Nucleotide Excision Repair Enzymes Research Articles

Abstract 1967: UvrABC excision of interstrand crosslink mitomycin C-DNA lesion induces double-stranded DNA breaks

Abstract Mitomycin C (MC) forms at least nine different covalent DNA adducts including an N2-2’-deoxyguanosine monoadduct (MC-mono-dG) and inter- and intrastrand bis-adducts with 2’-deoxyguanosine (dG) in vivo. Inter-strand cross-linked (ICL) dG-MC-dG-DNA adducts can prevent strand separation, and therefore, the MC antitumor activity is most likely derived from formation of ICL-MC adducts. In Escherichia coli cells, UvrABC, the nucleotide excision repair (NER) enzyme complex can repair ICL psoralen lesions that cause DNA bending. The mechanism and the consequence of NER of ICL dG-MC-dG lesions that do not induce DNA bending remain unclear. In order to compare the NER of MC-mono-dG with an ICL dG-MC-dG adduct, we constructed DNA fragments containing the adducts in the same DNA sequence context. We determined that UvrABC incises only at the strand containing MC-mono-dG adduct. In contrast, UvrABC makes three types of incisions on a site specific ICL dG-MC-dG adduct: type 1 (40 %), a single 5’ incision on one strand and 3’ incision on the other; type 2 (21 %), dual incisions on one strand and a single incision on the other strand; and type 3 (39%), dual incisions on both strand of both 5’ and 3’ sides of the ICL dG-MC-dG adduct. These three types of incisions occur with similar kinetics. An important consequence of these incisions is formation of DNA double-stranded breaks (DSB). Contrary to conventional model, we also found that UvrA, UvrA+UvrB and UvrA+UvrB+UvrC bind to MC-modified DNA specifically and form complexes that include UvrA. No UvrB- and UvrB+UvrCDNA complexes were observed. Our findings challenge the current UvrABC incision model of ICLs. We propose that NER of ICL dG-MC-dG adduct produces DSB, a lethal event that contributes to MC antitumor activity, and causes a non-homologous end-to-end joining that leads to DNA translocation and deletion mutations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1967.

DNA damages induced by trans, trans‐2,4‐decadienal (tt‐DDE), a component of cooking oil fume, in human bronchial epithelial cells

Epidemiological studies have demonstrated that cooking oil fumes (COF) are an environmental risk factor for the development of lung adenocarcinoma among nonsmoking females in Taiwan. Aside from polycyclic aromatic hydrocarbons, aldehydes, especially trans, trans-2,4-decadienal (tt-DDE) are found to be abundant in COF. Although there is indication that tt-DDE induces DNA damage, the precise role of tt-DDE in the induction of DNA damage in lung cells is still not clear. When we assessed DNA breaks with the Comet assay, we found that the DNA breaks induced by 1 muM tt-DDE in human bronchial epithelial cells (BEAS-2B) could be significantly reduced by antioxidants, suggesting that oxidative stress was involved. Indeed, when tt-DDE-treated cells were coincubated with endonuclease III/formamidopyrimidine-DNA glycosylase or with nuclear extract (NE), an enhancement of DNA breaks was observed at 1 hr after tt-DDE exposure. Furthermore, when NE was incubated with an antibody against 8-oxoguanine DNA glycosylase (anti-OGG1), a reduction in tt-DDE/NE-induced DNA breaks could be demonstrated. Since OGG1 is a specific repair enzyme for 8-oxo-deoxyguanosine (8-oxo-dG), these findings indicated that 8-oxo-dG was involved. On the other hand, when NE was incubated with antibodies against nucleotide excision repair enzymes, there was a significant reduction in tt-DDE/NE-induced DNA breaks at 4 hr after tt-DDE treatment. These observations indicate that, in addition to early oxidative DNA damage, nonoxidative DNA damage such as bulky adduct formation, was also induced by tt-DDE. Our study further affirms that tt-DDE is genotoxic to human lung cells and can increase carcinogenic risk.

