PubMed Central

2012-01-01

Background Occupational chromium exposure may induce DNA damage and lead to lung cancer and other work-related diseases. DNA repair gene polymorphisms, which may alter the efficiency of DNA repair, thus may contribute to genetic susceptibility of DNA damage. The aim of this study was to test the hypothesis that the genetic variations of 9 major DNA repair genes could modulate the hexavalent chromium (Cr (VI))-induced DNA damage. Findings The median (P25-P75) of Olive tail moment was 0.93 (0.58–1.79) for individuals carrying GG genotype of XRCC1 Arg399Gln (G/A), 0.73 (0.46–1.35) for GA heterozygote and 0.50 (0.43–0.93) for AA genotype. Significant difference was found among the subjects with three different genotypes (P = 0.048) after adjusting the confounding factors. The median of Olive tail moment of the subjects carrying A allele (the genotypes of AA and GA) was 0.66 (0.44–1.31), which was significantly lower than that of subjects with GG genotype (P = 0.043). The A allele conferred a significantly reduced risk of DNA damage with the OR of 0.39 (95% CI: 0.15–0.99, P = 0.048). No significant association was found between the XRCC1Arg194Trp, ERCC1 C8092A, ERCC5 His1104Asp, ERCC6 Gly399Asp, GSTP1 Ile105Val, OGG1 Ser326Cys, XPC Lys939Gln, XPD Lys751Gln and DNA damage. Conclusion The polymorphism of Arg399Gln in XRCC1 was associated with the Cr (VI)- induced DNA damage. XRCC1 Arg399Gln may serve as a genetic biomarker of susceptibility for Cr (VI)- induced DNA damage. PMID:22642904

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

PubMed

Bukowski, Karol; Woźniak, Katarzyna

2018-03-09

Genetic polymorphism is associated with the occurrence of at least 2 different alleles in the locus with a frequency higher than 1% in the population. Among polymorphisms we can find single nucleotide polymorphism (SNP) and polymorphism of variable number of tandem repeats. The presence of certain polymorphisms in genes encoding DNA repair enzymes is associated with the speed and efficiency of DNA repair and can protect or expose humans to the effects provoked by xenobiotics. Chemicals, such as lead, arsenic pesticides are considered to exhibit strong toxicity. There are many different polymorphisms in genes encoding DNA repair enzymes, which determine the speed and efficiency of DNA damage repair induced by these xenobiotics. In the case of lead, the influence of various polymorphisms, such as APE1 (apurinic/apyrimidinic endonuclease 1) (rs1130409), hOGG1 (human 8-oxoguanine glycosylase) (rs1052133), XRCC1 (X-ray repair cross-complementing protein group 1) (rs25487), XRCC1 (rs1799782) and XRCC3 (X-ray repair cross-complementing protein group 3) (rs861539) were described. For arsenic polymorphisms, such as ERCC2 (excision repair cross-complementing) (rs13181), XRCC3 (rs861539), APE1 (rs1130409) and hOGG1 (rs1052133) were examined. As to pesticides, separate and combined effects of polymorphisms in genes encoding DNA repair enzymes, such as XRCC1 (rs1799782), hOGG1 (rs1052133), XRCC4 (X-ray repair cross-complementing protein group 4) (rs28360135) and the gene encoding the detoxification enzyme PON1 paraoxonase (rs662) were reported. Med Pr 2018;69(2):225-235. This work is available in Open Access model and licensed under a CC BY-NC 3.0 PL license.

Genetic variation in DNA repair gene XRCC7 (G6721T) and susceptibility to breast cancer.

PubMed

Nasiri, Meysam; Saadat, Iraj; Omidvari, Shahpour; Saadat, Mostafa

2012-08-15

The human XRCC7 is a DNA double-strand break (DSBs) repair gene, involved in non-homologous end joining (NHEJ). It is speculated that DNA DSBs repair have an important role during development of breast cancer. The human XRCC7 is a NHEJ DSBs repair gene. Genetic variation G6721T of XRCC7 (rs7003908) is located in the intron 8 of the gene. This polymorphism may regulate splicing and cause mRNA instability. In the present study, we specifically investigated whether common G6721T genetic variant of XRCC7 was associated with an altered risk of breast cancer. The present study included 362 females with breast cancer. Age frequency-matched controls (362 persons) were randomly selected from the healthy female blood donors, according to the age distribution of the cases. Using RFLP-PCR based method, the polymorphism of XRCC7 was determined. The TG (OR=1.20, 95% CI: 0.83-1.74, P=0.320) and TT (OR=1.01, 95% CI: 0.67-1.53, P=0.933) genotypes had no significant effect on risk of breast cancer, in comparison with the GG genotype. Our present findings indicate that the TT and TG genotypes were not associated with an altered breast cancer risk. Copyright © 2012 Elsevier B.V. All rights reserved.

Structural and functional characterization of the PNKP–XRCC4–LigIV DNA repair complex

DOE PAGES

Aceytuno, R.  Daniel; Piett, Cortt G.; Havali-Shahriari, Zahra; ...

2017-04-27

Non-homologous end joining (NHEJ) repairs DNA double strand breaks in non-cycling eukaryotic cells. NHEJ relies on polynucleotide kinase/phosphatase (PNKP), which generates 5'-phosphate/3'-hydroxyl DNA termini that are critical for ligation by the NHEJ DNA ligase, LigIV. PNKP and LigIV require the NHEJ scaffolding protein, XRCC4. The PNKP FHA domain binds to the CK2-phosphorylated XRCC4 C-terminal tail, while LigIV uses its tandem BRCT repeats to bind the XRCC4 coiled-coil. Yet, the assembled PNKP-XRCC4-LigIV complex remains uncharacterized. Here, we report purification and characterization of a recombinant PNKP-XRCC4-LigIV complex. We show that the stable binding of PNKP in this complex requires XRCC4 phosphorylation andmore » that only one PNKP protomer binds per XRCC4 dimer. Small angle X-ray scattering (SAXS) reveals a flexiblemultistate complex that suggests that both the PNKP FHA and catalytic domains contact the XRCC4 coiled-coil and LigIV BRCT repeats. Hydrogen-deuterium exchange indicates protection of a surface on the PNKP phosphatase domain that may contact XRCC4-LigIV. Amutation on this surface (E326K) causes the hereditary neuro-developmental disorder, MCSZ. This mutation impairs PNKP recruitment to damaged DNA in human cells and provides a possible disease mechanism. Together, this work unveils multipoint contacts between PNKP and XRCC4-LigIV that regulate PNKP recruitment and activity within NHEJ.« less

Structural and functional characterization of the PNKP–XRCC4–LigIV DNA repair complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aceytuno, R.  Daniel; Piett, Cortt G.; Havali-Shahriari, Zahra

Non-homologous end joining (NHEJ) repairs DNA double strand breaks in non-cycling eukaryotic cells. NHEJ relies on polynucleotide kinase/phosphatase (PNKP), which generates 5'-phosphate/3'-hydroxyl DNA termini that are critical for ligation by the NHEJ DNA ligase, LigIV. PNKP and LigIV require the NHEJ scaffolding protein, XRCC4. The PNKP FHA domain binds to the CK2-phosphorylated XRCC4 C-terminal tail, while LigIV uses its tandem BRCT repeats to bind the XRCC4 coiled-coil. Yet, the assembled PNKP-XRCC4-LigIV complex remains uncharacterized. Here, we report purification and characterization of a recombinant PNKP-XRCC4-LigIV complex. We show that the stable binding of PNKP in this complex requires XRCC4 phosphorylation andmore » that only one PNKP protomer binds per XRCC4 dimer. Small angle X-ray scattering (SAXS) reveals a flexiblemultistate complex that suggests that both the PNKP FHA and catalytic domains contact the XRCC4 coiled-coil and LigIV BRCT repeats. Hydrogen-deuterium exchange indicates protection of a surface on the PNKP phosphatase domain that may contact XRCC4-LigIV. Amutation on this surface (E326K) causes the hereditary neuro-developmental disorder, MCSZ. This mutation impairs PNKP recruitment to damaged DNA in human cells and provides a possible disease mechanism. Together, this work unveils multipoint contacts between PNKP and XRCC4-LigIV that regulate PNKP recruitment and activity within NHEJ.« less

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fukuchi, Mikoto; Wanotayan, Rujira; Liu, Sicheng

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,more » 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.« less

Oxidative Stress in Mammalian Cells Impinges on the Cysteines Redox State of Human XRCC3 Protein and on Its Cellular Localization

PubMed Central

Girard, Pierre-Marie; Graindorge, Dany; Smirnova, Violetta; Rigolet, Pascal; Francesconi, Stefania; Scanlon, Susan; Sage, Evelyne

2013-01-01

In vertebrates, XRCC3 is one of the five Rad51 paralogs that plays a central role in homologous recombination (HR), a key pathway for maintaining genomic stability. While investigating the potential role of human XRCC3 (hXRCC3) in the inhibition of DNA replication induced by UVA radiation, we discovered that hXRCC3 cysteine residues are oxidized following photosensitization by UVA. Our in silico prediction of the hXRCC3 structure suggests that 6 out of 8 cysteines are potentially accessible to the solvent and therefore potentially exposed to ROS attack. By non-reducing SDS-PAGE we show that many different oxidants induce hXRCC3 oxidation that is monitored in Chinese hamster ovarian (CHO) cells by increased electrophoretic mobility of the protein and in human cells by a slight decrease of its immunodetection. In both cell types, hXRCC3 oxidation was reversed in few minutes by cellular reducing systems. Depletion of intracellular glutathione prevents hXRCC3 oxidation only after UVA exposure though depending on the type of photosensitizer. In addition, we show that hXRCC3 expressed in CHO cells localizes both in the cytoplasm and in the nucleus. Mutating all hXRCC3 cysteines to serines (XR3/S protein) does not affect the subcellular localization of the protein even after exposure to camptothecin (CPT), which typically induces DNA damages that require HR to be repaired. However, cells expressing mutated XR3/S protein are sensitive to CPT, thus highlighting a defect of the mutant protein in HR. In marked contrast to CPT treatment, oxidative stress induces relocalization at the chromatin fraction of both wild-type and mutated protein, even though survival is not affected. Collectively, our results demonstrate that the DNA repair protein hXRCC3 is a target of ROS induced by environmental factors and raise the possibility that the redox environment might participate in regulating the HR pathway. PMID:24116071

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

PubMed

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

2010-11-01

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/m³) and high (above 50 mg/m³)) 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/10⁹ Da), followed by high exposure group (0.72±0.81 SSB/10⁹ Da) and controls (0.65±0.82 SSB/10⁹ 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. Copyright © 2010 Elsevier Inc. All rights reserved.

Mutations in XRCC4 cause primordial dwarfism without causing immunodeficiency.

PubMed

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.

Characterization of the APLF FHA–XRCC1 phosphopeptide interaction and its structural and functional implications

PubMed Central

Kim, Kyungmin; Pedersen, Lars C.; Kirby, Thomas W.; DeRose, Eugene F.

2017-01-01

Abstract Aprataxin and PNKP-like factor (APLF) is a DNA repair factor containing a forkhead-associated (FHA) domain that supports binding to the phosphorylated FHA domain binding motifs (FBMs) in XRCC1 and XRCC4. We have characterized the interaction of the APLF FHA domain with phosphorylated XRCC1 peptides using crystallographic, NMR, and fluorescence polarization studies. The FHA–FBM interactions exhibit significant pH dependence in the physiological range as a consequence of the atypically high pK values of the phosphoserine and phosphothreonine residues and the preference for a dianionic charge state of FHA-bound pThr. These high pK values are characteristic of the polyanionic peptides typically produced by CK2 phosphorylation. Binding affinity is greatly enhanced by residues flanking the crystallographically-defined recognition motif, apparently as a consequence of non-specific electrostatic interactions, supporting the role of XRCC1 in nuclear cotransport of APLF. The FHA domain-dependent interaction of XRCC1 with APLF joins repair scaffolds that support single-strand break repair and non-homologous end joining (NHEJ). It is suggested that for double-strand DNA breaks that have initially formed a complex with PARP1 and its binding partner XRCC1, this interaction acts as a backup attempt to intercept the more error-prone alternative NHEJ repair pathway by recruiting Ku and associated NHEJ factors. PMID:29059378

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

PubMed

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.

Characterization of the APLF FHA-XRCC1 phosphopeptide interaction and its structural and functional implications.

PubMed

Kim, Kyungmin; Pedersen, Lars C; Kirby, Thomas W; DeRose, Eugene F; London, Robert E

2017-12-01

Aprataxin and PNKP-like factor (APLF) is a DNA repair factor containing a forkhead-associated (FHA) domain that supports binding to the phosphorylated FHA domain binding motifs (FBMs) in XRCC1 and XRCC4. We have characterized the interaction of the APLF FHA domain with phosphorylated XRCC1 peptides using crystallographic, NMR, and fluorescence polarization studies. The FHA-FBM interactions exhibit significant pH dependence in the physiological range as a consequence of the atypically high pK values of the phosphoserine and phosphothreonine residues and the preference for a dianionic charge state of FHA-bound pThr. These high pK values are characteristic of the polyanionic peptides typically produced by CK2 phosphorylation. Binding affinity is greatly enhanced by residues flanking the crystallographically-defined recognition motif, apparently as a consequence of non-specific electrostatic interactions, supporting the role of XRCC1 in nuclear cotransport of APLF. The FHA domain-dependent interaction of XRCC1 with APLF joins repair scaffolds that support single-strand break repair and non-homologous end joining (NHEJ). It is suggested that for double-strand DNA breaks that have initially formed a complex with PARP1 and its binding partner XRCC1, this interaction acts as a backup attempt to intercept the more error-prone alternative NHEJ repair pathway by recruiting Ku and associated NHEJ factors. Published by Oxford University Press on behalf of Nucleic Acids Research 2017.

A prospective study of XRCC1 haplotypes and their interaction with plasma carotenoids on breast cancer risk

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mohrenweiser, H W; Han, J; Hankinson, S E

2004-01-15

The XRCC1 protein is involved in the base excision repair pathway through interactions with other proteins. Polymorphisms in the XRCC1 gene may lead to variation in repair proficiency and confer inherited predisposition to cancer. We prospectively assessed the associations between polymorphisms and haplotypes in XRCC1 and breast cancer risk in a nested case-control study within the Nurses' Health Study (incident cases, n 1004; controls, n 1385). We further investigated gene-environment interactions between the XRCC1 variations and plasma carotenoids on breast cancer risk. We genotyped four haplotype-tagging single nucleotide polymorphisms Arg {sup 194}Trp, C26602T, Arg{sup 399}Gln, and Gln{sup 632}Gln in themore » XRCC1 gene. Five common haplotypes accounted for 99% of the chromosomes in the present study population of mostly Caucasian women. We observed a marginally significant reduction in the risk of breast cancer among {sup 194}Trp carriers. As compared with no-carriers, women with at least one {sup 194}Trp allele had a multivariate odds ratio of 0.79 (95% of the confidence interval, 0.60 -1.04). The inferred haplotype harboring the {sup 194}Trp allele was more common in controls than in cases (6.6 versus 5.3%, P 0.07). We observed that the Arg {sup 194}Trp modified the inverse associations of plasma -carotene level (P, ordinal test for interaction 0.02) and plasma -carotene level (P, ordinal test for interaction 0.003) with breast cancer risk. No suggestion of an interaction was observed between the Arg {sup 194}Trp and cigarette smoking. Our results suggest an inverse association between XRCC1 {sup 194}Trp allele and breast cancer risk. The findings of the effect modification of the Arg {sup 194}Trp on the relations of plasma -and -carotene levels with breast cancer risk suggest a potential protective effect of carotenoids in breast carcinogenesis by preventing oxidative DNA damage.« less

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stenerlöw, Bo; Karlsson, Karin H.; Radulescu, Irina

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 ofmore » 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.« less

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yin Ming; Liao Zhongxing; Liu Zhensheng

2011-11-01

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 SNPsmore » 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.« less

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chiang, Chien-I; Huang, Ya-Li; Chen, Wei-Jen

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 Lys751Glnmore » 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

XRCC3 polymorphism is associated with hypertension-induced left ventricular hypertrophy.

PubMed

Ariyandy, Andi; Sakai, Chiemi; Ishida, Mari; Mizuta, Ryusei; Miyagawa, Kiyoshi; Tashiro, Satoshi; Kinomura, Aiko; Hiraaki, Koji; Ueda, Keitaro; Yoshizumi, Masao; Ishida, Takafumi

2018-06-01

Deficiency of X-ray repair cross-complementing protein 3 (XRCC3), a DNA-damage repair molecule, and the 241Met variant of XRCC3 have been reported to increase endoreduplication, which induces polyploidy. The aims of this study were to determine the impact of the XRCC3 polymorphism on the incidence of hypertension-induced left ventricular hypertrophy (LVH) and to investigate the mechanisms underlying any potential relationship. Patients undergoing chronic hemodialysis (n = 77) were genotyped to assess for the XRCC3 Thr241Met polymorphism. The XRCC3 241Thr/Met genotype was more frequent in the LVH (+) group than in the LVH (-) group (42.3 vs. 13.7%, χ2 = 7.85, p = 0.0051). To investigate possible mechanisms underlying these observations, human XRCC3 cDNA of 241Thr or that of 241Met was introduced into cultured CHO cells. The surface area of CHO cells expressing XRCC3 241Met was larger than that expressing 241Thr. Spontaneous DNA double-strand breaks accumulated to a greater degree in NIH3T3 cells expressing 241Met (3T3-241Met) than in those expressing 241Thr (3T3-241Thr). DNA damage caused by radiation induced cell senescence more frequently in 3T3-241Met. The levels of basal and TNF-α-stimulated MCP-1 mRNA and protein secretion were higher in 3T3-241Met. Finally, FACS analysis revealed that the cell percentage in G2/M phase including polyploidy was significantly higher in 3T3-241Met than in 3T3-241Thr. Furthermore, the basal level of MCP-1 mRNA positively correlated with the cell percentage in G2/M phase and polyploidy. These data suggest that the XRCC3 241Met increases the risk of LVH via accumulation of DNA damage, thereby altering cell cycle progression and inducing cell senescence and a proinflammatory phenotype.

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

PubMed Central

Sujitha, S. P.; Kumar, D. Thirumal; Doss, C. George Priya; Aavula, K.; Ramesh, R.; Lakshmanan, S.; Gunasekaran, S.; Anilkumar, G.

2016-01-01

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. PMID:26824244

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

PubMed

Sujitha, S P; Kumar, D Thirumal; Doss, C George Priya; Aavula, K; Ramesh, R; Lakshmanan, S; Gunasekaran, S; Anilkumar, G

2016-01-01

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.

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

PubMed

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.

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

PubMed

Xue, Ping; Gao, Lin; Xiao, Sha; Zhang, Guopei; Xiao, Mingyang; Zhang, Qianye; Zheng, Xiao; Cai, Yuan; Jin, Cuihong; Yang, Jinghua; Wu, Shengwen; Lu, Xiaobo

2015-01-01

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

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

PubMed Central

Xiao, Sha; Zhang, Guopei; Xiao, Mingyang; Zhang, Qianye; Zheng, Xiao; Cai, Yuan; Jin, Cuihong; Yang, Jinghua; Wu, Shengwen; Lu, Xiaobo

2015-01-01

Objectives 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. Methods 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. Results 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

Association between genetic polymorphisms in the XRCC1, XRCC3, XPD, GSTM1, GSTT1, MSH2, MLH1, MSH3, and MGMT genes and radiosensitivity in breast cancer patients.

PubMed

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

2011-09-01

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). 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. 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). 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 involved in cellular response to radiotherapy. Genetic

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

PubMed

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.

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.

PubMed

Holmes, Amie L; Joyce, Kellie; Xie, Hong; Falank, Carolyne; Hinz, John M; Wise, John Pierce

2014-04-01

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. Copyright © 2014 Elsevier B.V. All rights reserved.

Mutations in the NHEJ component XRCC4 cause primordial dwarfism.

PubMed

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.

Mutations in the NHEJ Component XRCC4 Cause Primordial Dwarfism

PubMed Central

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.

2015-01-01

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. PMID:25728776

Interactions involving the Rad51 paralogs Rad51C and XRCC3 in human cells

NASA Technical Reports Server (NTRS)

Wiese, Claudia; Collins, David W.; Albala, Joanna S.; Thompson, Larry H.; Kronenberg, Amy; Schild, David; Chatterjee, A. (Principal Investigator)

2002-01-01

Homologous recombinational repair of DNA double-strand breaks and crosslinks in human cells is likely to require Rad51 and the five Rad51 paralogs (XRCC2, XRCC3, Rad51B/Rad51L1, Rad51C/Rad51L2 and Rad51D/Rad51L3), as has been shown in chicken and rodent cells. Previously, we reported on the interactions among these proteins using baculovirus and two- and three-hybrid yeast systems. To test for interactions involving XRCC3 and Rad51C, stable human cell lines have been isolated that express (His)6-tagged versions of XRCC3 or Rad51C. Ni2+-binding experiments demonstrate that XRCC3 and Rad51C interact in human cells. In addition, we find that Rad51C, but not XRCC3, interacts directly or indirectly with Rad51B, Rad51D and XRCC2. These results argue that there are at least two complexes of Rad51 paralogs in human cells (Rad51C-XRCC3 and Rad51B-Rad51C-Rad51D-XRCC2), both containing Rad51C. Moreover, Rad51 is not found in these complexes. X-ray treatment did not alter either the level of any Rad51 paralog or the observed interactions between paralogs. However, the endogenous level of Rad51C is moderately elevated in the XRCC3-overexpressing cell line, suggesting that dimerization between these proteins might help stabilize Rad51C.

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

PubMed

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.

The XRCC1 Arg194Trp polymorphism is significantly associated with lung adenocarcinoma: a case-control study in an Eastern European Caucasian group.

PubMed

Cătană, Andreea; Pop, Monica; Hincu, Bianca Domokos; Pop, Ioan V; Petrişor, Felicia M; Porojan, Mihai D; Popp, Radu A

2015-01-01

DNA repair plays an important role in maintaining the integrity of the genome by repairing DNA damage induced by carcinogens. 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 gene XRCC1 is involved in DNA repair. The purpose of our study was to investigate the association between XRCC1 Arg194Trp and Arg399Gln polymorphisms and the risk of lung cancer in a Romanian population. We recruited 222 healthy controls and 102 patients with lung cancer. Genotypes were determined by multiplex polymerase chain-reaction restriction fragment-length polymorphism. Statistical analysis (odds ratio, recessive model) revealed an increased risk for lung cancer for the homozygous 194Trp genotype (χ (2)=0.186, odds ratio 10.667, 95% confidence interval 1.309-86.933; P=0.007). Also, we found an association between the 194Trp allele and women with lung adenocarcinoma. In conclusion, the results of the study place the XRCC1 Arg194Trp polymorphism among independent risk factors for developing lung cancer.

Homologous recombination as a potential target for caffeine radiosensitization in mammalian cells: reduced caffeine radiosensitization in XRCC2 and XRCC3 mutants

NASA Technical Reports Server (NTRS)

Asaad, N. A.; Zeng, Z. C.; Guan, J.; Thacker, J.; Iliakis, G.

2000-01-01

The radiosensitizing effect of caffeine has been associated with the disruption of multiple DNA damage-responsive cell cycle checkpoints, but several lines of evidence also implicate inhibition of DNA repair. The role of DNA repair inhibition in caffeine radiosensitization remains uncharacterized, and it is unknown which repair process, or lesion, is affected. We show that a radiosensitive cell line, mutant for the RAD51 homolog XRCC2 and defective in homologous recombination repair (HRR), displays significantly diminished caffeine radiosensitization that can be restored by expression of XRCC2. Despite the reduced radiosensitization, caffeine effectively abrogates checkpoints in S and G2 phases in XRCC2 mutant cells indicating that checkpoint abrogation is not sufficient for radiosensitization. Another radiosensitive line, mutant for XRCC3 and defective in HRR, similarly shows reduced caffeine radiosensitization. On the other hand, a radiosensitive mutant (irs-20) of DNA-PKcs with a defect in non-homologous end-joining (NHEJ) is radiosensitized by caffeine to an extent comparable to wild-type cells. In addition, rejoining of radiation-induced DNA DSBs, that mainly reflects NHEJ, remains unaffected by caffeine in XRCC2 and XRCC3 mutants, or their wild-type counterparts. These observations suggest that caffeine targets steps in HRR but not in NHEJ and that abrogation of checkpoint response is not sufficient to explain radiosensitization. Indeed, immortalized fibroblasts from AT patients show caffeine radiosensitization despite the checkpoint defects associated with ATM mutation. We propose that caffeine radiosensitization is mediated by inhibition of stages in DNA DSB repair requiring HRR and that checkpoint disruption contributes by allowing these DSBs to transit into irreparable states. Thus, checkpoints may contribute to genomic stability by promoting error-free HRR.

XRCC1 Polymorphisms and Pancreatic Cancer: A Meta-Analysis

PubMed Central

Shen, Wei-dong; Chen, Hong-lin; Liu, Peng-fei

2011-01-01

Objective To assess the association between X-ray repair cross-complementating group 1 (XRCC1) polymorphisms and pancreatic cancer. Methods We searched MEDLINE, Web of Science and HuGE Navigator at June 2010, and then quantitatively summarized associations of the XRCC1 polymorphisms with pancreatic cancer risk using meta-analysis. Results Four studies with 1343 cases and 2302 controls were included. Our analysis found: at codon 194, the Trp allele did not decrease pancreatic cancer risk (Arg/Arg versus Trp/Trp: OR=0.97; 95% CI: 0.48-1.96; P=0.97; Arg/Arg versus Arg/Trp: OR=0.89; 95% CI: 0.70-1.13; P=0.55; Arg/Trp versus Trp/Trp: OR=1.06; 95% CI: 0.52-2.16; P=0.90); at codon 280, only a study showed a nonsignificant association between single nucleotide polymorphism with pancreatic cancer risk; at codon 399, the Gln allele also showed no significant effect on pancreatic cancer compared to Arg allele (Arg/Arg versus Gln/Gln: OR=0.94; 95% CI: 0.74-1.18; Arg/Arg versus Arg/Gln: OR=0.97; 95% CI: 0.83-1.13; Arg/Gln versus Gln/Gln: OR=0.97; 95% CI: 0.77-1.22). The shape of the funnel plot and the Egger’s test did not detect any publication bias. Conclusion There is no evidence that XRCC1 polymorphisms (Arg194Trp, Arg280His, and Arg399Gln) are associated with pancreatic cancer risk. PMID:23467456

The XRCC1 Arg194Trp polymorphism is significantly associated with lung adenocarcinoma: a case-control study in an Eastern European Caucasian group

PubMed Central

Cătană, Andreea; Pop, Monica; Hincu, Bianca Domokos; Pop, Ioan V; Petrişor, Felicia M; Porojan, Mihai D; Popp, Radu A

2015-01-01

DNA repair plays an important role in maintaining the integrity of the genome by repairing DNA damage induced by carcinogens. 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 gene XRCC1 is involved in DNA repair. The purpose of our study was to investigate the association between XRCC1 Arg194Trp and Arg399Gln polymorphisms and the risk of lung cancer in a Romanian population. We recruited 222 healthy controls and 102 patients with lung cancer. Genotypes were determined by multiplex polymerase chain-reaction restriction fragment-length polymorphism. Statistical analysis (odds ratio, recessive model) revealed an increased risk for lung cancer for the homozygous 194Trp genotype (χ2=0.186, odds ratio 10.667, 95% confidence interval 1.309–86.933; P=0.007). Also, we found an association between the 194Trp allele and women with lung adenocarcinoma. In conclusion, the results of the study place the XRCC1 Arg194Trp polymorphism among independent risk factors for developing lung cancer. PMID:26664136

Association of XRCC1 Trp194 allele with risk of breast cancer, and Ki67 protein status in breast tumor tissues

PubMed Central

Jalali, Chiya; Ghaderi, Bayazid; Amini, Sabrieh; Abdi, Mohammad; Roshani, Daem

2016-01-01

Objectives: To evaluate the role of this polymorphism as a risk factor for breast cancer in Kurdish patients and to investigate the possible association between Arg194Trp x-ray repair cross-complementing group 1 (XRCC1) gene polymorphisms with clinical and histopathological outcomes of patients with breast cancer. Methods: A total of 100 breast cancer patients and 200 cancer-free controls in Kurdish population of Kurdistan state admitted to Tohid Hospital, Sanandaj, Kurdistan, Iran between January 2012 and May 2015 were enrolled in this cross-sectional study. Tissue expression of estrogen receptor (ER), progesteron receptor (PR), human epidermal growth factor receptor 2 (Her2/neu), and Ki67 were evaluated by immunohistochemistry (IHC). The Arg194Trp genotypes were determined by polymerase chain reaction- restriction fragment length polymorphism method. Results: Our data showed that the risk for breast cancer increased significantly among the Trp variant of XRCC1. Statistically significant association was found between codon 194 polymorphisms and tissue expression of Ki67. Conclusion: The Trp allele of codon 194 XRCC1 is a potential risk factor for breast cancer in Kurdish ethnicity. Furthermore, effect of this polymorphism on clinical and histological features of breast cancer was significant. PMID:27279507

Xrcc2 deficiency sensitizes cells to apoptosis by MNNG and the alkylating anticancer drugs temozolomide, fotemustine and mafosfamide.

PubMed

Tsaryk, Roman; Fabian, Kerstin; Thacker, John; Kaina, Bernd

2006-08-08

DNA double-strand breaks (DSBs) are potent killing lesions, and inefficient repair of DSBs does not only lead to cell death but also to genomic instability and tumorigenesis. DSBs are repaired by non-homologous end-joining and homologous recombination (HR). A key player in HR is Xrcc2, a Rad51-like protein. Cells deficient in Xrcc2 are hypersensitive to X-rays and mitomycin C and display increased chromosomal aberration frequencies. In order to elucidate the role of Xrcc2 in resistance to anticancer drugs, we compared Xrcc2 knockout (Xrcc2-/-) mouse embryonic fibroblasts with the corresponding isogenic wild-type and Xrcc2 complemented knockout cells. We show that Xrcc2-/- cells are hypersensitive to the killing effect of the simple methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). They undergo apoptosis after MNNG treatment while necrosis is only marginally enhanced. Complementation of Xrcc2 deficient cells by Xrcc2 cDNA transfection conferred resistance to the cytotoxic and apoptosis-inducing effect of MNNG. The hypersensitivity of Xrcc2-/- cells to MNNG prompted us to investigate their killing and apoptotic response to various methylating, chloroethylating and crosslinking drugs used in anticancer therapy. Xrcc2 deficient cells were found to be hypersensitive to temozolomide, fotemustine and mafosfamide. They were also hypersensitive to cisplatin but not to taxol. The data reveal that Xrcc2 plays a role in the protection against a wide range of anticancer drugs and, therefore, suggest Xrcc2 to be a determinant of anticancer drug resistance. They also indicate that HR is involved in the processing of DNA damage induced by simple alkylating agents.

[Analysis for the association between genetic polymorphisms of XRCC1, XPD, XRCC3, CCND1 and the latency of the occupational chronic benzene poisoning].

PubMed

Xu, Jian-ning; Huang, Hui-long; Wang, Quan-kai; Wang, Ya-wen; Yang, Min; Zheng, Yu-xin

2007-03-01

To explore the association between genetic polymorphisms of XRCC1, XPD, XRCC3 and CCND1 and latency of occupational chronic benzene poisoning. 80 patients diagnosed with occupational chronic benzene poisoning were investigated. PCR-RFLP was applied to detect the single nucleotide polymorphisms of C26304T, G27466A, G28152A, G36189A of XRCC1, C22541A, C23591T, A35931C of XPD, C18067T of XRCC3 and G870A of CCND1. Their relationship with the latency of chronic benzene poisoning was analyzed by Kaplan-Meier method. The association of XRCC1 G27466A subgroup with the latency of chronic benzene poisoning was observed, as well as that of CCDN1G870A subgroup. The benzene-exposed workers with XRCC1 27466G/G homozygous wild genotype developed chronic benzene poisoning 6.9 years later than those had homozygous (27466A/A) or heterozygous (27466G/A) mutant alleles. On the other hand, the latency developing chronic benzene poisoning was longer in workers with homozygous (CCND1 870A/A) or heterozygous (CCND1 870G/A) mutant alleles than in those carrying 870G/G homozygous wild genotype (14.9 vs. 8.7 years). The polymorphisms of XRCC1 and CCND1 potentially modify the latency of the chronic benzene poisoning among workers exposed to benzene.

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

NASA Astrophysics Data System (ADS)

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.

DIFFERENTIAL ROLE OF BASE EXCISION REPAIR PROTEINS IN MEDIATING CISPLATIN CYTOTOXICITY

PubMed Central

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

2017-01-01

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. PMID:28110804

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

PubMed

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.

Prognostic significance of XRCC4 expression in hepatocellular carcinoma

PubMed Central

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

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wanotayan, Rujira; Fukuchi, Mikoto; Imamichi, Shoji

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.more » 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

XLS (c9orf142) is a new component of mammalian DNA double-stranded break repair.

PubMed

Craxton, A; Somers, J; Munnur, D; Jukes-Jones, R; Cain, K; Malewicz, M

2015-06-01

Repair of double-stranded DNA breaks (DSBs) in mammalian cells primarily occurs by the non-homologous end-joining (NHEJ) pathway, which requires seven core proteins (Ku70/Ku86, DNA-PKcs (DNA-dependent protein kinase catalytic subunit), Artemis, XRCC4-like factor (XLF), XRCC4 and DNA ligase IV). Here we show using combined affinity purification and mass spectrometry that DNA-PKcs co-purifies with all known core NHEJ factors. Furthermore, we have identified a novel evolutionary conserved protein associated with DNA-PKcs-c9orf142. Computer-based modelling of c9orf142 predicted a structure very similar to XRCC4, hence we have named c9orf142-XLS (XRCC4-like small protein). Depletion of c9orf142/XLS in cells impaired DSB repair consistent with a defect in NHEJ. Furthermore, c9orf142/XLS interacted with other core NHEJ factors. These results demonstrate the existence of a new component of the NHEJ DNA repair pathway in mammalian cells.

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

PubMed

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.

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

PubMed

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.

A unified view of base excision repair: lesion-dependent protein complexes regulated by post-translational modification

PubMed Central

Almeida, Karen H.; Sobol, Robert W.

2007-01-01

Base excision repair (BER) proteins act upon a significantly broad spectrum of DNA lesions that result from endogenous and exogenous sources. Multiple sub-pathways of BER (short-path or long-patch) and newly designated DNA repair pathways (e.g., SSBR and NIR) that utilize BER proteins complicate any comprehensive understanding of BER and its role in genome maintenance, chemotherapeutic response, neurodegeneration, cancer or aging. Herein, we propose a unified model of BER, comprised of three functional processes: Lesion Recognition/Strand Scission, Gap Tailoring and DNA Synthesis/Ligation, each represented by one or more multiprotein complexes and coordinated via the XRCC1/DNA Ligase III and PARP1 scaffold proteins. BER therefore may be represented by a series of repair complexes that assemble at the site of the DNA lesion and mediates repair in a coordinated fashion involving protein-protein interactions that dictate subsequent steps or sub-pathway choice. Complex formation is influenced by post-translational protein modifications that arise from the cellular state or the DNA damage response, providing an increase in specificity and efficiency to the BER pathway. In this review, we have summarized the reported BER protein-protein interactions and protein post-translational modifications and discuss the impact on DNA repair capacity and complex formation. PMID:17337257

XLS (c9orf142) is a new component of mammalian DNA double-stranded break repair

PubMed Central

Craxton, A; Somers, J; Munnur, D; Jukes-Jones, R; Cain, K; Malewicz, M

2015-01-01

Repair of double-stranded DNA breaks (DSBs) in mammalian cells primarily occurs by the non-homologous end-joining (NHEJ) pathway, which requires seven core proteins (Ku70/Ku86, DNA-PKcs (DNA-dependent protein kinase catalytic subunit), Artemis, XRCC4-like factor (XLF), XRCC4 and DNA ligase IV). Here we show using combined affinity purification and mass spectrometry that DNA-PKcs co-purifies with all known core NHEJ factors. Furthermore, we have identified a novel evolutionary conserved protein associated with DNA-PKcs—c9orf142. Computer-based modelling of c9orf142 predicted a structure very similar to XRCC4, hence we have named c9orf142—XLS (XRCC4-like small protein). Depletion of c9orf142/XLS in cells impaired DSB repair consistent with a defect in NHEJ. Furthermore, c9orf142/XLS interacted with other core NHEJ factors. These results demonstrate the existence of a new component of the NHEJ DNA repair pathway in mammalian cells. PMID:25941166

Unstabilized DNA breaks in HTLV-1 Tax expressing cells correlate with functional targeting of Ku80, not PKcs, XRCC4, or H2AX

PubMed Central

2012-01-01

Background Expression of the human T-cell leukemia virus type 1 (HTLV-1) Tax oncoprotein rapidily induces a significant increase of micronuclei (MN) and unstabilized DNA breaks in cells. Unstabilized DNA breaks can have free 3′-OH ends accessible to in situ addition of digoxygenin (DIG)-labeled dUTP using terminal deoxynucleotidyl transferase. In the present work, we used a GFP-Tax (green fluorescent protein) plasmid, which produces a functionally active GFP-tagged Tax protein, to detect the cellular target(s) for Tax which might mechanistically explain the clastogenic phenomenon. We examined the induction of MN and unstabilized DNA breaks in wild type cells and cells individually knocked out for Ku80, PKcs, XRCC4, and H2AX proteins. We also assessed in the same cells, the signal strengths produced by DIG-dUTP incorporation at the unstable DNA breaks in the presence and absence of Tax. Results Cells mutated for PKcs, XRCC4 and H2AX showed increased frequency of MN and unstabilized DNA breaks in response to the expression of Tax, while cells genetically mutated for Ku80 were refractory to Tax’s induction of these cytogenetic effects. Moreover, by measuring the size of DIG-dUTP incorporation signal, which indicates the extent of unstable DNA ends, we found that Tax induces larger signals than those in control cells. However, in xrs-6 cells deficient for Ku80, this Tax effect was not seen. Conclusions The data here demonstrate that clastogenic DNA damage in Tax expressing cells is explained by Tax targeting of Ku80, but not PKcs, XRCC4 or H2AX, which are all proteins directly or indirectly related to the non-homologous end-joining (NHEJ) repair system. Of note, the Ku80 protein plays an important role at the initial stage of the NHEJ repair system, protecting and stabilizing DNA-breaks. Accordingly, HTLV-1 Tax is shown to interfere with a normal cellular protective mechanism for stabilizing DNA breaks. These DNA breaks, unprotected by Ku80, are unstable and are

Requirement for Parp-1 and DNA ligases 1 or 3 but not of Xrcc1 in chromosomal translocation formation by backup end joining

PubMed Central

Soni, Aashish; Siemann, Maria; Grabos, Martha; Murmann, Tamara; Pantelias, Gabriel E.; Iliakis, George

2014-01-01

In mammalian cells, ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) are repaired in all phases of the cell cycle predominantly by classical, DNA-PK-dependent nonhomologous end joining (D-NHEJ). Homologous recombination repair (HRR) is functional during the S- and G2-phases, when a sister chromatid becomes available. An error-prone, alternative form of end joining, operating as backup (B-NHEJ) functions robustly throughout the cell cycle and particularly in the G2-phase and is thought to backup predominantly D-NHEJ. Parp-1, DNA-ligases 1 (Lig1) and 3 (Lig3), and Xrcc1 are implicated in B-NHEJ. Chromosome and chromatid translocations are manifestations of erroneous DSB repair and are crucial culprits in malignant transformation and IR-induced cell lethality. We analyzed shifts in translocation formation deriving from defects in D-NHEJ or HRR in cells irradiated in the G2-phase and identify B-NHEJ as the main DSB repair pathway backing up both of these defects at the cost of a large increase in translocation formation. Our results identify Parp-1 and Lig1 and 3 as factors involved in translocation formation and show that Xrcc1 reinforces the function of Lig3 in the process without being required for it. Finally, we demonstrate intriguing connections between B-NHEJ and DNA end resection in translocation formation and show that, as for D-NHEJ and HRR, the function of B-NHEJ facilitates the recovery from the G2-checkpoint. These observations advance our understanding of chromosome aberration formation and have implications for the mechanism of action of Parp inhibitors. PMID:24748665

Functional characterization of polymorphisms in DNA repair genes using cytogenetic challenge assays.

