DNA repair capacity: inconsistency between effect of over-expression of five NER genes and the correlation to mRNA levels in primary lymphocytes

We have previously shown that high DNA repair capacity protects psoriasis patients against chemically induced basal cell carcinoma [Dybdahl et al. Mutat. Res. 433 (1999) 15-22]. We have used the same study persons to investigate the correlation between expression of eight genes involved in nucleotide excision repair and DNA repair capacity. mRNA levels of XPA, XPB, XPC, XPD, XPF, XPG, CSB and ERCC1 in primary lymphocytes from 33 individuals were quantified by dot-blots and normalized to beta-actin. ERCC1 and XPD mRNA quantities were highly correlated (r=0.89; P<10(-11)) while XPA, XPB, XPC, XPG, XPFand CSB mRNAs were moderately correlated (r=0.2-0.7). Thus, the mRNA expressions seem to fall in at least two groups. There was a three to sevenfold variation in the expression levels of the mRNAs. This is in contrast to the more than a hundredfold variation in mRNA levels reported in cancer patients.DNA repair capacity was measured in a host cell reactivation assay, where primary lymphocytes were transfected with an UV-irradiated plasmid encoding firefly-luciferase. Only ERCC1 and XPD mRNA levels correlated with the DNA repair capacity (P<0.03). In order to see if ERCC1 or XPD activity was limiting for DNA repair, we cotransfected with plasmids encoding NER genes, thus over-expressing either XPB, XPC, XPD, CSB or ERCC1 in the host cell reactivation assay. Only XPB over-expression increased DNA repair capacity. Thus, there is no indication that neither XPD nor ERCC1 limits the DNA repair capacity. However, our results indicate that ERCC1 and XPD mRNA levels may be used as a proxy for DNA repair capacity in lymphocytes.     

Molecular analysis of DNA repair gene methylation and protein expression during chemical-induced rat lung carcinogenesis

A defective ratio between DNA damage and repair may result in the occurrence of a malignant phenotype. Previous studies have found that many genetic alterations in DNA repair genes occur frequently in lung cancer. However, the epigenetic mechanisms underlying this tumorigenesis are not clear. Herein, we have used a chemical-induced rat lung carcinogenesis model to study the evolution of methylation alterations of DNA repair genes BRCA1, ERCC1, XRCC1, and MLH1. Methylation-specific PCR and immunohistochemistry were used to analyze gene methylation status and protein expression during the progression of lung carcinogenesis. Promoter hypermethylation of BRCA1 was only detected in three samples of infiltrating carcinoma. CpG island hypermethylation of ERCC1, XRCC1, and MLH1 was found to increase gradually throughout lung carcinogenesis progression. Both the prevalence of at least one methylated gene and the average number of methylated genes were heightened in squamous metaplasia and dysplasia compared with normal tissue and hyperplasia, and was further increased in carcinoma in situ (CIS) and infiltrating carcinoma. Immunohistochemical analysis showed that BRCA1 and MLH1 protein expression decreased progressively during the stages of lung carcinogenesis, whereas ERCC1 and XRCC1 expression were only found in later stages. Although methylation levels were elevated for ERCC1 and XRCC1 during carcinogenesis, an inverse correlation with protein expression was found only for BRCA1 and MLH1. These results suggest that a continuous accumulation of DNA repair gene hypermethylation and the consequent protein alterations might be a vital molecular mechanism during the process of multistep chemical-induced rat lung carcinogenesis.     