HapMap-based study of the DNA repair gene ERCC2 and lung cancer susceptibility in a Chinese population

DNA repair genes have been proposed as candidate cancer susceptibility genes. The excision repair cross-complementing rodent repair deficiency, complementation group 2 (ERCC2)/xeroderma pigmentosum complementary group D (XPD) protein is considered to be a key enzyme in nucleotide excision repair (NER) pathway. To elucidate whether common ERCC2 variants are associated with lung cancer susceptibility, we conducted a case-control study consisting of 339 cases with primary lung cancer and 358 controls matched on age, gender and ethnicity in a Chinese population. Six haplotype tagging single-nucleotide polymorphisms (htSNPs) (rs238403, rs50871, rs3916840, rs238415, rs3916874 and rs1799787) from HapMap database were analyzed, which provide an almost complete coverage of the genetic variations in the ERCC2 gene. Although none of the six htSNPs was individually associated with lung cancer risk, we found that two ERCC2 haplotypes were associated with risk of lung cancer. Haplotype 4 defined by rs238403T-rs50871T-rs3916840C-rs238415C-rs3916874G-rs1799787C and haplotype 7 defined by rs238403C-rs50871G-rs3916840C-rs238415C-rs3916874G-rs1799787C were strongly associated with an increased risk of lung cancer [odds ratio, OR (95% confidence interval, CI) = 2.62 (1.53-4.50), P = 0.0003 for hap4; OR (95% CI) = 3.01 (1.36-6.63), P = 0.004 for hap7]. Furthermore, diplotype analyses also strengthened the significant associations of risk haplotype 4 [OR (95% CI) = 3.56 (2.12-5.87), P < 0.001] or risk haplotype 7 [OR (95% CI) = 3.38 (1.75-6.55), P < 0.001] and lung cancer. Analysis of linkage disequilibrium (LD) also confirmed that considerable LD exists between the pairs of the six htSNPs within ERCC2. These results suggested that the risk subhaplotypes cosegregate with one or more biologically functional polymorphisms. Our results provide evidence to support a role for ERCC2 in lung cancer development in a Chinese population.

ERCC6/CSB gene polymorphisms and lung cancer risk

Nucleotide excision repair (NER) enzymes are critical for the removal of bulky DNA adducts caused by environmental carcinogens such as smoking. Of them, Cockayne syndrome complementation group B (CSB), coded by ERCC6, recruits NER repair factors to the DNA damage site and plays an important role in the repair process. Genetic variants of ERCC6 may alter the regulation of DNA repair and therefore were hypothesized to be associated with altered risk of smoking-related lung cancer. To test this hypothesis, we genotyped eight tagging single nucleotide polymorphisms (tSNPs) and three potentially functional SNPs of ERCC6 in 500 incident lung cancer cases and 517 controls in a Chinese population. Single locus analyses showed that none of the single SNP alone had the significant main affect on the risk of lung cancer. However, the combined variant genotypes of the four loci with P trend approaching to 0.10 (rs2228526, rs4253160, rs12571445 and rs3793784) were associated with a significantly increased lung cancer risk (adjusted OR 1.35, 95% CI, 1.04–1.75 among subjects carrying three or more variant alleles), indicating that multiple loci in ERCC6 may jointly contribute to the susceptibility of lung cancer. These findings, if validated, may contribute to identify at-risk subjects in the general population for smoking-related lung cancer.

Recognition and incision of Cr(III) ligand-conjugated DNA adducts by the nucleotide excision repair proteins UvrABC: importance of the Cr(III)-purine moiety in the enzymatic reaction.