PubMed

Au, William W; Salama, Salama A; Sierra-Torres, Carlos H

2003-11-01

A major barrier to understanding the role of polymorphic DNA repair genes for environmental cancer is that the functions of variant genotypes are largely unknown. Using our cytogenetic challenge assays, we conducted an investigation to address the deficiency. Using X-rays or ultraviolet (UV) light, we irradiated blood lymphocytes from 80 nonsmoking donors to challenge the cells to repair the induced DNA damage, and we analyzed expression of chromosome aberrations (CA) specific to the inducing agents. We have genotyped polymorphic DNA repair genes preferentially involved with base excision repair (BER) and nucleotide excision repair (NER) activities (XRCC1, XRCC3, APE1, XPD) corresponding to the repair of X-ray- and UV light-induced DNA damage, respectively. We expected that defects in specific DNA repair pathways due to polymorphisms would cause corresponding increases of specific CA. From our data, XRCC1 399Gln and XRCC3 241Met were associated with significant increases in chromosome deletions compared with the corresponding homozygous wild types (18.27 1.1 vs 14.79 1.2 and 18.22 0.99 vs 14.20 1.39, respectively); XPD 312Asn and XPD 751Gln were associated with significant increases in chromatid breaks compared with wild types (16.09 1.36 vs 11.41 0.98 and 16.87 1.27 vs 10.54 0.87, respectively), p < 0.05. The data indicate that XRCC1 399Gln and XRCC3 241Met are significantly defective in BER, and the XPD 312Asn and XPD 751Gln are significantly defective in NER. In addition, the variant genotypes interact significantly, with limited overlap of the two different repair pathways.

Requirement for Parp-1 and DNA ligases 1 or 3 but not of Xrcc1 in chromosomal translocation formation by backup end joining.

PubMed

Soni, Aashish; Siemann, Maria; Grabos, Martha; Murmann, Tamara; Pantelias, Gabriel E; Iliakis, George

2014-06-01

In mammalian cells, ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) are repaired in all phases of the cell cycle predominantly by classical, DNA-PK-dependent nonhomologous end joining (D-NHEJ). Homologous recombination repair (HRR) is functional during the S- and G2-phases, when a sister chromatid becomes available. An error-prone, alternative form of end joining, operating as backup (B-NHEJ) functions robustly throughout the cell cycle and particularly in the G2-phase and is thought to backup predominantly D-NHEJ. Parp-1, DNA-ligases 1 (Lig1) and 3 (Lig3), and Xrcc1 are implicated in B-NHEJ. Chromosome and chromatid translocations are manifestations of erroneous DSB repair and are crucial culprits in malignant transformation and IR-induced cell lethality. We analyzed shifts in translocation formation deriving from defects in D-NHEJ or HRR in cells irradiated in the G2-phase and identify B-NHEJ as the main DSB repair pathway backing up both of these defects at the cost of a large increase in translocation formation. Our results identify Parp-1 and Lig1 and 3 as factors involved in translocation formation and show that Xrcc1 reinforces the function of Lig3 in the process without being required for it. Finally, we demonstrate intriguing connections between B-NHEJ and DNA end resection in translocation formation and show that, as for D-NHEJ and HRR, the function of B-NHEJ facilitates the recovery from the G2-checkpoint. These observations advance our understanding of chromosome aberration formation and have implications for the mechanism of action of Parp inhibitors. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Complementation of hypersensitivity to DNA interstrand crosslinking agents demonstrates that XRCC2 is a Fanconi anaemia gene.

PubMed

Park, Jung-Young; Virts, Elizabeth L; Jankowska, Anna; Wiek, Constanze; Othman, Mohamed; Chakraborty, Sujata C; Vance, Gail H; Alkuraya, Fowzan S; Hanenberg, Helmut; Andreassen, Paul R

2016-10-01

Fanconi anaemia (FA) is a heterogeneous inherited disorder clinically characterised by progressive bone marrow failure, congenital anomalies and a predisposition to malignancies. Determine, based on correction of cellular phenotypes, whether XRCC2 is a FA gene. Cells (900677A) from a previously identified patient with biallelic mutation of XRCC2, among other mutations, were genetically complemented with wild-type XRCC2. Wild-type XRCC2 corrects each of three phenotypes characteristic of FA cells, all related to the repair of DNA interstrand crosslinks, including increased sensitivity to mitomycin C (MMC), chromosome breakage and G2-M accumulation in the cell cycle. Further, the p.R215X mutant of XRCC2, which is harboured by the patient, is unstable. This provides an explanation for the pathogenesis of this mutant, as does the fact that 900677A cells have reduced levels of other proteins in the XRCC2-RAD51B-C-D complex. Also, FANCD2 monoubiquitination and foci formation, but not assembly of RAD51 foci, are normal in 900677A cells. Thus, XRCC2 acts late in the FA-BRCA pathway as also suggested by hypersensitivity of 900677A cells to ionising radiation. These cells also share milder sensitivities towards olaparib and formaldehyde with certain other FA cells. XRCC2/FANCU is a FA gene, as is another RAD51 paralog gene, RAD51C/FANCO. Notably, similar to a subset of FA genes that act downstream of FANCD2, biallelic mutation of XRCC2/FANCU has not been associated with bone marrow failure. Taken together, our results yield important insights into phenotypes related to FA and its genetic origins. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

SNPs of GSTM1, T1, P1, epoxide hydrolase and DNA repair enzyme XRCC1 and risk of urinary transitional cell carcinoma in southwestern Taiwan

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hsu, L.-I; Chiu, Allen W.; Huan, Steven K.

A hospital-based case-control study was conducted near a former black-foot disease (BFD)-endemic area in southwestern Taiwan to examine the possible risk factors and genetic susceptibility for urinary transitional cell carcinoma (TCC). A total of 221 patients with pathologically confirmed TCC and 223 age-sex-matched control subjects from urology outpatient clinics were recruited between 1998 and 2002. The results showed that residency in the BFD area and consumption of well water for more than 10 years was a strong factor on urinary cancer risk (odds ratio [OR],8.16, 95% confidence interval [CI],3.34-19.90, p < 0.0001). Dose response relationship between average arsenic concentration inmore » well water and TCC risk was also observed. Cigarette smoking played a relatively minor role in urinary carcinogenesis in this study. The GSTP1 Ile105Val A {yields} G polymorphism was significantly associated with cancer risk (A/G + G/G: OR = 0.60, 95%CI = 0.39-0.94, p = 0.02), and the effect of Val105 allele was largely confined to the subjects diagnosed earlier than 55 years old (A/G + G/G: OR,0.29; 95% CI, 0.09-0.87, p = 0.03). The results suggest that GSTP1 is a candidate for susceptibility locus and Ile105 allele may predispose individuals to early-onset urinary TCC. The GSTM1 null genotype was associated with tumors of high-invasiveness (OR,2.21; 95% CI, 1.34-4.73) as well as with early-onset TCC risk (OR,2.53; 95% CI, 0.97-6.59). Our preliminary results showed the XRCC1 Arg194Trp were associated with arsenic-related urinary TCC and the interaction between the genotype and the exposure was statistically significant. The modulating effect of the GSTM1, GSTT1, GSTP1 Ile105Val, EPHX Tyr113His and XRCC1 Arg280His on arsenic-related TCC risk was also suggestive. These observations implied that impaired metabolism of carcinogenic exposure as well as impaired DNA repair function play an important role in arsenic-related urinary transitional cell carcinogenesis.« less

Polymorphisms in DNA repair genes and MDR1 and the risk for non-Hodgkin lymphoma.

PubMed

Kim, Hee Nam; Kim, Nan Young; Yu, Li; Kim, Yeo-Kyeoung; Lee, Il-Kwon; Yang, Deok-Hwan; Lee, Je-Jung; Shin, Min-Ho; Park, Kyeong-Soo; Choi, Jin-Su; Kim, Hyeoung-Joon

2014-04-21

The damage caused by oxidative stress and exposure to cigarette smoke and alcohol necessitate DNA damage repair and transport by multidrug resistance-1 (MDR1). To explore the association between polymorphisms in these genes and non-Hodgkin lymphoma risk, we analyzed 15 polymorphisms of 12 genes in a population-based study in Korea (694 cases and 1700 controls). Four genotypes of DNA repair pathway genes (XRCC1 399 GA, OGG1 326 GG, BRCA1 871 TT, and WRN 787 TT) were associated with a decreased risk for NHL [odds ratio (OR)XRCC1 GA=0.80, p=0.02; OROGG1 GG=0.70, p=0.008; ORBRCA1 TT=0.71, p=0.048; ORWRN TT=0.68, p=0.01]. Conversely, the MGMT 115 CT genotype was associated with an increased risk for NHL (OR=1.25, p=0.04). In the MDR1 gene, the 1236 CC genotype was associated with a decreased risk for NHL (OR=0.74, p=0.04), and the 3435 CT and TT genotypes were associated with an increased risk (OR3435CT=1.50, p<0.0001; OR3435TT=1.43, p=0.02). These results suggest that polymorphisms in the DNA repair genes XRCC1, OGG1, BRCA1, WRN1, and MGMT and in the MDR1 gene may affect the risk for NHL in Korean patients.

[Relationship between XRCC3 gene polymorphism and susceptibility to lead poisoning in male lead-exposed workers].

PubMed

Liu, Xiang-quan; Zhang, Zhong

2013-06-01

To investigate the relationship between genetic polymorphism of X-ray repair cross-complementing gene 3 (XRCC3) and susceptibility to lead poisoning in male lead-exposed workers. Peripheral venous blood and morning urine samples were collected from 326 male lead-exposed workers in a storage battery factory in Fuzhou. Blood lead, urine lead, blood zinc protoporphyrin (ZPP), blood calcium, and blood iron were measured. The genotype of XRCC3 was determined by polymerase chain reaction-restriction fragment length polymorphism method. The relationship between XRCC3 gene polymorphism and susceptibility to lead poisoning in male lead-exposed workers was analyzed. Genetic polymorphism of XRCC3 was seen in the 326 subjects. The frequency distribution of XRCC3 genotypes, XRCC3-241CC (wild type), XRCC3-241CT (heterozygous mutation), and XRCC3-241TT (homozygous mutation), was in accordance with the Hardy-Weinberg equilibrium (P > 0.05). There were no significant differences in urine lead, blood ZPP, blood calcium, and blood iron between the lead-exposed workers with different XRCC3 genotypes (P > 0.05). The workers with XRCC3-241CT/TT had a significantly higher mean blood lead level than those with XRCC3-241CC (P < 0.05). With a blood lead level of 1.90 µmol/L as the cutoff value, the chi-square test and logistic regression analysis showed that the proportion of workers with XRCC3-241CT/TT was significantly higher than that of workers with XRCC3-241CC in the subjects with high blood leads (P < 0.05) and that the risk of high blood lead was significantly higher in the workers with XRCC3-241CT/TT than in those with XRCC3-241CC (OR = 2.34, 95%CI = 1.61 ∼ 5.13); the multivariate linear regression analysis showed that the workers with XRCC3-241CT/TT had high blood lead levels (β = 0.116, P < 0.05), the workers with smoking habit demonstrated marked lead absorption (β = 0.188, P < 0.05), good individual protection could reduce lead absorption (β = -0.247, P < 0.05), and the

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Seibold, Petra; Behrens, Sabine; Schmezer, Peter

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 Totalmore » 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.« less

Nuclear localization of the DNA repair scaffold XRCC1: Uncovering the functional role of a bipartite NLS

DOE PAGES

Kirby, Thomas W.; Gassman, Natalie R.; Smith, Cassandra E.; ...

2015-08-25

We have characterized the nuclear localization signal (NLS) of XRCC1 structurally using X-ray crystallography and functionally using fluorescence imaging. Crystallography and binding studies confirm the bipartite nature of the XRCC1 NLS interaction with Importin α (Impα) in which the major and minor binding motifs are separated by >20 residues, and resolve previous inconsistent determinations. Binding studies of peptides corresponding to the bipartite NLS, as well as its major and minor binding motifs, to both wild-type and mutated forms of Impα reveal pronounced cooperative binding behavior that is generated by the proximity effect of the tethered major and minor motifs ofmore » the NLS. The cooperativity stems from the increased local concentration of the second motif near its cognate binding site that is a consequence of the stepwise binding behavior of the bipartite NLS. We predict that the stepwise dissociation of the NLS from Impα facilitates unloading by providing a partially complexed intermediate that is available for competitive binding by Nup50 or the Importin β binding domain. This behavior gives a basis for meeting the intrinsically conflicting high affinity and high flux requirements of an efficient nuclear transport system.« less

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

PubMed Central

Fayaz, Shima; Fard-Esfahani, Pezhman; Fard-Esfahani, Armaghan; Mostafavi, Ehsan; Meshkani, Reza; Mirmiranpour, Hossein; Khaghani, Shahnaz

2012-01-01

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. PMID:22481871

An XRCC4 Splice Mutation Associated With Severe Short Stature, Gonadal Failure, and Early-Onset Metabolic Syndrome

PubMed Central

de Bruin, Christiaan; Mericq, Verónica; Andrew, Shayne F.; van Duyvenvoorde, Hermine A.; Verkaik, Nicole S.; Losekoot, Monique; Porollo, Aleksey; Garcia, Hernán; Kuang, Yi; Hanson, Dan; Clayton, Peter; van Gent, Dik C.; Wit, Jan M.; Hwa, Vivian

2015-01-01

Context: Severe short stature can be caused by defects in numerous biological processes including defects in IGF-1 signaling, centromere function, cell cycle control, and DNA damage repair. Many syndromic causes of short stature are associated with medical comorbidities including hypogonadism and microcephaly. Objective: To identify an underlying genetic etiology in two siblings with severe short stature and gonadal failure. Design: Clinical phenotyping, genetic analysis, complemented by in vitro functional studies of the candidate gene. Setting: An academic pediatric endocrinology clinic. Patients or Other Participants: Two adult siblings (male patient [P1] and female patient 2 [P2]) presented with a history of severe postnatal growth failure (adult heights: P1, −6.8 SD score; P2, −4 SD score), microcephaly, primary gonadal failure, and early-onset metabolic syndrome in late adolescence. In addition, P2 developed a malignant gastrointestinal stromal tumor at age 28. Intervention(s): Single nucleotide polymorphism microarray and exome sequencing. Results: Combined microarray analysis and whole exome sequencing of the two affected siblings and one unaffected sister identified a homozygous variant in XRCC4 as the probable candidate variant. Sanger sequencing and mRNA studies revealed a splice variant resulting in an in-frame deletion of 23 amino acids. Primary fibroblasts (P1) showed a DNA damage repair defect. Conclusions: In this study we have identified a novel pathogenic variant in XRCC4, a gene that plays a critical role in non-homologous end-joining DNA repair. This finding expands the spectrum of DNA damage repair syndromes to include XRCC4 deficiency causing severe postnatal growth failure, microcephaly, gonadal failure, metabolic syndrome, and possibly tumor predisposition. PMID:25742519

Complex formation by the human Rad51B and Rad51C DNA repair proteins and their activities in vitro

NASA Technical Reports Server (NTRS)

Lio, Yi-Ching; Mazin, Alexander V.; Kowalczykowski, Stephen C.; Chen, David J.

2003-01-01

The human Rad51 protein is essential for DNA repair by homologous recombination. In addition to Rad51 protein, five paralogs have been identified: Rad51B/Rad51L1, Rad51C/Rad51L2, Rad51D/Rad51L3, XRCC2, and XRCC3. To further characterize a subset of these proteins, recombinant Rad51, Rad51B-(His)(6), and Rad51C proteins were individually expressed employing the baculovirus system, and each was purified from Sf9 insect cells. Evidence from nickel-nitrilotriacetic acid pull-down experiments demonstrates a highly stable Rad51B.Rad51C heterodimer, which interacts weakly with Rad51. Rad51B and Rad51C proteins were found to bind single- and double-stranded DNA and to preferentially bind 3'-end-tailed double-stranded DNA. The ability to bind DNA was elevated with mixed Rad51 and Rad51C, as well as with mixed Rad51B and Rad51C, compared with that of the individual protein. In addition, both Rad51B and Rad51C exhibit DNA-stimulated ATPase activity. Rad51C displays an ATP-independent apparent DNA strand exchange activity, whereas Rad51B shows no such activity; this apparent strand exchange ability results actually from a duplex DNA destabilization capability of Rad51C. By analogy to the yeast Rad55 and Rad57, our results suggest that Rad51B and Rad51C function through interactions with the human Rad51 recombinase and play a crucial role in the homologous recombinational repair pathway.

Association between XRCC1 polymorphisms and laryngeal cancer susceptibility in a Chinese sample population.

PubMed

Wu, W Q; Zhang, L S; Liao, S P; Lin, X L; Zeng, J; Du, D

2016-10-05

Laryngeal cancer is the major malignant tumor affecting the upper respiratory tract. Previous studies have reported on the association between XRCC1 genetic polymorphisms and risk of laryngeal cancer, but with conflicting results. In this study, we attempted to assess the association between XRCC1 Arg194Trp, Arg280His and Arg399Gln polymorphisms and risk of laryngeal cancer in a Chinese population. A total of 126 laryngeal cancer patients and 254 control subjects were recruited to this study from the Second Medical College of Jinan University between December 2013 and May 2015. The XRCC1 Arg194Trp, Arg280His, and Arg399Gln polymorphic sites were genotyped by polymerase chain reaction-restriction fragment length polymorphism. Our results revealed a significant association between the AA genotype of XRCC1 Arg280His [odds ratio (OR) = 2.51, 95% confidence interval (CI) = 1.29-4.87, P = 0.01] and an increased risk of laryngeal cancer susceptibility compared to the GG genotype. Moreover, the A allele showed a higher risk of laryngeal cancer susceptibility compared to the G allele (OR = 1.63, 95%CI = 1.19-2.50, P = 0.002). In conclusion, the results of our study suggest that the AA genotype and A allele of the XRCC1 Arg280His polymorphism are associated with an increased laryngeal cancer risk in a Chinese population.

DNA repair genes polymorphisms and genetic susceptibility to Philadelphia-negative myeloproliferative neoplasms in a Portuguese population: The role of base excision repair genes polymorphisms.

PubMed

Azevedo, Ana P; Silva, Susana N; De Lima, João P; Reichert, Alice; Lima, Fernando; Júnior, Esmeraldina; Rueff, José

2017-06-01

The role of base excision repair (BER) genes in Philadelphia-negative (PN)-myeloproliferative neoplasms (MPNs) susceptibility was evaluated by genotyping eight polymorphisms [apurinic/apyrimidinic endodeoxyribonuclease 1, mutY DNA glycosylase, earlier mutY homolog ( E. coli ) (MUTYH), 8-oxoguanine DNA glycosylase 1, poly (ADP-ribose) polymerase (PARP) 1, PARP4 and X-ray repair cross-complementing 1 (XRCC1)] in a case-control study involving 133 Caucasian Portuguese patients. The results did not reveal a correlation between individual BER polymorphisms and PN-MPNs when considered as a whole. However, stratification for essential thrombocythaemia revealed i) borderline effect/tendency to increased risk when carrying at least one variant allele for XRCC1_399 single-nucleotide polymorphism (SNP); ii) decreased risk for Janus kinase 2-positive patients carrying at least one variant allele for XRCC1_399 SNP; and iii) decreased risk in females carrying at least one variant allele for MUTYH SNP. Combination of alleles demonstrated an increased risk to PN-MPNs for one specific haplogroup. These findings may provide evidence for gene variants in susceptibility to MPNs. Indeed, common variants in DNA repair genes may hamper the capacity to repair DNA, thus increasing cancer susceptibility.

DNA repair genes polymorphisms and genetic susceptibility to Philadelphia-negative myeloproliferative neoplasms in a Portuguese population: The role of base excision repair genes polymorphisms

PubMed Central

Azevedo, Ana P.; Silva, Susana N.; De Lima, João P.; Reichert, Alice; Lima, Fernando; Júnior, Esmeraldina; Rueff, José

2017-01-01

The role of base excision repair (BER) genes in Philadelphia-negative (PN)-myeloproliferative neoplasms (MPNs) susceptibility was evaluated by genotyping eight polymorphisms [apurinic/apyrimidinic endodeoxyribonuclease 1, mutY DNA glycosylase, earlier mutY homolog (E. coli) (MUTYH), 8-oxoguanine DNA glycosylase 1, poly (ADP-ribose) polymerase (PARP) 1, PARP4 and X-ray repair cross-complementing 1 (XRCC1)] in a case-control study involving 133 Caucasian Portuguese patients. The results did not reveal a correlation between individual BER polymorphisms and PN-MPNs when considered as a whole. However, stratification for essential thrombocythaemia revealed i) borderline effect/tendency to increased risk when carrying at least one variant allele for XRCC1_399 single-nucleotide polymorphism (SNP); ii) decreased risk for Janus kinase 2-positive patients carrying at least one variant allele for XRCC1_399 SNP; and iii) decreased risk in females carrying at least one variant allele for MUTYH SNP. Combination of alleles demonstrated an increased risk to PN-MPNs for one specific haplogroup. These findings may provide evidence for gene variants in susceptibility to MPNs. Indeed, common variants in DNA repair genes may hamper the capacity to repair DNA, thus increasing cancer susceptibility. PMID:28599464

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

PubMed Central

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

2017-01-01

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

MTHFR, TS and XRCC1 genetic variants may affect survival in patients with myelodysplastic syndromes treated with supportive care or azacitidine.

PubMed

Visani, G; Loscocco, F; Ruzzo, A; Galimberti, S; Graziano, F; Voso, M T; Giacomini, E; Finelli, C; Ciabatti, E; Fabiani, E; Barulli, S; Volpe, A; Magro, D; Piccaluga, P; Fuligni, F; Vignetti, M; Fazi, P; Piciocchi, A; Gabucci, E; Rocchi, M; Magnani, M; Isidori, A

2017-12-05

We evaluated the impact of genomic polymorphisms in folate-metabolizing, DNA synthesis and DNA repair enzymes on the clinical outcome of 108 patients with myelodysplastic syndromes (MDS) receiving best supportive care (BSC) or azacitidine. A statistically significant association between methylenetetrahydrofolate reductase (MTHFR) 677T/T, thymidylate synthase (TS) 5'-untranslated region (UTR) 3RG, TS 3'-UTR -6 bp/-6 bp, XRCC1 399G/G genotypes and short survival was found in patients receiving BSC by multivariate analysis (P<0.001; P=0.026; P=0.058; P=0.024). MTHFR 677T/T, TS 3'-UTR -6 bp/-6 bp and XRCC1 399G/G genotypes were associated with short survival in patients receiving azacitidine by multivariate analysis (P<0.001; P=0.004; P=0.002). We then performed an exploratory analysis to evaluate the effect of the simultaneous presence of multiple adverse variant genotypes. Interestingly, patients with ⩾1 adverse genetic variants had a short survival, independently from their International Prognostic Scoring System (IPSS) and therapy received. To our knowledge, this is the first study showing that polymorphisms in folate-metabolizing pathway, DNA synthesis and DNA repair genes could influence survival of MDS patients.The Pharmacogenomics Journal advance online publication, 5 December 2017; doi:10.1038/tpj.2017.48.

XRCC5 cooperates with p300 to promote cyclooxygenase-2 expression and tumor growth in colon cancers

PubMed Central

Hao, Jiajiao; Chen, Miao; Yu, Wendan; Guo, Wei; Chen, Yiming; Huang, Wenlin; Deng, Wuguo

2017-01-01

Cyclooxygenase (COX) is the rate-limiting enzyme in prostaglandins (PGs) biosynthesis. Previous studies indicate that COX-2, one of the isoforms of COX, is highly expressed in colon cancers and plays a key role in colon cancer carcinogenesis. Thus, searching for novel transcription factors regulating COX-2 expression will facilitate drug development for colon cancer. In this study, we identified XRCC5 as a binding protein of the COX-2 gene promoter in colon cancer cells with streptavidin-agarose pulldown assay and mass spectrometry analysis, and found that XRCC5 promoted colon cancer growth through modulation of COX-2 signaling. Knockdown of XRCC5 by siRNAs inhibited the growth of colon cancer cells in vitro and of tumor xenografts in a mouse model in vivo by suppressing COX-2 promoter activity and COX-2 protein expression. Conversely, overexpression of XRCC5 promoted the growth of colon cancer cells by activating COX-2 promoter and increasing COX-2 protein expression. Moreover, the role of p300 (a transcription co-activator) in acetylating XRCC5 to co-regulate COX-2 expression was also evaluated. Immunofluorescence assay and confocal microscopy showed that XRCC5 and p300 proteins were co-located in the nucleus of colon cancer cells. Co-immunoprecipitation assay also proved the interaction between XRCC5 and p300 in nuclear proteins of colon cancer cells. Cell viability assay indicated that the overexpression of wild-type p300, but not its histone acetyltransferase (HAT) domain deletion mutant, increased XRCC5 acetylation, thereby up-regulated COX-2 expression and promoted the growth of colon cancer cells. In contrast, suppression of p300 by a p300 HAT-specific inhibitor (C646) inhibited colon cancer cell growth by suppressing COX-2 expression. Taken together, our results demonstrated that XRCC5 promoted colon cancer growth by cooperating with p300 to regulate COX-2 expression, and suggested that the XRCC5/p300/COX-2 signaling pathway was a potential target in the

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

PubMed

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.

Susceptibility to Colorectal Cancer and Two Genetic Polymorphisms of XRCC4.

PubMed

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.

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

PubMed Central

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

2006-01-01

Poly(ADP-ribose)polymerase 1 (PARP-1) recognizes DNA strand interruptions in vivo and triggers its own modification as well as that of other proteins by the sequential addition of ADP-ribose to form polymers. This modification causes a release of PARP-1 from DNA ends and initiates a variety of responses including DNA repair. While PARP-1 has been firmly implicated in base excision and single strand break repair, its role in the repair of DNA double strand breaks (DSBs) remains unclear. Here, we show that PARP-1, probably together with DNA ligase III, operates in an alternative pathway of non-homologous end joining (NHEJ) that functions as backup to the classical pathway of NHEJ that utilizes DNA-PKcs, Ku, DNA ligase IV, XRCC4, XLF/Cernunnos and Artemis. PARP-1 binds to DNA ends in direct competition with Ku. However, in irradiated cells the higher affinity of Ku for DSBs and an excessive number of other forms of competing DNA lesions limit its contribution to DSB repair. When essential components of the classical pathway of NHEJ are absent, PARP-1 is recruited for DSB repair, particularly in the absence of Ku and non-DSB lesions. This form of DSB repair is sensitive to PARP-1 inhibitors. The results define the function of PARP-1 in DSB repair and characterize a candidate pathway responsible for joining errors causing genomic instability and cancer. PMID:17088286

Tankyrase1-mediated poly(ADP-ribosyl)ation of TRF1 maintains cell survival after telomeric DNA damage

PubMed Central

Yang, Lu; Sun, Luxi; Teng, Yaqun; Chen, Hao; Gao, Ying; Levine, Arthur S.; Nakajima, Satoshi

2017-01-01

Abstract Oxidative DNA damage triggers telomere erosion and cellular senescence. However, how repair is initiated at telomeres is largely unknown. Here, we found unlike PARP1-mediated Poly-ADP-Ribosylation (PARylation) at genomic damage sites, PARylation at telomeres is mainly dependent on tankyrase1 (TNKS1). TNKS1 is recruited to damaged telomeres via its interaction with TRF1, which subsequently facilitates the PARylation of TRF1 after damage. TNKS inhibition abolishes the recruitment of the repair proteins XRCC1 and polymerase β at damaged telomeres, while the PARP1/2 inhibitor only has such an effect at non-telomeric damage sites. The ANK domain of TNKS1 is essential for the telomeric damage response and TRF1 interaction. Mutation of the tankyrase-binding motif (TBM) on TRF1 (13R/18G to AA) disrupts its interaction with TNKS1 concomitant recruitment of TNKS1 and repair proteins after damage. Either TNKS1 inhibition or TBM mutated TRF1 expression markedly sensitizes cells to telomere oxidative damage as well as XRCC1 inhibition. Together, our data reveal a novel role of TNKS1 in facilitating SSBR at damaged telomeres through PARylation of TRF1, thereby protecting genome stability and cell viability. PMID:28160604

Domain mapping of the Rad51 paralog protein complexes

PubMed Central

Miller, Kristi A.; Sawicka, Dorota; Barsky, Daniel; Albala, Joanna S.

2004-01-01

The five human Rad51 paralogs are suggested to play an important role in the maintenance of genome stability through their function in DNA double-strand break repair. These proteins have been found to form two distinct complexes in vivo, Rad51B–Rad51C–Rad51D–Xrcc2 (BCDX2) and Rad51C–Xrcc3 (CX3). Based on the recent Pyrococcus furiosus Rad51 structure, we have used homology modeling to design deletion mutants of the Rad51 paralogs. The models of the human Rad51B, Rad51C, Xrcc3 and murine Rad51D (mRad51D) proteins reveal distinct N-terminal and C-terminal domains connected by a linker region. Using yeast two-hybrid and co-immunoprecipitation techniques, we have demonstrated that a fragment of Rad51B containing amino acid residues 1–75 interacts with the C-terminus and linker of Rad51C, residues 79–376, and this region of Rad51C also interacts with mRad51D and Xrcc3. We have also determined that the N-terminal domain of mRad51D, residues 4–77, binds to Xrcc2 while the C-terminal domain of mRad51D, residues 77–328, binds Rad51C. By this, we have identified the binding domains of the BCDX2 and CX3 complexes to further characterize the interaction of these proteins and propose a scheme for the three-dimensional architecture of the BCDX2 and CX3 paralog complexes. PMID:14704354

Normal development of mice lacking PAXX, the paralogue of XRCC4 and XLF.

PubMed

Gago-Fuentes, Raquel; Xing, Mengtan; Sæterstad, Siri; Sarno, Antonio; Dewan, Alisa; Beck, Carole; Bradamante, Stefano; Bjørås, Magnar; Oksenych, Valentyn

2018-03-01

DNA repair consists of several cellular pathways which recognize and repair damaged DNA. The classical nonhomologous DNA end-joining (NHEJ) pathway repairs double-strand breaks in DNA. It is required for maturation of both B and T lymphocytes by supporting V(D)J recombination as well as B-cell differentiation during class switch recombination (CSR). Inactivation of NHEJ factors Ku70, Ku80, XRCC4, DNA ligase 4, DNA-PKcs, and Artemis impairs V(D)J recombination and blocks lymphocyte development. Paralogue of XRCC4 and XLF (PAXX) is an accessory NHEJ factor that has a significant impact on the repair of DNA lesions induced by ionizing radiation in human, murine, and chicken cells. However, the role of PAXX during development is poorly understood. To determine the physiological role of PAXX, we deleted part of the Paxx promoter and the first two exons in mice. Further, we compared Paxx -knockout mice with wild-type (WT) and NHEJ-deficient controls including Ku80- and Dna-pkcs -null and severe combined immunodeficiency mice. Surprisingly, Paxx -deficient mice were not distinguishable from the WT littermates; they were the same weight and size, fertility status, had normal spleen, thymus, and bone marrow. Paxx -deficient mice had the same number of chromosomal and chromatid breaks as WT mice. Moreover, Paxx -deficient primary B lymphocytes had the same level of CSR as lymphocytes isolated from WT mice. We concluded that PAXX is dispensable for normal mouse development.

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

PubMed

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.

Evaluation of Prediction of Polymorphisms of DNA Repair Genes on the Efficacy of Platinum-Based Chemotherapy in Patients With Non-Small Cell Lung Cancer: A Network Meta-Analysis.

PubMed

Yu, Shao-Nan; Liu, Gui-Feng; Li, Xue-Feng; Fu, Bao-Hong; Dong, Li-Xin; Zhang, Shu-Hua

2017-12-01

This network meta-analysis (NMA) was conducted to compare the predictive value of 14 SNPs in eight DNA repair genes on the efficacy of platinum-based chemotherapy in patients with non-small cell lung cancer (NSCLC). These included ERCC1 (rs11615, rs3212986, rs3212948), XRCC1 (rs25487, rs25489, rs1799782), XPD (rs13181, rs1799793), XPG (rs1047768, rs17655), XPA (rs1800975), XRCC3 (rs861539), APE1 (rs3136820), and RRM1 (rs1042858). The PubMed and Cochrane library databases were reviewed from their inception to February 2017 and studies which met our inclusion criteria were included in our investigation. This network meta-analysis combines direct and indirect evidence to assess the predictive value of 14 SNPs in eight DNA repair genes on the efficacy of platinum-based chemotherapy in NSCLC. We evaluated the predictive value through the use of the odd ratios (OR) and drawing surface under the cumulative ranking curves (SUCRA). A total of 26 eligible cohort studies were enrolled in this NMA. The pairwise meta-analysis indicated that in terms of overall response ratio (ORR), ERCC1 (rs11615), XRCC1 (rs25487, rs1799782), and XPD (rs13181) polymorphisms are associated with the efficacy of platinum-based chemotherapy in NSCLC. The result of this NMA suggests that there is no significant difference in predictive value of 8 DNA repair genes on the efficacy of platinum-based chemotherapy in NSCLC patients. The rank of SUCRA values of the 14 SNPs in the eight DNA repair genes were: XPD (rs1799793)→ERCC1 (rs3212986)→XPA(rs1800975)→ERCC1(rs3212948)→XRCC1(rs25487)→XRCC3(rs861539)→APE1(rs3136820)→ERCC1(rs11615)→XRCC1(rs1799782)→RRM1(rs1042858)→XPD(rs13181)→XPG (rs1047768)→XPG(rs17655)→XRCC1(rs25489). ERCC1(rs11615), XRCC1(rs25487, rs1799782) and XPD(rs13181) polymorphisms were better predictors in evaluating the efficacy of platinum-based chemotherapy in NSCLC patients. J. Cell. Biochem. 118: 4782-4791, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley

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.

PubMed

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.

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

PubMed

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.

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

NASA Astrophysics Data System (ADS)

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.

Analysis and results of Ku and XRCC4 expression in hypopharyngeal cancer tissues treated with chemoradiotherapy

PubMed Central

HAYASHI, JYUNICHI; SAKATA, KOH-ICHI; SOMEYA, MASANORI; MATSUMOTO, YOSHIHISA; SATOH, MASAAKI; NAKATA, KENSEI; HORI, MASAKAZU; TAKAGI, MASARU; KONDOH, ATSUSHI; HIMI, TETSUO; HAREYAMA, MASATO

2012-01-01

DNA double-strand break (DSB) is one of the most serious forms of damage induced by ionizing irradiation. Non-homologous end-joining (NHEJ) is a key mechanism of DNA DSB repair. The immunohistochemical analysis of proteins involved in NHEJ may have potential as a predictive assay for tumor radiosensitivity. We examined the correlation between the expression of proteins involved in DNA DSB in biopsy specimens and the results of chemoradiotherapy in hypopharyngeal cancers. Fifty-seven patients with previously untreated squamous cell carcinoma of the hypopharynx were treated between March 2002 and December 2009. Most patients (75%) had stage III or IV disease. The chemotherapy consisted of cisplatin plus 5FU or S-1. A tumor dose of 50 Gy was usually administered to the primary tumor and regional lymph nodes. Doses of 10–20 Gy were usually added to the primary tumor with reduced fields after 50 Gy. The 5-year disease-free survival rate was 100% for patients in stage I, 90% in stage II, 64% in stage III and 50% in stage IV. In stages I-III, patients with a lower expression of Ku70 or XRCC4 tended to have better locoregional control. These results indicated that a lower expression of Ku70 or XRCC4 may be correlated with higher radiosensitivity. Two patients had distant metastasis alone, of which one had 0% expression of Ku70 and the other had 0% expression of Ku86. The absence of Ku70 or Ku86 expression indicates low DNA-PK activity. Low DNA-PK activity due to a low expression of Ku may result in the genetic alteration of cancer cells, leading to a higher tendency of distant metastasis. This finding suggests that proteins involved in NHEJ may have an impact on the treatment results of chemoradiotherapy in hypopharyngeal cancer. PMID:22807979

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Guo, Na; Du, Guanghua, E-mail: gh-du@impcas.ac.cn; Liu, Wenjing

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 themore » 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}.« less

Mapping of interaction domains between human repair proteins ERCC1 and XPF.

PubMed

de Laat, W L; Sijbers, A M; Odijk, H; Jaspers, N G; Hoeijmakers, J H

1998-09-15

ERCC1-XPF is a heterodimeric protein complexinvolved in nucleotide excision repair and recombinational processes. Like its homologous complex in Saccharomyces cerevisiae , Rad10-Rad1, it acts as a structure-specific DNA endonuclease, cleaving at duplex-single-stranded DNA junctions. In repair, ERCC1-XPF and Rad10-Rad1 make an incision on the the 5'-side of the lesion. No humans with a defect in the ERCC1 subunit of this protein complex have been identified and ERCC1-deficient mice suffer from severe developmental problems and signs of premature aging on top of a repair-deficient phenotype. Xeroderma pigmentosum group F patients carry mutations in the XPF subunit and generally show the clinical symptoms of mild DNA repair deficiency. All XP-F patients examined demonstrate reduced levels of XPF and ERCC1 protein, suggesting that proper complex formation is required for stability of the two proteins. To better understand the molecular and clinical consequences of mutations in the ERCC1-XPF complex, we decided to map the interaction domains between the two subunits. The XPF-binding domain comprises C-terminal residues 224-297 of ERCC1. Intriguingly, this domain resides outside the region of homology with its yeast Rad10 counterpart. The ERCC1-binding domain in XPF maps to C-terminal residues 814-905. ERCC1-XPF complex formation is established by a direct interaction between these two binding domains. A mutation from an XP-F patient that alters the ERCC1-binding domain in XPF indeed affects complex formation with ERCC1.

Mapping of interaction domains between human repair proteins ERCC1 and XPF.

PubMed Central

de Laat, W L; Sijbers, A M; Odijk, H; Jaspers, N G; Hoeijmakers, J H

1998-01-01

ERCC1-XPF is a heterodimeric protein complexinvolved in nucleotide excision repair and recombinational processes. Like its homologous complex in Saccharomyces cerevisiae , Rad10-Rad1, it acts as a structure-specific DNA endonuclease, cleaving at duplex-single-stranded DNA junctions. In repair, ERCC1-XPF and Rad10-Rad1 make an incision on the the 5'-side of the lesion. No humans with a defect in the ERCC1 subunit of this protein complex have been identified and ERCC1-deficient mice suffer from severe developmental problems and signs of premature aging on top of a repair-deficient phenotype. Xeroderma pigmentosum group F patients carry mutations in the XPF subunit and generally show the clinical symptoms of mild DNA repair deficiency. All XP-F patients examined demonstrate reduced levels of XPF and ERCC1 protein, suggesting that proper complex formation is required for stability of the two proteins. To better understand the molecular and clinical consequences of mutations in the ERCC1-XPF complex, we decided to map the interaction domains between the two subunits. The XPF-binding domain comprises C-terminal residues 224-297 of ERCC1. Intriguingly, this domain resides outside the region of homology with its yeast Rad10 counterpart. The ERCC1-binding domain in XPF maps to C-terminal residues 814-905. ERCC1-XPF complex formation is established by a direct interaction between these two binding domains. A mutation from an XP-F patient that alters the ERCC1-binding domain in XPF indeed affects complex formation with ERCC1. PMID:9722633

Genetic variants of SULT1A1 and XRCC1 genes and risk of lung cancer in Bangladeshi population.