Effect of carcinogenic acrolein on DNA repair and mutagenic susceptibility

Acrolein (Acr), a ubiquitous environmental contaminant, is a human carcinogen. Acr can react with DNA to form mutagenic α- and γ-hydroxy-1, N(2)-cyclic propano-2'-deoxyguanosine adducts (α-OH-Acr-dG and γ-OH-Acr-dG). We demonstrate here that Acr-dG adducts can be efficiently repaired by the nucleotide excision repair (NER) pathway in normal human bronchial epithelia (NHBE) and lung fibroblasts (NHLF). However, the same adducts were poorly processed in cell lysates isolated from Acr-treated NHBE and NHLF, suggesting that Acr inhibits NER. In addition, we show that Acr treatment also inhibits base excision repair and mismatch repair. Although Acr does not change the expression of XPA, XPC, hOGG1, PMS2 or MLH1 genes, it causes a reduction of XPA, XPC, hOGG1, PMS2, and MLH1 proteins; this effect, however, can be neutralized by the proteasome inhibitor MG132. Acr treatment further enhances both bulky and oxidative DNA damage-induced mutagenesis. These results indicate that Acr not only damages DNA but can also modify DNA repair proteins and further causes degradation of these modified repair proteins. We propose that these two detrimental effects contribute to Acr mutagenicity and carcinogenicity.     

Involvement of Nucleotide Excision and Mismatch Repair Mechanisms in Double Strand Break Repair

Living organisms are constantly threatened by environmental DNA-damaging agents, including UV and ionizing radiation (IR). Repair of various forms of DNA damage caused by IR is normally thought to follow lesion-specific repair pathways with distinct enzymatic machinery. DNA double strand break is one of the most serious kinds of damage induced by IR, which is repaired through double strand break (DSB) repair mechanisms, including homologous recombination (HR) and non-homologous end joining (NHEJ). However, recent studies have presented increasing evidence that various DNA repair pathways are not separated, but well interlinked. It has been suggested that non-DSB repair mechanisms, such as Nucleotide Excision Repair (NER), Mismatch Repair (MMR) and cell cycle regulation, are highly involved in DSB repairs. These findings revealed previously unrecognized roles of various non-DSB repair genes and indicated that a successful DSB repair requires both DSB repair mechanisms and non-DSB repair systems. One of our recent studies found that suppressed expression of non-DSB repair genes, such as XPA, RPA and MLH1, influenced the yield of IR induced micronuclei formation and/or chromosome aberrations, suggesting that these genes are highly involved in DSB repair and DSB-related cell cycle arrest, which reveals new roles for these gene products in the DNA repair network. In this review, we summarize current progress on the function of non-DSB repair-related proteins, especially those that participate in NER and MMR pathways, and their influence on DSB repair. In addition, we present our developing view that the DSB repair mechanisms are more complex and are regulated by not only the well known HR/NHEJ pathways, but also a systematically coordinated cellular network.Key Words: Ionizing radiation (IR), DNA damage, DSB repair, NER, MMR and cell cycle.     

Order of assembly of human DNA repair excision nuclease.

Human excision nuclease removes DNA damage by concerted dual incisions bracketing the lesion. The dual incisions are accomplished by sequential and partly overlapping actions of six repair factors, RPA, XPA, XPC, TFIIH, XPG, and XPF.ERCC1. Of these, RPA, XPA, and XPC have specific binding affinity for damaged DNA. To learn about the role of these three proteins in damage recognition and the order of assembly of the excision nuclease, we measured the binding affinities of XPA, RPA, and XPC to a DNA fragment containing a single (6-4) photoproduct and determined the rate of damage excision under a variety of reaction conditions. We found that XPC has the highest affinity to DNA and that RPA has the highest selectivity for damaged DNA. Under experimental conditions conducive to binding of either XPA + RPA or XPC to damaged DNA, the rate of damage removal was about 5-fold faster for reactions in which XPA + RPA was the first damage recognition factor presented to DNA compared with reactions in which XPC was the first protein that had the opportunity to bind to DNA. We conclude that RPA and XPA are the initial damage sensing factors of human excision nuclease.     

DNA repair gene polymorphisms and risk of early onset colorectal cancer in Lynch syndrome