Hexavalent chromium [Cr(VI)] is an ubiquitous environmental contaminant and a well-known etiological agent of human lung cancer. Inside human cells, Cr(VI) is reduced to Cr(III), which can conjugate with amino acids, ascorbic acids, and glutathiones in the cytoplasm. Conjugated and unconjugated Cr(III) can enter the nucleus to form adducts with DNA and electrostatically interact with the phosphate group of DNA. It has been found that in both human and Escherichia coli systems, Cr(III) ligand-conjugated DNA ternary adducts are efficiently repaired by the nucleotide excision repair (NER) pathway. In contrast, DNA adducts formed by unconjugated Cr(III) with DNA are repaired significantly less efficiently by the NER system. These results raise the possibility that the NER system repairs Cr(III) ligand-conjugated DNA adducts and biadducts such as Cr(III)-guanine-phosphate adducts but not Cr(III)-phosphate adducts. To test this hypothesis, we determined the cutting efficiency and the mode of cutting of DNA modified with tannin-conjugated Cr(III) by the E. coli NER enzymes UvrABC. Tannin compounds, gallic acid (GA), and ethyl gallate (EGA) can reduce Cr(VI) to Cr(III) to form Cr(III)-GA 2 and Cr(III)-EGA 2, respectively, which can interact with a single guanine or adenine base but not with the DNA phosphate backbone. We found that UvrABC is able to incise Cr(III)-GA 2- and Cr(III)-EGA 2-modified plasmid DNA, and the amount of incision increased as a function of tannin concentration used for modifications. In contrast, UvrABC nuclease does not incise GA- and EGA-modified plasmid DNA. Mapping the sequence specificity of Cr(III)-GA 2- and Cr(III)-EGA 2-DNA formation in the human p53 gene sequence by UvrABC nuclease cutting, we found that the sequence specificity for both adducts is the same but is much more selective than Cr(III)-guanine-DNA adducts. Together, these results suggest that NER proteins from E. coli recognize the purine-Cr(III) adduct but not the Cr(III)-backbone phosphate complex.

Influence of TCDD and natural Ah receptor agonists on benzo[a]pyrene-DNA adduct formation in the Caco-2 human colon cell line

Several compounds originating from cruciferous vegetables and citrus fruits bind to and activate the aryl hydrocarbon receptor (AhR). This receptor plays an important role in the toxicity of the known tumour promoter and potent AhR-agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). However, vegetables and fruits are generally considered as healthy. Therefore, besides the AhR activation, the natural AhR agonists (NAhRAs) are assumed to show other health-concerning effects. AhR activation induces several cytochrome P450 phase I enzymes involved, e.g. in the bioactivation of carcinogenic polycyclic aromatic hydrocarbons, like benzo[a]pyrene (BaP), and may as such stimulate DNA adduct formation of those compounds. Therefore, the influence of TCDD, indolo[3,2-b]carbazole (ICZ, an NAhRA originating from cruciferous vegetables) and an NAhRA-containing extract of grapefruit juice (GJE) on BaP-DNA adduct formation in the human Caco-2 cell line was studied. Also, we investigated if different effects of TCDD, ICZ and GJE on adduct formation could be related to the modulation of transcription of biotransformation- and DNA-repair enzymes. Co-exposure to high AhR-activating concentrations of both TCDD and ICZ significantly reduced the amount of BaP-DNA adducts at 0.1 microM BaP, while at higher concentrations of BaP no influence was observed. In contrast, exposure to 0.1 microM BaP combined with GJE showed a significant increase in BaP-DNA adducts, and a significant decrease at 0.3 and 1 microM BaP. These differences could not be related to transcription of the phase I and II enzymes CYP1A1, CYP1B1, NQO1, GSTP1 and UGT1A6 or to transcription of the nucleotide excision repair enzymes ERCC1, XPA, XPC, XPF and XPG. We conclude that ICZ showed a similar effect on BaP-DNA adduct formation than TCDD, while GJE influenced the adduct formation in a different way. The difference in the influence on adduct formation may be due to effects at the level of enzyme activity, rather than gene expression.

Dual role of NER in mutagenesis in Pseudomonas putida

Nucleotide excision repair (NER) is one of the most important repair systems which counteracts different forms of DNA damage either induced by various chemicals or irradiation. At the same time, less is known about the functions of NER in repair of DNA that is not exposed to exogenous DNA-damaging agents. We have investigated the role of NER in mutagenesis in Pseudomonas putida. The genome of this organism contains two uvrA genes, uvrA and uvrA2. Genetic studies on the effects of uvrA, uvrA2, uvrB and UvrC in mutagenic processes revealed that all of these genes are responsible for the repair of UV-induced DNA damage in P. putida. However, uvrA plays more important role in this process than uvrA2 since the deletion of uvrA2 gene had an effect on the UV-tolerance of bacteria only in the case when uvrA was also inactivated. Interestingly, the lack of functional uvrB, uvrC or uvrA2 gene reduced the frequency of stationary-phase mutations. The contribution of uvrA2, uvrB and uvrC to the mutagenesis appeared to be most significant in the case of 1-bp deletions whose emergence is dependent on error-prone DNA polymerase Pol IV. These data imply that NER has a dual role in mutagenesis in P. putida—besides functioning in repair of damaged DNA, NER is also important in generation of mutations. We hypothesize that NER enzymes may initiate gratuitous DNA repair and the following DNA repair synthesis might be mutagenic.