PubMed

Tasnim, Tasnova; Al-Mamun, Mir Md Abdullah; Nahid, Noor Ahmed; Islam, Md Reazul; Apu, Mohd Nazmul Hasan; Bushra, Most Umme; Rabbi, Sikder Nahidul Islam; Nahar, Zabun; Chowdhury, Jakir Ahmed; Ahmed, Maizbha Uddin; Islam, Mohammad Safiqul; Hasnat, Abul

2017-11-01

Lung cancer is one of the most frequently occurring cancers throughout the world as well as in Bangladesh. This study aimed to correlate the prognostic and/or predictive value of functional polymorphisms in SULT1A1 (rs9282861) and XRCC1 (rs25487) genes and lung cancer risk in Bangladeshi population. A case-control study was conducted which comprises 202 lung cancer patients and 242 healthy volunteers taking into account the age, sex, and smoking status. After isolation of genomic DNA, genotyping was done by polymerase chain reaction-restriction fragment length polymorphism method and the lung cancer risk was evaluated as odds ratio that was adjusted for age, sex, and smoking status. A significant association was found between SULT1A1 rs9282861 and XRCC1 rs25487 polymorphisms and lung cancer risk. In case of rs9282861 polymorphism, Arg/His (adjusted odds ratio = 5.06, 95% confidence interval = 3.05-8.41, p < 0.05) and His/His (adjusted odds ratio = 3.88, 95% confidence interval = 2.20-6.82, p < 0.05) genotypes were strongly associated with increased risk of lung cancer in comparison to the Arg/Arg genotype. In case of rs25487 polymorphism, Arg/Gln heterozygote (adjusted odds ratio = 4.57, 95% confidence interval = 2.79-7.46, p < 0.05) and Gln/Gln mutant homozygote (adjusted odds ratio = 4.99, 95% confidence interval = 2.66-9.36, p < 0.05) were also found to be significantly associated with increased risk of lung cancer. This study demonstrates that the presence of His allele and Gln allele in case of SULT1A1 rs9282861 and XRCC1 rs25487, respectively, involve in lung cancer prognosis in Bangladeshi population.

Bone morphogenetic protein (BMP)1-3 enhances bone repair.

PubMed

Grgurevic, Lovorka; Macek, Boris; Mercep, Mladen; Jelic, Mislav; Smoljanovic, Tomislav; Erjavec, Igor; Dumic-Cule, Ivo; Prgomet, Stefan; Durdevic, Dragan; Vnuk, Drazen; Lipar, Marija; Stejskal, Marko; Kufner, Vera; Brkljacic, Jelena; Maticic, Drazen; Vukicevic, Slobodan

2011-04-29

Members of the astacin family of metalloproteinases such as human bone morphogenetic protein 1 (BMP-1) regulate morphogenesis by processing precursors to mature functional extracellular matrix (ECM) proteins and several growth factors including TGFβ, BMP2, BMP4 and GFD8. We have recently discovered that BMP1-3 isoform of the Bmp-1 gene circulates in the human plasma and is significantly increased in patients with acute bone fracture. We hypothesized that circulating BMP1-3 might have an important role in bone repair and serve as a novel bone biomarker. When administered systemically to rats with a long bone fracture and locally to rabbits with a critical size defect of the ulna, recombinant human BMP1-3 enhanced bone healing. In contrast, neutralization of the endogenous BMP1-3 by a specific polyclonal antibody delayed the bone union. Invitro BMP1-3 increased the expression of collagen type I and osteocalcin in MC3T3-E(1) osteoblast like cells, and enhanced the formation of mineralized bone nodules from bone marrow mesenchymal stem cells. We suggest that BMP1-3 is a novel systemic regulator of bone repair. Copyright © 2011 Elsevier Inc. All rights reserved.

The Effect of Polymorphisms in DNA Repair Genes and Carcinogen Metabolizers on Leukocyte Telomere Length: A Cohort of Healthy Spanish Smokers.

PubMed

Verde, Zoraida; Reinoso-Barbero, Luis; Chicharro, Luis; Resano, Pilar; Sánchez-Hernández, Ignacio; Rodríguez González-Moro, Jose Miguel; Bandrés, Fernando; Gómez-Gallego, Félix; Santiago, Catalina

2016-04-01

Smoking implies exposure to carcinogenic agents that causes DNA damage, which could be suspected to enhance telomere attrition. To protect and deal with DNA damage, cells possess mechanisms that repair and neutralize harmful substances. Polymorphisms altering DNA repair capacity or carcinogen metabolism may lead to synergistic effects with tobacco carcinogen-induced shorter telomere length independently of cancer interaction. The aim of this study was to explore the association between leukocyte telomere length (LTL) and several genetic polymorphisms in DNA repair genes and carcinogen metabolizers in a cohort of healthy smokers. We evaluated the effect of six genetic polymorphisms in cytochrome P1A1 (Ile462Val), XRCC1 (Arg399Gln), APEX1 (Asp148Glu), XRCC3 (Thr241Met), and XPD (Asp312Asn; Lys751Gln) on LTL in a cohort of 145 healthy smokers in addition to smoking habits. Logistic regression analysis showed an association between XRCC1 399Gln allele and shorter telomere length (OR = 5.03, 95% CI = 1.08% to 23.36%). There were not association between the rest of polymorphisms analyzed and LTL. Continuous exposure to tobacco could overwhelm the DNA repair machinery, making the effect of the polymorphisms that reduce repair capacity more pronounced. Analyzing the function of smoking-induced DNA-repair genes and LTL is an important goal in order to identify therapeutic targets to treat smoking-induced diseases. © The Author 2015. Published by Oxford University Press on behalf of the Society for Research on Nicotine and Tobacco. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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

PubMed

Ganesan, Shanthi; Keating, Aileen F

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 6μM) for 24 or 48h. Cell viability was reduced (P<0.05) after 48h of exposure to 3 or 6μM PM. The NOR-G-OH DNA adduct was detected after 24h of 6μM PM exposure, while the more cytotoxic G-NOR-G DNA adduct was formed after 48h 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. Copyright © 2014 Elsevier Inc. All rights reserved.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

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 6more » 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.« less

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

PubMed

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.

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

PubMed

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.

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

PubMed Central

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

Association of Arg194Trp, Arg280His and Arg399Gln Polymorphisms in X-ray Repair Cross-Complementing Group 1 Gene and Risk of Differentiated Thyroid Carcinoma in Iran

PubMed Central

Fard-Esfahani, Pezhman; Fard-Esfahani, Armaghan; Fayaz, Shima; Ghanbarzadeh, Bahareh; Saidi, Parinaz; Mohabati, Reyhaneh; Bidoki, Seyed Kazem; Majdi, Mina

2011-01-01

Background: X-ray repair cross-complementing group 1 (XRCC1) gene is a DNA repair gene and its non-synonymous single nucleotide polymorphisms (SNP) may influence DNA repair capacity which has been considered as a modifying risk factor for cancer development. Methods: A case-control study was conducted to investigate impact of three frequently studied polymorphisms (Arg194Trp, Arg280His and Arg399Gln) on developing differentiated thyroid carcinoma (DTC). Results: Increased risks for DTC were shown in homozygous (odds ratio [OR]: 3.66, 95% confidence interval [CI]: 0.38-35.60) and in dominant trait (OR: 1.22, 95% CI: 1.64-2.32) of Arg194Trp genotype. Also, for Arg280His genotype, an increased risk for DTC was shown in dominant trait (OR: 1.42, 95% confidence interval [CI]: 0.76-2.68), while a mildly reduction of risk for DTC (OR: 0.77, 95% [CI]: 0.50-1.17) was estimated in dominant Gln genotype of Arg399Gln. Considering combinatory effects of Arg194Trp and Arg280His genotypes on DTC, the calculated OR and 95% CI for being heterozygous for one of Arg194Trp or Arg280His genotypes were 1.57 and 0.90-2.74, respectively. Conclusion: Genotyping of codons 194, 280 and 399 in XRCC1 gene may use in risk assessment of DTC. PMID:21987112

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

PubMed

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

2015-01-01

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

Combining heavy ion radiation and artificial microRNAs to target the homologous recombination repair gene efficiently kills human tumor cells.

PubMed

Zheng, Zhiming; Wang, Ping; Wang, Hongyan; Zhang, Xiangming; Wang, Minli; Cucinotta, Francis A; Wang, Ya

2013-02-01

Previously, we demonstrated that heavy ions kill more cells at the same dose than X-rays because DNA-clustered lesions produced by heavy ions affect nonhomologous end-joining (NHEJ) repair but not homologous recombination repair (HRR). We have also shown that our designed artificial microRNAs (amiRs) could efficiently target XRCC4 (an essential factor for NHEJ) or XRCC2 (an essential factor for HRR) and sensitize human tumor cells to X-rays. Based on these data, we were interested in testing the hypothesis that combining heavy ions and amiRs to target HRR but not NHEJ should more efficiently kill human tumor cells. Human tumor cell lines (U87MG, a brain tumor cell line, and A549, a lung cancer cell line) and their counterparts, overexpressed with amiR to target XRCC2, XRCC4 or both, were used in this study. Survival sensitivities were examined using a clonogenic assay after these cells were exposed to X-rays or heavy ions. In addition, these cell lines were subcutaneously injected into nude mice to form xenografts and the tumor size was compared after the tumor areas were exposed to X-rays or heavy ions. Although targeting either XRCC4 (NHEJ factor) or XRCC2 (HRR factor) sensitized the human tumor cells to X-rays, in vitro and the xenograft animal model, targeting only XRCC2 but not XRCC4 sensitized the human tumor cells to heavy ions in vitro and in the xenograft animal model. Combining heavy ions with targeting the HRR pathway, but not the NHEJ pathway, could significantly improve the efficiency of tumor cell death. Copyright © 2013 Elsevier Inc. All rights reserved.

Involvement of the Artemis Protein in the Relative Biological Efficiency Observed With the 76-MeV Proton Beam Used at the Institut Curie Proton Therapy Center in Orsay

DOE Office of Scientific and Technical Information (OSTI.GOV)

Calugaru, Valentin; Institut Curie, Centre Universitaire, Orsay; INSERM U612, Centre Universitaire, Orsay

2014-09-01

Purpose: Previously we showed that the relative biological efficiency for induced cell killing by the 76-MeV beam used at the Institut Curie Proton Therapy Center in Orsay increased with depth throughout the spread-out Bragg peak (SOBP). To investigate the repair pathways underlying this increase, we used an isogenic human cell model in which individual DNA repair proteins have been depleted, and techniques dedicated to precise measurements of radiation-induced DNA single-strand breaks (SSBs) and double-strand breaks (DSBs). Methods and Materials: The 3-Gy surviving fractions of HeLa cells individually depleted of Ogg1, XRCC1, and PARP1 (the base excision repair/SSB repair pathway) or ofmore » ATM, DNA-PKcs, XRCC4, and Artemis (nonhomologous end-joining pathway) were determined at the 3 positions previously defined in the SOBP. Quantification of incident SSBs and DSBs by the alkaline elution technique and 3-dimensional (3D) immunofluorescence of γ-H2AX foci, respectively, was performed in SQ20 B cells. Results: We showed that the amount of SSBs and DSBs depends directly on the particle fluence and that the increase in relative biological efficiency observed in the distal part of the SOBP is due to a subset of lesions generated under these conditions, leading to cell death via a pathway in which the Artemis protein plays a central role. Conclusions: Because therapies like proton or carbon beams are now being used to treat cancer, it is even more important to dissect the mechanisms implicated in the repair of the lesions generated by these particles. Additionally, alteration of the expression or activity of the Artemis protein could be a novel therapeutic tool before high linear energy transfer irradiation treatment.« less

Structural insights into NHEJ: building up an integrated picture of the dynamic DSB repair super complex, one component and interaction at a time

PubMed Central

Williams, Gareth J.; Hammel, Michal; Radhakrishnan, Sarvan Kumar; Ramsden, Dale; Lees-Miller, Susan P.; Tainer, John A.

2014-01-01

Non-homologous end joining (NHEJ) is the major pathway for repair of DNA double-strand breaks (DSBs) in human cells. NHEJ is also needed for V(D)J recombination and the development of T and B cells in vertebrate immune systems, and acts in both the generation and prevention of non-homologous chromosomal translocations, a hallmark of genomic instability and many human cancers. X-ray crystal structures, cryo-electron microscopy envelopes, and small angle X-ray scattering (SAXS) solution conformations and assemblies are defining most of the core protein components for NHEJ: Ku70/Ku80 heterodimer; the DNA dependent protein kinase catalytic subunit (DNA-PKcs); the structure-specific endonuclease Artemis along with polynucleotide kinase/phosphatase (PNKP), aprataxin and PNKP related protein (APLF); the scaffolding proteins XRCC4 and XLF (XRCC4-like factor); DNA polymerases, and DNA ligase IV (Lig IV). The dynamic assembly of multi-protein NHEJ complexes at DSBs is regulated in part by protein phosphorylation. The basic steps of NHEJ have been biochemically defined to require: 1) DSB detection by the Ku heterodimer with subsequent DNA-PKcs tethering to form the DNA-PKcs-Ku-DNA complex (termed DNA-PK), 2) lesion processing, and 3) DNA end ligation by Lig IV, which functions in complex with XRCC4 and XLF. The current integration of structures by combined methods is resolving puzzles regarding the mechanisms, coordination and regulation of these three basic steps. Overall, structural results suggest the NHEJ system forms a flexing scaffold with the DNA-PKcs HEAT repeats acting as compressible macromolecular springs suitable to store and release conformational energy to apply forces to regulate NHEJ complexes and the DNA substrate for DNA end protection, processing, and ligation. PMID:24656613

Parkinsonism-associated Protein DJ-1/Park7 Is a Major Protein Deglycase That Repairs Methylglyoxal- and Glyoxal-glycated Cysteine, Arginine, and Lysine Residues

PubMed Central

Richarme, Gilbert; Mihoub, Mouadh; Dairou, Julien; Bui, Linh Chi; Leger, Thibaut; Lamouri, Aazdine

2015-01-01

Glycation is an inevitable nonenzymatic covalent reaction between proteins and endogenous reducing sugars or dicarbonyls (methylglyoxal, glyoxal) that results in protein inactivation. DJ-1 was reported to be a multifunctional oxidative stress response protein with poorly defined function. Here, we show that human DJ-1 is a protein deglycase that repairs methylglyoxal- and glyoxal-glycated amino acids and proteins by acting on early glycation intermediates and releases repaired proteins and lactate or glycolate, respectively. DJ-1 deglycates cysteines, arginines, and lysines (the three major glycated amino acids) of serum albumin, glyceraldehyde-3-phosphate dehydrogenase, aldolase, and aspartate aminotransferase and thus reactivates these proteins. DJ-1 prevented protein glycation in an Escherichia coli mutant deficient in the DJ-1 homolog YajL and restored cell viability in glucose-containing media. These results suggest that DJ-1-associated Parkinsonism results from excessive protein glycation and establishes DJ-1 as a major anti-glycation and anti-aging protein. PMID:25416785

Combining Heavy Ion Radiation and Artificial MicroRNAs to Target the Homologous Recombination Repair Gene Efficiently Kills Human Tumor Cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zheng Zhiming; Department of Radiation Oncology, School of Medicine, Winship Cancer Institute, Emory University, Atlanta, Georgia; Wang Ping

2013-02-01

Purpose: Previously, we demonstrated that heavy ions kill more cells at the same dose than X-rays because DNA-clustered lesions produced by heavy ions affect nonhomologous end-joining (NHEJ) repair but not homologous recombination repair (HRR). We have also shown that our designed artificial microRNAs (amiRs) could efficiently target XRCC4 (an essential factor for NHEJ) or XRCC2 (an essential factor for HRR) and sensitize human tumor cells to X-rays. Based on these data, we were interested in testing the hypothesis that combining heavy ions and amiRs to target HRR but not NHEJ should more efficiently kill human tumor cells. Methods and Materials:more » Human tumor cell lines (U87MG, a brain tumor cell line, and A549, a lung cancer cell line) and their counterparts, overexpressed with amiR to target XRCC2, XRCC4 or both, were used in this study. Survival sensitivities were examined using a clonogenic assay after these cells were exposed to X-rays or heavy ions. In addition, these cell lines were subcutaneously injected into nude mice to form xenografts and the tumor size was compared after the tumor areas were exposed to X-rays or heavy ions. Results: Although targeting either XRCC4 (NHEJ factor) or XRCC2 (HRR factor) sensitized the human tumor cells to X-rays, in vitro and the xenograft animal model, targeting only XRCC2 but not XRCC4 sensitized the human tumor cells to heavy ions in vitro and in the xenograft animal model. Conclusions: Combining heavy ions with targeting the HRR pathway, but not the NHEJ pathway, could significantly improve the efficiency of tumor cell death.« less

Disruption of nucleotide excision repair by the human T-cell leukemia virus type 1 Tax protein.

PubMed

Kao, S Y; Marriott, S J

1999-05-01

The Tax protein of human T-cell leukemia virus type 1 (HTLV-1) is a transcriptional transactivator and viral oncogene. Since cellular transformation has been frequently linked to alterations in genome stability, we investigated the effect of Tax on nucleotide excision repair (NER), a prominent cellular DNA repair pathway. Cells expressing Tax exhibited a reduced capacity for NER as measured by unscheduled DNA synthesis and host cell reactivation assays. The cellular proliferating cell nuclear antigen (PCNA) gene product regulates DNA replication and repair pathways, including NER. Since Tax activates transcription of the PCNA promoter, we investigated whether this activity contributes to the reduction of NER. Tax increased endogenous PCNA protein expression, and analysis of Tax mutant proteins demonstrated that the reduction in NER correlated with Tax transactivation of PCNA gene expression. Direct overexpression of PCNA also reduced NER. We propose that overexpression of PCNA, and disruption of NER induced by Tax, predisposes cells to accumulate DNA damage and contributes to HTLV-1 transformation.

DNA repair pathways and mitochondrial DNA mutations in gastrointestinal carcinogenesis.

PubMed

Basso, Daniela; Navaglia, Filippo; Fogar, Paola; Zambon, Carlo-Federico; Greco, Eliana; Schiavon, Stefania; Fasolo, Michela; Stranges, Alessia; Falda, Alessandra; Padoan, Andrea; Fadi, Elisa; Pedrazzoli, Sergio; Plebani, Mario

2007-05-01

This work focuses on the main DNA repair pathways, highlighting their role in gastrointestinal carcinogenesis and the role of mitochondrial DNA (mtDNA), mutations being described in several tumor types, including those of the gastrointestinal tract. The mismatch repair (MMR) system is inherently altered in patients with hereditary non-polyposis colorectal cancer, and plays a role in carcinogenesis in a subset of sporadic colorectal, gastric and esophageal cancers. Alterations in homologous recombination (HR) and non-homologous end-joining (NHEJ) also contribute to the development of pancreatic cancer. Gene polymorphisms of some X-ray cross-complementing (XRCCs), cofactor proteins involved in the base excision repair pathway, have been investigated in relation to gastric, colorectal and pancreatic cancer. Yet only one polymorphism, XRCC1 Arg194Trp, appears to be involved in smoking-related cancers and in early onset pancreatic cancer. Although evidence in the literature indicates that mtDNA somatic mutations play a role in gastric and colorectal carcinogenesis, no sound conclusions have yet been drawn regarding this issue in pancreatic cancer, although an mtDNA variant at 16519 is believed to worsen the outcome of pancreatic cancer patients, possibly because it is involved in altering cellular metabolism.

An Intrinsically Disordered APLF Links Ku, DNA-PKcs, and XRCC4-DNA Ligase IV in an Extended Flexible Non-homologous End Joining Complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hammel, Michal; Yu, Yaping; Radhakrishnan, Sarvan K.

DNA double-strand break (DSB) repair by non-homologous end joining (NHEJ) in human cells is initiated by Ku heterodimer binding to a DSB, followed by recruitment of core NHEJ factors including DNA-dependent protein kinase catalytic subunit (DNA-PKcs), XRCC4-like factor (XLF), and XRCC4 (X4)-DNA ligase IV (L4). Ku also interacts with accessory factors such as aprataxin and polynucleotide kinase/phosphatase-like factor (APLF). But, how these factors interact to tether, process, and ligate DSB ends while allowing regulation and chromatin interactions remains enigmatic. Here, small angle X-ray scattering (SAXS) and mutational analyses show APLF is largely an intrinsically disordered protein that binds Ku, Ku/DNA-PKcsmore » (DNA-PK), and X4L4 within an extended flexible NHEJ core complex. X4L4 assembles with Ku heterodimers linked to DNA-PKcs via flexible Ku80 C-terminal regions (Ku80CTR) in a complex stabilized through APLF interactions with Ku, DNA-PK, and X4L4. Our collective results unveil the solution architecture of the six-protein complex and suggest cooperative assembly of an extended flexible NHEJ core complex that supports APLF accessibility while possibly providing flexible attachment of the core complex to chromatin. The resulting dynamic tethering furthermore, provides geometric access of L4 catalytic domains to the DNA ends during ligation and of DNA-PKcs for targeted phosphorylation of other NHEJ proteins as well as trans-phosphorylation of DNA-PKcs on the opposing DSB without disrupting the core ligation complex. Overall the results shed light on evolutionary conservation of Ku, X4, and L4 activities, while explaining the observation that Ku80CTR and DNA-PKcs only occur in a subset of higher eukaryotes.« less

An Intrinsically Disordered APLF Links Ku, DNA-PKcs, and XRCC4-DNA Ligase IV in an Extended Flexible Non-homologous End Joining Complex

DOE PAGES

Hammel, Michal; Yu, Yaping; Radhakrishnan, Sarvan K.; ...

2016-11-14

DNA double-strand break (DSB) repair by non-homologous end joining (NHEJ) in human cells is initiated by Ku heterodimer binding to a DSB, followed by recruitment of core NHEJ factors including DNA-dependent protein kinase catalytic subunit (DNA-PKcs), XRCC4-like factor (XLF), and XRCC4 (X4)-DNA ligase IV (L4). Ku also interacts with accessory factors such as aprataxin and polynucleotide kinase/phosphatase-like factor (APLF). But, how these factors interact to tether, process, and ligate DSB ends while allowing regulation and chromatin interactions remains enigmatic. Here, small angle X-ray scattering (SAXS) and mutational analyses show APLF is largely an intrinsically disordered protein that binds Ku, Ku/DNA-PKcsmore » (DNA-PK), and X4L4 within an extended flexible NHEJ core complex. X4L4 assembles with Ku heterodimers linked to DNA-PKcs via flexible Ku80 C-terminal regions (Ku80CTR) in a complex stabilized through APLF interactions with Ku, DNA-PK, and X4L4. Our collective results unveil the solution architecture of the six-protein complex and suggest cooperative assembly of an extended flexible NHEJ core complex that supports APLF accessibility while possibly providing flexible attachment of the core complex to chromatin. The resulting dynamic tethering furthermore, provides geometric access of L4 catalytic domains to the DNA ends during ligation and of DNA-PKcs for targeted phosphorylation of other NHEJ proteins as well as trans-phosphorylation of DNA-PKcs on the opposing DSB without disrupting the core ligation complex. Overall the results shed light on evolutionary conservation of Ku, X4, and L4 activities, while explaining the observation that Ku80CTR and DNA-PKcs only occur in a subset of higher eukaryotes.« less

Polymorphisms in DNA double-strand break repair genes and risk of breast cancer: two population-based studies in USA and Poland, and meta-analyses.

PubMed

García-Closas, Montserrat; Egan, Kathleen M; Newcomb, Polly A; Brinton, Louise A; Titus-Ernstoff, Linda; Chanock, Stephen; Welch, Robert; Lissowska, Jolanta; Peplonska, Beata; Szeszenia-Dabrowska, Neonila; Zatonski, Witold; Bardin-Mikolajczak, Alicja; Struewing, Jeffery P

2006-05-01

The double-strand break DNA repair pathway has been implicated in breast carcinogenesis. We evaluated the association between 19 polymorphisms in seven genes in this pathway (XRCC2, XRCC3, BRCA2, ZNF350, BRIP1, XRCC4, LIG4) and breast cancer risk in two population-based studies in USA (3,368 cases and 2,880 controls) and Poland (1,995 cases and 2,296 controls). These data suggested weak associations with breast cancer risk for XRCC3 T241M and IVS7-14A>G (pooled odds ratio (95% confidence interval): 1.18 (1.04-1.34) and 0.85 (0.73-0.98) for homozygous variant vs wild-type genotypes, respectively), and for an uncommon variant in ZNF350 S472P (1.24 (1.05-1.48)), with no evidence for study heterogeneity. The remaining variants examined had no significant relationships to breast cancer risk. Meta-analyses of studies in Caucasian populations, including ours, provided some support for a weak association for homozygous variants for XRCC3 T241M (1.16 (1.04-1.30); total of 10,979 cases and 10,423 controls) and BRCA2 N372H (1.13 (1.10-1.28); total of 13,032 cases and 13,314 controls), and no support for XRCC2 R188H (1.06 (0.59-1.91); total of 8,394 cases and 8,404 controls). In conclusion, the genetic variants evaluated are unlikely to have a substantial overall association with breast cancer risk; however, weak associations are possible for XRCC3 (T241M and IVS7-14A>G), BRCA2 N372H, and ZNF350 S472P. Evaluation of potential underlying gene-gene interactions or associations in population subgroups will require even larger sample sizes.

The Major DNA Repair Pathway after Both Proton and Carbon-Ion Radiation is NHEJ, but the HR Pathway is More Relevant in Carbon Ions

PubMed Central

Gerelchuluun, Ariungerel; Manabe, Eri; Ishikawa, Takaaki; Sun, Lue; Itoh, Kazuya; Sakae, Takeji; Suzuki, Kenshi; Hirayama, Ryoichi; Asaithamby, Aroumougame; Chen, David J.; Tsuboi, Koji

2017-01-01

The purpose of this study was to identify the roles of non-homologous end-joining (NHEJ) or homologous recombination (HR) pathways in repairing DNA double-strand breaks (DSBs) induced by exposure to high-energy protons and carbon ions (C ions) versus gamma rays in Chinese hamster cells. Two Chinese hamster cell lines, ovary AA8 and lung fibroblast V79, as well as various mutant sublines lacking DNA-PKcs (V3), X-ray repair cross-complementing protein-4 [XRCC4 (XR1), XRCC3 (irs1SF) and XRCC2 (irs1)] were exposed to gamma rays (137Cs), protons (200 MeV; 2.2 keV/μm) and C ions (290 MeV; 50 keV/μm). V3 and XR1 cells lack the NHEJ pathway, whereas irs1 and irs1SF cells lack the HR pathway. After each exposure, survival was measured using a clonogenic survival assay, in situ DSB induction was evaluated by immunocytochemical analysis of histone H2AX phosphorylation at serine 139 (γ-H2AX foci) and chromosome aberrations were examined using solid staining. The findings from this study showed that clonogenic survival clearly depended on the NHEJ and HR pathway statuses, and that the DNA-PKcs−/− cells (V3) were the most sensitive to all radiation types. While protons and γ rays yielded almost the same biological effects, C-ion exposure greatly enhanced the sensitivity of wild-type and HR-deficient cells. However, no significant enhancement of sensitivity in cell killing was seen after C-ion irradiation of NHEJ deficient cells. Decreases in the number of γ-H2AX foci after irradiation occurred more slowly in the NHEJ deficient cells. In particular, V3 cells had the highest number of residual γ-H2AX foci at 24 h after C-ion irradiation. Chromosomal aberrations were significantly higher in both the NHEJ- and HR-deficient cell lines than in wild-type cell lines in response to all radiation types. Protons and gamma rays induced the same aberration levels in each cell line, whereas C ions introduced higher but not significantly different aberration levels. Our results

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Paez, David, E-mail: dpaez@santpau.cat; Salazar, Juliana; Pare, Laia

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 polymerasemore » 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

Mismatch repair proteins recruited to ultraviolet light-damaged sites lead to degradation of licensing factor Cdt1 in the G1 phase.

PubMed

Tanaka, Miyuki; Takahara, Michiyo; Nukina, Kohei; Hayashi, Akiyo; Sakai, Wataru; Sugasawa, Kaoru; Shiomi, Yasushi; Nishitani, Hideo

2017-04-03

Cdt1 is rapidly degraded by CRL4 Cdt2 E3 ubiquitin ligase after UV (UV) irradiation. Previous reports revealed that the nucleotide excision repair (NER) pathway is responsible for the rapid Cdt1-proteolysis. Here, we show that mismatch repair (MMR) proteins are also involved in the degradation of Cdt1 after UV irradiation in the G1 phase. First, compared with the rapid (within ∼15 min) degradation of Cdt1 in normal fibroblasts, Cdt1 remained stable for ∼30 min in NER-deficient XP-A cells, but was degraded within ∼60 min. The delayed degradation was also dependent on PCNA and CRL4 Cdt2 . The MMR proteins Msh2 and Msh6 were recruited to the UV-damaged sites of XP-A cells in the G1 phase. Depletion of these factors with small interfering RNAs prevented Cdt1 degradation in XP-A cells. Similar to the findings in XP-A cells, depletion of XPA delayed Cdt1 degradation in normal fibroblasts and U2OS cells, and co-depletion of Msh6 further prevented Cdt1 degradation. Furthermore, depletion of Msh6 alone delayed Cdt1 degradation in both cell types. When Cdt1 degradation was attenuated by high Cdt1 expression, repair synthesis at the damaged sites was inhibited. Our findings demonstrate that UV irradiation induces multiple repair pathways that activate CRL4 Cdt2 to degrade its target proteins in the G1 phase of the cell cycle, leading to efficient repair of DNA damage.

Expression of DNA repair proteins MSH2, MLH1 and MGMT in human benign and malignant thyroid lesions: An immunohistochemical study

PubMed Central

Giaginis, Constantinos; Michailidi, Christina; Stolakis, Vasileios; Alexandrou, Paraskevi; Tsourouflis, Gerasimos; Klijanienko, Jerzy; Delladetsima, Ioanna; Theocharis, Stamatios

2011-01-01

Summary Background DNA repair is a major defense mechanism, which contributes to the maintenance of genetic sequence, and minimizes cell death, mutation rates, replication errors, DNA damage persistence and genomic instability. Alterations in the expression levels of proteins participating in DNA repair mechanisms have been associated with several aspects of cancer biology. The present study aimed to evaluate the clinical significance of DNA repair proteins MSH2, MLH1 and MGMT in benign and malignant thyroid lesions. Material/Methods MSH2, MLH1 and MGMT protein expression was assessed immunohistochemically on paraffin-embedded thyroid tissues from 90 patients with benign and malignant lesions. Results The expression levels of MLH1 was significantly upregulated in cases with malignant compared to those with benign thyroid lesions (p=0.038). The expression levels of MGMT was significantly downregulated in malignant compared to benign thyroid lesions (p=0.001). Similar associations for both MLH1 and MGMT between cases with papillary carcinoma and hyperplastic nodules were also noted (p=0.014 and p=0.026, respectively). In the subgroup of malignant thyroid lesions, MSH2 downregulation was significantly associated with larger tumor size (p=0.031), while MLH1 upregulation was significantly associated with the presence of lymphatic and vascular invasion (p=0.006 and p=0.002, respectively). Conclusions Alterations in the mismatch repair proteins MSH2 and MLH1 and the direct repair protein MGMT may result from tumor development and/or progression. Further studies are recommended to draw definite conclusions on the clinical significance of DNA repair proteins in thyroid neoplasia. PMID:21358597

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

PubMed

Reinardy, Helena C; Syrett, James R; Jeffree, Ross A; Henry, Theodore B; Jha, Awadhesh N

2013-01-15

Although cobalt (Co) is an environmental contaminant of surface waters in both radioactive (e.g. (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(50)) in larval zebrafish exposed (96 h) to 0-50 mg l(-1) Co was 35.3 ± 1.1 (95%C.I.) mg l(-1) Co. Adult zebrafish were exposed (13 d) to sub-lethal (0-25 mg l(-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(-1)) and embryo survival to hatching decreased (60% fertilised eggs survived, 25 mg l(-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(-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(-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. Copyright © 2012 Elsevier B.V. All rights reserved.

Predictive assessment in pharmacogenetics of XRCC1 gene on clinical outcomes of advanced lung cancer patients treated with platinum-based chemotherapy

PubMed Central

Yuan, Zhengrong; Li, Jiao; Hu, Ruiqi; Jiao, Yang; Han, Yingying; Weng, Qiang

2015-01-01

Published data have shown inconsistent results about the pharmacogenetics of XRCC1 gene on clinical outcomes of advanced lung cancer patients treated with platinum-based chemotherapy. This meta-analysis aimed to summarize published findings and provide more reliable association. A total of 53 eligible studies including 7433 patients were included. Patients bearing the favorable TrpTrp and TrpArg genotypes of Arg194Trp were more likely to better response rates to platinum-based chemotherapy compared to those with the unfavorable ArgArg genotype (TrpTrp+TrpArg vs. ArgArg: odds ratio (OR) = 2.02, 95% CI, 1.66–2.45). The GlnGln and GlnArg genotypes of Arg399Gln were significantly associated with the poorer response rates compared to those with the ArgArg genotype (GlnGln +GlnArg vs. ArgArg: OR = 0.68, 95% CI, 0.54–0.86). The GlnGln genotype might be more closely associated with shorter survival time and higher risks of death for patients (GlnGln vs. ArgArg: hazard ratio (HR) = 1.14, 95% CI, 0.75–1.75). Our cumulative meta-analyses indicated a distinct apparent trend toward a better response rate for Arg194Trp, but a poorer response rate in Arg399Gln. These findings indicate a predictive role of XRCC1 polymorphisms in clinical outcomes. The use of XRCC1 polymorphisms as predictive factor of clinical outcomes in personalized chemotherapy treatment requires further verification from large well-designed pharmacogenetics studies. PMID:26585370

Application of the microfluidic-assisted replication track analysis to measure DNA repair in human and mouse cells.

PubMed

Welcsh, Piri; Kehrli, Keffy; Lazarchuk, Pavlo; Ladiges, Warren; Sidorova, Julia

2016-10-01

Functional studies of the roles that DNA helicases play in human cells have benefited immensely from DNA fiber (or single molecule) technologies, which enable us to discern minute differences in behaviors of individual replication forks in genomic DNA in vivo. DNA fiber technologies are a group of methods that use different approaches to unravel and stretch genomic DNA to its contour length, and display it on a glass surface in order to immuno-stain nucleoside analog incorporation into DNA to reveal tracks (or tracts) of replication. We have previously adopted a microfluidic approach to DNA stretching and used it to analyze DNA replication. This method was introduced under the moniker maRTA or microfluidic-assisted Replication Track Analysis, and we have since used it to analyze roles of the RECQ helicases WRN and BLM, and other proteins in normal and perturbed replication. Here we describe a novel application of maRTA to detect and measure repair of DNA damage produced by three different agents relevant to etiology or therapy of cancer: methyl-methanesulfonate, UV irradiation, and mitomycin C. Moreover, we demonstrate the utility of this method by analyzing DNA repair in cells with reduced levels of WRN or of the base excision repair protein XRCC1. Copyright © 2016 Elsevier Inc. All rights reserved.

Homologous Recombination Repair Protects Against Particulate Chromate-induced Chromosome Instability in Chinese Hamster Cells

PubMed Central

Stackpole, Megan M.; Wise, Sandra S.; Duzevik, Eliza Grlickova; Munroe, Ray C.; Thompson, W. Douglas; Thacker, John; Thompson, Larry H.; Hinz, John M.; Wise, John Pierce

2008-01-01

Particulate hexavalent chromium [Cr(VI)] compounds are well-established human carcinogens. Cr(VI)-induced tumors are characterized by chromosomal instability (CIN); however, the mechanisms of this effect are unknown. We investigated the hypothesis that homologous recombination (HR) repair of DNA double strand breaks protect cells from Cr(VI)-induced CIN by focusing on the XRCC3 and RAD51C genes, which play an important role in cellular resistance to DNA double strand breaks. We used Chinese hamster cells defective in each HR gene (irs3 for RAD51C and irs1SF for XRCC3) and compared with their wildtype parental and cDNA-complemented controls. We found that the intracellular Cr ion levels varied among the cell lines after particulate chromate treatment. Importantly, accounting for differences in Cr ion levels, we discovered that XRCC3 and RAD51C cells treated with lead chromate had increased cytotoxicity and chromosomal aberrations, relative to wild-type and cDNA-complimented cells. We also observed the emergence of high levels of chromatid exchanges in the two mutant cell lines. For example, 1 ug/cm2 lead chromate induced 20 and 32 exchanges in XRCC3- and RAD51C-deficient cells, respectively, whereas no exchanges were detected in the wildtype and cDNA-complemented cells. These observations suggest that HR protects cells from Cr(VI)-induced CIN, consistent with the ability of particulate Cr(VI) to induce double strand breaks. PMID:17662313

XRCC4 suppresses medulloblastomas with recurrent translocations in p53-deficient mice

PubMed Central

Yan, Catherine T.; Kaushal, Dhruv; Murphy, Michael; Zhang, Yu; Datta, Abhishek; Chen, Changzhong; Monroe, Brianna; Mostoslavsky, Gustavo; Coakley, Kristen; Gao, Yijie; Mills, Kevin D.; Fazeli, Alex P.; Tepsuporn, Suprawee; Hall, Giles; Mulligan, Richard; Fox, Edward; Bronson, Roderick; De Girolami, Umberto; Lee, Charles; Alt, Frederick W.

2006-01-01

Inactivation of the XRCC4 nonhomologous end-joining factor in the mouse germ line leads to embryonic lethality, in association with apoptosis of newly generated, postmitotic neurons. We now show that conditional inactivation of the XRCC4 in nestin-expressing neuronal progenitor cells, although leading to no obvious phenotype in a WT background, leads to early onset of neuronally differentiated medulloblastomas (MBs) in a p53-deficient background. A substantial proportion of the XRCC4/p53-deficient MBs have high-level N-myc gene amplification, often intrachromosomally in the context of complex translocations or other alterations of chromosome 12, on which N-myc resides, or extrachromosomally within double minutes. In addition, most XRCC4/p53-deficient MBs harbor clonal translocations of chromosome 13, which frequently involve chromosome 6 as a partner. One copy of the patched gene (Ptc), which lies on chromosome 13, was deleted in all tested XRCC4/p53-deficient MBs in the context of translocations or interstitial deletions. In addition, Cyclin D2, a chromosome 6 gene, was amplified in a subset of tumors. Notably, amplification of Myc-family or Cyclin D2 genes and deletion of Ptc also have been observed in human MBs. We therefore conclude that, in neuronal cells of mice, the nonhomologous end-joining pathway plays a critical role in suppressing genomic instability that, in a p53-deficient background, routinely contributes to genesis of MBs with recurrent chromosomal alterations. PMID:16670198

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.

PubMed

Devi, K Rekha; Ahmed, Jishan; Narain, Kanwar; Mukherjee, Kaustab; Majumdar, Gautam; Chenkual, Saia; Zonunmawia, Jason C

2017-12-01

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 < .05 for R/W-W/W genotype). Moreover, it was found that tryptophan allele (W/W genotype) at codon 194 of X-ray repair cross complementary 1A (Arg194Trp) gene significantly increased the risk of breast cancer in premenopausal females (crude odds ratio = 1.66, 95

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

PubMed Central

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 < .05 for R/W-W/W genotype). Moreover, it was found that tryptophan allele (W/W genotype) at codon 194 of X-ray repair cross complementary 1A (Arg194Trp) gene significantly increased the risk of breast cancer in premenopausal females (crude odds ratio = 1

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

PubMed

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.

Mutational analysis of the human nucleotide excision repair gene ERCC1.

PubMed Central

Sijbers, A M; van der Spek, P J; Odijk, H; van den Berg, J; van Duin, M; Westerveld, A; Jaspers, N G; Bootsma, D; Hoeijmakers, J H

1996-01-01

The human DNA repair protein ERCC1 resides in a complex together with the ERCC4, ERCC11 and XP-F correcting activities, thought to perform the 5' strand incision during nucleotide excision repair (NER). Its yeast counterpart, RAD1-RAD10, has an additional engagement in a mitotic recombination pathway, probably required for repair of DNA cross-links. Mutational analysis revealed that the poorly conserved N-terminal 91 amino acids of ERCC1 are dispensable for both repair functions, in contrast to a deletion of only four residues from the C-terminus. A database search revealed a strongly conserved motif in this C-terminus sharing sequence homology with many DNA break processing proteins, indicating that this part is primarily required for the presumed structure-specific endonuclease activity of ERCC1. Most missense mutations in the central region give rise to an unstable protein (complex). Accordingly, we found that free ERCC1 is very rapidly degraded, suggesting that protein-protein interactions provide stability. Survival experiments show that the removal of cross-links requires less ERCC1 than UV repair. This suggests that the ERCC1-dependent step in cross-link repair occurs outside the context of NER and provides an explanation for the phenotype of the human repair syndrome xeroderma pigmentosum group F. PMID:8811092

Hemoglobin level and XRCC1 polymorphisms to select patients with locally advanced rectal cancer candidate for neoadjuvant chemoradiotherapy with concurrent capecitabine and a platinum salt.