DNA repair plays a pivotal role in maintaining genomic integrity with over 130 genes involved in various repair pathways that include base excision repair, nucleotide excision repair, double strand break repair and DNA mismatch repair. Polymorphisms within genes that are involved in these processes have been widely reported to be associated with cancer susceptibility in an extensive range of malignancies that include colorectal cancer (CRC). Lynch syndrome is caused by inherited germline mutations in DNA mismatch repair genes, predominantly in MLH1 and MSH2, that predispose to a variety of epithelial malignancies, most notably CRC. Despite being a relatively well understood hereditary cancer syndrome there remain several questions in relation to genetic influences on disease expression. Since Lynch syndrome is associated with a breakdown in DNA mismatch repair variation in other DNA repair genes may influence disease expression. In this report we have genotyped 424 Australian and Polish Lynch syndrome participants for eight common DNA repair gene polymorphisms to assess any association with the age of CRC onset. The DNA repair gene SNPs included in the study were: BRCA2 (rs11571653), MSH3 (rs26279), Lig4 (rs1805386), OGG1 (rs1052133), XRCC1 (rs25487), XRCC2 (rs3218536 and rs1799793) and XRCC3 (rs861539). Cox multi-variant regression modelling failed to provide any convincing evidence of an effect in any of the polymorphisms analysed. The data suggest that polymorphisms in DNA repair genes do not contribute to cancer risk in a population of CRC patients who are at increased risk of disease as a result in a deficiency of DNA mismatch repair.     

Polymorphisms of DNA repair genes are associated with renal cell carcinoma

DNA repair gene alterations have been shown to cause a reduction in DNA repair capacity and may influence an individual's susceptibility to carcinogenesis. Single nucleotide polymorphisms (SNPs) of DNA repair genes have been shown to cause a reduction in repair activity. We hypothesized that SNPs of DNA repair genes may be a risk factor for renal cell carcinoma (RCC). To test this hypothesis, DNA samples from 112 cases of renal cell cancer and healthy controls (n=180) were analyzed by PCR-RFLP to determine the genotypic frequency of six different polymorphic loci on five DNA repair genes (XRCC1, XPC, ERCC1, XRCC3, and XRCC7). The chi(2) test was applied to compare the genotype frequency between patients and controls. We found that the frequency of 399Gln variant at XRCC1 Arg399Gln was significantly higher in RCC cases than in controls (OR=2.83, 95%CI=1.24-6.49, P=0.01). The frequency of T-A haplotype of XRCC1 194 Trp and XRCC1 399Gln was significantly higher in RCC than controls. No differences in genotypes were observed at the other sites. This is the first report on SNPs of DNA repair genes in renal cell carcinoma that suggests XRCC1 399Gln polymorphism may be a risk factor for RCC. Our present data suggest that the XRCC1 399Gln allele may be linked to susceptibility for RCC.     

Analysis of DNA repair gene polymorphisms in glioblastoma

Background

Glioblastoma is the most common and aggressive primary brain tumor in adults. Despite several factors such as ionizing radiation exposure or rare genetic syndromes have been associated with the development of glioblastoma, no underlying cause has been identified for the majority of cases. We thus aimed to investigate the role of DNA repair polymorphisms in modulating glioblastoma risk.

Methods

Genotypic and allelic frequencies of seven common polymorphisms in DNA repair genes involved in nucleotide excision repair (ERCC1 rs11615, ERCC2 rs13181, ERCC6 rs4253079), base excision repair (APEX1 rs1130409, XRCC1 rs25487), double-strand break repair (XRCC3 rs861539) and mismatch repair (MLH1 rs1800734) pathways were analyzed in 115 glioblastoma patients and 200 healthy controls. Haplotype analysis was also performed for ERCC1 rs11615 and ERCC2 rs13181 polymorphisms, located on the same chromosomal region (19q13.32).

Results

Our results indicated that carriers of the ERCC2 Gln/Gln genotype were associated with a lower glioblastoma risk (OR = 0.32, 95% CI 0.12–0.89; P = 0.028), whereas carriers of the MLH1 AA genotype were associated with an increased risk of glioblastoma (OR = 3.14, 95% CI 1.09–9.06; P = 0.034). Furthermore, the haplotype containing the C allele of ERCC2 rs13181 polymorphism and the T allele of ERCC1 rs11615 polymorphism was significantly associated with a protective effect of developing glioblastoma (OR = 0.34, 95% CI 0.16–0.71; P = 0.004).

Conclusions

These results pointed out that MLH1 rs1800734 and ERCC2 rs13181 polymorphisms might constitute glioblastoma susceptibility factors, and also suggested that the chromosomal region 19q could be important in glioblastoma pathogenesis.     