Base excision repair pathway is involved in the repair of lesions generated by flavonoid‐enriched fractions of pepper tree (Schinus terebinthifolius, Raddi) stem bark

Cell-free and bacterial assays indicate that flavonoid-enriched fractions and the flavonoids of pepper tree stem bark from Schinus terebinthifolius Raddi have genotoxic rather than antigenotoxic properties. In the present report, we have examined the ability of flavonoid-enriched fractions to damage plasmid DNA and the repair pathways involved in the recognition of these DNA lesions. High concentrations of two flavonoid-enriched fractions were able to break phosphodiester bonds in DNA. In addition, studies using bacterial strains deficient in nucleotide excision repair and base excision repair (BER) enzymes indicated that the flavonoid-enriched fractions generated lesions that were substrates for enzymes belonging to the BER pathway. In addition, in vitro studies indicated that the DNA damage produced by the flavonoid-enriched fractions was also a substrate for exonuclease III and that the phosphodiester breakage was amplified by copper ions. These results indicate that flavonoids from the pepper tree (Schinus terebinthifolius, Raddi) generate lesions on DNA that are potential targets of FPG and MutY glycosylase from the BER pathway. Chromatographic and spectral analyses helped to support the hypothesis that the flavonoids of the Brazilian pepper tree bark are the main factors involved in the fraction's damage potential. The isolated flavonoids from Fraction II were also tested in vitro and support the oxidative damage potential of these flavonoids.

UvrA and UvrB enhance mutations induced by oxidized deoxyribonucleotides

Oxidatively damaged DNA precursors (deoxyribonucleotides) are formed by reactive oxygen species. After the damaged DNA precursors are incorporated into DNA, they might be removed by DNA repair enzymes. In this study, to examine whether a nucleotide excision repair enzyme, Escherichia coli UvrABC, could suppress the mutations induced by oxidized deoxyribonucleotides in vivo, oxidized DNA precursors, 8-hydroxy-2′-deoxyguanosine 5′-triphosphate and 2-hydroxy-2′-deoxyadenosine 5′-triphosphate, were introduced into uvrA, uvrB, and uvrC E. coli strains, and mutations in the chromosomal rpoB gene were analyzed. Unexpectedly, these oxidized DNA precursors induced mutations only slightly in the uvrA and uvrB strains. In contrast, effect of the uvrC-deficiency was not observed. Next, mutT, mutT/ uvrA, and mutT/ uvrB E. coli strains were treated with H 2O 2, and the rpoB mutant frequencies were calculated. The frequency of the H 2O 2-induced mutations was increased in all of the strains tested; however, the increase was three- to four-fold lower in the mutT/ uvrA and mutT/ uvrB strains than in the mutT strain. Thus, UvrA and UvrB are involved in the enhancement, but not in the suppression, of the mutations induced by these oxidized deoxyribonucleotides. These results suggest a novel role for UvrA and UvrB in the processing of oxidative damage.

Prediction of response to chemotherapy by ERCC1 immunohistochemistry and ERCC1 polymorphism in ovarian cancer