PubMed

Formica, Vincenzo; Benassi, Michaela; Del Vecchio Blanco, Giovanna; Doldo, Elena; Martano, Laura; Portarena, Ilaria; Nardecchia, Antonella; Lucchetti, Jessica; Morelli, Cristina; Giudice, Emilia; Rossi, Piero; Anselmo, Alessandro; Sileri, Pierpaolo; Sica, Giuseppe; Orlandi, Augusto; Santoni, Riccardo; Roselli, Mario

2018-05-02

A platinum salt (oxaliplatin or cisplatin) is widely used to enhance chemoradation (CRT) response. The potential of cisplatin in neoadjuvant CRT for locally advanced rectal cancer (LARC) has not been fully investigated. Consecutive patients with histologically confirmed LARC were treated with standard pelvic radiotherapy and concurrent cisplatin plus capecitabine (CisCape CRT). Surgery and eight cycles of adjuvant FOLFOX4 were offered to all patients after CRT. Common biochemical variables and key germline genetic polymorphisms were analyzed as predictors of pathological complete response (pCR). Fifty-one patients were enrolled. pCR (regression AJCC grade 0) was documented in 7 patients (14%), nearly complete response (AJCC grade 1) in 10 pts. There was a strong association between disease-free survival and AJCC grade (p 0.0047). Grade 3-4 toxicities (mainly diarrhea) was observed in 41% of patients. Among all analyzed variables, baseline hemoglobin (Hb) was significantly associated with AJCC grade 0-1 response (p 0.027). As for the pharmacogenetic analysis, XRCC1 rs25487 polymorphism was significantly associated with AJCC grade 0-1, Odds Ratio 25.8, p 0.049. AJCC grade 0-1 response rate for patients with high Hb and/or XRCC1 rs25487 G/G genotype was as high as 57%. Baseline Hb and XRCC1 polymorphisms are valuable selection criteria for the CisCape CRT regimen, given its otherwise meaningful toxicity.

Stimulation of lactate receptor (HCAR1) affects cellular DNA repair capacity.

PubMed

Wagner, Waldemar; Kania, Katarzyna D; Ciszewski, Wojciech M

2017-04-01

Numerous G-protein coupled receptors have been reported to enhance cancer cell survival and resistance to clinically used chemotherapeutics. Recently, hydroxycarboxylic acid receptor 1 (HCAR1) was shown to drive lactate-dependent enhancement of cell survival and metastasis in pancreatic and breast cancers. Furthermore, our previous study confirmed the involvement of HCAR1 in lactate-related enhancement of DNA repair in cervical cancer cells. In the present study, we examined the possible mechanisms of HCAR1-mediated enhancement of DNA repair capacity. We observed that the HCAR1 agonist dihydroxybenzoic acid (DHBA) up-regulated BRCA1 (breast cancer type 1 susceptibility protein) and NBS1 (Nijmegen breakage syndrome 1) expression in HeLa cells. Moreover, HCAR1 silencing decreased mRNA and protein levels of BRCA1 by 30% and 20%, respectively. Immunocytochemical analyses of BRCA1, nibrin and DNA-PKcs indicated an increased accumulation of these proteins in cell nuclei after DHBA stimulation. Subsequently, these changes in the DNA repair protein levels translated into an enhanced DNA repair rate after doxorubicin treatment, as shown by γ-H2AX and comet assay experiments. In contrast, the down-regulation of HCAR1 decreased the efficiency of DNA repair. Finally, we observed the abrogation of DHBA-driven BRCA1 protein up-regulation and enhanced DNA repair following the preincubation of cells with the PKC inhibitor Gö6983. Taken together, our data indicate that lactate receptor/HCAR1 expression in cervical carcinoma cells may contribute to the modulation of cellular DNA repair mechanisms. Copyright © 2017 Elsevier B.V. All rights reserved.

The RSF1 histone-remodelling factor facilitates DNA double-strand break repair by recruiting centromeric and Fanconi Anaemia proteins.

PubMed

Pessina, Fabio; Lowndes, Noel F

2014-05-01

ATM is a central regulator of the cellular responses to DNA double-strand breaks (DSBs). Here we identify a biochemical interaction between ATM and RSF1 and we characterise the role of RSF1 in this response. The ATM-RSF1 interaction is dependent upon both DSBs and ATM kinase activity. Together with SNF2H/SMARCA5, RSF1 forms the RSF chromatin-remodelling complex. Although RSF1 is specific to the RSF complex, SNF2H/SMARCA5 is a catalytic subunit of several other chromatin-remodelling complexes. Although not required for checkpoint signalling, RSF1 is required for efficient repair of DSBs via both end-joining and homology-directed repair. Specifically, the ATM-dependent recruitment to sites of DSBs of the histone fold proteins CENPS/MHF1 and CENPX/MHF2, previously identified at centromeres, is RSF1-dependent. In turn these proteins recruit and regulate the mono-ubiquitination of the Fanconi Anaemia proteins FANCD2 and FANCI. We propose that by depositing CENPS/MHF1 and CENPX/MHF2, the RSF complex either directly or indirectly contributes to the reorganisation of chromatin around DSBs that is required for efficient DNA repair.

The RSF1 Histone-Remodelling Factor Facilitates DNA Double-Strand Break Repair by Recruiting Centromeric and Fanconi Anaemia Proteins

PubMed Central

Pessina, Fabio; Lowndes, Noel F.

2014-01-01

ATM is a central regulator of the cellular responses to DNA double-strand breaks (DSBs). Here we identify a biochemical interaction between ATM and RSF1 and we characterise the role of RSF1 in this response. The ATM–RSF1 interaction is dependent upon both DSBs and ATM kinase activity. Together with SNF2H/SMARCA5, RSF1 forms the RSF chromatin-remodelling complex. Although RSF1 is specific to the RSF complex, SNF2H/SMARCA5 is a catalytic subunit of several other chromatin-remodelling complexes. Although not required for checkpoint signalling, RSF1 is required for efficient repair of DSBs via both end-joining and homology-directed repair. Specifically, the ATM-dependent recruitment to sites of DSBs of the histone fold proteins CENPS/MHF1 and CENPX/MHF2, previously identified at centromeres, is RSF1-dependent. In turn these proteins recruit and regulate the mono-ubiquitination of the Fanconi Anaemia proteins FANCD2 and FANCI. We propose that by depositing CENPS/MHF1 and CENPX/MHF2, the RSF complex either directly or indirectly contributes to the reorganisation of chromatin around DSBs that is required for efficient DNA repair. PMID:24800743

Interdependence of DNA mismatch repair proteins MLH1 and MSH2 in apoptosis in human colorectal carcinoma cell lines.

PubMed

Hassen, Samar; Ali, Akhtar A; Kilaparty, Surya P; Al-Anbaky, Qudes A; Majeed, Waqar; Boman, Bruce M; Fields, Jeremy Z; Ali, Nawab

2016-01-01

The mammalian DNA mismatch repair (MMR) system consists of a number of proteins that play important roles in repair of base pair mismatch mutations and in maintenance of genomic integrity. A defect in this system can cause genetic instability, which can lead to carcinogenesis. For instance, a germline mutation in one of the mismatch repair proteins, especially MLH1 or MSH2, is responsible for hereditary non-polyposis colorectal cancer. These MMR proteins also play an important role in the induction of apoptosis. Accordingly, altered expression of or a defect in MLH1 or MSH2 may confer resistance to anti-cancer drugs used in chemotherapy. We hypothesized that the ability of these two MMR proteins to regulate apoptosis are interdependent. Moreover, a defect in either one may confer resistance to chemotherapy by an inability to trigger apoptosis. To this end, we studied three cell lines-SW480, LoVo, and HTC116. These cell lines were selected based on their differential expression of MLH1 and MSH2 proteins. SW480 expresses both MLH1 and MSH2; LoVo expresses only MLH1 but not MSH2; HCT116 expresses only MSH2 but not MLH1 protein. MTT assays, a measure of cytotoxicity, showed that there were different cytotoxic effects of an anti-cancer drug, etoposide, on these cell lines, effects that were correlated with the MMR status of the cells. Cells that are deficient in MLH1 protein (HCT116 cells) were resistant to the drug. Cells that express both MLH1 and MSH2 proteins (SW480 cells) showed caspase-3 cleavage, an indicator of apoptosis. Cells that lack MLH1 (HCT116 cells) did not show any caspase-3 cleavage. Expression of full-length MLH1 protein was decreased in MMR proficient (SW480) cells during apoptosis; it remained unchanged in cells that lack MSH2 (LoVo cells). The expression of MSH2 protein remained unchanged during apoptosis both in MMR proficient (SW480) and deficient (HCT116) cells. Studies on translocation of MLH1 protein from nucleus to cytosolic fraction, an

Influence of allelic Variations of hypoxia-related and DNA repair genes on patient outcome and toxicity in head and neck cancer treated with radiotherapy plus cetuximab.

PubMed

Muñoz, Carmen; Caballero, Miguel; Hakim, Sofia; Verger, Eugenia; Grau, Juan Jose

2016-08-01

Although cetuximab plus radiotherapy is a standard treatment for patients with inoperable head and neck squamous cell carcinoma (HNSCC), its efficacy varies greatly among individuals. To identify predictive markers of efficacy, we examined the effects of single nucleotide polymorphisms (SNPs) in hypoxia-related and DNA repair genes on the clinical outcome and occurrence of skin toxicity. We analyzed 61 consecutive patients with HNSCC for the presence of specific SNPs (HIF-1α, HIF-2α, HIF-1β, VHL, FIH-1, XRCC1, and XRCC5). The results were then correlated with time to progression (TTP), overall survival (OS), and toxicity (epithelitis, mucositis, and folliculitis). The median TTP and OS were better in patients with severe vs mild mucositis (17 vs 7 months, p = 0.03; and 26 vs 12 months, p = 0.016, respectively) and folliculitis (10 vs 7 months, p = 0.01, and 26 vs 10 months, p < 0.001, respectively). Patients with the HIF-1α CT/TT genotype had better OS than those with the wild-type HIF-1α CC genotype (28 vs 13 months, p = 0.035). Patients with the XRCC5 GG/AA genotype had longer TTP than patients with the XRCC5 AG genotype (11 vs 7 months, p = 0.035). Severe skin toxicity and SNPs of HIF-1α and XRCC5 were associated with different outcomes among patients treated with radiotherapy plus cetuximab.

Targeting MPS1 Enhances Radiosensitization of Human Glioblastoma by Modulating DNA Repair Proteins

PubMed Central

Maachani, Uday B.; Kramp, Tamalee; Hanson, Ryan; Zhao, Shuping; Celiku, Orieta; Shankavaram, Uma; Colombo, Riccardo; Caplen, Natasha J.; Camphausen, Kevin; Tandle, Anita

2015-01-01

To ensure faithful chromosome segregation, cells use the spindle assembly checkpoint (SAC), which can be activated in aneuploid cancer cells. Targeting the components of SAC machinery required for the growth of aneuploid cells may offer a cancer cell specific therapeutic approach. In this study, the effects of inhibiting Monopolar spindle 1, MPS1 (TTK), an essential SAC kinase, on the radiosensitization of glioblastoma (GBM) cells was analyzed. Clonogenic survival was used to determine the effects of the MPS1 inhibitor, NMS-P715 on radiosensitivity in multiple model systems including: GBM cell lines, a normal astrocyte and a normal fibroblast cell line. DNA double strand breaks (DSBs) were evaluated using γH2AX foci and cell death was measured by mitotic catastrophe evaluation. Transcriptome analysis was performed via unbiased microarray expression profiling. Tumor xenografts grown from GBM cells were used in tumor growth delay studies. Inhibition of MPS1 activity resulted in reduced GBM cell proliferation. Further, NMS-P715 enhanced the radiosensitivity of GBM cells by decreased repair of DSBs and induction of post-radiation mitotic catastrophe. MNS-P715 in combination with fractionated doses of radiation significantly enhanced the tumor growth delay. Molecular profiling of MPS1 silenced GBM cells showed an altered expression of transcripts associated with DNA damage, repair and replication including the DNA-dependent protein kinase (PRKDC/DNAPK). Next, inhibition of MPS1 blocked two important DNA repair pathways. In conclusion, these results not only highlight a role for MPS1 kinase in DNA repair and as prognostic marker but also indicate it as a viable option in glioblastoma therapy. PMID:25722303

Repair of traumatized mammalian hair cells via sea anemone repair proteins.

PubMed

Tang, Pei-Ciao; Smith, Karen Müller; Watson, Glen M

2016-08-01

Mammalian hair cells possess only a limited ability to repair damage after trauma. In contrast, sea anemones show a marked capability to repair damaged hair bundles by means of secreted repair proteins (RPs). Previously, it was found that recovery of traumatized hair cells in blind cavefish was enhanced by anemone-derived RPs; therefore, the ability of anemone RPs to assist recovery of damaged hair cells in mammals was tested here. After a 1 h incubation in RP-enriched culture media, uptake of FM1-43 by experimentally traumatized murine cochlear hair cells was restored to levels comparable to those exhibited by healthy controls. In addition, RP-treated explants had significantly more normally structured hair bundles than time-matched traumatized control explants. Collectively, these results indicate that anemone-derived RPs assist in restoring normal function and structure of experimentally traumatized hair cells of the mouse cochlea. © 2016. Published by The Company of Biologists Ltd.

Brca2 (XRCC11) Deficiency Results in Radioresistant DNA Synthesis and a Higher Frequency of Spontaneous Deletions

PubMed Central

Kraakman-van der Zwet, Maria; Overkamp, Wilhelmina J. I.; van Lange, Rebecca E. E.; Essers, Jeroen; van Duijn-Goedhart, Annemarie; Wiggers, Ingrid; Swaminathan, Srividya; van Buul, Paul P. W.; Errami, Abdellatif; Tan, Raoul T. L.; Jaspers, Nicolaas G. J.; Sharan, Shyam K.; Kanaar, Roland; Zdzienicka, Małgorzata Z.

2002-01-01

We show here that the radiosensitive Chinese hamster cell mutant (V-C8) of group XRCC11 is defective in the breast cancer susceptibility gene Brca2. The very complex phenotype of V-C8 cells is complemented by a single human chromosome 13 providing the BRCA2 gene, as well as by the murine Brca2 gene. The Brca2 deficiency in V-C8 cells causes hypersensitivity to various DNA-damaging agents with an extreme sensitivity toward interstrand DNA cross-linking agents. Furthermore, V-C8 cells show radioresistant DNA synthesis after ionizing radiation, suggesting that Brca2 deficiency affects cell cycle checkpoint regulation. In addition, V-C8 cells display tremendous chromosomal instability and a high frequency of abnormal centrosomes. The mutation spectrum at the hprt locus showed that the majority of spontaneous mutations in V-C8 cells are deletions, in contrast to wild-type V79 cells. A mechanistic explanation for the genome instability phenotype of Brca2-deficient cells is provided by the observation that the nuclear localization of the central DNA repair protein in homologous recombination, Rad51, is reduced in V-C8 cells. PMID:11756561

Transected sciatic nerve repair by diode laser protein soldering.

PubMed

Fekrazad, Reza; Mortezai, Omid; Pedram, MirSepehr; Kalhori, Katayoun Am; Joharchi, Khojasteh; Mansoori, Korosh; Ebrahimi, Roja; Mashhadiabbas, Fatemeh

2017-08-01

Despite advances in microsurgical techniques, repair of peripheral nerve injuries (PNI) is still a major challenge in regenerative medicine. The standard treatment for PNI includes suturing and anasthomosis of the transected nerve. The objective of this study was to compare neurorraphy (nerve repair) using standard suturingto diode laser protein soldering on the functional recovery of transected sciatic nerves. Thirty adult male Fischer-344 Wistar rats were randomly assigned to 3 groups: 1. The control group, no repair, 2. the standard of care suture group, and 3. The laser/protein solder group. For all three groups, the sciatic nerve was transected and the repair was done immediately. For the suture repair group, 10.0 prolene suture was used and for the laser/protein solder group a diode laser (500mW output power) in combination with bovine serum albumen and indocyanine green dye was used. Behavioral assessment by sciatic functional index was done on all rats biweekly. At 12weeks post-surgery, EMG recordings were done on all the rats and the rats were euthanized for histological evaluation of the sciatic nerves. The one-way ANOVA test was used for statistical analysis. The average time required to perform the surgery was significantly shorter for the laser-assisted nerve repair group compared to the suture group. The EMG evaluation revealed no difference between the two groups. Based on the sciatic function index the laser group was significantly better than the suture group after 12weeks (p<0.05). Histopathologic evaluation indicated that the epineurium recovery was better in the laser group (p<0.05). There was no difference in the inflammation between the suture and laser groups. Based on this evidence, laser/protein nerve soldering is a more efficient and efficacious method for repair of nerve injury compared to neurorraphy using standard suturing methods. Copyright © 2017 Elsevier B.V. All rights reserved.

Targeting MPS1 Enhances Radiosensitization of Human Glioblastoma by Modulating DNA Repair Proteins.

PubMed

Maachani, Uday Bhanu; Kramp, Tamalee; Hanson, Ryan; Zhao, Shuping; Celiku, Orieta; Shankavaram, Uma; Colombo, Riccardo; Caplen, Natasha J; Camphausen, Kevin; Tandle, Anita

2015-05-01

To ensure faithful chromosome segregation, cells use the spindle assembly checkpoint (SAC), which can be activated in aneuploid cancer cells. Targeting the components of SAC machinery required for the growth of aneuploid cells may offer a cancer cell-specific therapeutic approach. In this study, the effects of inhibiting Monopolar spindle 1, MPS1 (TTK), an essential SAC kinase, on the radiosensitization of glioblastoma (GBM) cells were analyzed. Clonogenic survival was used to determine the effects of the MPS1 inhibitor NMS-P715 on radiosensitivity in multiple model systems, including GBM cell lines, a normal astrocyte, and a normal fibroblast cell line. DNA double-strand breaks (DSB) were evaluated using γH2AX foci, and cell death was measured by mitotic catastrophe evaluation. Transcriptome analysis was performed via unbiased microarray expression profiling. Tumor xenografts grown from GBM cells were used in tumor growth delay studies. Inhibition of MPS1 activity resulted in reduced GBM cell proliferation. Furthermore, NMS-P715 enhanced the radiosensitivity of GBM cells by decreased repair of DSBs and induction of postradiation mitotic catastrophe. NMS-P715 in combination with fractionated doses of radiation significantly enhanced the tumor growth delay. Molecular profiling of MPS1-silenced GBM cells showed an altered expression of transcripts associated with DNA damage, repair, and replication, including the DNA-dependent protein kinase (PRKDC/DNAPK). Next, inhibition of MPS1 blocked two important DNA repair pathways. In conclusion, these results not only highlight a role for MPS1 kinase in DNA repair and as prognostic marker but also indicate it as a viable option in glioblastoma therapy. Inhibition of MPS1 kinase in combination with radiation represents a promising new approach for glioblastoma and for other cancer therapies. ©2015 American Association for Cancer Research.

Rare, protein-truncating variants in ATM, CHEK2 and PALB2, but not XRCC2, are associated with increased breast cancer risks

PubMed Central

Decker, Brennan; Allen, Jamie; Luccarini, Craig; Pooley, Karen A; Shah, Mitul; Bolla, Manjeet K; Wang, Qin; Ahmed, Shahana; Baynes, Caroline; Conroy, Don M; Brown, Judith; Luben, Robert; Ostrander, Elaine A; Pharoah, Paul DP; Dunning, Alison M; Easton, Douglas F

2017-01-01

Background Breast cancer (BC) is the most common malignancy in women and has a major heritable component. The risks associated with most rare susceptibility variants are not well estimated. To better characterise the contribution of variants in ATM, CHEK2, PALB2 and XRCC2, we sequenced their coding regions in 13 087 BC cases and 5488 controls from East Anglia, UK. Methods Gene coding regions were enriched via PCR, sequenced, variant called and filtered for quality. ORs for BC risk were estimated separately for carriers of truncating variants and of rare missense variants, which were further subdivided by functional domain and pathogenicity as predicted by four in silico algorithms. Results Truncating variants in PALB2 (OR=4.69, 95% CI 2.27 to 9.68), ATM (OR=3.26; 95% CI 1.82 to 6.46) and CHEK2 (OR=3.11; 95% CI 2.15 to 4.69), but not XRCC2 (OR=0.94; 95% CI 0.26 to 4.19) were associated with increased BC risk. Truncating variants in ATM and CHEK2 were more strongly associated with risk of oestrogen receptor (ER)-positive than ER-negative disease, while those in PALB2 were associated with similar risks for both subtypes. There was also some evidence that missense variants in ATM, CHEK2 and PALB2 may contribute to BC risk, but larger studies are necessary to quantify the magnitude of this effect. Conclusions Truncating variants in PALB2 are associated with a higher risk of BC than those in ATM or CHEK2. A substantial risk of BC due to truncating XRCC2 variants can be excluded. PMID:28779002

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

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 6more » (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.« less

COMPREHENSIVE ANALYSES OF DNA REPAIR PATHWAYS, SMOKING, AND BLADDER CANCER RISK IN LOS ANGELES AND SHANGHAI

PubMed Central

Corral, Roman; Lewinger, Juan Pablo; Berg, David Van Den; Joshi, Amit D.; Yuan, Jian-Min; Gago-Dominguez, Manuela; Cortessis, Victoria K.; Pike, Malcolm C.; Conti, David V.; Thomas, Duncan C.; Edlund, Christopher K.; Gao, Yu-Tang; Xiang, Yong-Bing; Zhang, Wei; Su, Yu-Chen; Stern, Mariana C.

2014-01-01

Tobacco smoking is a bladder cancer risk factor and a source of carcinogens that induce DNA damage to urothelial cells. Using data and samples from 988 cases and 1,004 controls enrolled in the Los Angeles County Bladder Cancer Study and the Shanghai Bladder Cancer Study we investigated associations between bladder cancer risk and 632 tagSNPs that comprehensively capture genetic variation in 28 DNA repair genes from four DNA repair pathways: base excision repai, nucleotide excision repair (NER), non-homologous end-joining (NHEJ), and homologous recombination repair (HHR). Odds ratios (ORs) and 95% confidence intervals (CIs) for each tagSNP were corrected for multiple testing for all SNPs within each gene using pACT, and for genes within each pathway and across pathways with Bonferroni. Gene and pathway summary estimates were obtained using ARTP. We observed an association between bladder cancer and POLB rs7832529 (BER) (pACT = 0.003; ppathway = 0.021) among all, and SNPs in XPC (NER) and OGG1 (BER) among Chinese men and women, respectively. The NER pathway showed an overall association with risk among Chinese males (ARTP NER p = 0.034). The XRCC6 SNP rs2284082 (NHEJ), also in LD with SREBF2, showed an interaction with smoking (Smoking status interaction pgene = 0.001, ppathway = 0.008, poverall = 0.034). Our findings support a role in bladder carcinogenesis for regions that map close to or within BER (POLB, OGG1) and NER genes (XPC). A SNP that tags both the XRCC6 and SREBF2 genes strongly modifies the association between bladder cancer risk and smoking. PMID:24382701

Expression of domains for protein-protein interaction of nucleotide excision repair proteins modifies cancer cell sensitivity to platinum derivatives and genomic stability.

PubMed

Jordheim, Lars Petter; Cros-Perrial, Emeline; Matera, Eva-Laure; Bouledrak, Karima; Dumontet, Charles

2014-10-01

Nucleotide excision repair (NER) is involved in the repair of DNA damage caused by platinum derivatives and has been shown to decrease the cytotoxic activity of these drugs. Because protein-protein interactions are essential for NER activity, we transfected human cancer cell lines (A549 and HCT116) with plasmids coding the amino acid sequences corresponding to the interacting domains between excision repair cross-complementation group 1 (ERCC1) and xeroderma pigmentosum, complementation group A (XPA), as well as ERCC1 and xeroderma pigmentosum, complementation group F (XPF), all NER proteins. Using the 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and annexin V staining, we showed that transfected A549 cells were sensitized 1.2-2.2-fold to carboplatin and that transfected HCT116 cells were sensitized 1.4-5.4-fold to oxaliplatin in vitro. In addition, transfected cells exhibited modified in vivo sensitivity to the same drugs. Finally, in particular cell models of the interaction between ERCC1 and XPF, DNA repair was decreased, as evidenced by increased phosphorylation of the histone 2AX after exposure to mitomycin C, and genomic instability was increased, as determined by comparative genomic hybridization studies. The results indicate that the interacting peptides act as dominant negatives and decrease NER activity through inhibition of protein-protein interactions. © 2014 Wiley Publishing Asia Pty Ltd.

A 3' untranslated region polymorphism rs2304277 in the DNA repair pathway gene OGG1 is a novel risk modulator for urothelial bladder carcinoma.

PubMed

Ahmed, Tayyaba; Nawaz, Saira; Noreen, Rabia; Bangash, Kashif Sardar; Rauf, Abdur; Younis, Muhammad; Anwar, Khursheed; Khawaja, Muhammad Athar; Azam, Maleeha; Qureshi, Abid Ali; Akhter, Saeed; Kiemeney, Lambertus A; Qamar, Raheel; Ali, Syeda Hafiza Benish

2018-03-01

Altered DNA repair capacity may affect an individual's susceptibility to cancers due to compromised genomic integrity. This study was designed to elucidate the association of selected polymorphisms in DNA repair genes with urothelial bladder carcinoma (UBC). OGG1 rs1052133 and rs2304277, XRCC1 rs1799782 and rs25487, XRCC3 rs861539, XPC rs2228001, and XPD rs13181 were genotyped using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in 200 UBC cases and 200 controls. We found association of OGG1 rs2304277 [odds ratio (OR) GG = 3.55, 95% confidence interval (CI) = 1.79-7.06] and XPC rs2228001 (OR AC = 2.38, 95% CI = 1.43-3.94) with UBC. In stratified analysis with respect to smoking status, OGG1 rs2304277 and XPC rs2228001 exhibited increased risk in smokers [(rs2304277 OR GG = 4.96, 95% CI = 1.51-16.30) (rs2228001 OR AC = 2.19, 95% CI = 1.02-4.72)] as well as nonsmokers [(rs2304277 OR GG = 2.95, 95% CI = 1.26-6.90) (rs2228001 OR AC = 2.57, 95% CI = 1.31-5.04)]. These polymorphisms were also associated with both low-grade [(rs2304277 OR GG = 3.73, 95% CI = 1.72-8.09) (rs2228001 OR AC = 2.18, 95% CI = 1.21-3.92)] and high-grade tumors [(rs2304277 OR GG = 3.45, 95% CI = 1.52-7.80) (rs2228001 OR AC = 2.81, 95% CI = 1.48-5.33)] as well as with non-muscle-invasive bladder cancer [(rs2304277 OR GG = 4.03, 95% CI = 1.87-8.67) (rs2228001 OR AC = 2.14, 95% CI = 1.20-3.81)] and muscle-invasive bladder cancer [(rs2304277 OR GG = 3.06, 95%CI = 1.31-7.13) (rs2228001 OR AC = 2.95, 95%CI = 1.51-5.75)]. This is the first study on DNA repair gene polymorphisms and UBC in the Pakistani population. It identifies OGG1 rs2304277 and replicates XPC rs2228001 as significant modulators of UBC susceptibility. © 2017 John Wiley & Sons Ltd/University College London.

Prereplicative repair of oxidized bases in the human genome is mediated by NEIL1 DNA glycosylase together with replication proteins

PubMed Central

Hegde, Muralidhar L.; Hegde, Pavana M.; Bellot, Larry J.; Mandal, Santi M.; Hazra, Tapas K.; Li, Guo-Min; Boldogh, Istvan; Tomkinson, Alan E.; Mitra, Sankar

2013-01-01

Base oxidation by endogenous and environmentally induced reactive oxygen species preferentially occurs in replicating single-stranded templates in mammalian genomes, warranting prereplicative repair of the mutagenic base lesions. It is not clear how such lesions (which, unlike bulky adducts, do not block replication) are recognized for repair. Furthermore, strand breaks caused by base excision from ssDNA by DNA glycosylases, including Nei-like (NEIL) 1, would generate double-strand breaks during replication, which are not experimentally observed. NEIL1, whose deficiency causes a mutator phenotype and is activated during the S phase, is present in the DNA replication complex isolated from human cells, with enhanced association with DNA in S-phase cells and colocalization with replication foci containing DNA replication proteins. Furthermore, NEIL1 binds to 5-hydroxyuracil, the oxidative deamination product of C, in replication protein A-coated ssDNA template and inhibits DNA synthesis by DNA polymerase δ. We postulate that, upon encountering an oxidized base during replication, NEIL1 initiates prereplicative repair by acting as a “cowcatcher” and preventing nascent chain growth. Regression of the stalled replication fork, possibly mediated by annealing helicases, then allows lesion repair in the reannealed duplex. This model is supported by our observations that NEIL1, whose deficiency slows nascent chain growth in oxidatively stressed cells, is stimulated by replication proteins in vitro. Furthermore, deficiency of the closely related NEIL2 alone does not affect chain elongation, but combined NEIL1/2 deficiency further inhibits DNA replication. These results support a mechanism of NEIL1-mediated prereplicative repair of oxidized bases in the replicating strand, with NEIL2 providing a backup function. PMID:23898192

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cooper, Karen L.; Dashner, Erica J.; Tsosie, Ranalda

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.more » 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.« less

[Expression of DNA mismatch repair protein in endometrial carcinomas and its correlation with clinicopathologic features].

PubMed

Bi, R; Tu, X Y; Xiao, Y X; Shan, B E; Wang, H Y; Cai, X; Zhou, X Y; Yang, W T

2016-05-08

To study the expression of mismatch repair protein in a series of endometrial carcinomas and its correlation with clinicopathologic features. The clinical data of 150 consecutive cases of endometrial carcinoma were collected during the period from December, 2014 to August, 2015 in Fudan University Cancer Center. Morphologic features including tumor infiltrating lymphocytes (TIL), peritumoral lymphocytes and tumor heterogeneity were reviewed. Immunohistochemistry for expression of mismatch repair proteins was performed. The correlation with clinicopathologic features was analyzed. Loss of mismatch repair protein expression was observed in 43 cases (28.7%), including loss of MLH1/PMS2 in 27 cases (18%), loss of MSH2/MSH6 in 7 cases (4.7%), loss of MSH6 in 6 cases (4%) and loss of PMS2 in 3 cases (2%). There were 23.3% and 27.1% of mismatch repair protein-deficient endometrial carcinomas in women under and above 50 years of age, respectively, which was not statistically significant. Amongst the 12 cases with family history of tumors, 4 of the 6 mismatch repair protein-deficient cases were under 50 years of age, which was higher than that in the 6 cases with mismatch repair protein expression (P=0.014). The mismatch repair protein-deficient group showed significantly more prominent TIL and peritumoral lymphocytes than protein-expression group (P=0.033 and <0.001). Moreover, there were also significant differences in depth of myometrial invasion and occurrence of synchronous malignancy (2 cases of ovarian clear cell carcinoma and 1 case of colonic carcinoma) between the two groups (P=0.039 and 0.022). However, there were no significant differences in lymph node metastasis, tumor heterogeneity, lower uterine segment involvement and tumor stage between the two groups. Prominent TIL and peritumoral lymphocytes characteristically occur in mismatch repair protein-deficient endometrial carcinomas. Patient age does not significantly correlate with the loss of mismatch repair

Distinct kinetics of DNA repair protein accumulation at DNA lesions and cell cycle-dependent formation of γH2AX- and NBS1-positive repair foci.

PubMed

Suchánková, Jana; Kozubek, Stanislav; Legartová, Soňa; Sehnalová, Petra; Küntziger, Thomas; Bártová, Eva

2015-12-01

The DNA damage response is a fundamental, well-regulated process that occurs in the genome to recognise DNA lesions. Here, we studied kinetics of proteins involved in DNA repair pathways and their recruitment to DNA lesions during the cell cycle. In non-irradiated and irradiated cells, we analysed the distribution pattern and spatiotemporal dynamics of γH2AX, 53BP1, BMI1, MDC1, NBS1, PCNA, coilin and BRCA1 proteins. We observed that spontaneous and irradiation-induced foci (IRIF) demonstrated a high abundance of phosphorylated H2AX, which was consistent with 53BP1 and BMI1 protein accumulation. However, NBS1 and MDC1 proteins were recruited to nuclear bodies (NBs) to a lesser extent. Irradiation by γ-rays significantly increased the number of 53BP1- and γH2AX-positive IRIF, but cell cycle-dependent differences were only observed for γH2AX-positive foci in both non-irradiated and γ-irradiated cells. In non-irradiated cells, the G2 phase was characterised by an increased number of spontaneous γH2AX-foci; this increase was more pronounced after γ-irradiation. Cells in G2 phase had the highest number of γH2AX-positive foci. Similarly, γ-irradiation increased the number of NBS1-positive NBs only in G2 phase. Moreover, NBS1 accumulated in nucleoli after γ-irradiation showed the slowest recovery after photobleaching. Analysis of protein accumulation kinetics at locally induced DNA lesions showed that in HeLa cells, BMI1, PCNA and coilin were rapidly recruited to the lesions, 10-15 s after UVA-irradiation, whereas among the other proteins studied, BRCA1 demonstrated the slowest recruitment: BRCA1 appeared at the lesion 20 min after local micro-irradiation by UVA laser. We show that the kinetics of the accumulation of selected DNA repair-related proteins is protein specific at locally induced DNA lesions, and that the formation of γH2AX- and NBS1-positive foci, but not 53BP1-positive NBs, is cell cycle dependent in HeLa cells. Moreover, γH2AX is the most

SNPs in DNA repair or oxidative stress genes and late subcutaneous fibrosis in patients following single shot partial breast irradiation

PubMed Central

2012-01-01

Background The aim of this study was to evaluate the potential association between single nucleotide polymorphisms related response to radiotherapy injury, such as genes related to DNA repair or enzymes involved in anti-oxidative activities. The paper aims to identify marker genes able to predict an increased risk of late toxicity studying our group of patients who underwent a Single Shot 3D-CRT PBI (SSPBI) after BCS (breast conserving surgery). Methods A total of 57 breast cancer patients who underwent SSPBI were genotyped for SNPs (single nucleotide polymorphisms) in XRCC1, XRCC3, GST and RAD51 by Pyrosequencing technology. Univariate analysis (ORs and 95% CI) was performed to correlate SNPs with the risk of developing ≥ G2 fibrosis or fat necrosis. Results A higher significant risk of developing ≥ G2 fibrosis or fat necrosis in patients with: polymorphic variant GSTP1 (Ile105Val) (OR = 2.9; 95%CI, 0.88-10.14, p = 0.047). Conclusions The presence of some SNPs involved in DNA repair or response to oxidative stress seem to be able to predict late toxicity. Trial Registration ClinicalTrials.gov: NCT01316328 PMID:22272830

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

PubMed

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.

Lifespan and Stress Resistance in Drosophila with Overexpressed DNA Repair Genes

PubMed Central

Shaposhnikov, Mikhail; Proshkina, Ekaterina; Shilova, Lyubov; Zhavoronkov, Alex; Moskalev, Alexey

2015-01-01

DNA repair declines with age and correlates with longevity in many animal species. In this study, we investigated the effects of GAL4-induced overexpression of genes implicated in DNA repair on lifespan and resistance to stress factors in Drosophila melanogaster. Stress factors included hyperthermia, oxidative stress, and starvation. Overexpression was either constitutive or conditional and either ubiquitous or tissue-specific (nervous system). Overexpressed genes included those involved in recognition of DNA damage (homologs of HUS1, CHK2), nucleotide and base excision repair (homologs of XPF, XPC and AP-endonuclease-1), and repair of double-stranded DNA breaks (homologs of BRCA2, XRCC3, KU80 and WRNexo). The overexpression of different DNA repair genes led to both positive and negative effects on lifespan and stress resistance. Effects were dependent on GAL4 driver, stage of induction, sex, and role of the gene in the DNA repair process. While the constitutive/neuron-specific and conditional/ubiquitous overexpression of DNA repair genes negatively impacted lifespan and stress resistance, the constitutive/ubiquitous and conditional/neuron-specific overexpression of Hus1, mnk, mei-9, mus210, and WRNexo had beneficial effects. This study demonstrates for the first time the effects of overexpression of these DNA repair genes on both lifespan and stress resistance in D. melanogaster. PMID:26477511

Rare, protein-truncating variants in ATM, CHEK2 and PALB2, but not XRCC2, are associated with increased breast cancer risks.

PubMed

Decker, Brennan; Allen, Jamie; Luccarini, Craig; Pooley, Karen A; Shah, Mitul; Bolla, Manjeet K; Wang, Qin; Ahmed, Shahana; Baynes, Caroline; Conroy, Don M; Brown, Judith; Luben, Robert; Ostrander, Elaine A; Pharoah, Paul Dp; Dunning, Alison M; Easton, Douglas F

2017-11-01

Breast cancer (BC) is the most common malignancy in women and has a major heritable component. The risks associated with most rare susceptibility variants are not well estimated. To better characterise the contribution of variants in ATM , CHEK2 , PALB2 and XRCC2 , we sequenced their coding regions in 13 087 BC cases and 5488 controls from East Anglia, UK. Gene coding regions were enriched via PCR, sequenced, variant called and filtered for quality. ORs for BC risk were estimated separately for carriers of truncating variants and of rare missense variants, which were further subdivided by functional domain and pathogenicity as predicted by four in silico algorithms. Truncating variants in PALB2 (OR=4.69, 95% CI 2.27 to 9.68), ATM (OR=3.26; 95% CI 1.82 to 6.46) and CHEK2 (OR=3.11; 95% CI 2.15 to 4.69), but not XRCC2 (OR=0.94; 95% CI 0.26 to 4.19) were associated with increased BC risk. Truncating variants in ATM and CHEK2 were more strongly associated with risk of oestrogen receptor (ER)-positive than ER-negative disease, while those in PALB2 were associated with similar risks for both subtypes. There was also some evidence that missense variants in ATM , CHEK2 and PALB2 may contribute to BC risk, but larger studies are necessary to quantify the magnitude of this effect. Truncating variants in PALB2 are associated with a higher risk of BC than those in ATM or CHEK2 . A substantial risk of BC due to truncating XRCC2 variants can be excluded. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Prospective assessment of XRCC3, XPD and Aurora kinase A single-nucleotide polymorphisms in advanced lung cancer.

PubMed

Provencio, M; Camps, C; Cobo, M; De las Peñas, R; Massuti, B; Blanco, R; Alberola, V; Jimenez, U; Delgado, J R; Cardenal, F; Tarón, M; Ramírez, J L; Sanchez, A; Rosell, R

2012-12-01

New therapeutic approaches are being developed based on findings that several genetic abnormalities underlying non-small-cell lung cancer (NSCLC) can influence chemosensitivity. The identification of molecular markers, useful for therapeutic decisions in lung cancer, is thus crucial for disease management. The present study evaluated single-nucleotide polymorphisms (SNPs) in XRCC3, XPD and Aurora kinase A in NSCLC patients in order to assess whether these biomarkers were able to predict the outcomes of the patients. The Spanish Lung Cancer Group prospectively assessed this clinical study. Eligible patients had histologically confirmed stage IV or IIIB (with malignant pleural effusion) NSCLC, which had not previously been treated with chemotherapy, and a World Health Organization performance status (PS) of 0-1. Patients received intravenous doses of vinorelbine 25 mg/m(2) on days 1 and 8, and cisplatin 75 mg/m(2) on day 1, every 21 days for a maximum of 6 cycles. Venous blood was collected from each, and genomic DNA was isolated. SNPs in XRCC3 T241M, XPD K751Q, XPD D312N, AURORA 91, AURORA 169 were assessed. The study included 180 patients. Median age was 62 years; 87 % were male; 34 % had PS 0; and 83 % had stage IV disease. The median number of cycles was 4. Time to progression was 5.1 months (95 % CI, 4.2-5.9). Overall median survival was 8.6 months (95 % CI, 7.1-10.1). There was no significant association between SNPs in XRCC3 T241M, XPD K751Q, XPD D312N, AURORA 91, AURORA 169 in outcome or toxicity. Our findings indicate that SNPs in XRCC3, XPD or Aurora kinase A cannot predict outcomes in advanced NSCLC patients treated with platinum-based chemotherapy.

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

PubMed

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.

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

PubMed Central

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

2016-01-01

ABSTRACT 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. PMID:27463890

Variable continental distribution of polymorphisms in the coding regions of DNA-repair genes.