Epidermal growth factor and ionizing radiation up-regulate the DNA repair genes XRCC1 and ERCC1 in DU145 and LNCaP prostate carcinoma through MAPK signaling

This work examined the importance of radiation-induced and ligand-induced EGFR-ERK signaling for the regulation of DNA repair proteins XRCC1 and ERCC1 in prostate carcinoma cells, DU145 (TP53(mut)), displaying EGFR-TGFA-dependent autocrine growth and high MAPK (ERK1/2) activity, and LNCaP (TP53(wt)) cells expressing low constitutive levels of ERK1/2 activity. Using quantitative RT-PCR and Western analyses, we determined that ionizing radiation activated the DNA repair genes XRCC1 and ERCC1 in an ERK1/2-dependent fashion for each cell line. After irradiation, a rapid increase followed by a decrease in ERK1/2 activity preceded the increase in XRCC1/ERCC1 expression in DU145 cells, while only the rapid decrease in ERK1/2 preceded the increase in XRCC1/ERCC1 expression in LNCaP cells. Administration of EGF, however, markedly increased the up-regulation of phospho-ERK, ERCC1 and XRCC1 in both cell lines. Although the EGFR inhibitor tyrphostin (AG-1478) and the MEK inhibitor PD90859 both attenuated EGF-induced levels of the ERCC1 and XRCC1 protein, PD98059 blocked the induction of ERCC1 and XRCC1 by radiation more effectively in both cell lines. Inhibition of ERK at a level that reduced the up-regulation of DNA repair led to the persistence of apurinic/apyrimidinic (AP) sites of DNA damage and increased cell killing. Taken together, these data imply a complex control of DNA repair activation that may be more generally dependent on MAPK (ERK1/2) signaling than was previously noted. These data provide novel insights into the capacity of the EGFR-ERK signaling to modulate DNA repair in cancer cells and into the functional significance of this signaling.     

Properties and applications of human DNA repair genes

The importance of understanding DNA repair processes is discussed in terms of the origins of human cancer. Several human repair genes have been mapped to specific human chromosomes using somatic cell hybrids. It is noteworthy that 3 of these genes lie in the same region of chromosome 19: genes ERCC1 and ERCC2, which are involved in nucleotide excision repair, and XRCC1, which is involved in the repair of strand breaks. The genes XRCC1 and ERCC2 were cloned from cosmid libraries prepared from DNA transformants of the CHO mutants EM9 and UV5, respectively. Analysis of the cDNA sequence of ERCC2 showed that the protein encoded by this gene is highly homologous (73%) to the RAD3 repair protein in the yeast Saccharomyces cerevisiae. Thus, the known properties of RAD3 combined with the high homology provide the first insight about the biochemical role of a human repair protein involved in the incision step of nucleotide excision repair. So far XRCC1 is the only cloned mammalian gene involved in repairing damage from ionizing radiation. The UV5 mutant line was also applied to problems in environmental mutagenesis by introducing the mouse cytochrome P(3)450 (P450IA2 subfamily) gene for metabolic activation of aromatic amines. We show in a rapid differential cytotoxicity assay with 2 compounds found in cooked beef (IQ, 2-amino-3-methylimidazo[4,5-f]quinoline and PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine) that this gene is efficiently expressed in the transformed UV5P3 cells. Reversion of the repair deficiency in these cells will give a matched pair of cell lines that are metabolically proficient and repair deficient. Such lines will provide a rapid assay for genotoxic heterocyclic amines requiring activation.     

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

ABSTRACT: 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.     

A cell cycle-specific requirement for the XRCC1 BRCT II domain during mammalian DNA strand break repair

XRCC1 protein is essential for viability in mammals and is required for efficient DNA single-strand break repair and genetic stability following DNA base damage. We report here that XRCC1-dependent strand break repair in G(1) phase of the cell cycle is abolished by mutations created within the XRCC1 BRCT domain that interact with DNA ligase III. In contrast, XRCC1-dependent DNA strand break repair in S phase is largely unaffected by these mutations. These data describe a cell cycle-specific role for a BRCT domain, and we conclude that the XRCC1-DNA ligase III complex is required for DNA strand break repair in G(1) phase of the cell cycle but is dispensable for this process in S phase. The S-phase DNA repair process can remove both strand breaks induced in S phase and those that persist from G(1) and can in part compensate for lack of repair in G(1). This process correlates with the appearance of XRCC1 nuclear foci that colocalize with Rad51 and may thus function in concert with homologous recombination.     