5568 Background: The response of tumor cells to platinum-based drugs involves DNA repair mechanisms. Platinum-DNA adducts are repaired by nucleotide excision repair (NER) enzymes that recognize the DNA damage and excise the platinum-DNA adducts from the injured DNA strand. Excision repair cross-complementation group 1 (ERCC1) is one of the genes that encode the proteins of the NER complex and several studies have linked ERCC1 to platinum resistance in cell lines and in human cancers. Cells with a high repair capacity, e.g. high level of ERCC1 expression may therefore be resistant to platinum-based chemotherapy, and conversely, polymorphisms within encoding DNA repair enzymes or low repair capacity may confer sensitivity. A common single nucleotide polymorphism (SNP) of ERCC1 at codon 118 have been proposed to impair ERCC1 translation and reduce ERCC1 protein expression and consequently influence the response to platinum based chemotherapy. The aim of this study was to evaluate ERCC1 expression and ERCC1 118 polymorphism in epithelial ovarian cancer and the potential association with response to platinum-based chemotherapy. Methods: Formalin-fixed, paraffin-embedded tissue sections from 159 patients with epithelial ovarian cancer, FIGO stage IIb-IV, were used for immunohistochemistry (monoclonal mouse antibody, clone 8F1, Neomarkers). ERCC1 codon 118 SNP genotyping was performed by real time PCR, Taqman SNP genotyping assay. Results: ERCC1 protein overexpression was found in 37.7 % of the tumors. The response rate (normalization of CA125 during platinum-based chemotherapy) was 63.6 % (35/55) in patients with ERCC1-negative tumors compared to only 35.6 % (16/45) in patients with ERCC1-positive tumors. (p = 0.0052, χ2). Furthermore increasing immunohistochemical score (H-score) was associated with poorer response to chemotherapy. (p = 0.0006, Spearman`s). The T/T genotype (44 %) showed better response to chemotherapy than C/C + C/T (15 % + 41 %) variants (p=0.042). Conclusions: Patients with ERCC1-negative tumors appear to have significantly better response to platinumbased chemotherapy compared to patients with ERCC1-positive tumors. In addition the TT genotype seems to be favorable towards better response to platinum-based chemotherapy. No significant financial relationships to disclose.

Diallyl sulfide induces the expression of nucleotide excision repair enzymes in the breast of female ACI rats

Diethylstilbestrol (DES) causes DNA adducts resulting in breast cancer, whereas diallyl sulfide (DAS) inhibits cancer formation. We hypothesize that DAS induces the expression of nucleotide excision repair genes. To test this hypothesis, female ACI rats were treated for 4 days with corn oil, DES, DAS, and DAS/DES (50 mg/kg). The expression of P53, Gadd45a, PCNA, and DNA polymerase delta was analyzed by real-time PCR. DES decreased the expression of P53, Gadd45a and PCNA. DAS and DAS/DES increased the expression of all four genes. These results suggest that DAS enhances the ability of breast tissue to repair DNA damage thus preventing cancer.

Recognition and Incision of Oxidative Intrastrand Cross-Link Lesions by UvrABC Nuclease

Nucleotide excision repair (NER) is a repair pathway that removes a variety of bulky DNA lesions in both prokaryotic and eukaryotic cells. The perturbation of DNA helix structure caused by the oxidative intrastrand lesions could render them good substrates for the NER pathway. Here we employed Escherichia coli NER enzymes, i.e., UvrA, UvrB, and UvrC, to examine the incision efficiency of duplex DNA carrying three different oxidative intrastrand cross-link lesions, that is, G[8-5]C, G[8-5m]mC, and G[8-5m]T, and two dithymine photoproducts, namely, the cis,syn-cyclobutane pyrimidine dimer (T[c,s]T) and the pyrimidine(6-4)pyrimidone product (T[6-4]T). Our results showed that T[6-4]T was the best substrate for UvrA binding, followed by G[8-5]C, G[8-5m]mC, and G[8-5m]T, and then by T[c,s]T. The efficiencies of the UvrABC incisions of these lesions were consistent with their UvrA binding affinities: the stronger the binding to UvrA, the higher the rate of incision. In addition, flanking DNA sequences appeared to have little effect on the binding affinity of UvrA for G[8-5]C as AG[8-5]CA was only slightly preferred over CG[8-5]CG. Consistently, these two sequences exhibited almost no difference in incision rates. Furthermore, we investigated the thermal stability of dodecameric duplexes containing G[8-5m]mC or G[8-5m]T, and our results revealed that these two lesions destabilized the duplex, due to an increase in the free energy for duplex formation at 37 degrees C, by approximately 5.4 and 3.6 kcal/mol, respectively. The destabilizations to the DNA helix caused by those lesions, for the most part, are correlated with the binding affinities of UvrA and incision rates of UvrABC. Taken together, the results from this study suggest that oxidative intrastrand lesions might be substrates for NER enzymes in vivo.