PubMed

Mathonnet, Géraldine; Labuda, Damian; Meloche, Caroline; Wambach, Tina; Krajinovic, Maja; Sinnett, Daniel

2003-01-01

DNA-repair pathways are critical for maintaining the integrity of the genetic material by protecting against mutations due to exposure-induced damages or replication errors. Polymorphisms in the corresponding genes may be relevant in genetic epidemiology by modifying individual cancer susceptibility or therapeutic response. We report data on the population distribution of potentially functional variants in XRCC1, APEX1, ERCC2, ERCC4, hMLH1, and hMSH3 genes among groups representing individuals of European, Middle Eastern, African, Southeast Asian and North American descent. The data indicate little interpopulation differentiation in some of these polymorphisms and typical FST values ranging from 10 to 17% at others. Low FST was observed in APEX1 and hMSH3 exon 23 in spite of their relatively high minor allele frequencies, which could suggest the effect of balancing selection. In XRCC1, hMSH3 exon 21 and hMLH1 Africa clusters either with Middle East and Europe or with Southeast Asia, which could be related to the demographic history of human populations, whereby human migrations and genetic drift rather than selection would account for the observed differences.

The Functions of BRCA2 in Homologous Recombinational Repair

DTIC Science & Technology

2005-07-01

human Rad5l, Rad51B and Rad51C proteins 5 homogeneity using spermidine precipitation and sequential column chromatography with hydroxyapatite , Q-Sepharose...G. G., Albala, J. S., Shen, Z., ovary mutants for XRCC4, Ku86, and DNA-PKcs (the XR -1 and Schild, D. (1998) Nucleic Acids Res. 26, 1179-1184 oh 11

Upper tract urothelial carcinomas: frequency of association with mismatch repair protein loss and lynch syndrome.

PubMed

Harper, Holly L; McKenney, Jesse K; Heald, Brandie; Stephenson, Andrew; Campbell, Steven C; Plesec, Thomas; Magi-Galluzzi, Cristina

2017-01-01

Increased risk for upper tract urothelial carcinoma is described in patients with Lynch syndrome, caused by germline mutations in mismatch repair genes. We aimed to identify the frequency of mismatch repair protein loss in upper tract urothelial carcinoma and its potential for identifying an association with Lynch syndrome. We queried our database to identify upper tract urothelial carcinomas. Patients were cross-referenced for history of colorectal carcinoma or other common Lynch syndrome-associated neoplasms to enrich for potential Lynch syndrome cases. Tumor histopathologic characteristics were reviewed and each case was analyzed for loss of mismatch repair proteins, MLH1, MSH2, MSH6, and PMS2, by immunohistochemistry. Of 444 patients with upper tract urothelial carcinoma, a subset of 215 (encompassing 30 with upper tract urothelial carcinoma and another common Lynch syndrome-associated neoplasm) was analyzed for loss of mismatch repair protein expression. Of 30 patients with Lynch syndrome-associated neoplasms, six had documented Lynch syndrome, including two with Muir-Torre syndrome. Mismatch repair protein loss was identified in 7% of total upper tract urothelial carcinomas and 30% of patients with Lynch syndrome-associated neoplasms (including all patients with Lynch syndrome/Muir-Torre syndrome). Of patients without history of Lynch syndrome-associated neoplasms, 5 of 184 (2.7%) had loss of mismatch repair protein expression. Twelve cases with mismatch repair protein loss demonstrated loss of MSH2 and MSH6, and 2 had isolated loss of MSH6. MLH1 and PMS2 expression were consistently retained. Although increased intratumoral lymphocytes, inverted growth, pushing tumor-stromal interface, and lack of nuclear pleomorphism were more commonly seen in cases with mismatch repair protein loss, only intratumoral lymphocytes and presence of pushing borders were statistically significant. MLH1 and PMS2 testing appear to have little utility in upper tract urothelial

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

NASA Astrophysics Data System (ADS)

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.

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.

PubMed

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

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

PubMed Central

Cooper, Karen L.; Dashner, Erica J.; Tsosie, Ranalda; Cho, Young Mi; Lewis, Johnnye

2015-01-01

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. PMID:26627003

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

PubMed

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; <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. Copyright © 2015 Elsevier Inc. All rights reserved.

Base excision repair genes and risk of lung cancer among San Francisco Bay Area Latinos and African-Americans

PubMed Central

Chang, Jeffrey S.; Wrensch, Margaret R.; Hansen, Helen M.; Sison, Jennette D.; Aldrich, Melinda C.; Quesenberry, Charles P.; Seldin, Michael F.; Kelsey, Karl T.; Wiencke, John K.

2009-01-01

Base excision repair (BER) is the primary DNA damage repair mechanism for repairing small base lesions resulting from oxidation and alkylation damage. This study examines the association between 24 single-nucleotide polymorphisms (SNPs) belonging to five BER genes (XRCC1, APEX1, PARP1, MUTYH and OGG1) and lung cancer among Latinos (113 cases and 299 controls) and African-Americans (255 cases and 280 controls). The goal was to evaluate the differences in genetic contribution to lung cancer risk by ethnic groups. Analyses of individual SNPs and haplotypes were performed using unconditional logistic regressions adjusted for age, sex and genetic ancestry. Four SNPs among Latinos and one SNP among African-Americans were significantly (P < 0.05) associated with either risk of all lung cancer or non-small cell lung cancer (NSCLC). However, only the association between XRCC1 Arg399Gln (rs25487) and NSCLC among Latinos (odds ratio associated with every copy of Gln = 1.52; 95% confidence interval: 1.01–2.28) had a false-positive report probability of <0.5. Arg399Gln is a SNP with some functional evidence and has been shown previously to be an important SNP associated with lung cancer, mostly for Asians. Since the analyses were adjusted for genetic ancestry, the observed association between Arg399Gln and NSCLC among Latinos is unlikely to be confounded by population stratification; however, this result needs to be confirmed by additional studies among the Latino population. This study suggests that there are genetic differences in the association between BER pathway and lung cancer between Latinos and African-Americans. PMID:19029194

DNA repair properties of Escherichia coli tif-1, recAo281 and lexA1 strains deficient in single-strand DNA binding protein.

PubMed

Whittier, R F; Chase, J W

1983-01-01

Mutations affecting single-strand DNA binding protein (SSB) impair induction of mutagenic (SOS) repair. To further investigate the role of SSB in SOS induction and DNA repair, isogenic strains were constructed combining the ssb+, ssb-1 or ssb-113 alleles with one or more mutations known to alter regulation of damage inducible functions. As is true in ssb+ strains tif-1 (recA441) was found to allow thermal induction of prophage lambda + and Weigle reactivation in ssb-1 and ssb-113 strains. Furthermore, tif-1 decreased the UV sensitivity of the ssb-113 strain slightly and permitted UV induction of prophage lambda + at 30 degrees C. Strains carrying the recAo281 allele were also constructed. This mutation causes high constitutive levels of RecA protein synthesis and relieves much of the UV sensitivity conferred by lexA- alleles without restoring SOS (error-prone) repair. In contrast, the recAo281 allele failed to alleviate the UV sensitivity associated with either ssb- mutation. In a lexA1 recAo281 background the ssb-1 mutation increased the extent of postirradiation DNA degradation and concommitantly increased UV sensitivity 20-fold to the level exhibited by a recA1 strain. The ssb-113 mutation also increased UV sensitivity markedly in this background but did so without greatly increasing postirradiation DNA degradation. These results suggest a direct role for SSB in recombinational repair apart from and in addition to its role in facilitating induction of the recA-lexA regulon.

Protein Repair l-Isoaspartyl Methyltransferase1 Is Involved in Both Seed Longevity and Germination Vigor in Arabidopsis[W

PubMed Central

Ogé, Laurent; Bourdais, Gildas; Bove, Jérôme; Collet, Boris; Godin, Béatrice; Granier, Fabienne; Boutin, Jean-Pierre; Job, Dominique; Jullien, Marc; Grappin, Philippe

2008-01-01

The formation of abnormal amino acid residues is a major source of spontaneous age-related protein damage in cells. The protein l-isoaspartyl methyltransferase (PIMT) combats protein misfolding resulting from l-isoaspartyl formation by catalyzing the conversion of abnormal l-isoaspartyl residues to their normal l-aspartyl forms. In this way, the PIMT repair enzyme system contributes to longevity and survival in bacterial and animal kingdoms. Despite the discovery of PIMT activity in plants two decades ago, the role of this enzyme during plant stress adaptation and in seed longevity remains undefined. In this work, we have isolated Arabidopsis thaliana lines exhibiting altered expression of PIMT1, one of the two genes encoding the PIMT enzyme in Arabidopsis. PIMT1 overaccumulation reduced the accumulation of l-isoaspartyl residues in seed proteins and increased both seed longevity and germination vigor. Conversely, reduced PIMT1 accumulation was associated with an increase in the accumulation of l-isoaspartyl residues in the proteome of freshly harvested dry mature seeds, thus leading to heightened sensitivity to aging treatments and loss of seed vigor under stressful germination conditions. These data implicate PIMT1 as a major endogenous factor that limits abnormal l-isoaspartyl accumulation in seed proteins, thereby improving seed traits such as longevity and vigor. The PIMT repair pathway likely works in concert with other anti-aging pathways to actively eliminate deleterious protein products, thus enabling successful seedling establishment and strengthening plant proliferation in natural environments. PMID:19011119

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

PubMed Central

Kopera, Huira C.; Moldovan, John B.; Morrish, Tammy A.; Moran, John V.

2011-01-01

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. PMID:21940498

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

PubMed

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.

Human mismatch repair protein hMutLα is required to repair short slipped-DNAs of trinucleotide repeats.

PubMed

Panigrahi, Gagan B; Slean, Meghan M; Simard, Jodie P; Pearson, Christopher E

2012-12-07

Mismatch repair (MMR) is required for proper maintenance of the genome by protecting against mutations. The mismatch repair system has also been implicated as a driver of certain mutations, including disease-associated trinucleotide repeat instability. We recently revealed a requirement of hMutSβ in the repair of short slip-outs containing a single CTG repeat unit (1). The involvement of other MMR proteins in short trinucleotide repeat slip-out repair is unknown. Here we show that hMutLα is required for the highly efficient in vitro repair of single CTG repeat slip-outs, to the same degree as hMutSβ. HEK293T cell extracts, deficient in hMLH1, are unable to process single-repeat slip-outs, but are functional when complemented with hMutLα. The MMR-deficient hMLH1 mutant, T117M, which has a point mutation proximal to the ATP-binding domain, is defective in slip-out repair, further supporting a requirement for hMLH1 in the processing of short slip-outs and possibly the involvement of hMHL1 ATPase activity. Extracts of hPMS2-deficient HEC-1-A cells, which express hMLH1, hMLH3, and hPMS1, are only functional when complemented with hMutLα, indicating that neither hMutLβ nor hMutLγ is sufficient to repair short slip-outs. The resolution of clustered short slip-outs, which are poorly repaired, was partially dependent upon a functional hMutLα. The joint involvement of hMutSβ and hMutLα suggests that repeat instability may be the result of aberrant outcomes of repair attempts.

Heat shock protein 70 stimulation of the deoxyribonucleic acid base excision repair enzyme polymerase β

PubMed Central

Mendez, Frances; Kozin, Elliott; Bases, Robert

2003-01-01

Base excision repair (BER) of damaged deoxyribonucleic acid (DNA) is a multistep process during which potentially lethal abasic sites temporarily exist. Repair of these lesions is greatly stimulated by heat shock protein 70 (Hsp70), which enhances strand incision and removal of the abasic sites by human apurinic-apyrimidinic endonuclease (HAP1). The resulting single-strand gaps must then be filled in. Here, we show that Hsp70 and its 48- and 43-kDa N-terminal domains greatly stimulated filling in the single-strand gaps by DNA polymerase β, a novel finding that extends the role of Hsps in DNA repair. Incorporation of deoxyguanosine monophosphate (dGMP) to fill in single-strand gaps in DNA phagemid pBKS by DNA polymerase β was stimulated by Hsp70. Truncated proteins derived from the C-terminus of Hsp70 as well as unrelated proteins were less effective, but proteins derived from the N-terminus of Hsp70 remained efficient stimulators of DNA polymerase β repair of DNA single-strand gaps. In agreement with these results, repair of a gap in a 30-bp oligonucleotide by polymerase β also was strongly stimulated by Hsp70 although not by a truncated protein from the C-terminus of Hsp70. Sealing of the repaired site in the oligonucleotide by human DNA ligase 1 was not specifically stimulated by Hsp-related proteins. Results presented here now implicate and extend the role of Hsp70 as a partner in the enzymatic repair of damaged DNA. The participation of Hsp70 jointly with base excision enzymes improves repair efficiency by mechanisms that are not yet understood. PMID:14627201

DNA repair variants and breast cancer risk.

PubMed

Grundy, Anne; Richardson, Harriet; Schuetz, Johanna M; Burstyn, Igor; Spinelli, John J; Brooks-Wilson, Angela; Aronson, Kristan J

2016-05-01

A functional DNA repair system has been identified as important in the prevention of tumour development. Previous studies have hypothesized that common polymorphisms in DNA repair genes could play a role in breast cancer risk and also identified the potential for interactions between these polymorphisms and established breast cancer risk factors such as physical activity. Associations with breast cancer risk for 99 single nucleotide polymorphisms (SNPs) from genes in ten DNA repair pathways were examined in a case-control study including both Europeans (644 cases, 809 controls) and East Asians (299 cases, 160 controls). Odds ratios in both additive and dominant genetic models were calculated separately for participants of European and East Asian ancestry using multivariate logistic regression. The impact of multiple comparisons was assessed by correcting for the false discovery rate within each DNA repair pathway. Interactions between several breast cancer risk factors and DNA repair SNPs were also evaluated. One SNP (rs3213282) in the gene XRCC1 was associated with an increased risk of breast cancer in the dominant model of inheritance following adjustment for the false discovery rate (P < 0.05), although no associations were observed for other DNA repair SNPs. Interactions of six SNPs in multiple DNA repair pathways with physical activity were evident prior to correction for FDR, following which there was support for only one of the interaction terms (P < 0.05). No consistent associations between variants in DNA repair genes and breast cancer risk or their modification by breast cancer risk factors were observed. © 2016 Wiley Periodicals, Inc.

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

PubMed

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.

The Linker Histone GH1-HMGA1 Is Involved in Telomere Stability and DNA Damage Repair1[OPEN

PubMed Central

Charbonnel, Cyril; Benyahya, Fatiha; Butter, Falk

2018-01-01

Despite intensive searches, few proteins involved in telomere homeostasis have been identified in plants. Here, we used pull-down assays to identify potential telomeric interactors in the model plant species Arabidopsis (Arabidopsis thaliana). We identified the candidate protein GH1-HMGA1 (also known as HON4), an uncharacterized linker histone protein of the High Mobility Group Protein A (HMGA) family in plants. HMGAs are architectural transcription factors and have been suggested to function in DNA damage repair, but their precise biological roles remain unclear. Here, we show that GH1-HMGA1 is required for efficient DNA damage repair and telomere integrity in Arabidopsis. GH1-HMGA1 mutants exhibit developmental and growth defects, accompanied by ploidy defects, increased telomere dysfunction-induced foci, mitotic anaphase bridges, and degraded telomeres. Furthermore, mutants have a higher sensitivity to genotoxic agents such as mitomycin C and γ-irradiation. Our work also suggests that GH1-HMGA1 is involved directly in the repair process by allowing the completion of homologous recombination. PMID:29622687

Fanconi Anemia Proteins, DNA Interstrand Crosslink Repair Pathways, and Cancer Therapy

PubMed Central

Andreassen, Paul R.; Ren, Keqin

2016-01-01

DNA interstrand crosslinkers, a chemically diverse group of compounds which also induce alkylation of bases and DNA intrastrand crosslinks, are extensively utilized for cancer therapy. Understanding the cellular response to DNA damage induced by these agents is critical for more effective utilization of these compounds and for the identification of novel therapeutic targets. Importantly, the repair of DNA interstrand crosslinks (ICLs) involves many distinct DNA repair pathways, including nucleotide excision repair, translesion synthesis (TLS), and homologous recombination (HR). Additionally, proteins implicated in the pathophysiology of the multigenic disease Fanconi anemia (FA) have a role in the repair of ICLs that is not well understood. Cells from FA patients are hypersensitive to agents that induce ICLs, therefore FA proteins are potentially novel therapeutic targets. Here we will review current research directed at identifying FA genes and understanding the function of FA proteins in DNA damage responses. We will also examine interactions of FA proteins with other repair proteins and pathways, including signaling networks, which are potentially involved in ICL repair. Potential approaches to the modulation of FA protein function to enhance therapeutic outcome will be discussed. Also, mutation of many genes that encode proteins involved in ICL repair, including FA genes, increases susceptibility to cancer. A better understanding of these pathways is therefore critical for the design of individualized therapies tailored to the genetic profile of a particular malignancy. For this purpose, we will also review evidence for the association of mutation of FA genes with cancer in non-FA patients. PMID:19200054

Sufficient Amounts of Functional HOP2/MND1 Complex Promote Interhomolog DNA Repair but Are Dispensable for Intersister DNA Repair during Meiosis in Arabidopsis[W

PubMed Central

Uanschou, Clemens; Ronceret, Arnaud; Von Harder, Mona; De Muyt, Arnaud; Vezon, Daniel; Pereira, Lucie; Chelysheva, Liudmila; Kobayashi, Wataru; Kurumizaka, Hitoshi; Schlögelhofer, Peter; Grelon, Mathilde

2013-01-01

During meiosis, homologous recombination (HR) is essential to repair programmed DNA double-strand breaks (DSBs), and a dedicated protein machinery ensures that the homologous chromosome is favored over the nearby sister chromatid as a repair template. The HOMOLOGOUS-PAIRING PROTEIN2/MEIOTIC NUCLEAR DIVISION PROTEIN1 (HOP2/MND1) protein complex has been identified as a crucial factor of meiotic HR in Arabidopsis thaliana, since loss of either MND1 or HOP2 results in failure of DNA repair. We isolated two mutant alleles of HOP2 (hop2-2 and hop2-3) that retained the capacity to repair meiotic DSBs via the sister chromatid but failed to use the homologous chromosome. We show that in these alleles, the recombinases RADIATION SENSITIVE51 (RAD51) and DISRUPTED MEIOTIC cDNA1 (DMC1) are loaded, but only the intersister DNA repair pathway is activated. The hop2-2 phenotype is correlated with a decrease in HOP2/MND1 complex abundance. In hop2-3, a truncated HOP2 protein is produced that retains its ability to bind to DMC1 and DNA but forms less stable complexes with MND1 and fails to efficiently stimulate DMC1-driven D-loop formation. Genetic analyses demonstrated that in the absence of DMC1, HOP2/MND1 is dispensable for RAD51-mediated intersister DNA repair, while in the presence of DMC1, a minimal amount of functional HOP2/MND1 is essential to drive intersister DNA repair. PMID:24363313

Environmental exposure and HPV infection may act synergistically to induce lung tumorigenesis in nonsmokers

PubMed Central

Cheng, Ya-Wen; Lin, Frank Cheau-Feng; Chen, Chih-Yi; Hsu, Nan-Yung

2016-01-01

Most studies of lung tumorigenesis have focused on smokers rather than nonsmokers. In this study, we used human papillomavirus (HPV)-positive and HPV-negative lung cancer cells to test the hypothesis that HPV infection synergistically increases DNA damage induced by exposure to the carcinogen benzo[a]pyrene (B[a]P), and contributes to lung tumorigenesis in nonsmokers. DNA adduct levels induced by B[a]P in HPV-positive cells were significantly higher than in HPV-negative cells. The DNA adduct formation was dependent on HPV E6 oncoprotein expression. Gene and protein expression of two DNA repair genes, XRCC3 and XRCC5, were lower in B[a]P-treated E6-positive cells than in E6-negative lung cancer cells. The reduced expression was also detected immunohistochemically and was caused by increased promoter hypermethylation. Moreover, mutations of p53 and epidermal growth factor receptor (EGFR) genes in lung cancer patients were associated with XRCC5 inactivation. In sum, our study indicates that HPV E6-induced promoter hypermethylation of the XRCC3 and XRCC5 DNA repair genes and the resultant decrease in their expression increases B[a]P-induced DNA adducts and contributes to lung tumorigenesis in nonsmokers. PMID:26918347

Probing the substrate specificity of the bacterial Pnkp/Hen1 RNA repair system using synthetic RNAs

PubMed Central

Zhang, Can; Chan, Chio Mui; Wang, Pei; Huang, Raven H.

2012-01-01

Ribotoxins cleave essential RNAs involved in protein synthesis as a strategy for cell killing. RNA repair systems exist in nature to counteract the lethal actions of ribotoxins, as first demonstrated by the RNA repair system from bacteriophage T4 25 yr ago. Recently, we found that two bacterial proteins, named Pnkp and Hen1, form a stable complex and are able to repair ribotoxin-cleaved tRNAs in vitro. However, unlike the well-studied T4 RNA repair system, the natural RNA substrates of the bacterial Pnkp/Hen1 RNA repair system are unknown. Here we present comprehensive RNA repair assays with the recombinant Pnkp/Hen1 proteins from Anabaena variabilis using a total of 33 different RNAs as substrates that might mimic various damaged forms of RNAs present in living cells. We found that unlike the RNA repair system from bacteriophage T4, the bacterial Pnkp/Hen1 RNA repair system exhibits broad substrate specificity. Based on the experimental data presented here, a model of preferred RNA substrates of the Pnkp/Hen1 repair system is proposed. PMID:22190744

HYDROGEL-BASED NANOCOMPOSITES OF THERAPEUTIC PROTEINS FOR TISSUE REPAIR

PubMed Central

Zhu, Suwei; Segura, Tatiana

2014-01-01

The ability to design artificial extracellular matrices as cell instructive scaffolds has opened the door to technologies capable of studying cell fates in vitro and to guide tissue repair in vivo. One main component of the design of artificial extracellular matrices is the incorporation of protein-based biochemical cues to guide cell phenotypes and multicellular organizations. However, promoting the long-term bioactivity, controlling the bioavailability and understanding how the physical presentations of these proteins impacts cellular fates are among the challenges of the field. Nanotechnolgy has advanced to meet the challenges of protein therapeutics. For example, the approaches to incorporating proteins into tissue repairing scaffolds have ranged from bulk encapsulations to smart nanodepots that protect proteins from degradations and allow opportunities for controlled release. PMID:24778979

HYDROGEL-BASED NANOCOMPOSITES OF THERAPEUTIC PROTEINS FOR TISSUE REPAIR.

PubMed

Zhu, Suwei; Segura, Tatiana

2014-05-01

The ability to design artificial extracellular matrices as cell instructive scaffolds has opened the door to technologies capable of studying cell fates in vitro and to guide tissue repair in vivo . One main component of the design of artificial extracellular matrices is the incorporation of protein-based biochemical cues to guide cell phenotypes and multicellular organizations. However, promoting the long-term bioactivity, controlling the bioavailability and understanding how the physical presentations of these proteins impacts cellular fates are among the challenges of the field. Nanotechnolgy has advanced to meet the challenges of protein therapeutics. For example, the approaches to incorporating proteins into tissue repairing scaffolds have ranged from bulk encapsulations to smart nanodepots that protect proteins from degradations and allow opportunities for controlled release.

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

PubMed Central

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

NASA Technical Reports Server (NTRS)

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.

An integrated molecular analysis of lung adenocarcinomas identifies potential therapeutic targets among TTF1-negative tumors including DNA repair proteins and Nrf2

PubMed Central

Cardnell, Robert J.G.; Behrens, Carmen; Diao, Lixia; Fan, YouHong; Tang, Ximing; Tong, Pan; John D., Minna; Mills, Gordon B.; Heymach, John V.; Wistuba, Ignacio I.; Wang, Jing; Byers., Lauren A.

2015-01-01

Purpose Thyroid transcription factor-1 (TTF1) immunohistochemistry (IHC) is used clinically to differentiate primary lung adenocarcinomas (LUAD) from squamous lung cancers and metastatic adenocarcinomas from other primary sites. However, a subset of LUAD (15-20%) does not express TTF1 and TTF1-negative patients have worse clinical outcomes. As there are no established targeted agents with activity in TTF1-negative LUAD, we performed an integrated molecular analysis to identify potential therapeutic targets. Experimental Design Using two clinical LUAD cohorts (274 tumors), one from our institution (PROSPECT) and the TCGA, we interrogated proteomic profiles (by reverse-phase protein array (RPPA)), gene expression, and mutational data. Drug response data from 74 cell lines were used to validate potential therapeutic agents. Results Strong correlations were observed between TTF1 IHC and TTF1 measurements by RPPA (Rho=0.57, p<0.001) and gene expression (NKX2-1, Rho=0.61, p<0.001). Established driver mutations (e.g. BRAF and EGFR) were associated with high TTF1 expression. In contrast, TTF1-negative LUAD had a higher frequency of inactivating KEAP1 mutations (p=0.001). Proteomic profiling identified increased expression of DNA repair proteins (e.g., Chk1 and the DNA repair score) and suppressed PI3K/MAPK signaling among TTF1-negative tumors, with differences in total proteins confirmed at the mRNA level. Cell line analysis showed drugs targeting DNA repair to be more active in TTF1-low cell lines. Conclusions Combined genomic and proteomic analyses demonstrated infrequent alteration of validated lung cancer targets (including the absence of BRAF mutations in TTF1-negative LUAD), but identified novel potential targets for TTF1-negative LUAD includingKEAP1/Nrf2 and DNA repair pathways. PMID:25878335

A Synthetic Lethal Screen Identifies DNA Repair Pathways that Sensitize Cancer Cells to Combined ATR Inhibition and Cisplatin Treatments

PubMed Central

Mohni, Kareem N.; Thompson, Petria S.; Luzwick, Jessica W.; Glick, Gloria G.; Pendleton, Christopher S.; Lehmann, Brian D.; Pietenpol, Jennifer A.; Cortez, David

2015-01-01

The DNA damage response kinase ATR may be a useful cancer therapeutic target. ATR inhibition synergizes with loss of ERCC1, ATM, XRCC1 and DNA damaging chemotherapy agents. Clinical trials have begun using ATR inhibitors in combination with cisplatin. Here we report the first synthetic lethality screen with a combination treatment of an ATR inhibitor (ATRi) and cisplatin. Combination treatment with ATRi/cisplatin is synthetically lethal with loss of the TLS polymerase ζ and 53BP1. Other DNA repair pathways including homologous recombination and mismatch repair do not exhibit synthetic lethal interactions with ATRi/cisplatin, even though loss of some of these repair pathways sensitizes cells to cisplatin as a single-agent. We also report that ATRi strongly synergizes with PARP inhibition, even in homologous recombination-proficient backgrounds. Lastly, ATR inhibitors were able to resensitize cisplatin-resistant cell lines to cisplatin. These data provide a comprehensive analysis of DNA repair pathways that exhibit synthetic lethality with ATR inhibitors when combined with cisplatin chemotherapy, and will help guide patient selection strategies as ATR inhibitors progress into the cancer clinic. PMID:25965342

P53 protein in proliferation, repair and apoptosis of cells.

PubMed

Wawryk-Gawda, Ewelina; Chylińska-Wrzos, Patrycja; Lis-Sochocka, Marta; Chłapek, Katarzyna; Bulak, Kamila; Jędrych, Marian; Jodłowska-Jędrych, Barbara

2014-05-01

The p53 protein is an important factor of many intra- and extracellular processes. This protein regulates the repair of cellular DNA and induces apoptosis. It is also responsible for the regulation of the senescence and the cell entering the subsequent stages of the cellular cycle. The protein p53 is also involved in inhibiting angiogenesis and the induction of oxidative shock. In our study, we examined the activity of p53 protein in the uterine epithelial cells in rats treated with cladribine. Its action is mainly based on apoptosis induction. We compared the activity of p53 protein in cells with a high apoptosis index and in cells with active repair mechanisms and high proliferation index. We observed stronger p53 protein expression in the epithelial cells of the materials taken 24 h after the last dose of 2-CdA associated with the active process of apoptosis and inhibition of proliferation. After 4 weeks from the last dose of cladribine, the stronger expression of p53 protein was associated with both the existing changes in the cell's genome, the effects of the ongoing repair mechanisms, as well as the high proliferation activity.

DNA encoding a DNA repair protein

DOEpatents

Petrini, John H.; Morgan, William Francis; Maser, Richard Scott; Carney, James Patrick

2006-08-15

An isolated and purified DNA molecule encoding a DNA repair protein, p95, is provided, as is isolated and purified p95. Also provided are methods of detecting p95 and DNA encoding p95. The invention further provides p95 knock-out mice.

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

PubMed

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.

Nuclear translocation of p21{sup WAF1/CIP1} protein prior to its cytosolic degradation by UV enhances DNA repair and survival

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Ji Young; Kim, Hee Suk; Kim, Joo Young

2009-12-25

We previously reported that UV induced rapid proteasomal degradation of p21 protein in an ubiquitination-independent manner. Here, UV-induced p21 proteolysis was found to occur in the cytosol. Before cytosolic degradation, however, p21 protein translocated to and transiently accumulated in the nucleus. Nuclear translocation of p21 was not required for its degradation, but rather promoted DNA repair and cell survival. Overexpression of the wild type p21, but not the one with defective nuclear localization signal (NLS), reduced UV-induced DNA damage and cell death. Some of p21 protein translocated to the nucleus were associated with chromatin-bound PCNA and saved from UV-induced proteolysis.more » These data together show that p21 translocates to the nucleus to participate in DNA repair, while the rest is rapidly degraded in the cytosol. We propose that our findings reflect a mechanism to facilitate removal of damaged cells, enhancing DNA repair at the same time.« less

Repair of Nerve Cell Membrance Damage by Calcium-Dependent, Membrane-Binding Proteins

DTIC Science & Technology

2013-09-01

In acute spinal cord injury the plasma membranes of spinal neurons are torn allowing high concentrations of calcium to enter the cytoplasm, activating...repairing the cell membrane as soon as the increase in intracellular calcium is sensed by calcium -binding proteins. If these repair mechanisms can be...testing the hypothesis that the action of copine, a human calcium -dependent-membrane-binding protein, in model systems can promote a stable repair of

Zinc finger transcription factor CASZ1 interacts with histones, DNA repair proteins and recruits NuRD complex to regulate gene transcription.

PubMed

Liu, Zhihui; Lam, Norris; Thiele, Carol J

2015-09-29

The zinc finger transcription factor CASZ1 has been found to control neural fate-determination in flies, regulate murine and frog cardiac development, control murine retinal cell progenitor expansion and function as a tumor suppressor gene in humans. However, the molecular mechanism by which CASZ1 regulates gene transcription to exert these diverse biological functions has not been described. Here we identify co-factors that are recruited by CASZ1b to regulate gene transcription using co-immunoprecipitation (co-IP) and mass spectrometry assays. We find that CASZ1b binds to the nucleosome remodeling and histone deacetylase (NuRD) complex, histones and DNA repair proteins. Mutagenesis of the CASZ1b protein assay demonstrates that the N-terminus of CASZ1b is required for NuRD binding, and a poly(ADP-ribose) binding motif in the CASZ1b protein is required for histone H3 and DNA repair proteins binding. The N-terminus of CASZ1b fused to an artificial DNA-binding domain (GAL4DBD) causes a significant repression of transcription (5xUAS-luciferase assay), which could be blocked by treatment with an HDAC inhibitor. Realtime PCR results show that the transcriptional activity of CASZ1b mutants that abrogate NuRD or histone H3/DNA binding is significantly decreased. This indicates a model in which CASZ1b binds to chromatin and recruits NuRD complexes to orchestrate epigenetic-mediated transcriptional programs.

Nucleotide Excision Repair Proteins Rapidly Accumulate but Fail to Persist in Human XP-E (DDB2 Mutant) Cells

PubMed Central

Oh, Kyu-Seon; Imoto, Kyoko; Emmert, Steffen; Tamura, Deborah; DiGiovanna, John J.; Kraemer, Kenneth. H.

2011-01-01

The XP-E DNA damage binding protein (DDB2) is involved in early recognition of global genome DNA damage during DNA nucleotide excision repair (NER). We found that skin fibroblasts from 4 newly reported XP-E patients with numerous skin cancers and DDB2 mutations had slow repair of 6-4 photoproducts (6-4PP) and markedly reduced repair of cyclobutane pyrimidine dimers (CPD). NER proteins (XPC, XPB, XPG, XPA, and XPF) co-localized to CPD and 6-4PP positive regions immediately (< 0.1h) after localized UV irradiation in cells from the XP-E patients and normal controls. While these proteins persist in normal cells, surprisingly, within 0.5h these repair proteins were no longer detectable at the sites of DNA damage in XP-E cells. Our results indicate that DDB2 is not required for the rapid recruitment of NER proteins to sites of UV photoproducts or for partial repair of 6-4PP but is essential for normal persistence of these proteins for CPD photoproduct removal. PMID:21388382

Novel solid protein solder designs for laser-assisted tissue repair.

PubMed

McNally, K M; Sorg, B S; Welch, A J

2000-01-01

Previous studies have shown that the application of chromophore-enhanced albumin protein solders to augment laser tissue repairs significantly improves repair strength, enhances edge co-optation, and reduces thermal tissue injury. These investigations are furthered with this in vitro study conducted to assess a new range of specially designed chromophore-enhanced solid protein solders manufactured and tested for application during laser-assisted tissue repair. The experimental study was divided into three parts. In the first part of the study, the creation of a chromophore concentration gradient across the thickness of the solid protein solder was investigated as a means to improve control of the heat source gradient through the solder during laser irradiation. In the second part of the study, predenaturation of the solid protein solder was investigated as a means for enhancing the stability of the solder in physiological fluids before irradiation. Finally, in the third part of the study, the feasibility of using synthetic polymers as a scaffold for traditional albumin protein solder mixes was investigated as a means of improving the flexibility of the solder. Uniform denaturation across the thickness of the solder was achieved by controlling the chromophore concentration gradient, thus ensuring stable solder-tissue fusion when the specimen was submerged in a hydrated environment. Predenaturation of the solid protein solder significantly reduced the solubility of the solder, and consequently, improved the handling characteristics of the solder. The solder-doped polymer membranes were flexible enough to be wrapped around tissue, whereas their solid nature avoided problems associated with "runaway" of the less viscous liquid solders currently used by researchers. In addition, the solder-doped polymer membranes could be easily tailored to a wide range of geometries suitable to many clinical applications. The novel solid protein solder designs presented here add a new

RTEL1 contributes to DNA replication and repair and telomere maintenance.

PubMed

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.

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

PubMed

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

2016-11-20

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

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

PubMed Central

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

2016-01-01

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

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

EPA Science Inventory

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

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

PubMed Central

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

2015-01-01

Background: 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. Materials and methods: We evaluated 20 SNPs of 703 glioma cases (338 astrocytoma cases, 122 glioblastoma cases) and 635 controls in a Han Chinese population using χ2 test and genetic model analysis. Results: 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). Conclusions: 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. PMID:26328260

Endonuclease-independent LINE-1 retrotransposition at mammalian telomeres.

PubMed

Morrish, Tammy A; Garcia-Perez, José Luis; Stamato, Thomas D; Taccioli, Guillermo E; Sekiguchi, JoAnn; Moran, John V

2007-03-08

Long interspersed element-1 (LINE-1 or L1) elements are abundant, non-long-terminal-repeat (non-LTR) retrotransposons that comprise approximately 17% of human DNA. The average human genome contains approximately 80-100 retrotransposition-competent L1s (ref. 2), and they mobilize by a process that uses both the L1 endonuclease and reverse transcriptase, termed target-site primed reverse transcription. We have previously reported an efficient, endonuclease-independent L1 retrotransposition pathway (EN(i)) in certain Chinese hamster ovary (CHO) cell lines that are defective in the non-homologous end-joining (NHEJ) pathway of DNA double-strand-break repair. Here we have characterized EN(i) retrotransposition events generated in V3 CHO cells, which are deficient in DNA-dependent protein kinase catalytic subunit (DNA-PKcs) activity and have both dysfunctional telomeres and an NHEJ defect. Notably, approximately 30% of EN(i) retrotransposition events insert in an orientation-specific manner adjacent to a perfect telomere repeat (5'-TTAGGG-3'). Similar insertions were not detected among EN(i) retrotransposition events generated in controls or in XR-1 CHO cells deficient for XRCC4, an NHEJ factor that is required for DNA ligation but has no known function in telomere maintenance. Furthermore, transient expression of a dominant-negative allele of human TRF2 (also called TERF2) in XRCC4-deficient XR-1 cells, which disrupts telomere capping, enables telomere-associated EN(i) retrotransposition events. These data indicate that L1s containing a disabled endonuclease can use dysfunctional telomeres as an integration substrate. The findings highlight similarities between the mechanism of EN(i) retrotransposition and the action of telomerase, because both processes can use a 3' OH for priming reverse transcription at either internal DNA lesions or chromosome ends. Thus, we propose that EN(i) retrotransposition is an ancestral mechanism of RNA-mediated DNA repair associated with non

Small-molecule inhibitors of APE1 DNA repair function: an overview.

PubMed

Al-Safi, Rasha I; Odde, Srinivas; Shabaik, Yumna; Neamati, Nouri

2012-01-01

APE1 is a multifaceted protein that orchestrates multiple activities in the cell, one of which is the preservation of genomic integrity; a vital process that takes place in the context of the base excision repair (BER) pathway. Studies have implicated APE1 in rendering cancerous cells less vulnerable to the effects of DNA-damaging agents that are commonly used for the treatment of cancer. Furthermore, suppression of APE1 expression in cancer cell lines is accompanied by the potentiation of the activity of cytotoxic agents. As a result, major efforts have been directed towards the identification of small-molecule inhibitors of this DNA-repair enzyme. Herein, we review all patented small-molecule APE1 inhibitors reported prior to 2011. Unfortunately, the potency and selectivity of many of the reported inhibitors were not disclosed by the original authors, and at present it is unclear if APE1 is a bona fide target for many of the purported inhibitors. Moreover, cellular activity and toxicity of many inhibitors remain to be established. Since this is the first comprehensive review of small molecule APE1 inhibitors, we present all compounds reported to inhibit APE1 activity with an IC50 value ≤ 25 μM. Efforts towards a careful validation and optimization of these compounds are warranted. Furthermore, we explore potential allosteric drug-binding sites on the protein as an alternative approach for modulating the activity of this multifunctional protein. In addition, we give an overview of APE2, as well as other APE1 homologues in some disease-causing pathogens. Finally, given the universal importance of DNA repair, as well as the considerable conservation of repair proteins across all living organisms, we propose targeting the AP endonuclease activity of pathogens by the compounds discussed in this review, thereby expanding their therapeutic potential and application.

FAN1 acts with FANCI-FANCD2 to promote DNA interstrand cross-link repair.

PubMed

Liu, Ting; Ghosal, Gargi; Yuan, Jingsong; Chen, Junjie; Huang, Jun

2010-08-06

Fanconi anemia (FA) is caused by mutations in 13 Fanc genes and renders cells hypersensitive to DNA interstrand cross-linking (ICL) agents. A central event in the FA pathway is mono-ubiquitylation of the FANCI-FANCD2 (ID) protein complex. Here, we characterize a previously unrecognized nuclease, Fanconi anemia-associated nuclease 1 (FAN1), that promotes ICL repair in a manner strictly dependent on its ability to accumulate at or near sites of DNA damage and that relies on mono-ubiquitylation of the ID complex. Thus, the mono-ubiquitylated ID complex recruits the downstream repair protein FAN1 and facilitates the repair of DNA interstrand cross-links.