Nucleotide Excision Repair Gene Polymorphisms and Risk of Advanced Colorectal Adenoma:XPCPolymorphisms Modify Smoking-Related Risk

Nucleotide excision repair enzymes remove bulky damage caused by environmental agents, including carcinogenic polycyclic aromatic hydrocarbons found in cigarette smoke, a risk factor for colorectal adenoma. Among participants randomized to the screening arm of the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial, we studied the risk of advanced colorectal adenoma in relation to cigarette smoking and selected single nucleotide polymorphisms (SNP) in the nucleotide excision repair pathway. Cases (n = 772) were subjects with left-sided advanced adenoma (>1 cm in size, high-grade dysplasia, or villous characteristics). Controls (n = 777) were screen-negative for left-sided polyps by sigmoidoscopy. DNA was extracted from blood samples and 15 common nonsynonymous SNPs in seven-nucleotide excision repair genes [XPC, RAD23B (hHR23B), CSB (ERCC6), XPD (ERCC2), CCNH, XPF (ERCC4), and XPG (ERCC5)] were genotyped. None of the studied SNPs were independently associated with advanced adenoma risk. Smoking was related to adenoma risk and XPC polymorphisms (R492H, A499V, K939Q) modified these effects (P(interaction) from 0.03-0.003). Although the three XPC variants were in linkage disequilibrium, a multivariate logistic regression tended to show independent protective effects for XPC 499V (P(trend) = 0.06), a finding supported by haplotype analysis (covariate-adjusted global permutation P = 0.03). Examining a spectrum of polymorphic variants in nucleotide excision repair genes, we found evidence that smoking-associated risks for advanced colorectal adenoma are modified by polymorphisms in XPC, particularly haplotypes containing XPC 499V.

Activity of nucleotide excision repair enzymes for oxanine cross-link lesions

Nitric oxide and nitrous acid induce deamination of DNA bases, resulting in uracil, hypoxanthine, xanthine, and oxanine (Oxa) as major damage. Oxa reacts further with polyamines and DNA binding proteins, generating bulky cross-link adducts. Recently we have shown Oxa and cross-link adducts are potentially genotoxic lesions. In the present study, we have assessed the role of base excision repair (BER) and nucleotide excision repair (NER) systems in the repair of Oxa and Oxa-spermine (Oxa-Sp) cross-link adducts. Oxa was very poorly removed from DNA by both BER glycosylases and NER enzymes, whereas Oxa-Sp was efficiently excised by E. coli and human NER enzymes.

ERCC1 Codon 118 Polymorphism Is a Predictive Factor for the Tumor Response to Oxaliplatin/5-Fluorouracil Combination Chemotherapy in Patients with Advanced Colorectal Cancer

The aim of our study was to assess whether the polymorphism of the nucleotide excision repair enzyme, excision repair cross-complementing rodent repair deficiency, complementation group 1 (ERCC1), had an effect on the tumor response in patients treated with standard chemotherapy regimens for a metastatic colorectal cancer. We have studied the synonymous polymorphism that causes a single nucleotide change C to T at codon 118 converting a codon of common usage (AAC) to a less used codon (AAT), both coding asparagine. This change results in a decreased ERCC1 gene expression, which impairs repair activity. Ninety-one patients with a median age of 55.1 years treated for a metastatic colorectal cancer were included in our retrospective study. The ERCC1 polymorphism was analyzed in the normal tissue of all patients. Twenty (22%) were homozygous for AAC codon (C/C genotype), 30 were (33%) homozygous for AAT codon (T/T genotype), and 41 (45%) were heterozygous (C/T genotype). The objective response rate to oxaliplatin in combination with 5-fluorouracil (5-FU) was significantly higher in the T/T genotype group compared with the C/T and the C/C genotype groups (61.9%, 42.3%, and 21.4%, respectively; P = 0.018). By contrast, no significant difference was observed when patients were treated with either 5-FU alone (45%, 29.2%, and 33.3%, respectively; P = 0.407) or in combination with irinotecan (46.1%, 25.0%, and 27.3%, respectively; P = 0.305). Our observations allowed us to define the first useful predictive criterion for oxaliplatin/5-FU response in patients with metastatic colorectal cancer.

Polymorphisms in ERCC2 and overall survival (OS) in early stage non-small cell lung cancer (NSCLC)

9524 Background: ERCC2 is an important enzyme in nucleotide excision repair. Polymorphisms in the ERCC2 gene may affect DNA repair capacity and prognosis. Methods: We evaluated the relationship between the ERCC2 Asp312Asn and Lys751Gln polymorphisms and OS among surgically resected Stage I and II NSCLC patients. Genotyping was done using modified PCR-RFLP methods, Kaplan-Meier methods and log-rank test were used to compare OS by polymorphism status. Cox proportional hazards models were used to adjust for possible confounding variables. Results: There were 405 patients in this study. Median age 69 yrs; 51% male; 52% Stage IA, 29% IB, 4% IIA, 15% IIB. 63% lobectomy; 23% wedge resection; 7% pneumonectomy; 3% sleeve lobectomy; 3% bilobectomy; 1% other. 49% adenocarcinoma; 29% squamous; 12% BAC; 4% large cell; 6% other NSCLC. Genotype frequencies were in Hardy-Weinberg equilibrium: Asp/Asp 40%, Asp/Asn 45%, Asn/Asn 15%; Lys/Lys 44%, Lys/Gln 41%, Gln/Gln 15%. The two polymorphisms were linked. Median follow-up time was 5.9 yrs. There were 209 deaths. Median survival time (MST) was longer among patients carrying the variant Asn or Gln allele: MST Arg/Arg 5.4 yrs; Arg/Asn or Asn/Asn 7.5 yrs (log-rank test, p = 0.06); MST Lys/Lys 5.0 yrs; Lys/Gln or Gln/Gln 7.6 yrs (log-rank test, p < 0.01). On univariate analysis, age, sex, stage, BAC histology, and the Lys751Gln polymorphism were significantly associated with OS, while the Arg312Asn polymorphism had a borderline association. After adjusting for age, sex, stage, and BAC histology, carrying the Asn or Gln variant alleles was associated with improved OS (HR 0.72, 95% CI 0.55–0.95, p = 0.02; HR 0.63, 95% CI 0.47–0.83, p < 0.01; respectively). Conclusions: Carrying the variant allele of the ERCC2 Arg312Asn or Lys751Gln polymorphism is associated with improved OS in surgically resected Stage I/II NSCLC. Supported by NIH grants CA074386, CA092824, 5T32CA071345. No significant financial relationships to disclose.

Repair activity of base and nucleotide excision repair enzymes for guanine lesions induced by nitrosative stress

Nitric oxide (NO) induces deamination of guanine, yielding xanthine and oxanine (Oxa). Furthermore, Oxa reacts with polyamines and DNA binding proteins to form cross-link adducts. Thus, it is of interest how these lesions are processed by DNA repair enzymes in view of the genotoxic mechanism of NO. In the present study, we have examined the repair capacity for Oxa and Oxa–spermine cross-link adducts (Oxa–Sp) of enzymes involved in base excision repair (BER) and nucleotide excision repair (NER) to delineate the repair mechanism of nitrosative damage to guanine. Oligonucleotide substrates containing Oxa and Oxa–Sp were incubated with purified BER and NER enzymes or cell-free extracts (CFEs), and the damage-excising or DNA-incising activity was compared with that for control (physiological) substrates. The Oxa-excising activities of Escherichia coli and human DNA glycosylases and HeLa CFEs were 0.2–9% relative to control substrates, implying poor processing of Oxa by BER. In contrast, DNA containing Oxa–Sp was incised efficiently by UvrABC nuclease and SOS-induced E.coli CFEs, suggesting a role of NER in ameliorating genotoxic effects associated with nitrosative stress. Analyses of the activity of CFEs from NER-proficient and NER-deficient human cells on Oxa–Sp DNA confirmed further the involvement of NER in the repair of nitrosative DNA damage.

The response of Cryptosporidium parvum to UV light

Ultraviolet (UV) light is being considered as a disinfectant by the water industry because it appears to be very effective for controlling potential waterborne pathogens, including Cryptosporidium parvum. However, many organisms have mechanisms such as nucleotide excision repair and photolyase enzymes for repairing UV-induced DNA damage and regaining preirradiation levels of infectivity or population density. Genes encoding UV repair proteins exist in C. parvum, so the parasite should be able to regain infectivity following exposure to UV. Nevertheless, there is an increasing body of evidence that the organism is unable to reactivate following UV irradiation. This paper describes the effective inactivation of C. parvum by UV light, identifies nucleotide excision repair genes in the C. parvum and Cryptosporidium hominis genomes and discusses the inability of UV-exposed oocysts to regain infectivity.