Poly(ADP-Ribose) Polymerase-1 and DNA-Dependent Protein Kinase Have Equivalent Roles in Double Strand Break Repair Following Ionizing Radiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mitchell, Jody; Smith, Graeme; Curtin, Nicola J., E-mail: n.j.curtin@ncl.ac.u

2009-12-01

Purpose: Radiation-induced DNA double strand breaks (DSBs) are predominantly repaired by nonhomologous end joining (NHEJ), involving DNA-dependent protein kinase (DNA-PK). Poly(ADP-ribose) polymerase-1 (PARP-1), well characterized for its role in single strand break repair, may also facilitate DSB repair. We investigated the activation of these enzymes by differing DNA ends and their interaction in the cellular response to ionizing radiation (IR). Methods and Materials: The effect of PARP and DNA-PK inhibitors (KU-0058684 and NU7441) on repair of IR-induced DSBs was investigated in DNA-PK and PARP-1 proficient and deficient cells by measuring gammaH2AX foci and neutral comets. Complementary in vitro enzyme kineticsmore » assays demonstrated the affinities of DNA-PK and PARP-1 for DSBs with varying DNA termini. Results: DNA-PK and PARP-1 both promoted the fast phase of resolution of IR-induced DSBs in cells. Inactivation of both enzymes was not additive, suggesting that PARP-1 and DNA-PK cooperate within the same pathway to promote DSB repair. The affinities of the two enzymes for oligonucleotides with blunt, 3' GGG or 5' GGG overhanging termini were similar and overlapping (K{sub dapp} = 2.6-6.4nM for DNA-PK; 1.7-4.5nM for PARP-1). DNA-PK showed a slightly greater affinity for overhanging DNA and was significantly more efficient when activated by a 5' GGG overhang. PARP-1 had a preference for blunt-ended DNA and required a separate factor for efficient stimulation by a 5' GGG overhang. Conclusion: DNA-PK and PARP-1 are both required in a pathway facilitating the fast phase of DNA DSB repair.« less

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

PubMed

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.

Antibody specific for a DNA repair protein

DOEpatents

Petrini, John H.; Morgan, William Francis; Maser, Richard Scott; Carney, James Patrick

2006-07-11

An isolated and purified DNA molecule encoding a DNA repair protein, p95, is provided, as is isolated and purified p95. Also provided are methods of detecting p95 and DNA encoding p95. The invention further provides p95 knock-out mice.

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

PubMed

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.

SAW1 is required for SDSA double-strand break repair in S. cerevisiae.

PubMed

Diamante, Graciel; Phan, Claire; Celis, Angie S; Krueger, Jonas; Kelson, Eric P; Fischhaber, Paula L

2014-03-14

SAW1, coding for Saw1, is required for single-strand annealing (SSA) DNA double-strand break (DSB) repair in Saccharomycescerevisiae. Saw1 physically associates with Rad1 and Rad52 and recruits the Rad1-Rad10 endonuclease. Herein we show by fluorescence microscopy that SAW1 is similarly required for recruitment of Rad10 to sites of Synthesis-Dependent Strand Annealing (SDSA) and associates with sites of SDSA repair in a manner temporally overlapped with Rad10. The magnitude of induction of colocalized Saw1-CFP/Rad10-YFP/DSB-RFP foci in SDSA is more dramatic in S and G2 phase cells than in M phase, consistent with the known mechanism of SDSA. We observed a substantial fraction of foci in which Rad10 was localized to the repair site without Saw1, but few DSB sites that contained Saw1 without Rad10. Together these data are consistent with a model in which Saw1 recruits Rad1-Rad10 to SDSA sites, possibly even binding as a protein-protein complex, but departs the repair site in advance of Rad1-Rad10. Copyright © 2014 Elsevier Inc. All rights reserved.

Host DNA repair proteins in response to Pseudomonas aeruginosa in lung epitehlial cells and in mice

USDA-ARS?s Scientific Manuscript database

Host DNA damage and DNA repair response to bacterial infections and its significance are not fully understood. Here, we demonstrate that infection by Gram-negative bacterium P. aeruginosa significantly altered the expression and enzymatic activity of base excision DNA repair protein OGG1 in lung epi...

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, Jeong-Min; Choi, Ji Ye; Yi, Joo Mi

2015-06-05

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

Expression of ERCC1, RRM1, TUBB3 in correlation with apoptosis repressor ARC, DNA mismatch repair proteins and p53 in liver metastasis of colorectal cancer.

PubMed

Tóth, Csaba; Sükösd, Farkas; Valicsek, Erzsébet; Herpel, Esther; Schirmacher, Peter; Renner, Marcus; Mader, Christoph; Tiszlavicz, László; Kriegsmann, Jörg

2017-11-01

Liver metastasis in colorectal cancer is common and the primary treatment is chemotherapy. To date, there is no routinely used test in clinical practice to predict the effectiveness of conventional chemotherapy. Therefore, biomarkers with predictive value for conventional chemotherapy would be of considerable benefit in treatment planning. We analysed three proteins [excision repair cross-complementing 1 (ERCC1), ribonucleoside-diphosphate reductase 1 (RRM1) and class III β-tubulin (TUBB3)] in colorectal cancer liver metastasis. We used tissue microarray slides with 101 liver metastasis samples, stained for ERCC1, RRM1 and TUBB3 and established scoring systems (fitted for tissue microarray) for each protein. In statistical analysis, we compared the expression of ERCC1, RRM1 and TUBB3 to mismatch proteins (MLH1, MSH2, MSH6 and PMS2), p53 and to apoptosis repressor protein (ARC). Statistically significant correlations were found between ERCC1, TUBB3 and MLH1, MSH2 and RRM1 and MSH2, MSH6. Noteworthy, our analysis revealed a strong significant correlation between cytoplasmic ARC expression and RRM1, TUBB3 (p=0.000 and p=0.001, respectively), implying an additional role of TUBB3 and RRM1 not only in therapy resistance, but also in the apoptotic machinery. Our data strengthens the importance of ERCC1, TUBB3 and RRM1 in the prediction of chemotherapy effectiveness and suggest new functional connections in DNA repair, microtubule network and apoptotic signaling (i.e. ARC protein). In conclusion, we showed the importance and need of predictive biomarkers in metastasized colorectal cancer and pointed out the relevance not only of single predictive markers but also of their interactions with other known and newly explored relations between different signaling pathways.

Two familial ALS proteins function in prevention/repair of transcription-associated DNA damage.

PubMed

Hill, Sarah J; Mordes, Daniel A; Cameron, Lisa A; Neuberg, Donna S; Landini, Serena; Eggan, Kevin; Livingston, David M

2016-11-29

Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron dysfunction disease that leads to paralysis and death. There is currently no established molecular pathogenesis pathway. Multiple proteins involved in RNA processing are linked to ALS, including FUS and TDP43, and we propose a disease mechanism in which loss of function of at least one of these proteins leads to an accumulation of transcription-associated DNA damage contributing to motor neuron cell death and progressive neurological symptoms. In support of this hypothesis, we find that FUS or TDP43 depletion leads to increased sensitivity to a transcription-arresting agent due to increased DNA damage. Thus, these proteins normally contribute to the prevention or repair of transcription-associated DNA damage. In addition, both FUS and TDP43 colocalize with active RNA polymerase II at sites of DNA damage along with the DNA damage repair protein, BRCA1, and FUS and TDP43 participate in the prevention or repair of R loop-associated DNA damage, a manifestation of aberrant transcription and/or RNA processing. Gaining a better understanding of the role(s) that FUS and TDP43 play in transcription-associated DNA damage could shed light on the mechanisms underlying ALS pathogenesis.

Two familial ALS proteins function in prevention/repair of transcription-associated DNA damage

PubMed Central

Hill, Sarah J.; Mordes, Daniel A.; Cameron, Lisa A.; Neuberg, Donna S.; Landini, Serena; Eggan, Kevin; Livingston, David M.

2016-01-01

Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron dysfunction disease that leads to paralysis and death. There is currently no established molecular pathogenesis pathway. Multiple proteins involved in RNA processing are linked to ALS, including FUS and TDP43, and we propose a disease mechanism in which loss of function of at least one of these proteins leads to an accumulation of transcription-associated DNA damage contributing to motor neuron cell death and progressive neurological symptoms. In support of this hypothesis, we find that FUS or TDP43 depletion leads to increased sensitivity to a transcription-arresting agent due to increased DNA damage. Thus, these proteins normally contribute to the prevention or repair of transcription-associated DNA damage. In addition, both FUS and TDP43 colocalize with active RNA polymerase II at sites of DNA damage along with the DNA damage repair protein, BRCA1, and FUS and TDP43 participate in the prevention or repair of R loop-associated DNA damage, a manifestation of aberrant transcription and/or RNA processing. Gaining a better understanding of the role(s) that FUS and TDP43 play in transcription-associated DNA damage could shed light on the mechanisms underlying ALS pathogenesis. PMID:27849576

Posttranslational Protein Modifications in Type 1 Diabetes - Genetic Studies with PCMT1, the Repair Enzyme Protein Isoaspartate Methyltransferase (PIMT) Encoding Gene

PubMed Central

Wägner, Ana M.; Cloos, Paul; Bergholdt, Regine; Eising, Stefanie; Brorsson, Caroline; Stalhut, Martin; Christgau, Stephan; Nerup, Jørn; Pociot, Flemming

2008-01-01

BACKGROUND: Posttranslational protein modifications have been implicated in the development of autoimmunity. Protein L-isoaspartate (D-aspartate) O-methyltransferase (PIMT) repairs modified proteins and is encoded by PCMT1, located in a region linked to type 1 diabetes (T1D), namely IDDM5. AIM: To evaluate the association between genetic variations in the PCMT1 gene and T1D. METHODS: Firstly, PCMT1 was sequenced in 26 patients with T1D (linked to IDDM5) and 10 control subjects. The variations found in PCMT1 were then tested (alone and interacting with a functional polymorphism in SUMO4 and with HLA) for association with T1D in 253 families (using transmission disequilibrium test). In a third step, the association of the functional variation in PCMT1 (rs4816) with T1D was analyzed in 778 T1D patients and 749 controls (using chi-square test). In vitro promoter activity was assessed by transfecting INS-1E cells with PCMT1 promoter constructs and a reporter gene, with or without cytokine stimulation. RESULTS: Four polymorphisms in complete linkage disequilibrium were identified in PCMT1 (5' to the gene (rs11155676), exon 5 (rs4816) and exon 8 (rs7818 and rs4552)). In the whole cohort of 253 families, the allele associated with increased PIMT enzyme activity (rs4816, allele A) was less frequently transmitted to the affected than to the non-affected offspring (46% vs. 53%, p = 0.099). This finding was even more evident in the subset of families where the proband had high-risk SUMO4 (p = 0.069) or low-risk HLA (p = 0.086). Surprisingly, in the case-control study with 778 cases and 749 controls, an inverse trend was found (40.36% of patients and 36.98% of controls had the allele, p = 0.055). PCMT1 promoter activity increased with cytokine stimulation, but no differences were detected between the constructs adjacent to rs11155676. CONCLUSION: PCMT1 was virtually associated with T1D in groups defined by other risk genes (SUMO4 and HLA). A general association in a not further

WHERE MULTIFUNCTIONAL DNA REPAIR PROTEINS MEET: MAPPING THE INTERACTION DOMAINS BETWEEN XPG AND WRN

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rangaraj, K.; Cooper, P.K.; Trego, K.S.

The rapid recognition and repair of DNA damage is essential for the maintenance of genomic integrity and cellular survival. Multiple complex and interconnected DNA damage responses exist within cells to preserve the human genome, and these repair pathways are carried out by a specifi c interplay of protein-protein interactions. Thus a failure in the coordination of these processes, perhaps brought about by a breakdown in any one multifunctional repair protein, can lead to genomic instability, developmental and immunological abnormalities, cancer and premature aging. This study demonstrates a novel interaction between two such repair proteins, Xeroderma pigmentosum group G protein (XPG)more » and Werner syndrome helicase (WRN), that are both highly pleiotropic and associated with inherited genetic disorders when mutated. XPG is a structure-specifi c endonuclease required for the repair of UV-damaged DNA by nucleotide excision repair (NER), and mutations in XPG result in the diseases Xeroderma pigmentosum (XP) and Cockayne syndrome (CS). A loss of XPG incision activity results in XP, whereas a loss of non-enzymatic function(s) of XPG causes CS. WRN is a multifunctional protein involved in double-strand break repair (DSBR), and consists of 3’–5’ DNA-dependent helicase, 3’–5’ exonuclease, and single-strand DNA annealing activities. Nonfunctional WRN protein leads to Werner syndrome, a premature aging disorder with increased cancer incidence. Far Western analysis was used to map the interacting domains between XPG and WRN by denaturing gel electrophoresis, which separated purifi ed full length and recombinant XPG and WRN deletion constructs, based primarily upon the length of each polypeptide. Specifi c interacting domains were visualized when probed with the secondary protein of interest which was then detected by traditional Western analysis using the antibody of the secondary protein. The interaction between XPG and WRN was mapped to the C

Replication Protein A (RPA) deficiency activates the Fanconi anemia DNA repair pathway.

PubMed

Jang, Seok-Won; Jung, Jin Ki; Kim, Jung Min

2016-09-01

The Fanconi anemia (FA) pathway regulates DNA inter-strand crosslink (ICL) repair. Despite our greater understanding of the role of FA in ICL repair, its function in the preventing spontaneous genome instability is not well understood. Here, we show that depletion of replication protein A (RPA) activates the FA pathway. RPA1 deficiency increases chromatin recruitment of FA core complex, leading to FANCD2 monoubiquitination (FANCD2-Ub) and foci formation in the absence of DNA damaging agents. Importantly, ATR depletion, but not ATM, abolished RPA1 depletion-induced FANCD2-Ub, suggesting that ATR activation mediated FANCD2-Ub. Interestingly, we found that depletion of hSSB1/2-INTS3, a single-stranded DNA-binding protein complex, induces FANCD2-Ub, like RPA1 depletion. More interestingly, depletion of either RPA1 or INTS3 caused increased accumulation of DNA damage in FA pathway deficient cell lines. Taken together, these results indicate that RPA deficiency induces activation of the FA pathway in an ATR-dependent manner, which may play a role in the genome maintenance.

AHNAK1 and AHNAK2 are costameric proteins: AHNAK1 affects transverse skeletal muscle fiber stiffness

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marg, Andreas, E-mail: andreas.marg@mdc-berlin.de; Haase, Hannelore; Neumann, Tanja

2010-10-08

Research highlights: {yields} AHNAK1 and AHNAK2 are costameric proteins. {yields} Intact membrane repair in AHNAK1-deficient mice. {yields} AHNAK1{sup -/-} single fibers have a higher transverse stiffness. -- Abstract: The AHNAK scaffold PDZ-protein family is implicated in various cellular processes including membrane repair; however, AHNAK function and subcellular localization in skeletal muscle are unclear. We used specific AHNAK1 and AHNAK2 antibodies to analyzed the detailed localization of both proteins in mouse skeletal muscle. Co-localization of AHNAK1 and AHNAK2 with vinculin clearly demonstrates that both proteins are components of the costameric network. In contrast, no AHNAK expression was detected in the T-tubulemore » system. A laser wounding assay with AHNAK1-deficient fibers suggests that AHNAK1 is not involved in membrane repair. Using atomic force microscopy (AFM), we observed a significantly higher transverse stiffness of AHNAK1{sup -/-} fibers. These findings suggest novel functions of AHNAK proteins in skeletal muscle.« less

DNA repair polymorphisms in B-cell chronic lymphocytic leukemia in sufferers of Chernobyl Nuclear Power Plant accident.

PubMed

Abramenko, Iryna; Bilous, Nadiia; Chumak, Anatolyi; Kostin, Alexey; Martina, Zoya; Dyagil, Iryna

2012-01-01

An association between DNA repair gene polymorphisms, environmental factors, and development of some types of cancer has been suggested by several studies. Chronic lymphocytic leukemia (CLL) is the most common form of leukemia in the clean-up workers of the Chernobyl Nuclear Power Plant (NPP) accident and it has some specific features. Therefore, we have studied the possible differences in DNA repair gene polymorphisms in CLL patients depending on ionizing radiation (IR) exposure history and their clinical characterictics. Arg399Gln XRCC1, Thr241Met XRCC3, and Lys751Gln XPD polymorphisms were studied in 64 CLL patients, exposed to IR due to the Chernobyl NPP accident, 114 IR-non-exposed CLL patients, and 103 sex- and age-matched IR-exposed controls using polymerase chain reaction-restriction fragment-length polymorphism analysis. All investigated polymorphisms were equally distributed between two groups of CLL patients and IR-exposed controls, except that that there was a significant reduction of the common homozygous Lys/Lys XPD genotype among IR-exposed CLL patients (23.7%) compared with IR-exposed controls (45.6%), OR = 0.37; 95% CI = 0.18-0.75; (P = 0.005). The number of IR-non-exposed CLL patients (37.4%) with the Lys/Lys XPD genotype was also decreased compared to IR-exposed controls, although this difference was not significant (P = 0.223). These preliminary data suggest a possible modifying role of Lys751Gln XPD polymorphism for the development of CLL, expecially in radiation-exposed persons.

The yeast MSH1 gene is not involved in DNA repair or recombination during meiosis.

PubMed

Sia, Elaine A; Kirkpatrick, David T

2005-02-03

Six strong homologs of the bacterial MutS DNA mismatch repair (MMR) gene have been identified in the yeast Saccharomyces cerevisiae. With the exception of the MSH1 gene, the involvement of each homolog in DNA repair and recombination during meiosis has been determined previously. Five of the homologs have been demonstrated to act in meiotic DNA repair (MSH2, MSH3, MSH6 and MSH4) and/or meiotic recombination (MSH4 and MSH5). Unfortunately the loss of mitochondrial function that results from deletion of MSH1 disrupts meiotic progression, precluding an analysis of MSH1 function in meiotic DNA repair and recombination. However, the recent identification of two separation-of-function alleles of MSH1 that interfere with protein function but still maintain functional mitochondria allow the meiotic activities of MSH1 to be determined. We show that the G776D and F105A alleles of MSH1 exhibit no defects in meiotic recombination, repair base-base mismatches and large loop mismatches efficiently during meiosis, and have high levels of spore viability. These data indicate that the MSH1 protein, unlike other MutS homologs in yeast, plays no role in DNA repair or recombination during meiosis.

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

PubMed

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.

Evidence that MEK1 positively promotes interhomologue double-strand break repair

PubMed Central

Terentyev, Yaroslav; Johnson, Rebecca; Neale, Matthew J.; Khisroon, Muhammad; Bishop-Bailey, Anna; Goldman, Alastair S. H.

2010-01-01

During meiosis there is an imperative to create sufficient crossovers for homologue segregation. This can be achieved during repair of programmed DNA double-strand breaks (DSBs), which are biased towards using a homologue rather than sister chromatid as a repair template. Various proteins contribute to this bias, one of which is a meiosis specific kinase Mek1. It has been proposed that Mek1 establishes the bias by creating a barrier to sister chromatid repair, as distinct from enforcing strand invasion with the homologue. We looked for evidence that Mek1 positively stimulates strand invasion of the homologue. This was done by analysing repair of DSBs induced by the VMA1-derived endonuclease (VDE) and flanked by directly repeated sequences that can be used for intrachromatid single-strand annealing (SSA). SSA competes with interhomologue strand invasion significantly more successfully when Mek1 function is lost. We suggest the increase in intrachromosomal SSA reflects an opportunistic default repair pathway due to loss of a MEK1 stimulated bias for strand invasion of the homologous chromosome. Making use of an inhibitor sensitive mek1-as1 allele, we found that Mek1 function influences the repair pathway throughout the first4–5 h of meiosis. Perhaps reflecting a particular need to create bias for successful interhomologue events before chromosome pairing is complete. PMID:20223769

Relationship of the Xeroderma Pigmentosum Group E DNA Repair Defect to the Chromatin and DNA Binding Proteins UV-DDB and Replication Protein A

PubMed Central

Rapić Otrin, Vesna; Kuraoka, Isao; Nardo, Tiziana; McLenigan, Mary; Eker, A. P. M.; Stefanini, Miria; Levine, Arthur S.; Wood, Richard D.

1998-01-01

Cells from complementation groups A through G of the heritable sun-sensitive disorder xeroderma pigmentosum (XP) show defects in nucleotide excision repair of damaged DNA. Proteins representing groups A, B, C, D, F, and G are subunits of the core recognition and incision machinery of repair. XP group E (XP-E) is the mildest form of the disorder, and cells generally show about 50% of the normal repair level. We investigated two protein factors previously implicated in the XP-E defect, UV-damaged DNA binding protein (UV-DDB) and replication protein A (RPA). Three newly identified XP-E cell lines (XP23PV, XP25PV, and a line formerly classified as an XP variant) were defective in UV-DDB binding activity but had levels of RPA in the normal range. The XP-E cell extracts did not display a significant nucleotide excision repair defect in vitro, with either UV-irradiated DNA or a uniquely placed cisplatin lesion used as a substrate. Purified UV-DDB protein did not stimulate repair of naked DNA by DDB− XP-E cell extracts, but microinjection of the protein into DDB− XP-E cells could partially correct the repair defect. RPA stimulated repair in normal, XP-E, or complemented extracts from other XP groups, and so the effect of RPA was not specific for XP-E cell extracts. These data strengthen the connection between XP-E and UV-DDB. Coupled with previous results, the findings suggest that UV-DDB has a role in the repair of DNA in chromatin. PMID:9584159

Small RNA-mediated repair of UV-induced DNA lesions by the DNA DAMAGE-BINDING PROTEIN 2 and ARGONAUTE 1

PubMed Central

Schalk, Catherine; Cognat, Valérie; Graindorge, Stéfanie; Vincent, Timothée; Voinnet, Olivier; Molinier, Jean

2017-01-01

As photosynthetic organisms, plants need to prevent irreversible UV-induced DNA lesions. Through an unbiased, genome-wide approach, we have uncovered a previously unrecognized interplay between Global Genome Repair and small interfering RNAs (siRNAs) in the recognition of DNA photoproducts, prevalently in intergenic regions. Genetic and biochemical approaches indicate that, upon UV irradiation, the DNA DAMAGE-BINDING PROTEIN 2 (DDB2) and ARGONAUTE 1 (AGO1) of Arabidopsis thaliana form a chromatin-bound complex together with 21-nt siRNAs, which likely facilitates recognition of DNA damages in an RNA/DNA complementary strand-specific manner. The biogenesis of photoproduct-associated siRNAs involves the noncanonical, concerted action of RNA POLYMERASE IV, RNA-DEPENDENT RNA POLYMERASE-2, and DICER-LIKE-4. Furthermore, the chromatin association/dissociation of the DDB2-AGO1 complex is under the control of siRNA abundance and DNA damage signaling. These findings reveal unexpected nuclear functions for DCL4 and AGO1, and shed light on the interplay between small RNAs and DNA repair recognition factors at damaged sites. PMID:28325872

Dimethylarsinic acid in drinking water changed the morphology of urinary bladder but not the expression of DNA repair genes of bladder transitional epithelium in F344 rats.

PubMed

Wang, Amy; Wolf, Douglas C; Sen, Banalata; Knapp, Geremy W; Holladay, Steven D; Huckle, William R; Caceci, Thomas; Robertson, John L

2009-06-01

Inorganic arsenic increases urinary bladder transitional cell carcinoma in humans. In F344 rats, dimethylarsinic acid (DMA[V]) increases transitional cell carcinoma. Arsenic-induced inhibition of DNA repair has been reported in cultured cell lines and in lymphocytes of arsenic-exposed humans, but it has not been studied in urinary bladder. Should inhibition of DNA damage repair in transitional epithelium occur, it may contribute to carcinogenesis or cocarcinogenesis. We investigated morphology and expression of DNA repair genes in F344 rat transitional cells following up to 100 ppm DMA(V) in drinking water for four weeks. Mitochondria were very sensitive to DMA(V), and swollen mitochondria appeared to be the main source of vacuoles in the transitional epithelium. Real-time reverse transcriptase polymerase chain reaction (Real-Time RT PCR) showed the mRNA levels of tested DNA repair genes, ataxia telangectasia mutant (ATM), X-ray repair cross-complementing group 1 (XRCC1), excision repair cross-complementing group 3/xeroderma pigmentosum B (ERCC3/XPB), and DNA polymerase beta (Polbeta), were not altered by DMA(V). These data suggested that either DMA(V) does not affect DNA repair in the bladder or DMA(V) affects DNA repair without affecting baseline mRNA levels of repair genes. The possibility remains that DMA(V) may lower damage-induced increases in repair gene expression or cause post-translational modification of repair enzymes.

Trichomonas vaginalis Repair of Iron Centres Proteins: The Different Role of Two Paralogs.

PubMed

Nobre, Lígia S; Meloni, Dionigia; Teixeira, Miguel; Viscogliosi, Eric; Saraiva, Lígia M

2016-06-01

Trichomonas vaginalis, the causative parasite of one of the most prevalent sexually transmitted diseases is, so far, the only protozoan encoding two putative Repair of Iron Centres (RIC) proteins. Homologs of these proteins have been shown to protect bacteria from the chemical stress imposed by mammalian immunity. In this work, the biochemical and functional characterisation of the T. vaginalis RICs revealed that the two proteins have different properties. Expression of ric1 is induced by nitrosative stress but not by hydrogen peroxide, while ric2 transcription remained unaltered under similar conditions. T. vaginalis RIC1 contains a di-iron centre, but RIC2 apparently does not. Only RIC1 resembles bacterial RICs on spectroscopic profiling and repairing ability of oxidatively-damaged iron-sulfur clusters. Unexpectedly, RIC2 was found to bind DNA plasmid and T. vaginalis genomic DNA, a function proposed to be related with its leucine zipper domain. The two proteins also differ in their cellular localization: RIC1 is expressed in the cytoplasm only, and RIC2 occurs both in the nucleus and cytoplasm. Therefore, we concluded that the two RIC paralogs have different roles in T. vaginalis, with RIC2 showing an unprecedented DNA binding ability when compared with all other until now studied RICs. Copyright © 2016 Elsevier GmbH. All rights reserved.

The unstructured linker arms of Mlh1-Pms1 are important for interactions with DNA during mismatch repair

PubMed Central

Plys, Aaron J.; Rogacheva, Maria V.; Greene, Eric C.; Alani, Eric

2012-01-01

DNA mismatch repair (MMR) models have proposed that MSH proteins identify DNA polymerase errors while interacting with the DNA replication fork. MLH proteins (primarily Mlh1-Pms1 in baker’s yeast) then survey the genome for lesion-bound MSH proteins. The resulting MSH-MLH complex formed at a DNA lesion initiates downstream steps in repair. MLH proteins act as dimers and contain long (20 – 30 nanometers) unstructured arms that connect two terminal globular domains. These arms can vary between 100 to 300 amino acids in length, are highly divergent between organisms, and are resistant to amino acid substitutions. To test the roles of the linker arms in MMR, we engineered a protease cleavage site into the Mlh1 linker arm domain of baker’s yeast Mlh1-Pms1. Cleavage of the Mlh1 linker arm in vitro resulted in a defect in Mlh1-Pms1 DNA binding activity, and in vivo proteolytic cleavage resulted in a complete defect in MMR. We then generated a series of truncation mutants bearing Mlh1 and Pms1 linker arms of varying lengths. This work revealed that MMR is greatly compromised when portions of the Mlh1 linker are removed, whereas repair is less sensitive to truncation of the Pms1 linker arm. Purified complexes containing truncations in Mlh1 and Pms1 linker arms were analyzed and found to have differential defects in DNA binding that also correlated with the ability to form a ternary complex with Msh2-Msh6 and mismatch DNA. These observations are consistent with the unstructured linker domains of MLH proteins providing distinct interactions with DNA during MMR. PMID:22659005

Atl1 regulates choice between global genome and transcription-coupled repair of O(6)-alkylguanines.

PubMed

Latypov, Vitaly F; Tubbs, Julie L; Watson, Amanda J; Marriott, Andrew S; McGown, Gail; Thorncroft, Mary; Wilkinson, Oliver J; Senthong, Pattama; Butt, Amna; Arvai, Andrew S; Millington, Christopher L; Povey, Andrew C; Williams, David M; Santibanez-Koref, Mauro F; Tainer, John A; Margison, Geoffrey P

2012-07-13

Nucleotide excision repair (NER) has long been known to remove DNA lesions induced by chemical carcinogens, and the molecular mechanism has been partially elucidated. Here we demonstrate that in Schizosaccharomyces pombe a DNA recognition protein, alkyltransferase-like 1 (Atl1), can play a pivotal role in selecting a specific NER pathway, depending on the nature of the DNA modification. The relative ease of dissociation of Atl1 from DNA containing small O(6)-alkylguanines allows accurate completion of global genome repair (GGR), whereas strong Atl1 binding to bulky O(6)-alkylguanines blocks GGR, stalls the transcription machinery, and diverts the damage to transcription-coupled repair. Our findings redraw the initial stages of the NER process in those organisms that express an alkyltransferase-like gene and raise the question of whether or not O(6)-alkylguanine lesions that are poor substrates for the alkyltransferase proteins in higher eukaryotes might, by analogy, signal such lesions for repair by NER. Copyright © 2012 Elsevier Inc. All rights reserved.

Genetic Variation in DNA Repair Genes and Prostate Cancer Risk: Results from a Population-Based Study

PubMed Central

Agalliu, Ilir; Kwon, Erika M; Salinas, Claudia A.; Koopmeiners, Joseph S.; Ostrander, Elaine A.; Stanford, Janet L.

2009-01-01

Objective DNA repair pathways are crucial to prevent accumulation of DNA damage and maintain genomic stability. Alterations of this pathway have been reported in many cancers. An increase in oxidative DNA damage or decrease of DNA repair capacity with aging or due to germline genetic variation may affect prostate cancer risk. Methods Pooled data from two population-based studies (1,457 cases and 1,351 controls) were analyzed to examine associations between 28 SNPs in 9 DNA repair genes (APEX1, BRCA2, ERCC2, ERCC4, MGMT, MUTYH, OGG1, XPC, and XRCC1) and prostate cancer risk. We also explored whether associations varied by smoking, by family history or clinical features of prostate cancer. Results There were no associations between these SNPs and overall risk of prostate cancer. Risks did not vary either by smoking or by family history of prostate cancer. Although, two SNPs in BRCA2 (rs144848, rs1801406) and two SNPs in ERCC2 (rs1799793, rs13181) showed stronger associations with high Gleason score or advanced stage tumors when comparing homozygous men carrying the minor vs. major allele, results were not statistically significantly different between clinically aggressive and non-aggressive tumors. Conclusion Overall this study found no associations between prostate cancer and the SNPs in DNA repair genes. Given the complexity of this pathway and its crucial role in maintenance of genomic stability a pathway-based analysis of all 150 genes in DNA repair pathways, as well as exploration of gene-environment interactions may be warranted. PMID:19902366

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

PubMed

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

2010-11-01

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

Role of Saccharomyces cerevisiae Msh2 and Msh3 repair proteins in double-strand break-induced recombination

PubMed Central

Sugawara, Neal; Pâques, Frédéric; Colaiácovo, Mónica; Haber, James E.

1997-01-01

When gene conversion is initiated by a double-strand break (DSB), any nonhomologous DNA that may be present at the ends must be removed before new DNA synthesis can be initiated. In Saccharomyces cerevisiae, removal of nonhomologous ends depends not only on the nucleotide excision repair endonuclease Rad1/Rad10 but also on Msh2 and Msh3, two proteins that are required to correct mismatched bp. These proteins have no effect when DSB ends are homologous to the donor, either in the kinetics of recombination or in the proportion of gene conversions associated with crossing-over. A second DSB repair pathway, single-strand annealing also requires Rad1/Rad10 and Msh2/Msh3, but reveals a difference in their roles. When the flanking homologous regions that anneal are 205 bp, the requirement for Msh2/Msh3 is as great as for Rad1/Rad10; but when the annealing partners are 1,170 bp, Msh2/Msh3 have little effect, while Rad1/Rad10 are still required. Mismatch repair proteins Msh6, Pms1, and Mlh1 are not required. We suggest Msh2 and Msh3 recognize not only heteroduplex loops and mismatched bp, but also branched DNA structures with a free 3′ tail. PMID:9256462

Role of Saccharomyces cerevisiae Msh2 and Msh3 repair proteins in double-strand break-induced recombination.

PubMed

Sugawara, N; Pâques, F; Colaiácovo, M; Haber, J E

1997-08-19

When gene conversion is initiated by a double-strand break (DSB), any nonhomologous DNA that may be present at the ends must be removed before new DNA synthesis can be initiated. In Saccharomyces cerevisiae, removal of nonhomologous ends depends not only on the nucleotide excision repair endonuclease Rad1/Rad10 but also on Msh2 and Msh3, two proteins that are required to correct mismatched bp. These proteins have no effect when DSB ends are homologous to the donor, either in the kinetics of recombination or in the proportion of gene conversions associated with crossing-over. A second DSB repair pathway, single-strand annealing also requires Rad1/Rad10 and Msh2/Msh3, but reveals a difference in their roles. When the flanking homologous regions that anneal are 205 bp, the requirement for Msh2/Msh3 is as great as for Rad1/Rad10; but when the annealing partners are 1,170 bp, Msh2/Msh3 have little effect, while Rad1/Rad10 are still required. Mismatch repair proteins Msh6, Pms1, and Mlh1 are not required. We suggest Msh2 and Msh3 recognize not only heteroduplex loops and mismatched bp, but also branched DNA structures with a free 3' tail.

Warthin tumors do not have microsatellite instability and express normal DNA mismatch repair proteins.

PubMed

Hunt, Jennifer L

2006-01-01

Warthin tumors are controversial entities with a poorly understood etiology. Although some investigators have suggested a neoplastic origin, others have supported a developmental anomaly. A recent study described the absence of staining for hMLH1 and hMSH2 proteins in the epithelial component of Warthin tumors, suggesting that they arise secondary to defects in the DNA mismatch repair system. To determine if Warthin tumors exhibit evidence of DNA mismatch repair defects. Immunostains for hMLH1 and hMSH2 were performed using a standard approach. Microdissection of the epithelial component was followed by DNA extraction from the tissue fragments. Polymerase chain reaction and capillary electrophoresis analyses were performed for the following 5 National Cancer Institute-recommended microsatellites: D2s123, D5s346, D17s250, BAT25, and BAT26. Twelve patients with Warthin tumors were included. The immunostains for hMLH1 and hMSH2 showed preserved expression in the nuclei of the epithelial component of all Warthin tumors. No microsatellite instability was detected, and no loss of heterozygosity was seen. These results are not concordant with previously reported results showing loss of expression of the hMLH1 and hMSH2 DNA mismatch repair enzymes in the epithelial component of Warthin tumors. Furthermore, no microsatellite instability was detected in the 5 loci tested for each tumor in this series. These data demonstrate that Warthin tumors do not have evidence of DNA mismatch repair defects at the genomic or protein expression level.

Direct and indirect roles of RECQL4 in modulating base excision repair capacity

PubMed Central

Schurman, Shepherd H.; Hedayati, Mohammad; Wang, ZhengMing; Singh, Dharmendra K.; Speina, Elzbieta; Zhang, Yongqing; Becker, Kevin; Macris, Margaret; Sung, Patrick; Wilson, David M.; Croteau, Deborah L.; Bohr, Vilhelm A.

2009-01-01

RECQL4 is a human RecQ helicase which is mutated in approximately two-thirds of individuals with Rothmund–Thomson syndrome (RTS), a disease characterized at the cellular level by chromosomal instability. BLM and WRN are also human RecQ helicases, which are mutated in Bloom and Werner's syndrome, respectively, and associated with chromosomal instability as well as premature aging. Here we show that primary RTS and RECQL4 siRNA knockdown human fibroblasts accumulate more H2O2-induced DNA strand breaks than control cells, suggesting that RECQL4 may stimulate repair of H2O2-induced DNA damage. RTS primary fibroblasts also accumulate more XRCC1 foci than control cells in response to endogenous or induced oxidative stress and have a high basal level of endogenous formamidopyrimidines. In cells treated with H2O2, RECQL4 co-localizes with APE1, and FEN1, key participants in base excision repair. Biochemical experiments indicate that RECQL4 specifically stimulates the apurinic endonuclease activity of APE1, the DNA strand displacement activity of DNA polymerase β, and incision of a 1- or 10-nucleotide flap DNA substrate by Flap Endonuclease I. Additionally, RTS cells display an upregulation of BER pathway genes and fail to respond like normal cells to oxidative stress. The data herein support a model in which RECQL4 regulates both directly and indirectly base excision repair capacity. PMID:19567405

ERCC1 isoform expression and DNA repair in non-small-cell lung cancer.

PubMed

Friboulet, Luc; Olaussen, Ken André; Pignon, Jean-Pierre; Shepherd, Frances A; Tsao, Ming-Sound; Graziano, Stephen; Kratzke, Robert; Douillard, Jean-Yves; Seymour, Lesley; Pirker, Robert; Filipits, Martin; André, Fabrice; Solary, Eric; Ponsonnailles, Florence; Robin, Angélique; Stoclin, Annabelle; Dorvault, Nicolas; Commo, Frédéric; Adam, Julien; Vanhecke, Elsa; Saulnier, Patrick; Thomale, Jürgen; Le Chevalier, Thierry; Dunant, Ariane; Rousseau, Vanessa; Le Teuff, Gwénaël; Brambilla, Elisabeth; Soria, Jean-Charles

2013-03-21

The excision repair cross-complementation group 1 (ERCC1) protein is a potential prognostic biomarker of the efficacy of cisplatin-based chemotherapy in non-small-cell lung cancer (NSCLC). Although several ongoing trials are evaluating the level of expression of ERCC1, no consensus has been reached regarding a method for evaluation. We used the 8F1 antibody to measure the level of expression of ERCC1 protein by means of immunohistochemical analysis in a validation set of samples obtained from 494 patients in two independent phase 3 trials (the National Cancer Institute of Canada Clinical Trials Group JBR.10 and the Cancer and Leukemia Group B 9633 trial from the Lung Adjuvant Cisplatin Evaluation Biology project). We compared the results of repeated staining of the entire original set of samples obtained from 589 patients in the International Adjuvant Lung Cancer Trial Biology study, which had led to the initial correlation between the absence of ERCC1 expression and platinum response, with our previous results in the same tumors. We mapped the epitope recognized by 16 commercially available ERCC1 antibodies and investigated the capacity of the different ERCC1 isoforms to repair platinum-induced DNA damage. We were unable to validate the predictive effect of immunostaining for ERCC1 protein. The discordance in the results of staining for ERCC1 suggested a change in the performance of the 8F1 antibody since 2006. We found that none of the 16 antibodies could distinguish among the four ERCC1 protein isoforms, whereas only one isoform produced a protein that had full capacities for nucleotide excision repair and cisplatin resistance. Immunohistochemical analysis with the use of currently available ERCC1 antibodies did not specifically detect the unique functional ERCC1 isoform. As a result, its usefulness in guiding therapeutic decision making is limited. (Funded by Eli Lilly and others.).

Genetic polymorphisms of genes involved in DNA repair and metabolism influence micronucleus frequencies in human peripheral blood lymphocytes.

PubMed

Dhillon, Varinderpal S; Thomas, Philip; Iarmarcovai, G; Kirsch-Volders, Micheline; Bonassi, Stefano; Fenech, Michael

2011-01-01

The cytokinesis-block micronucleus cytome (CBMNCyt) assay is a widely used technique for measuring DNA damage in human populations. The formation of micronuclei (MN) in dividing cells can result from chromosome breakage due to unrepaired or mis-repaired DNA lesions or chromosome malsegregation due to mitotic malfunction. The sensitivity of the MN assay to polymorphisms in various genes involved in DNA repair, activation/deactivation of carcinogens/chemicals/drugs/alcohol, folate metabolism pathway and micronutrient transport has been extensively reported in the literature. MN frequency is also an important index for determining DNA repair efficiency phenotype (including mis-repair), response to environmental exposure and identifying various dietary factors required for optimal genome stability. The aim of the present study is to review the reported in vivo associations between genotype and MN frequency in humans taking into considerations the presence of interactions with nutrients levels and/or exposure to genotoxins. One hundred and eleven publications linking MN frequency in peripheral blood lymphocytes to gene polymorphism were retrieved from PubMed. After applying exclusion criteria, only 37 studies were evaluated in the present review. Polymorphisms in XRCC1 (Arg280His), ERCC2 (Lys751Gln), CYP2E1 (c1/c2) and MTR (A2756G) were consistently associated with the MN formation. These results contribute substantial evidence to the hypothesis that genotype may influence MN frequency in human cells.

Disruption of DNA repair in cancer cells by ubiquitination of a destabilising dimerization domain of nucleotide excision repair protein ERCC1

PubMed Central

Yang, Lanlan; Ritchie, Ann-Marie; Melton, David W.