ERCC2 Genotypes and a Corresponding Haplotype Are Linked with Breast Cancer Risk in a German Population

Abstract The polygenic concept of breast cancer susceptibility calls for the identification of genetic variants that contribute to breast cancer risk. Reduced DNA repair proficiencies in women with breast cancer pointed to a possible role of DNA repair enzymes in the risk to develop the disease. The nucleotide excision repair enzyme encoded by the excision repair cross-complementing group 2 gene ERCC2 (formerly XPD) known to cause skin cancer by germ line mutations has multiple regulatory cellular functions, including nucleotide excision repair, basal transcription, cell cycle control, and apoptosis. ERCC2 polymorphisms ERCC2_6540_G&amp;gt;A (Asp312Asn) and ERCC2_18880_A&amp;gt;C (Lys751Gln) within the coding region of this evolutionarily highly conserved gene have been of functional relevance and therefore are potential candidates to confer breast cancer susceptibility. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, we analyzed genotype frequencies in constitutional DNA of study participants of a German case-control study that included 688 cases of incident breast cancer and 724 population-based, age-matched controls. We identified ERCC2_6540_GG (Asp312Asp) as an at-risk genotype [odds ratio (OR), 2.06; 95% confidence interval (95% CI), 1.39-3.07]. The ERCC2_6540_GG-associated breast cancer risk was even higher in women who were also carriers of the ERCC2_18880_CC (Gln751Gln) genotype (OR, 3.69; 95% CI, 1.76-7.74). We identified ERCC2_6540_G/ERCC2_18880_C (Asp312/Gln751) as the most potent risk-conferring haplotype (OR, 3.49; 95% CI, 2.30-5.28). To our knowledge, this is the first study assigning breast cancer risk to both the ERCC2 genotype encoding Asp312Asp and the haplotype encoding Asp312/Gln751.

Single Nucleotide Polymorphisms of DNA Synthetic and Repair Enzymes and Risk of Childhood Acute Lymphoblastic Leukemia.

Although the outcome of children with acute lymphoblastic leukemia (ALL) has been improving, the origin of this disease is still unknown. It is likely that individual genetic susceptibility and environmental exposure play a role in carcinogenesis. To maintain normal hematopoiesis, DNA synthesis and repair systems need to be intact. Folate metabolism, producing purines and pyrimidines, is the source of nucleotides while base excision and nucleotide excision repair enzymes, i.e. XRCC1 and XPD, have important roles in protection of DNA damage. Single nucleotide polymorphisms (SNPs) of enzymes involving these processes alter their enzymatic structures and functions. We hypothesized that SNPs of folate metabolic enzymes including methylenetetrahydrofolate reductase (MTHFR 677 C>T; 1298 A>C), methionine synthase (MS 2756 A>G), and tandem repeats of thymidylate synthase (TS 2R or 3R) and of DNA repair enzymes (XRCC1 26304 C>T; 27466 G>A; 28152 G>A and XPD 23591 G>A; 35931 A>C) could effect the risk of ALL in children. We performed genotyping of these SNPs in 108 ALL and 317 normal controls by using PCR RFLP method. Genotype frequencies of folate metabolic enzymes SNPs were not different between cases and controls. However, TS 2R allele showed a trend to decrease risk of ALL (OR=0.66; 95% CI 0.41–1.07). We observed a 4.5 fold reduction in the risk of ALL in individuals with the XRCC1 26304 TT genotype (OR=0.22; 95% CI 0.5–0.96). XRCC1 26304 T allele showed a protective effect to this disease (OR=0.67; 95% CI 0.47–0.97). In contrast, individuals with XRCC1 28152 A allele had an increased risk of ALL (OR=1.67; 95% CI 1.2–2.33) compared to those with G allele. XRCC1 28152 GA and GA+AA genotypes were significantly higher among patients with ALL compared to controls (OR=2.17; 95% CI 1.37–3.45 and OR=2.18; 95%CI 1.38–3.42). No association between genotype or allele frequencies of both XPD SNPs and risk of ALL was shown in this study. In conclusion, our data demonstrated that SNPs of DNA repair enzymes (XRCC1 26304 C>T and 28152 G>A) but not folate-dependent DNA synthetic enzymes may influence risk of childhood ALL.