2017-01-01

DNA repair pathways present in all cells serve to preserve genome stability, but in cancer cells they also act reduce the efficacy of chemotherapy. The endonuclease ERCC1-XPF has an important role in the repair of DNA damage caused by a variety of chemotherapeutic agents and there has been intense interest in the use of ERCC1 as a predictive marker of therapeutic response in non-small cell lung carcinoma, squamous cell carcinoma and ovarian cancer. We have previously validated ERCC1 as a therapeutic target in melanoma, but all small molecule ERCC1-XPF inhibitors reported to date have lacked sufficient potency and specificity for clinical use. In an alternative approach to prevent the repair activity of ERCC1-XPF, we investigated the mechanism of ERCC1 ubiquitination and found that the key region was the C-terminal (HhH)2 domain which heterodimerizes with XPF. This ERCC1 region was modified by non-conventional lysine-independent, but proteasome-dependent polyubiquitination, involving Lys33 of ubiquitin and a linear ubiquitin chain. XPF was not polyubiquitinated and its expression was dependent on presence of ERCC1, but not vice versa. To our surprise we found that ERCC1 can also homodimerize through its C-terminal (HhH)2 domain. We exploited the ability of a peptide containing this C-terminal domain to destabilise both endogenous ERCC1 and XPF in human melanoma cells and fibroblasts, resulting in reductions of up to 85% in nucleotide excision repair and near two-fold increased sensitivity to DNA damaging agents. We suggest that the ERCC1 (HhH)2 domain could be used in an alternative strategy to treat cancer. PMID:28903417

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

NASA Astrophysics Data System (ADS)

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.

A novel regulatory circuit in base excision repair involving AP endonuclease 1, Creb1 and DNA polymerase β

PubMed Central

Pei, De-Sheng; Yang, Xiao-Jie; Liu, Wei; Guikema, Jeroen E. J.; Schrader, Carol E.; Strauss, Phyllis R.

2011-01-01

DNA repair is required to maintain genome stability in stem cells and early embryos. At critical junctures, oxidative damage to DNA requires the base excision repair (BER) pathway. Since early zebrafish embryos lack the major polymerase in BER, DNA polymerase ß, repair proceeds via replicative polymerases, even though there is ample polb mRNA. Here, we report that Polb protein fails to appear at the appropriate time in development when AP endonuclease 1 (Apex), the upstream protein in BER, is knocked down. Because polb contains a Creb1 binding site, we examined whether knockdown of Apex affects creb1. Apex knockdown results in loss of Creb1 and Creb complex members but not Creb1 phosphorylation. This effect is independent of p53. Although both apex and creb1 mRNA rescue Creb1 and Polb after Apex knockdown, Apex is not a co-activator of creb1 transcription. This observation has broad significance, as similar results occur when Apex is inhibited in B cells from apex+/− mice. These results describe a novel regulatory circuit involving Apex, Creb1 and Polb and provide a mechanism for lethality of Apex loss in higher eukaryotes. PMID:21172930

Forkhead-Associated Domain of Yeast Xrs2, a Homolog of Human Nbs1, Promotes Nonhomologous End Joining Through Interaction With a Ligase IV Partner Protein, Lif1

PubMed Central

Matsuzaki, Kenichiro; Shinohara, Akira; Shinohara, Miki

2008-01-01

DNA double-strand breaks (DSB) are repaired through two different pathways, homologous recombination (HR) and nonhomologous end joining (NHEJ). Yeast Xrs2, a homolog of human Nbs1, is a component of the Mre11-Rad50-Xrs2 (MRX) complex required for both HR and NHEJ. Previous studies showed that the N-terminal forkhead-associated (FHA) domain of Xrs2/Nbs1 in yeast is not involved in HR, but is likely to be in NHEJ. In this study, we showed that the FHA domain of Xrs2 plays a critical role in efficient DSB repair by NHEJ. The FHA domain of Xrs2 specifically interacts with Lif1, a component of the ligase IV complex, Dnl4-Nej1-Lif1 (DNL). Lif1, which is phosphorylated in vivo, contains two Xrs2-binding regions. Serine 383 of Lif1 plays an important role in the interaction with Xrs2 as well as in NHEJ. Interestingly, the phospho-mimetic substitutions of serine 383 enhance the NHEJ activity of Lif1. Our results suggest that the phosphorylation of Lif1 at serine 383 is recognized by the Xrs2 FHA domain, which in turn may promote recruitment of the DNL complex to DSB for NHEJ. The interaction between Xrs2 and Lif1 through the FHA domain is conserved in humans; the FHA domain Nbs1 interacts with Xrcc4, a Lif1 homolog of human. PMID:18458108

The Nucleotide Excision Repair Pathway Limits L1 Retrotransposition

PubMed Central

Servant, Geraldine; Streva, Vincent A.; Derbes, Rebecca S.; Wijetunge, Madushani I.; Neeland, Marc; White, Travis B.; Belancio, Victoria P.; Roy-Engel, Astrid M.; Deininger, Prescott L.

2017-01-01

Long interspersed elements 1 (L1) are active mobile elements that constitute almost 17% of the human genome. They amplify through a “copy-and-paste” mechanism termed retrotransposition, and de novo insertions related to these elements have been reported to cause 0.2% of genetic diseases. Our previous data demonstrated that the endonuclease complex ERCC1-XPF, which cleaves a 3′ DNA flap structure, limits L1 retrotransposition. Although the ERCC1-XPF endonuclease participates in several different DNA repair pathways, such as single-strand annealing, or in telomere maintenance, its recruitment to DNA lesions is best characterized in the nucleotide excision repair (NER) pathway. To determine if the NER pathway prevents the insertion of retroelements in the genome, we monitored the retrotransposition efficiencies of engineered L1 elements in NER-deficient cells and in their complemented versions. Core proteins of the NER pathway, XPD and XPA, and the lesion binding protein, XPC, are involved in limiting L1 retrotransposition. In addition, sequence analysis of recovered de novo L1 inserts and their genomic locations in NER-deficient cells demonstrated the presence of abnormally large duplications at the site of insertion, suggesting that NER proteins may also play a role in the normal L1 insertion process. Here, we propose new functions for the NER pathway in the maintenance of genome integrity: limitation of insertional mutations caused by retrotransposons and the prevention of potentially mutagenic large genomic duplications at the site of retrotransposon insertion events. PMID:28049704

Dysfunction of methionine sulfoxide reductases to repair damaged proteins by nickel nanoparticles.

PubMed

Feng, Po-Hao; Huang, Ya-Li; Chuang, Kai-Jen; Chen, Kuan-Yuan; Lee, Kang-Yun; Ho, Shu-Chuan; Bien, Mauo-Ying; Yang, You-Lan; Chuang, Hsiao-Chi

2015-07-05

Protein oxidation is considered to be one of the main causes of cell death, and methionine is one of the primary targets of reactive oxygen species (ROS). However, the mechanisms by which nickel nanoparticles (NiNPs) cause oxidative damage to proteins remain unclear. The objective of this study is to investigate the effects of NiNPs on the methionine sulfoxide reductases (MSR) protein repairing system. Two physically similar nickel-based nanoparticles, NiNPs and carbon-coated NiNP (C-NiNPs; control particles), were exposed to human epithelial A549 cells. Cell viability, benzo(a)pyrene diolepoxide (BPDE) protein adducts, methionine oxidation, MSRA and B3, microtubule-associated protein 1A/1B-light chain 3 (LC3) and extracellular signal-regulated kinase (ERK) phosphorylation were investigated. Exposure to NiNPs led to a dose-dependent reduction in cell viability and increased BPDE protein adduct production and methionine oxidation. The methionine repairing enzymatic MSRA and MSRB3 production were suppressed in response to NiNP exposure, suggesting the oxidation of methionine to MetO by NiNP was not reversed back to methionine. Additionally, LC3, an autophagy marker, was down-regulated by NiNPs. Both NiNP and C-NiNP caused ERK phosphorylation. LC3 was positively correlated with MSRA (r = 0.929, p < 0.05) and MSRB3 (r = 0.893, p < 0.05). MSR was made aberrant by NiNP, which could lead to the dysfunction of autophagy and ERK phosphorylation. The toxicological consequences may be dependent on the chemical characteristics of the nanoparticles. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Genetic variation in the base excision repair pathway and bladder cancer risk.

PubMed

Figueroa, Jonine D; Malats, Núria; Real, Francisco X; Silverman, Debra; Kogevinas, Manolis; Chanock, Stephen; Welch, Robert; Dosemeci, Mustafa; Tardón, Adonina; Serra, Consol; Carrato, Alfredo; García-Closas, Reina; Castaño-Vinyals, Gemma; Rothman, Nathaniel; García-Closas, Montserrat

2007-04-01

Genetic polymorphisms in DNA repair genes may impact individual variation in DNA repair capacity and alter cancer risk. In order to examine the association of common genetic variation in the base-excision repair (BER) pathway with bladder cancer risk, we analyzed 43 single nucleotide polymorphisms (SNPs) in 12 BER genes (OGG1, MUTYH, APEX1, PARP1, PARP3, PARP4, XRCC1, POLB, POLD1, PCNA, LIG1, and LIG3). Using genotype data from 1,150 cases of urinary bladder transitional cell carcinomas and 1,149 controls from the Spanish Bladder Cancer Study we estimated odds ratios (ORs) and 95% confidence intervals (CIs) adjusting for age, gender, region and smoking status. SNPs in three genes showed significant associations with bladder cancer risk: the 8-oxoG DNA glycosylase gene (OGG1), the Poly (ADP-ribose) polymerase family member 1 (PARP1) and the major gap filling polymerase-beta (POLB). Subjects who were heterozygous or homozygous variant for an OGG1 SNP in the promoter region (rs125701) had significantly decreased bladder cancer risk compared to common homozygous: OR (95%CI) 0.78 (0.63-0.96). Heterozygous or homozygous individuals for the functional SNP PARP1 rs1136410 (V762A) or for the intronic SNP POLB rs3136717 were at increased risk compared to those homozygous for the common alleles: 1.24 (1.02-1.51) and 1.30 (1.04-1.62), respectively. In summary, data from this large case-control study suggested bladder cancer risk associations with selected BER SNPs, which need to be confirmed in other study populations.

Reduced transforming growth factor β1 (TGF-β1) in the repair of airway epithelial cells of children with asthma.

PubMed

Ling, Kak-Ming; Sutanto, Erika N; Iosifidis, Thomas; Kicic-Starcevich, Elizabeth; Looi, Kevin; Garratt, Luke W; Martinovich, Kelly M; Lannigan, Francis J; Knight, Darryl A; Stick, Stephen M; Kicic, Anthony

2016-10-01

Evidence into the role of TGF-β1 in airway epithelial repair in asthma is still controversial. This study tested the hypothesis that the reduced TGF-β1 levels previously observed in paediatric asthmatic airway epithelial cells directly contribute to the dysregulated repair seen in these cells. Primary airway epithelial cells (pAEC) from children with asthma (n = 16) and non-asthmatic subjects (n = 20) were isolated, and subcultured for investigation of TGF-β1 gene and protein via quantitative polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Expression of other associated genes such as integrins αvβ6, αvβ8 and MT1-MMP were also tested. Small interfering RNA (siRNA) was employed to assess the role of TGF-β1 during wound repair. TGF-β1 gene and protein expression were significantly downregulated in asthmatic pAEC over the course of repair, compared with cells from non-asthmatic children. Messenger RNA (mRNA) expression of TGF-β1 was also directly implicated in non-asthmatic and asthmatic pAEC proliferation over their quiescent counterparts. Small interfering RNA-mediated knockdown of TGF-β1 compromised repair in non-asthmatic pAEC and exacerbated the dysregulated repair seen in asthmatic pAEC. Expression of major TGF-β1 activators of epithelial cells, integrin αvβ6 and αvβ8 was also measured and there was no difference in αvβ6 gene expression between the two cohorts. Although integrin αvβ8 gene expression was significantly higher in asthmatic pAEC, the expression of MT1-MMP (MMP14) which facilitates the αvβ8 mediated TGF-β1 activation was significantly downregulated. Our data has highlighted the importance of TGF-β1 in pAEC wound repair in vitro. The significantly lower levels seen in asthmatic pAEC subsequently contributes to the dysregulated repair observed in these cells. © 2016 Asian Pacific Society of Respirology.

XRCC2 Polymorphisms and Environmental Factors Predict High Risk of Colorectal Cancer.

PubMed

Wang, Lijie; Ma, Junxun; Yang, Bo; Jing, Fangfang; Hu, Yi

2018-05-07

BACKGROUND This case-control study aimed to analyze the association of [i]XRCC2[/i] polymorphisms (rs3218408 and rs3218384) with colorectal cancer (CRC) risk. The interaction of [i]XRCC2[/i] polymorphisms with environmental factors was investigated as well. MATERIAL AND METHODS We enrolled 147 CRC patients and 114 healthy individuals into the study. Polymerase chain reaction (PCR)-sequencing method was performed to detect rs3218408 and rs3218384 polymorphisms. Hardy-Weinberg equilibrium (HWE) was checked in the control group. Odds ratio (OR) with 95% confidence interval (CI) represented the risk of CRC. Cross-table method was used in analyzing the interaction effects. RESULTS Compared to the control group, the frequency of smokers was much higher in the case group ([i]P[/i]<0.001). A similar result was observed in drinkers (55.8% [i]vs.[/i] 40.4%, [i]P[/i]=0.013). Dietary habits of all subjects were investigated as well, showing that CRC patients ate fewer vegetables than did healthy controls (P<0.001). In the analysis of polymorphisms, rs3218408 appeared to be an independent risk factor of CRC (GG: OR=2.048, 95%CI=1.032-4.061; G allele: OR=1.445, 95%CI=1.019-2.049). There were 68 (76.4%) C allele carriers (rs3218384) among smokers, which was higher than the number of G allele carriers ([i]P[/i]<0.001). A similar outcome was observed for alcohol drinkers ([i]P[/i]=0.048), which suggests a relationship of rs3218384 with smoking and drinking. Further analysis indicated that interaction of rs3218384 with smoking increased the risk of CRC (GG and smoking: OR=3.250, 95%CI=1.235-8.556; GC+CC and smoking: OR=2.167, 95%CI=1.175-3.996). CONCLUSIONS We found that rs3218408 was related with increased risk of CRC, and the interaction of rs3218384 with smoking increased the risk of CRC.

Characterization of the human SNM1A and SNM1B/Apollo DNA repair exonucleases.

PubMed

Sengerová, Blanka; Allerston, Charles K; Abu, Mika; Lee, Sook Y; Hartley, Janet; Kiakos, Konstantinos; Schofield, Christopher J; Hartley, John A; Gileadi, Opher; McHugh, Peter J

2012-07-27

Human SNM1A and SNM1B/Apollo have both been implicated in the repair of DNA interstrand cross-links (ICLs) by cellular studies, and SNM1B is also required for telomere protection. Here, we describe studies on the biochemical characterization of the SNM1A and SNM1B proteins. The results reveal some fundamental differences in the mechanisms of the two proteins. Both SNM1A and SNM1B digest double-stranded and single-stranded DNA with a 5'-to-3' directionality in a reaction that is stimulated by divalent cations, and both nucleases are inhibited by the zinc chelator o-phenanthroline. We find that SNM1A has greater affinity for single-stranded DNA over double-stranded DNA that is not observed with SNM1B. Although both proteins demonstrate a low level of processivity on low molecular weight DNA oligonucleotide substrates, when presented with high molecular weight DNA, SNM1A alone is rendered much more active, being capable of digesting kilobase-long stretches of DNA. Both proteins can digest past ICLs induced by the non-distorting minor groove cross-linking agent SJG-136, albeit with SNM1A showing a greater capacity to achieve this. This is consistent with the proposal that SNM1A and SNM1B might exhibit some redundancy in ICL repair. Together, our work establishes differences in the substrate selectivities of SNM1A and SNM1B that are likely to be relevant to their in vivo roles and which might be exploited in the development of selective inhibitors.

Methods to alter levels of a DNA repair protein

DOEpatents

Petrini, John H.; Morgan, William Francis; Maser, Richard Scott; Carney, James Patrick

2006-10-17

An isolated and purified DNA molecule encoding a DNA repair protein, p95, is provided, as is isolated and purified p95. Also provided are methods of detecting p95 and DNA encoding p95. The invention further provides p95 knock-out mice.

Laser-activated solid protein bands for peripheral nerve repair: an vivo study.

PubMed

Lauto, A; Trickett, R; Malik, R; Dawes, J M; Owen, E R

1997-01-01

Severed tibial nerves in rats were repaired using a novel technique, utilizing a semiconductor diode-laser-activated protein solder applied longitudinally across the join. Welding was produced by selective laser denaturation of solid solder bands containing the dye indocyanine green. An in vivo study, using 48 adult male Wistar rats, compared conventional microsuture-repaired tibial nerves with laser solder-repaired nerves. Nerve repairs were characterised immediately after surgery and after 3 months. Successful regeneration with average compound muscle action potentials of 2.5 +/- 0.5 mV and 2.7 +/- 0.3 mV (mean and standard deviation) was demonstrated for the laser-soldered nerves and the sutured nerves, respectively. Histopathology confirmed comparable regeneration of axons in laser- and suture-operated nerves. The laser-based nerve repair technique was easier and faster than microsuture repair, minimising manipulation damage to the nerve.

Poly(ADP-ribose) Contributes to an Association between Poly(ADP-ribose) Polymerase-1 and Xeroderma Pigmentosum Complementation Group A in Nucleotide Excision Repair*

PubMed Central

King, Brenee S.; Cooper, Karen L.; Liu, Ke Jian; Hudson, Laurie G.

2012-01-01

Exposure to ultraviolet radiation (UVR) promotes the formation of UVR-induced, DNA helix distorting photolesions such as (6-4) pyrimidine-pyrimidone photoproducts and cyclobutane pyrimidine dimers. Effective repair of such lesions by the nucleotide excision repair (NER) pathway is required to prevent DNA mutations and chromosome aberrations. Poly(ADP-ribose) polymerase-1 (PARP-1) is a zinc finger protein with well documented involvement in base excision repair. PARP-1 is activated in response to DNA damage and catalyzes the formation of poly(ADP-ribose) subunits that assist in the assembly of DNA repair proteins at sites of damage. In this study, we present evidence for PARP-1 contributions to NER, extending the knowledge of PARP-1 function in DNA repair beyond the established role in base excision repair. Silencing the PARP-1 protein or inhibiting PARP activity leads to retention of UVR-induced photolesions. PARP activation following UVR exposure promotes association between PARP-1 and XPA, a central protein in NER. Administration of PARP inhibitors confirms that poly(ADP-ribose) facilitates PARP-1 association with XPA in whole cell extracts, in isolated chromatin complexes, and in vitro. Furthermore, inhibition of PARP activity decreases UVR-stimulated XPA chromatin association, illustrating that these relationships occur in a meaningful context for NER. These results provide a mechanistic link for PARP activity in the repair of UVR-induced photoproducts. PMID:23038248

MOF phosphorylation by ATM regulates 53BP1-mediated DSB repair pathway choice

PubMed Central

Gupta, Arun; Hunt, Clayton R.; Hegdec, Muralidhar L.; Chakraborty, Sharmistha; Udayakumar, Durga; Horikoshi, Nobuo; Singh1, Mayank; Ramnarain, Deepti B.; Hittelman, Walter N.; Namjoshi, Sarita; Asaithamby, Aroumougame; Hazra, Tapas K.; Ludwig, Thomas; Pandita, Raj K.; Tyler, Jessica K.; Pandita, Tej K.

2014-01-01

Cell cycle phase is a critical determinant of the choice between DNA damage repair by non-homologous end joining (NHEJ) or homologous recombination (HR). Here we report that DSBs induce ATM-dependent MOF (a histone H4 acetyl-transferase) phosphorylation (p-T392-MOF) and that phosphorylated MOF co-localizes with γ-H2AX, ATM, and 53BP1 foci. Mutation of the phosphorylation site (MOF-T392A) impedes DNA repair in S- and G2-phase but not G1-phase cells. Expression of MOF-T392A also reverses the reduction in DSB associated 53BP1 seen in wild type S/G2-phase cells, resulting in enhanced 53BP1 and reduced BRCA1 association. Decreased BRCA1 levels at DSB sites correlates with defective repairosome formation, reduced HR repair and decreased cell survival following irradiation. These data support a model whereby ATM mediated MOF-T392 phosphorylation modulates 53BP1 function to facilitate the subsequent recruitment of HR repair proteins, uncovering a regulatory role for MOF in DSB repair pathway choice during S/G2-phase. PMID:24953651

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

NASA Technical Reports Server (NTRS)

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.

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

PubMed

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

2012-05-25

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

Cellular and molecular consequences of defective Fanconi anemia proteins in replication-coupled DNA repair: mechanistic insights

PubMed Central

Thompson, Larry H.; Hinz, John M.

2009-01-01

The Fanconi anemia (FA) molecular network consists of 15 “FANC” proteins, of which 13 are associated with mutations in patients with this cancer-prone chromosome instability disorder. Whereas historically the common phenotype associated with FA mutations is marked sensitivity to DNA interstrand crosslinking agents, the literature supports a more global role for FANC proteins in coping with diverse stresses encountered by replicative polymerases. We have attempted to reconcile and integrate numerous observations into a model in which FANC proteins coordinate the following physiological events during DNA crosslink repair: (a) activating a FANCM-ATR-dependent S-phase checkpoint; (b) mediating enzymatic replication-fork breakage and crosslink unhooking; (c) filling the resulting gap by translesion synthesis (TLS) by error-prone polymerase(s); and (d) restoring the resulting one-ended double-strand break by homologous recombination repair (HRR). The FANC core subcomplex (FANCA, B, C, E, F, G, L, FAAP100) promotes TLS for both crosslink and non-crosslink damage such as spontaneous oxidative base damage, UV-C photoproducts, and alkylated bases. TLS likely helps prevent stalled replication forks from breaking, thereby maintaining chromosome continuity. Diverse DNA damages and replication inhibitors result in monoubiquitination of the FANCD2-FANCI complex by the FANCL ubiquitin ligase activity of the core subcomplex upon its recruitment to chromatin by the FANCM-FAAP24 heterodimeric translocase. We speculate that this translocase activity acts as the primary damage sensor and helps remodel blocked replication forks to facilitate checkpoint activation and repair. Monoubiquitination of FANCD2-FANCI is needed for promoting HRR, in which the FANCD1/BRCA2 and FANCN/PALB2 proteins act at an early step. We conclude that the core subcomplex is required for both TLS and HRR occurring separately for non-crosslink damages and for both events during crosslink repair. The FANCJ

DNA repair factor XPC is modified by SUMO-1 and ubiquitin following UV irradiation

PubMed Central

Wang, Qi-En; Zhu, Qianzheng; Wani, Gulzar; El-Mahdy, Mohamed A.; Li, Jinyou; Wani, Altaf A.

2005-01-01

Nucleotide excision repair (NER) is the major DNA repair process that removes diverse DNA lesions including UV-induced photoproducts. There are more than 20 proteins involved in NER. Among them, XPC is thought to be one of the first proteins to recognize DNA damage during global genomic repair (GGR), a sub-pathway of NER. In order to study the mechanism through which XPC participates in GGR, we investigated the possible modifications of XPC protein upon UV irradiation in mammalian cells. Western blot analysis of cell lysates from UV-irradiated normal human fibroblast, prepared by direct boiling in an SDS lysis buffer, showed several anti-XPC antibody-reactive bands with molecular weight higher than the original XPC protein. The reciprocal immunoprecipitation and siRNA transfection analysis demonstrated that XPC protein is modified by SUMO-1 and ubiquitin. By using several NER-deficient cell lines, we found that DDB2 and XPA are required for UV-induced XPC modifications. Interestingly, both the inactivation of ubiquitylation and the treatment of proteasome inhibitors quantitatively inhibited the UV-induced XPC modifications. Furthermore, XPC protein is degraded significantly following UV irradiation in XP-A cells in which sumoylation of XPC does not occur. Taken together, we conclude that XPC protein is modified by SUMO-1 and ubiquitin following UV irradiation and these modifications require the functions of DDB2 and XPA, as well as the ubiquitin–proteasome system. Our results also suggest that at least one function of UV-induced XPC sumoylation is related to the stabilization of XPC protein. PMID:16030353

E2F1 interactions with hHR23A inhibit its degradation and promote DNA repair.

PubMed

Singh, Randeep K; Dagnino, Lina

2016-05-03

Nucleotide excision repair (NER) is a major mechanism for removal of DNA lesions induced by exposure to UV radiation in the epidermis. Recognition of damaged DNA sites is the initial step in their repair, and requires multiprotein complexes that contain XPC and hHR23 proteins, or their orthologues. A variety of transcription factors are also involved in NER, including E2F1. In epidermal keratinocytes, UV exposure induces E2F1 phosphorylation, which allows it to recruit various NER factors to sites of DNA damage. However, the relationship between E2F1 and hHR23 proteins vis-à-vis NER has remained unexplored. We now show that E2F1 and hHR23 proteins can interact, and this interaction stabilizes E2F1, inhibiting its proteasomal degradation. Reciprocally, E2F1 regulates hHR23A subcellular localization, recruiting it to sites of DNA photodamage. As a result, E2F1 and hHR23A enhance DNA repair following exposure to UV radiation, contributing to genomic stability in the epidermis.

E2F1 interactions with hHR23A inhibit its degradation and promote DNA repair

PubMed Central

Singh, Randeep K.; Dagnino, Lina

2016-01-01

Nucleotide excision repair (NER) is a major mechanism for removal of DNA lesions induced by exposure to UV radiation in the epidermis. Recognition of damaged DNA sites is the initial step in their repair, and requires multiprotein complexes that contain XPC and hHR23 proteins, or their orthologues. A variety of transcription factors are also involved in NER, including E2F1. In epidermal keratinocytes, UV exposure induces E2F1 phosphorylation, which allows it to recruit various NER factors to sites of DNA damage. However, the relationship between E2F1 and hHR23 proteins vis-à-vis NER has remained unexplored. We now show that E2F1 and hHR23 proteins can interact, and this interaction stabilizes E2F1, inhibiting its proteasomal degradation. Reciprocally, E2F1 regulates hHR23A subcellular localization, recruiting it to sites of DNA photodamage. As a result, E2F1 and hHR23A enhance DNA repair following exposure to UV radiation, contributing to genomic stability in the epidermis. PMID:27028861

Laser-activated protein bands for peripheral nerve repair

NASA Astrophysics Data System (ADS)

Lauto, Antonio; Trickett, Rodney I.; Malik, Richard; Dawes, Judith M.; Owen, Earl R.

1996-01-01

A 100 micrometer core optical fiber-coupled 75 mW diode laser operating at a wavelength of 800 nm has been used in conjunction with a protein solder to stripe weld severed rat tibial nerves, reducing the long operating time required for microsurgical nerve repair. Welding is produced by selective laser denaturation of the protein based solder which contains the dye indocyanine green. Operating time for laser soldering was 10 plus or minus 5 min. (n equals 24) compared to 23 plus or minus 9 min (n equals 13) for microsuturing. The laser solder technique resulted in patent welds with a tensile strength of 15 plus or minus 5 g, while microsutured nerves had a tensile strength of 40 plus or minus 10 g. Histopathology of the laser soldered nerves, conducted immediately after surgery, displayed solder adhesion to the outer membrane with minimal damage to the inner axons of the nerves. An in vivo study, with a total of fifty-seven adult male wistar rats, compared laser solder repaired tibial nerves to conventional microsuture repair. Twenty-four laser soldered nerves and thirteen sutured nerves were characterized at three months and showed successful regeneration with average compound muscle action potentials (CMAP) of 2.4 plus or minus 0.7 mV and 2.7 plus or minus 0.8 mV respectively. Histopathology of the in vivo study, confirmed the comparable regeneration of axons in laser and suture operated nerves. A faster, less damaging and long lasting laser based anastomotic technique is presented.

Excess single-stranded DNA inhibits meiotic double-strand break repair.

PubMed

Johnson, Rebecca; Borde, Valérie; Neale, Matthew J; Bishop-Bailey, Anna; North, Matthew; Harris, Sheila; Nicolas, Alain; Goldman, Alastair S H

2007-11-01

During meiosis, self-inflicted DNA double-strand breaks (DSBs) are created by the protein Spo11 and repaired by homologous recombination leading to gene conversions and crossovers. Crossover formation is vital for the segregation of homologous chromosomes during the first meiotic division and requires the RecA orthologue, Dmc1. We analyzed repair during meiosis of site-specific DSBs created by another nuclease, VMA1-derived endonuclease (VDE), in cells lacking Dmc1 strand-exchange protein. Turnover and resection of the VDE-DSBs was assessed in two different reporter cassettes that can repair using flanking direct repeat sequences, thereby obviating the need for a Dmc1-dependent DNA strand invasion step. Access of the single-strand binding complex replication protein A, which is normally used in all modes of DSB repair, was checked in chromatin immunoprecipitation experiments, using antibody against Rfa1. Repair of the VDE-DSBs was severely inhibited in dmc1Delta cells, a defect that was associated with a reduction in the long tract resection required to initiate single-strand annealing between the flanking repeat sequences. Mutants that either reduce Spo11-DSB formation or abolish resection at Spo11-DSBs rescued the repair block. We also found that a replication protein A component, Rfa1, does not accumulate to expected levels at unrepaired single-stranded DNA (ssDNA) in dmc1Delta cells. The requirement of Dmc1 for VDE-DSB repair using flanking repeats appears to be caused by the accumulation of large quantities of ssDNA that accumulate at Spo11-DSBs when Dmc1 is absent. We propose that these resected DSBs sequester both resection machinery and ssDNA binding proteins, which in wild-type cells would normally be recycled as Spo11-DSBs repair. The implication is that repair proteins are in limited supply, and this could reflect an underlying mechanism for regulating DSB repair in wild-type cells, providing protection from potentially harmful effects of overabundant

Excess Single-Stranded DNA Inhibits Meiotic Double-Strand Break Repair

PubMed Central

Bishop-Bailey, Anna; North, Matthew; Harris, Sheila; Nicolas, Alain; Goldman, Alastair S. H

2007-01-01

During meiosis, self-inflicted DNA double-strand breaks (DSBs) are created by the protein Spo11 and repaired by homologous recombination leading to gene conversions and crossovers. Crossover formation is vital for the segregation of homologous chromosomes during the first meiotic division and requires the RecA orthologue, Dmc1.We analyzed repair during meiosis of site-specific DSBs created by another nuclease, VMA1-derived endonuclease (VDE), in cells lacking Dmc1 strand-exchange protein. Turnover and resection of the VDE-DSBs was assessed in two different reporter cassettes that can repair using flanking direct repeat sequences, thereby obviating the need for a Dmc1-dependent DNA strand invasion step. Access of the single-strand binding complex replication protein A, which is normally used in all modes of DSB repair, was checked in chromatin immunoprecipitation experiments, using antibody against Rfa1. Repair of the VDE-DSBs was severely inhibited in dmc1Δ cells, a defect that was associated with a reduction in the long tract resection required to initiate single-strand annealing between the flanking repeat sequences. Mutants that either reduce Spo11-DSB formation or abolish resection at Spo11-DSBs rescued the repair block. We also found that a replication protein A component, Rfa1, does not accumulate to expected levels at unrepaired single-stranded DNA (ssDNA) in dmc1Δ cells. The requirement of Dmc1 for VDE-DSB repair using flanking repeats appears to be caused by the accumulation of large quantities of ssDNA that accumulate at Spo11-DSBs when Dmc1 is absent. We propose that these resected DSBs sequester both resection machinery and ssDNA binding proteins, which in wild-type cells would normally be recycled as Spo11-DSBs repair. The implication is that repair proteins are in limited supply, and this could reflect an underlying mechanism for regulating DSB repair in wild-type cells, providing protection from potentially harmful effects of overabundant repair

Alpha-phellandrene-induced DNA damage and affect DNA repair protein expression in WEHI-3 murine leukemia cells in vitro.

PubMed

Lin, Jen-Jyh; Wu, Chih-Chung; Hsu, Shu-Chun; Weng, Shu-Wen; Ma, Yi-Shih; Huang, Yi-Ping; Lin, Jaung-Geng; Chung, Jing-Gung

2015-11-01

Although there are few reports regarding α-phellandrene (α-PA), a natural compound from Schinus molle L. essential oil, there is no report to show that α-PA induced DNA damage and affected DNA repair associated protein expression. Herein, we investigated the effects of α-PA on DNA damage and repair associated protein expression in murine leukemia cells. Flow cytometric assay was used to measure the effects of α-PA on total cell viability and the results indicated that α-PA induced cell death. Comet assay and 4,6-diamidino-2-phenylindole dihydrochloride staining were used for measuring DNA damage and condensation, respectively, and the results indicated that α-PA induced DNA damage and condensation in a concentration-dependent manner. DNA gel electrophoresis was used to examine the DNA damage and the results showed that α-PA induced DNA damage in WEHI-3 cells. Western blotting assay was used to measure the changes of DNA damage and repair associated protein expression and the results indicated that α-PA increased p-p53, p-H2A.X, 14-3-3-σ, and MDC1 protein expression but inhibited the protein of p53, MGMT, DNA-PK, and BRCA-1. © 2014 Wiley Periodicals, Inc.

A mutation in EXO1 defines separable roles in DNA mismatch repair and post-replication repair

PubMed Central

Tran, Phuoc T.; Fey, Julien P.; Erdeniz, Naz; Gellon, Lionel; Boiteux, Serge; Liskay, R. Michael

2007-01-01

Replication forks stall at DNA lesions or as a result of an unfavorable replicative environment. These fork stalling events have been associated with recombination and gross chromosomal rearrangements. Recombination and fork bypass pathways are the mechanisms accountable for restart of stalled forks. An important lesion bypass mechanism is the highly conserved post-replication repair (PRR) pathway that is composed of error-prone translesion and error-free bypass branches. EXO1 codes for a Rad2p family member nuclease that has been implicated in a multitude of eukaryotic DNA metabolic pathways that include DNA repair, recombination, replication, and telomere integrity. In this report, we show EXO1 functions in the MMS2 error-free branch of the PRR pathway independent of the role of EXO1 in DNA mismatch repair (MMR). Consistent with the idea that EXO1 functions independently in two separate pathways, we defined a domain of Exo1p required for PRR distinct from those required for interaction with MMR proteins. We then generated a point mutant exo1 allele that was defective for the function of Exo1p in MMR due to disrupted interaction with Mlh1p, but still functional for PRR. Lastly, by using a compound exo1 mutant that was defective for interaction with Mlh1p and deficient for nuclease activity, we provide further evidence that Exo1p plays both structural and catalytic roles during MMR. PMID:17602897

Characterization of the Human SNM1A and SNM1B/Apollo DNA Repair Exonucleases*

PubMed Central

Sengerová, Blanka; Allerston, Charles K.; Abu, Mika; Lee, Sook Y.; Hartley, Janet; Kiakos, Konstantinos; Schofield, Christopher J.; Hartley, John A.; Gileadi, Opher; McHugh, Peter J.

2012-01-01

Human SNM1A and SNM1B/Apollo have both been implicated in the repair of DNA interstrand cross-links (ICLs) by cellular studies, and SNM1B is also required for telomere protection. Here, we describe studies on the biochemical characterization of the SNM1A and SNM1B proteins. The results reveal some fundamental differences in the mechanisms of the two proteins. Both SNM1A and SNM1B digest double-stranded and single-stranded DNA with a 5′-to-3′ directionality in a reaction that is stimulated by divalent cations, and both nucleases are inhibited by the zinc chelator o-phenanthroline. We find that SNM1A has greater affinity for single-stranded DNA over double-stranded DNA that is not observed with SNM1B. Although both proteins demonstrate a low level of processivity on low molecular weight DNA oligonucleotide substrates, when presented with high molecular weight DNA, SNM1A alone is rendered much more active, being capable of digesting kilobase-long stretches of DNA. Both proteins can digest past ICLs induced by the non-distorting minor groove cross-linking agent SJG-136, albeit with SNM1A showing a greater capacity to achieve this. This is consistent with the proposal that SNM1A and SNM1B might exhibit some redundancy in ICL repair. Together, our work establishes differences in the substrate selectivities of SNM1A and SNM1B that are likely to be relevant to their in vivo roles and which might be exploited in the development of selective inhibitors. PMID:22692201

Structure of the human DNA-repair protein RAD52 containing surface mutations.

PubMed

Saotome, Mika; Saito, Kengo; Onodera, Keiichi; Kurumizaka, Hitoshi; Kagawa, Wataru

2016-08-01

The Rad52 protein is a eukaryotic single-strand DNA-annealing protein that is involved in the homologous recombinational repair of DNA double-strand breaks. The isolated N-terminal half of the human RAD52 protein (RAD52(1-212)) forms an undecameric ring structure with a surface that is mostly positively charged. In the present study, it was found that RAD52(1-212) containing alanine mutations of the charged surface residues (Lys102, Lys133 and Glu202) is highly amenable to crystallization. The structure of the mutant RAD52(1-212) was solved at 2.4 Å resolution. The structure revealed an association between the symmetry-related RAD52(1-212) rings, in which a partially unfolded, C-terminal region of RAD52 extended into the DNA-binding groove of the neighbouring ring in the crystal. The alanine mutations probably reduced the surface entropy of the RAD52(1-212) ring and stabilized the ring-ring association observed in the crystal.

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

PubMed

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.

Cell micro-irradiation with MeV protons counted by an ultra-thin diamond membrane

NASA Astrophysics Data System (ADS)

Barberet, Philippe; Pomorski, Michal; Muggiolu, Giovanna; Torfeh, Eva; Claverie, Gérard; Huss, Cédric; Saada, Samuel; Devès, Guillaume; Simon, Marina; Seznec, Hervé

2017-12-01

We report the development of thin single crystal diamond membranes suitable for dose control in targeted cell irradiation experiments with a proton microbeam. A specific design was achieved to deliver single protons with a hit detection efficiency approaching 100%. The membranes have thicknesses between 1.8 and 3 μm and are used as vacuum windows on the microbeam line. The impact of these transmission detectors on the microbeam spot size is estimated by Monte-Carlo simulations, indicating that a beam lateral resolution below 2 μm is achieved. This is confirmed by experiments showing the accumulation online of X-ray Repair Cross-Complementing protein 1 (XRCC1)-Green Fluorescent Protein (GFP) at DNA damaged sites in living cells.

Mdt1 Facilitates Efficient Repair of Blocked DNA Double-Strand Breaks and Recombinational Maintenance of Telomeres▿

PubMed Central

Pike, Brietta L.; Heierhorst, Jörg

2007-01-01

DNA recombination plays critical roles in DNA repair and alternative telomere maintenance. Here we show that absence of the SQ/TQ cluster domain-containing protein Mdt1 (Ybl051c) renders Saccharomyces cerevisiae particularly hypersensitive to bleomycin, a drug that causes 3′-phospho-glycolate-blocked DNA double-strand breaks (DSBs). mdt1Δ also hypersensitizes partially recombination-defective cells to camptothecin-induced 3′-phospho-tyrosyl protein-blocked DSBs. Remarkably, whereas mdt1Δ cells are unable to restore broken chromosomes after bleomycin treatment, they efficiently repair “clean” endonuclease-generated DSBs. Epistasis analyses indicate that MDT1 acts in the repair of bleomycin-induced DSBs by regulating the efficiency of the homologous recombination pathway as well as telomere-related functions of the KU complex. Moreover, mdt1Δ leads to severe synthetic growth defects with a deletion of the recombination facilitator and telomere-positioning factor gene CTF18 already in the absence of exogenous DNA damage. Importantly, mdt1Δ causes a dramatic shift from the usually prevalent type II to the less-efficient type I pathway of recombinational telomere maintenance in the absence of telomerase in liquid senescence assays. As telomeres resemble protein-blocked DSBs, the results indicate that Mdt1 acts in a novel blocked-end-specific recombination pathway that is required for the efficiency of both drug-induced DSB repair and telomerase-independent telomere maintenance. PMID:17636027

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

PubMed

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

1998-11-20

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

Contributions of recombination and repair proteins to telomere maintenance in telomerase-positive and negative Ustilago maydis.

PubMed

Yu, Eun Young; Hsu, Min; Holloman, William K; Lue, Neal F

2018-01-01

Homologous recombination and repair factors are known to promote both telomere replication and recombination-based telomere extension. Herein, we address the diverse contributions of several recombination/repair proteins to telomere maintenance in Ustilago maydis, a fungus that bears strong resemblance to mammals with respect to telomere regulation and recombination mechanisms. In telomerase-positive U. maydis, deletion of rad51 and blm separately caused shortened but stably maintained telomeres, whereas deletion of both engendered similar telomere loss, suggesting that the repair proteins help to resolve similar problems in telomere replication. In telomerase-negative cells, the loss of Rad51 or Brh2 caused accelerated senescence and failure to generate survivors on semi-solid medium. However, slow growing survivors can be isolated through continuous liquid culturing, and these survivors exhibit type II-like as well as ALT-like telomere features. In contrast, the trt1Δ blmΔ double mutant gives rise to survivors as readily as the trt1Δ single mutant, and like the single mutant survivors, exhibit almost exclusively type I-like telomere features. In addition, we observed direct physical interactions between Blm and two telomere-binding proteins, which may thus recruit or regulate Blm at telomeres. Our findings provide the basis for further analyzing the interplays between telomerase, telomere replication, and telomere recombination. © 2017 John Wiley & Sons Ltd.

ERCC1 function in nuclear excision and interstrand crosslink repair pathways is mediated exclusively by the ERCC1-202 isoform

PubMed Central

Friboulet, Luc; Postel-Vinay, Sophie; Sourisseau, Tony; Adam, Julien; Stoclin, Annabelle; Ponsonnailles, Florence; Dorvault, Nicolas; Commo, Frédéric; Saulnier, Patrick; Salome-Desmoulez, Sophie; Pottier, Géraldine; André, Fabrice; Kroemer, Guido; Soria, Jean Charles; Olaussen, Ken André

2013-01-01

ERCC1 (excision repair cross-complementation group 1) plays essential roles in the removal of DNA intrastrand crosslinks by nucleotide excision repair, and that of DNA interstrand crosslinks by the Fanconi anemia (FA) pathway and homology-directed repair processes (HDR). The function of ERCC1 thus impacts on the DNA damage response (DDR), particularly in anticancer therapy when DNA damaging agents are employed. ERCC1 expression has been proposed as a predictive biomarker of the response to platinum-based therapy. However, the assessment of ERCC1 expression in clinical samples is complicated by the existence of 4 functionally distinct protein isoforms, which differently impact on DDR. Here, we explored the functional competence of each ERCC1 protein isoform and obtained evidence that the 202 isoform is the sole one endowed with ERCC1 activity in DNA repair pathways. The ERCC1 isoform 202 interacts with RPA, XPA, and XPF, and XPF stability requires expression of the ERCC1 202 isoform (but none of the 3 others). ERCC1-deficient non-small cell lung cancer cells show abnormal mitosis, a phenotype reminiscent of the FA phenotype that can be rescued by isoform 202 only. Finally, we could not observe any dominant-negative interaction between ERCC1 isoforms. These data suggest that the selective assessment of the ERCC1 isoform 202 in clinical samples should accurately reflect the DDR-related activity of the gene and hence constitute a useful biomarker for customizing anticancer therapies. PMID:24036546

Diadenosine 5', 5'''-P(1),P(4)-tetraphosphate (Ap4A) is synthesized in response to DNA damage and inhibits the initiation of DNA replication.

PubMed

Marriott, Andrew S; Copeland, Nikki A; Cunningham, Ryan; Wilkinson, Mark C; McLennan, Alexander G; Jones, Nigel J

2015-09-01

The level of intracellular diadenosine 5', 5'''-P(1),P(4)-tetraphosphate (Ap4A) increases several fold in mammalian cells treated with non-cytotoxic doses of interstrand DNA-crosslinking agents such as mitomycin C. It is also increased in cells lacking DNA repair proteins including XRCC1, PARP1, APTX and FANCG, while >50-fold increases (up to around 25 μM) are achieved in repair mutants exposed to mitomycin C. Part of this induced Ap4A is converted into novel derivatives, identified as mono- and di-ADP-ribosylated Ap4A. Gene knockout experiments suggest that DNA ligase III is primarily responsible for the synthesis of damage-induced Ap4A and that PARP1 and PARP2 can both catalyze its ADP-ribosylation. Degradative proteins such as aprataxin may also contribute to the increase. Using a cell-free replication system, Ap4A was found to cause a marked inhibition of the initiation of DNA replicons, while elongation was unaffected. Maximum inhibition of 70-80% was achieved with 20 μM Ap4A. Ap3A, Ap5A, Gp4G and ADP-ribosylated Ap4A were without effect. It is proposed that Ap4A acts as an important inducible ligand in the DNA damage response to prevent the replication of damaged DNA. Copyright © 2015 Elsevier B.V. All rights reserved.

The Molybdenum Cofactor Biosynthetic Protein Cnx1 Complements Molybdate-Repairable Mutants, Transfers Molybdenum to the Metal Binding Pterin, and Is Associated with the Cytoskeleton

PubMed Central

Schwarz, Günter; Schulze, Jutta; Bittner, Florian; Eilers, Thomas; Kuper, Jochen; Bollmann, Gabriele; Nerlich, Andrea; Brinkmann, Henner; Mendel, Ralf R.

2000-01-01

Molybdenum (Mo) plays an essential role in the active site of all eukaryotic Mo-containing enzymes. In plants, Mo enzymes are important for nitrate assimilation, phytohormone synthesis, and purine catabolism. Mo is bound to a unique metal binding pterin (molybdopterin [MPT]), thereby forming the active Mo cofactor (Moco), which is highly conserved in eukaryotes, eubacteria, and archaebacteria. Here, we describe the function of the two-domain protein Cnx1 from Arabidopsis in the final step of Moco biosynthesis. Cnx1 is constitutively expressed in all organs and in plants grown on different nitrogen sources. Mo-repairable cnxA mutants from Nicotiana plumbaginifolia accumulate MPT and show altered Cnx1 expression. Transformation of cnxA mutants and the corresponding Arabidopsis chl-6 mutant with cnx1 cDNA resulted in functional reconstitution of their Moco deficiency. We also identified a point mutation in the Cnx1 E domain of Arabidopsis chl-6 that causes the molybdate-repairable phenotype. Recombinant Cnx1 protein is capable of synthesizing Moco. The G domain binds and activates MPT, whereas the E domain is essential for activating Mo. In addition, Cnx1 binds to the cytoskeleton in the same way that its mammalian homolog gephyrin does in neuronal cells, which suggests a hypothetical model for anchoring the Moco-synthetic machinery by Cnx1 in plant cells. PMID:11148290

BRCA1 interacts directly with the Fanconi anemia protein FANCA.

PubMed

Folias, Alexandra; Matkovic, Mara; Bruun, Donald; Reid, Sonja; Hejna, James; Grompe, Markus; D'Andrea, Alan; Moses, Robb

2002-10-01

Fanconi anemia (FA) is a rare autosomal recessive disease characterized by skeletal defects, anemia, chromosomal instability and increased risk of leukemia. At the cellular level FA is characterized by increased sensitivity to agents forming interstrand crosslinks (ICL) in DNA. Six FA genes have been cloned and interactions among individual FANC proteins have been found. The FANCD2 protein co-localizes in nuclear foci with the BRCA1 protein following DNA damage and during S-phase, requiring the FANCA, C, E and G proteins to do so. This finding may reflect a direct role for the BRCA1 protein in double strand break (DSB) repair and interaction with the FANC proteins. Therefore interactions between BRCA1 and the FANC proteins were investigated. Among the known FANC proteins, we find evidence for direct interaction only between the FANCA protein and BRCA1. The evidence rests on three different tests: yeast two-hybrid analysis, coimmunoprecipitation from in vitro synthesis, and coimmunoprecipitation from cell extracts. The amino terminal portion of FANCA and the central part (aa 740-1083) of BRCA1 contain the sites of interaction. The interaction does not depend on DNA damage, thus FANCA and BRCA1 are constitutively interacting. The demonstrated interaction directly connects BRCA1 to the FA pathway of DNA repair.

Viral interference with DNA repair by targeting of the single-stranded DNA binding protein RPA.

PubMed

Banerjee, Pubali; DeJesus, Rowena; Gjoerup, Ole; Schaffhausen, Brian S

2013-10-01

Correct repair of damaged DNA is critical for genomic integrity. Deficiencies in DNA repair are linked with human cancer. Here we report a novel mechanism by which a virus manipulates DNA damage responses. Infection with murine polyomavirus sensitizes cells to DNA damage by UV and etoposide. Polyomavirus large T antigen (LT) alone is sufficient to sensitize cells 100 fold to UV and other kinds of DNA damage. This results in activated stress responses and apoptosis. Genetic analysis shows that LT sensitizes via the binding of its origin-binding domain (OBD) to the single-stranded DNA binding protein replication protein A (RPA). Overexpression of RPA protects cells expressing OBD from damage, and knockdown of RPA mimics the LT phenotype. LT prevents recruitment of RPA to nuclear foci after DNA damage. This leads to failure to recruit repair proteins such as Rad51 or Rad9, explaining why LT prevents repair of double strand DNA breaks by homologous recombination. A targeted intervention directed at RPA based on this viral mechanism could be useful in circumventing the resistance of cancer cells to therapy.

SPOC1 modulates DNA repair by regulating key determinants of chromatin compaction and DNA damage response

PubMed Central

Mund, Andreas; Schubert, Tobias; Staege, Hannah; Kinkley, Sarah; Reumann, Kerstin; Kriegs, Malte; Fritsch, Lauriane; Battisti, Valentine; Ait-Si-Ali, Slimane; Hoffbeck, Anne-Sophie; Soutoglou, Evi; Will, Hans

2012-01-01

Survival time-associated plant homeodomain (PHD) finger protein in Ovarian Cancer 1 (SPOC1, also known as PHF13) is known to modulate chromatin structure and is essential for testicular stem-cell differentiation. Here we show that SPOC1 is recruited to DNA double-strand breaks (DSBs) in an ATM-dependent manner. Moreover, SPOC1 localizes at endogenous repair foci, including OPT domains and accumulates at large DSB repair foci characteristic for delayed repair at heterochromatic sites. SPOC1 depletion enhances the kinetics of ionizing radiation-induced foci (IRIF) formation after γ-irradiation (γ-IR), non-homologous end-joining (NHEJ) repair activity, and cellular radioresistance, but impairs homologous recombination (HR) repair. Conversely, SPOC1 overexpression delays IRIF formation and γH2AX expansion, reduces NHEJ repair activity and enhances cellular radiosensitivity. SPOC1 mediates dose-dependent changes in chromatin association of DNA compaction factors KAP-1, HP1-α and H3K9 methyltransferases (KMT) GLP, G9A and SETDB1. In addition, SPOC1 interacts with KAP-1 and H3K9 KMTs, inhibits KAP-1 phosphorylation and enhances H3K9 trimethylation. These findings provide the first evidence for a function of SPOC1 in DNA damage response (DDR) and repair. SPOC1 acts as a modulator of repair kinetics and choice of pathways. This involves its dose-dependent effects on DNA damage sensors, repair mediators and key regulators of chromatin structure. PMID:23034801

Histone H1 functions as a stimulatory factor in backup pathways of NHEJ

PubMed Central

Rosidi, Bustanur; Wang, Minli; Wu, Wenqi; Sharma, Aparna; Wang, Huichen; Iliakis, George

2008-01-01

DNA double-strand breaks (DSBs) induced in the genome of higher eukaryotes by ionizing radiation (IR) are predominantly removed by two pathways of non-homologous end-joining (NHEJ) termed D-NHEJ and B-NHEJ. While D-NHEJ depends on the activities of the DNA-dependent protein kinase (DNA-PK) and DNA ligase IV/XRCC4/XLF, B-NHEJ utilizes, at least partly, DNA ligase III/XRCC1 and PARP-1. Using in vitro end-joining assays and protein fractionation protocols similar to those previously applied for the characterization of DNA ligase III as an end-joining factor, we identify here histone H1 as an additional putative NHEJ factor. H1 strongly enhances DNA-end joining and shifts the product spectrum from circles to multimers. While H1 enhances the DNA-end-joining activities of both DNA Ligase IV and DNA Ligase III, the effect on ligase III is significantly stronger. Histone H1 also enhances the activity of PARP-1. Since histone H1 has been shown to counteract D-NHEJ, these observations and the known functions of the protein identify it as a putative alignment factor operating preferentially within B-NHEJ. PMID:18250087

Emerging Roles of the Nucleolus in Regulating the DNA Damage Response: The Noncanonical DNA Repair Enzyme APE1/Ref-1 as a Paradigmatical Example

PubMed Central

Antoniali, Giulia; Lirussi, Lisa; Poletto, Mattia

2014-01-01

Abstract Significance: 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. Recent Advances: 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. Critical Issues: 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. Future Directions: 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. Antioxid. Redox Signal. 20, 621–639. PMID:23879289

C-Terminal Fluorescent Labeling Impairs Functionality of DNA Mismatch Repair Proteins

PubMed Central

Brieger, Angela; Plotz, Guido; Hinrichsen, Inga; Passmann, Sandra; Adam, Ronja; Zeuzem, Stefan

2012-01-01

The human DNA mismatch repair (MMR) process is crucial to maintain the integrity of the genome and requires many different proteins which interact perfectly and coordinated. Germline mutations in MMR genes are responsible for the development of the hereditary form of colorectal cancer called Lynch syndrome. Various mutations mainly in two MMR proteins, MLH1 and MSH2, have been identified so far, whereas 55% are detected within MLH1, the essential component of the heterodimer MutLα (MLH1 and PMS2). Most of those MLH1 variants are pathogenic but the relevance of missense mutations often remains unclear. Many different recombinant systems are applied to filter out disease-associated proteins whereby fluorescent tagged proteins are frequently used. However, dye labeling might have deleterious effects on MutLα's functionality. Therefore, we analyzed the consequences of N- and C-terminal fluorescent labeling on expression level, cellular localization and MMR activity of MutLα. Besides significant influence of GFP- or Red-fusion on protein expression we detected incorrect shuttling of single expressed C-terminal GFP-tagged PMS2 into the nucleus and found that C-terminal dye labeling impaired MMR function of MutLα. In contrast, N-terminal tagged MutLαs retained correct functionality and can be recommended both for the analysis of cellular localization and MMR efficiency. PMID:22348133

Traditional Chinese Medicine Curcumin Sensitizes Human Colon Cancer to Radiation by Altering the Expression of DNA Repair-related Genes.

PubMed

Yang, Guangen; Qiu, Jianming; Wang, Dong; Tao, Yong; Song, Yihuan; Wang, Hongtao; Tang, Juping; Wang, Xing; Sun, Y U; Yang, Zhijian; Hoffman, Robert M

2018-01-01

The aim of the present study was to investigate the radio-sensitizing efficacy of curcumin, a traditional Chinese medicine (TCM) on colon cancer cells in vitro and in vivo. Human colon cancer HT-29 cells were treated with curcumin (2.5 μM), irradiation (10 Gy) and the combination of irradiation and curcumin. Cell proliferation was assessed using the MTT assay. Apoptotic cells were detected by Annexin V-PE/7-AAD analysis. PCR was performed to determine differential-expression profiling of 95 DNA-repair genes in irradiated cells and cells treated with both irradiation and curcumin. Differentially-expressed genes were confirmed by Western blotting. In vivo radio-sensitizing efficacy of curcumin was assessed in a xenograft mouse model of HT-29 colon cancer. Curcumin was administrated daily by intraperitoneal injection at 20 mg/kg/dose. Mice received irradiation (10 Gy) twice weekly. Apoptosis of the cancer cells following treatment was determined by TUNEL staining. Irradiation induced proliferation inhibition and apoptosis of HT-29 cells in vitro. Concurrent curcumin treatment sensitized the HT-29 tumor to irradiation (p<0.01). DNA repair-related genes CCNH and XRCC5 were upregulated and LIG4 and PNKP downregulated by the combination of curcumin and irradiation compared with irradiation alone (p<0.05). Combined treatment of curcumin and irradiation resulted in a significantly greater tumor-growth inhibition and apoptosis compared to irradiation treatment alone (p<0.01). Curcumin sensitizes human colon cancer in vitro and in vivo to radiation. Downregulation of LIG4 and PNKP and upregulation of XRCC5 and CCNH DNA-repair-related genes were involved in the radio-sensitizing efficacy of curcumin in colon cancer. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Small-Molecule Inhibitors Targeting DNA Repair and DNA Repair Deficiency in Research and Cancer Therapy.

PubMed

Hengel, Sarah R; Spies, M Ashley; Spies, Maria

2017-09-21

To maintain stable genomes and to avoid cancer and aging, cells need to repair a multitude of deleterious DNA lesions, which arise constantly in every cell. Processes that support genome integrity in normal cells, however, allow cancer cells to develop resistance to radiation and DNA-damaging chemotherapeutics. Chemical inhibition of the key DNA repair proteins and pharmacologically induced synthetic lethality have become instrumental in both dissecting the complex DNA repair networks and as promising anticancer agents. The difficulty in capitalizing on synthetically lethal interactions in cancer cells is that many potential targets do not possess well-defined small-molecule binding determinates. In this review, we discuss several successful campaigns to identify and leverage small-molecule inhibitors of the DNA repair proteins, from PARP1, a paradigm case for clinically successful small-molecule inhibitors, to coveted new targets, such as RAD51 recombinase, RAD52 DNA repair protein, MRE11 nuclease, and WRN DNA helicase. Copyright © 2017 Elsevier Ltd. All rights reserved.

Ntg1p, the base excision repair protein, generates mutagenic intermediates in yeast mitochondrial DNA.

PubMed

Phadnis, Naina; Mehta, Reema; Meednu, Nida; Sia, Elaine A

2006-07-13

Mitochondrial DNA is predicted to be highly prone to oxidative damage due to its proximity to free radicals generated by oxidative phosphorylation. Base excision repair (BER) is the primary repair pathway responsible for repairing oxidative damage in nuclear and mitochondrial genomes. In yeast mitochondria, three N-glycosylases have been identified so far, Ntg1p, Ogg1p and Ung1p. Ntg1p, a broad specificity N-glycosylase, takes part in catalyzing the first step of BER that involves the removal of the damaged base. In this study, we examined the role of Ntg1p in maintaining yeast mitochondrial genome integrity. Using genetic reporters and assays to assess mitochondrial mutations, we found that loss of Ntg1p suppresses mitochondrial point mutation rates, frameshifts and recombination rates. We also observed a suppression of respiration loss in the ntg1-Delta cells in response to ultraviolet light exposure implying an overlap between BER and UV-induced damage in the yeast mitochondrial compartment. Over-expression of the BER AP endonuclease, Apn1p, did not significantly affect the mitochondrial mutation rate in the presence of Ntg1p, whereas Apn1p over-expression in an ntg1-Delta background increased the frequency of mitochondrial mutations. In addition, loss of Apn1p also suppressed mitochondrial point mutations. Our work suggests that both Ntg1p and Apn1p generate mutagenic intermediates in the yeast mitochondrial genome.

Using Atomic Force Microscopy to Characterize the Conformational Properties of Proteins and Protein-DNA Complexes That Carry Out DNA Repair.

PubMed

LeBlanc, Sharonda; Wilkins, Hunter; Li, Zimeng; Kaur, Parminder; Wang, Hong; Erie, Dorothy A

2017-01-01

Atomic force microscopy (AFM) is a scanning probe technique that allows visualization of single biomolecules and complexes deposited on a surface with nanometer resolution. AFM is a powerful tool for characterizing protein-protein and protein-DNA interactions. It can be used to capture snapshots of protein-DNA solution dynamics, which in turn, enables the characterization of the conformational properties of transient protein-protein and protein-DNA interactions. With AFM, it is possible to determine the stoichiometries and binding affinities of protein-protein and protein-DNA associations, the specificity of proteins binding to specific sites on DNA, and the conformations of the complexes. We describe methods to prepare and deposit samples, including surface treatments for optimal depositions, and how to quantitatively analyze images. We also discuss a new electrostatic force imaging technique called DREEM, which allows the visualization of the path of DNA within proteins in protein-DNA complexes. Collectively, these methods facilitate the development of comprehensive models of DNA repair and provide a broader understanding of all protein-protein and protein-nucleic acid interactions. The structural details gleaned from analysis of AFM images coupled with biochemistry provide vital information toward establishing the structure-function relationships that govern DNA repair processes. © 2017 Elsevier Inc. All rights reserved.

Phenotypic Analysis of ATM Protein Kinase in DNA Double-Strand Break Formation and Repair.

PubMed

Mian, Elisabeth; Wiesmüller, Lisa

2017-01-01

Ataxia telangiectasia mutated (ATM) encodes a serine/threonine protein kinase, which is involved in various regulatory processes in mammalian cells. Its best-known role is apical activation of the DNA damage response following generation of DNA double-strand breaks (DSBs). When DSBs appear, sensor and mediator proteins are recruited, activating transducers such as ATM, which in turn relay a widespread signal to a multitude of downstream effectors. ATM mutation causes Ataxia telangiectasia (AT), whereby the disease phenotype shows differing characteristics depending on the underlying ATM mutation. However, all phenotypes share progressive neurodegeneration and marked predisposition to malignancies at the organismal level and sensitivity to ionizing radiation and chromosome aberrations at the cellular level. Expression and localization of the ATM protein can be determined via western blotting and immunofluorescence microscopy; however, detection of subtle alterations such as resulting from amino acid exchanges rather than truncating mutations requires functional testing. Previous studies on the role of ATM in DSB repair, which connects with radiosensitivity and chromosomal stability, gave at first sight contradictory results. To systematically explore the effects of clinically relevant ATM mutations on DSB repair, we engaged a series of lymphoblastoid cell lines (LCLs) derived from AT patients and controls. To examine DSB repair both in a quantitative and qualitative manners, we used an EGFP-based assay comprising different substrates for distinct DSB repair mechanisms. In this way, we demonstrated that particular signaling defects caused by individual ATM mutations led to specific DSB repair phenotypes. To explore the impact of ATM on carcinogenic chromosomal aberrations, we monitored chromosomal breakage at a breakpoint cluster region hotspot within the MLL gene that has been associated with therapy-related leukemia. PCR-based MLL-breakage analysis of HeLa cells

ATM-independent, high-fidelity nonhomologous end joining predominates in human embryonic stem cells

PubMed Central

Adams, Bret R.; Hawkins, Amy J.; Povirk, Lawrence F.; Valerie, Kristoffer

2010-01-01

We recently demonstrated that human embryonic stem cells (hESCs) utilize homologous recombination repair (HRR) as primary means of double-strand break (DSB) repair. We now show that hESCs also use nonhomologous end joining (NHEJ). NHEJ kinetics were several-fold slower in hESCs and neural progenitors (NPs) than in astrocytes derived from hESCs. ATM and DNA-PKcs inhibitors were ineffective or partially effective, respectively, at inhibiting NHEJ in hESCs, whereas progressively more inhibition was seen in NPs and astrocytes. The lack of any major involvement of DNA-PKcs in NHEJ in hESCs was supported by siRNA-mediated DNA-PKcs knockdown. Expression of a truncated XRCC4 decoy or XRCC4 knock-down reduced NHEJ by more than half suggesting that repair is primarily canonical NHEJ. Poly(ADP-ribose) polymerase (PARP) was dispensable for NHEJ suggesting that repair is largely independent of backup NHEJ. Furthermore, as hESCs differentiated a progressive decrease in the accuracy of NHEJ was observed. Altogether, we conclude that NHEJ in hESCs is largely independent of ATM, DNA-PKcs, and PARP but dependent on XRCC4 with repair fidelity several-fold greater than in astrocytes. PMID:20844317

The DNA-dependent protein kinase: a multifunctional protein kinase with roles in DNA double strand break repair and mitosis

PubMed Central

Jette, Nicholas; Lees-Miller, Susan P.

2015-01-01

The DNA-dependent protein kinase (DNA-PK) is a serine/threonine protein kinase composed of a large catalytic subunit (DNA-PKcs) and the Ku70/80 heterodimer. Over the past two decades, significant progress has been made in elucidating the role of DNA-PK in non-homologous end joining (NHEJ), the major pathway for repair of ionizing radiation-induced DNA double strand breaks in human cells and recently, additional roles for DNA-PK have been reported. In this review, we will describe the biochemistry, structure and function of DNA-PK, its roles in DNA double strand break repair and its newly described roles in mitosis and other cellular processes. PMID:25550082

Laser-activated protein solder for peripheral nerve repair

NASA Astrophysics Data System (ADS)

Trickett, Rodney I.; Lauto, Antonio; Dawes, Judith M.; Owen, Earl R.

1995-05-01

A 100 micrometers core optical fiber-coupled 75 mW diode laser operating at a wavelength of 800 nm has been used in conjunction with a protein solder to stripe weld severed rat tibial nerves, reducing the long operating time required for microsurgical nerve repair. Welding is produced by selective laser denaturation of the albumin based solder which contains the dye indocyanine green. Operating time for laser soldering was 10 +/- 5 min. (n equals 20) compared to 23 +/- 9 min. (n equals 10) for microsuturing. The laser solder technique resulted in patent welds with a tensile strength of 15 +/- 5 g, while microsutured nerves had a tensile strength of 40 +/- 10 g. Histopathology of the laser soldered nerves, conducted immediately after surgery, displayed solder adhesion to the outer membrane with minimal damage to the inner axons of the nerves. An in vivo study is under way comparing laser solder repaired tibial nerves to conventional microsuture repair. At the time of submission 15 laser soldered nerves and 7 sutured nerves were characterized at 3 months and showed successful regeneration with compound muscle action potentials of 27 +/- 8 mV and 29 +/- 8 mW respectively. A faster, less damaging and long lasting laser based anastomotic technique is presented.

MOF phosphorylation by ATM regulates 53BP1-mediated double-strand break repair pathway choice.

PubMed

Gupta, Arun; Hunt, Clayton R; Hegde, Muralidhar L; Chakraborty, Sharmistha; Chakraborty, Sharmistha; Udayakumar, Durga; Horikoshi, Nobuo; Singh, Mayank; Ramnarain, Deepti B; Hittelman, Walter N; Namjoshi, Sarita; Asaithamby, Aroumougame; Hazra, Tapas K; Ludwig, Thomas; Pandita, Raj K; Tyler, Jessica K; Pandita, Tej K

2014-07-10

Cell-cycle phase is a critical determinant of the choice between DNA damage repair by nonhomologous end-joining (NHEJ) or homologous recombination (HR). Here, we report that double-strand breaks (DSBs) induce ATM-dependent MOF (a histone H4 acetyl-transferase) phosphorylation (p-T392-MOF) and that phosphorylated MOF colocalizes with γ-H2AX, ATM, and 53BP1 foci. Mutation of the phosphorylation site (MOF-T392A) impedes DNA repair in S and G2 phase but not G1 phase cells. Expression of MOF-T392A also blocks the reduction in DSB-associated 53BP1 seen in wild-type S/G2 phase cells, resulting in enhanced 53BP1 and reduced BRCA1 association. Decreased BRCA1 levels at DSB sites correlates with defective repairosome formation, reduced HR repair, and decreased cell survival following irradiation. These data support a model whereby ATM-mediated MOF-T392 phosphorylation modulates 53BP1 function to facilitate the subsequent recruitment of HR repair proteins, uncovering a regulatory role for MOF in DSB repair pathway choice during S/G2 phase. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

PARP1 impact on DNA repair of platinum adducts: preclinical and clinical read-outs.

PubMed

Olaussen, Ken A; Adam, Julien; Vanhecke, Elsa; Vielh, Philippe; Pirker, Robert; Friboulet, Luc; Popper, Helmut; Robin, Angélique; Commo, Fréderic; Thomale, Jürgen; Kayitalire, Louis; Filipits, Martin; Le Chevalier, Thierry; André, Fabrice; Brambilla, Elisabeth; Soria, Jean-Charles

2013-05-01

Evaluation of DNA repair proteins might provide meaningful information in relation to prognosis and chemotherapy efficacy in Non-Small Cell Lung Cancer (NSCLC) patients. The role of Poly(ADP-Ribose) Polymerase (PARP) in DNA repair of platinum adducts has not been firmly established. We used a DNA repair functional test based on antibody recognition of cisplatin intrastrand platinum adducts on DNA. We evaluated the effect of PARP inhibition on DNA repair functionality in a panel of cisplatin cell lines treated by the clinical-grade pharmacological inhibitor CEP8983 (a 4-methoxy-carbazole derivate) and the commercially available inhibitor PJ34 (phenanthridinone). We determined PARP1 protein expression in whole tumor sections from the International Adjuvant Lung cancer Trial (IALT)-bio study and tested a 3-marker PARP1/MSH2/ERCC1 algorithm combining PARP1 tumor status with previously published data. Chemosensitivity of cisplatin in NSCLC cell lines was correlated with the accumulation of cisplatin DNA adducts (P=0.0004). Further, the pharmacological inhibition of PARP induced a 1.7 to 2.3-fold increase in platinum adduct accumulation (24h) in A549 cell line suggesting a slow-down of platinum DNA-adduct repair capacity. In parallel, PARP1 inhibition increased the sensitivity to cisplatin treatment. In patient samples, PARP1 expression levels did not influence patient survival or the effect of platinum-based post-operative chemotherapy in the global IALT-bio population (interaction P=0.79). Among cases with high expression of all three markers (triple positive), untreated patients had prolonged survival with a median DFS of 7.8 years, (HR=0.34, 95%CI [0.19-0.61], adjusted P=0.0003) compared to triple negative patients (1.4 years). Remarkably, triple positive patients suffered from a detrimental effect (4.9-year reduction of median DFS) by post-operative cisplatin-based chemotherapy (HR=1.79, 95%CI [1.01-3.17], adjusted P=0.04, chemotherapy vs. control). Combinatorial

Replication protein A: directing traffic at the intersection of replication and repair.

PubMed

Oakley, Greg G; Patrick, Steve M

2010-06-01

Since the initial discovery of replication protein A (RPA) as a DNA replication factor, much progress has been made on elucidating critical roles for RPA in other DNA metabolic pathways. RPA has been shown to be required for DNA replication, DNA repair, DNA recombination, and the DNA damage response pathway with roles in checkpoint activation. This review summarizes the current understanding of RPA structure, phosphorylation and protein-protein interactions in mediating these DNA metabolic processes.

DNA-repair protein hHR23a alters its protein structure upon binding proteasomal subunit S5a

PubMed Central

Walters, Kylie J.; Lech, Patrycja J.; Goh, Amanda M.; Wang, Qinghua; Howley, Peter M.

2003-01-01

The Rad23 family of proteins, including the human homologs hHR23a and hHR23b, stimulates nucleotide excision repair and has been shown to provide a novel link between proteasome-mediated protein degradation and DNA repair. In this work, we illustrate how the proteasomal subunit S5a regulates hHR23a protein structure. By using NMR spectroscopy, we have elucidated the structure and dynamic properties of the 40-kDa hHR23a protein and show it to contain four structured domains connected by flexible linker regions. In addition, we reveal that these domains interact in an intramolecular fashion, and by using residual dipolar coupling data in combination with chemical shift perturbation analysis, we present the hHR23a structure. By itself, hHR23a adopts a closed conformation defined by the interaction of an N-terminal ubiquitin-like domain with two ubiquitin-associated domains. Interestingly, binding of the proteasomal subunit S5a disrupts the hHR23a interdomain interactions and thereby causes it to adopt an opened conformation. PMID:14557549

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

PubMed

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.

Dynamic binding of replication protein a is required for DNA repair

PubMed Central

Chen, Ran; Subramanyam, Shyamal; Elcock, Adrian H.; Spies, Maria; Wold, Marc S.

2016-01-01

Replication protein A (RPA), the major eukaryotic single-stranded DNA (ssDNA) binding protein, is essential for replication, repair and recombination. High-affinity ssDNA-binding by RPA depends on two DNA binding domains in the large subunit of RPA. Mutation of the evolutionarily conserved aromatic residues in these two domains results in a separation-of-function phenotype: aromatic residue mutants support DNA replication but are defective in DNA repair. We used biochemical and single-molecule analyses, and Brownian Dynamics simulations to determine the molecular basis of this phenotype. Our studies demonstrated that RPA binds to ssDNA in at least two modes characterized by different dissociation kinetics. We also showed that the aromatic residues contribute to the formation of the longer-lived state, are required for stable binding to short ssDNA regions and are needed for RPA melting of partially duplex DNA structures. We conclude that stable binding and/or the melting of secondary DNA structures by RPA is required for DNA repair, including RAD51 mediated DNA strand exchange, but is dispensable for DNA replication. It is likely that the binding modes are in equilibrium and reflect dynamics in the RPA–DNA complex. This suggests that dynamic binding of RPA to DNA is necessary for different cellular functions. PMID:27131385

Modeling non-homologous end joining.

PubMed

Li, Yongfeng; Cucinotta, Francis A

2011-08-21

Non-homologous end joining (NHEJ) is an important DNA repair pathway for DNA double-strand breaks. Several proteins, including Ku, DNA-PKcs, Artemis, XRCC4/Ligase IV and XLF, are involved in the NHEJ for the DNA damage detection, DNA free end processing and ligation. The classical model of NHEJ is a sequential model in which DNA-PKcs is first recruited by the Ku bound DNA prior to any other repair proteins. Recent experimental study (McElhinny et al., 2000; Costantini et al., 2007; Mari et al., 2006; Yano and Chen, 2008) suggested that the recruitment ordering is not crucial. In this work, by proposing a mathematical model in terms of biochemical reaction network and performing stability and related analysis, we demonstrate theoretically that if DSB repair pathway independent of DNA-PKcs exists, then the classical sequential model and new two-phase model are essentially indistinguishable in the sense that DSB can be repaired thoroughly in both models when the repair proteins are sufficient. Published by Elsevier Ltd.

Polymorphic trial in oxidative damage of arsenic exposed Vietnamese.

PubMed

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

2011-10-15

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. Published by Elsevier Inc.

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

PubMed Central

Grindel, Annemarie; Guggenberger, Bianca; Eichberger, Lukas; Pöppelmeyer, Christina; Gschaider, Michaela; Tosevska, Anela; Mare, George; Briskey, David; Brath, Helmut; Wagner, Karl-Heinz

2016-01-01

Background 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. Methods 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. Results 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. Conclusion 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

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

PubMed

Grindel, Annemarie; Guggenberger, Bianca; Eichberger, Lukas; Pöppelmeyer, Christina; Gschaider, Michaela; Tosevska, Anela; Mare, George; Briskey, David; Brath, Helmut; Wagner, Karl-Heinz

2016-01-01

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 treatment

BRCA Mutations, DNA Repair Deficiency, and Ovarian Aging1

PubMed Central

Oktay, Kutluk; Turan, Volkan; Titus, Shiny; Stobezki, Robert; Liu, Lin

2015-01-01

Oocyte aging has a significant impact on reproductive outcomes both quantitatively and qualitatively. However, the molecular mechanisms underlying the age-related decline in reproductive success have not been fully addressed. BRCA is known to be involved in homologous DNA recombination and plays an essential role in double-strand DNA break repair. Given the growing body of laboratory and clinical evidence, we performed a systematic review on the current understanding of the role of DNA repair in human reproduction. We find that BRCA mutations negatively affect ovarian reserve based on convincing evidence from in vitro and in vivo results and prospective studies. Because decline in the function of the intact gene occurs at an earlier age, women with BRCA1 mutations exhibit accelerated ovarian aging, unlike those with BRCA2 mutations. However, because of the still robust function of the intact allele in younger women and because of the masking of most severe cases by prophylactic oophorectomy or cancer, it is less likely one would see an effect of BRCA mutations on fertility until later in reproductive age. The impact of BRCA2 mutations on reproductive function may be less visible because of the delayed decline in the function of normal BRCA2 allele. BRCA1 function and ataxia-telangiectasia-mutated (ATM)-mediated DNA repair may also be important in the pathogenesis of age-induced increase in aneuploidy. BRCA1 is required for meiotic spindle assembly, and cohesion function between sister chromatids is also regulated by ATM family member proteins. Taken together, these findings strongly suggest the implication of BRCA and DNA repair malfunction in ovarian aging. PMID:26224004

UCHL3 Regulates Topoisomerase-Induced Chromosomal Break Repair by Controlling TDP1 Proteostasis.

PubMed

Liao, Chunyan; Beveridge, Ryan; Hudson, Jessica J R; Parker, Jacob D; Chiang, Shih-Chieh; Ray, Swagat; Ashour, Mohamed E; Sudbery, Ian; Dickman, Mark J; El-Khamisy, Sherif F

2018-06-12

Genomic damage can feature DNA-protein crosslinks whereby their acute accumulation is utilized to treat cancer and progressive accumulation causes neurodegeneration. This is typified by tyrosyl DNA phosphodiesterase 1 (TDP1), which repairs topoisomerase-mediated chromosomal breaks. Although TDP1 levels vary in multiple clinical settings, the mechanism underpinning this variation is unknown. We reveal that TDP1 is controlled by ubiquitylation and identify UCHL3 as the deubiquitylase that controls TDP1 proteostasis. Depletion of UCHL3 increases TDP1 ubiquitylation and turnover rate and sensitizes cells to TOP1 poisons. Overexpression of UCHL3, but not a catalytically inactive mutant, suppresses TDP1 ubiquitylation and turnover rate. TDP1 overexpression in the topoisomerase therapy-resistant rhabdomyosarcoma is driven by UCHL3 overexpression. In contrast, UCHL3 is downregulated in spinocerebellar ataxia with axonal neuropathy (SCAN1), causing elevated levels of TDP1 ubiquitylation and faster turnover rate. These data establish UCHL3 as a regulator of TDP1 proteostasis and, consequently, a fine-tuner of protein-linked DNA break repair. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Aberrant topoisomerase-1 DNA lesions are pathogenic in neurodegenerative genome instability syndromes.

PubMed

Katyal, Sachin; Lee, Youngsoo; Nitiss, Karin C; Downing, Susanna M; Li, Yang; Shimada, Mikio; Zhao, Jingfeng; Russell, Helen R; Petrini, John H J; Nitiss, John L; McKinnon, Peter J

2014-06-01

DNA damage is considered to be a prime factor in several spinocerebellar neurodegenerative diseases; however, the DNA lesions underpinning disease etiology are unknown. We observed the endogenous accumulation of pathogenic topoisomerase-1 (Top1)-DNA cleavage complexes (Top1ccs) in murine models of ataxia telangiectasia and spinocerebellar ataxia with axonal neuropathy 1. We found that the defective DNA damage response factors in these two diseases cooperatively modulated Top1cc turnover in a non-epistatic and ATM kinase-independent manner. Furthermore, coincident neural inactivation of ATM and DNA single-strand break repair factors, including tyrosyl-DNA phosphodiesterase-1 or XRCC1, resulted in increased Top1cc formation and excessive DNA damage and neurodevelopmental defects. Notably, direct Top1 poisoning to elevate Top1cc levels phenocopied the neuropathology of the mouse models described above. Our results identify a critical endogenous pathogenic lesion associated with neurodegenerative syndromes arising from DNA repair deficiency, indicating that genome integrity is important for preventing disease in the nervous system.

DNA mismatch repair proteins MLH1 and PMS2 can be imported to the nucleus by a classical nuclear import pathway.

PubMed

de Barros, Andrea C; Takeda, Agnes A S; Dreyer, Thiago R; Velazquez-Campoy, Adrian; Kobe, Boštjan; Fontes, Marcos R M

2018-03-01

MLH1 and PMS2 proteins form the MutLα heterodimer, which plays a major role in DNA mismatch repair (MMR) in humans. Mutations in MMR-related proteins are associated with cancer, especially with colon cancer. The N-terminal region of MutLα comprises the N-termini of PMS2 and MLH1 and, similarly, the C-terminal region of MutLα is composed by the C-termini of PMS2 and MLH1, and the two are connected by linker region. The nuclear localization sequences (NLSs) necessary for the nuclear transport of the two proteins are found in this linker region. However, the exact NLS sequences have been controversial, with different sequences reported, particularly for MLH1. The individual components are not imported efficiently, presumably due to their C-termini masking their NLSs. In order to gain insights into the nuclear transport of these proteins, we solved the crystal structures of importin-α bound to peptides corresponding to the supposed NLSs of MLH1 and PMS2 and performed isothermal titration calorimetry to study their binding affinities. Both putative MLH1 and PMS2 NLSs can bind to importin-α as monopartite NLSs, which is in agreement with some previous studies. However, MLH1-NLS has the highest affinity measured by a natural NLS peptide, suggesting a major role of MLH1 protein in nuclear import compared to PMS2. Finally, the role of MLH1 and PMS2 in the nuclear transport of the MutLα heterodimer is discussed. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.