Biochemical analysis of the damage recognition process in nucleotide excision repair

XPA, XPC-hHR23B, RPA, and TFIIH all are the damage recognition proteins essential for the early stage of nucleotide excision repair. Nonetheless, it is not clear how these proteins work together at the damaged DNA site. To get insight into the molecular mechanism of damage recognition, we carried out a comprehensive analysis on the interaction between damage recognition proteins and their assembly on damaged DNA. XPC physically interacted with XPA, but failed to stabilize the XPA-damaged DNA complex. Instead, XPC-hHR23B was effectively displaced from the damaged DNA by the combined action of RPA and XPA. A mutant RPA lacking the XPA interaction domain failed to displace XPC-hHR23B from damaged DNA, suggesting that XPA and RPA cooperate with each other to destabilize the XPC-hHR23B-damaged DNA complex. Interestingly, the presence of hHR23B significantly increased RPA/XPA-mediated displacement of XPC from damaged DNA, suggesting that hHR23B may modulate the binding of XPC to damaged DNA. Together, our results suggest that damage recognition occurs in a multistep process such that XPC-hHR23B initiates damage recognition, which was replaced by combined action of XPA and RPA. XPA and RPA, once forming a complex at the damage site, would likely work with TFIIH, XPG, and ERCC1-XPF for dual incision.     

Are genetic polymorphisms in OGG1, XRCC1 and XRCC3 genes predictive for the DNA strand break repair phenotype and genotoxicity in workers exposed to low dose ionising radiations?

Identification of higher risk individuals carrying genetic polymorphisms responsible for reduced DNA repair capacity has substantial preventive implications as these individuals could be targeted for cancer prevention. We have conducted a study to assess the predictivity of the OGG1, XRCC1 and XRCC3 genotypes and the in vitro single strand break repair phenotype for the induction of genotoxic effects. At the population level, a significant contribution of the OGG1 genotypes to the in vitro DNA strand break repair capacity was found. At an individual level, the OGG1 variants Ser/Cys and Cys/Cys genotypes showed a slower in vitro DNA repair than the Ser/Ser OGG1genotype. A multivariate analysis performed with genotypes, age, cumulative dose, exposure status and smoking as independent variables indicated that in the control population, repair capacity is influenced by age and OGG1 polymorphisms. In the exposed population, DNA damage is greater in older men and in smokers. Repair capacity is slower in individuals with Ser/Cys or Cys/Cys OGG1 genotypes compared to those with the Ser/Ser OGG1 genotype. Micronuclei (MN) frequencies increased with age and the cumulative dose of gamma-rays. Analysis of the total population revealed that genetic polymorphisms in XRCC1 resulted in higher residual DNA (RDNA) values and the Met/Met variant of XRCC3 resulted in an increased frequency of micronuclei. The analysis confirms that MN frequencies are reliable biomarkers for the assessment of genetic effects in workers exposed to ionising radiation (IR). A combined analysis of the three genotypes, OGG1, XRCC1 and XRCC3 polymorphisms is advised in order to assess individual susceptibility to ionising radiation. As an alternative or complement, the in vitro DNA strand break repair phenotype which integrates several repair pathways is recommended. Smokers with OGG1 polymorphisms who are exposed to ionising radiation represent a specific population requiring closer medical surveillance because of their increased mutagenic/carcinogenic risk.     

Repair of large insertion/deletion heterologies in human nuclear extracts is directed by a 5' single-strand break and is independent of the mismatch repair system

The repair of 12-, 27-, 62-, and 216-nucleotide unpaired insertion/deletion heterologies has been demonstrated in nuclear extracts of human cells. When present in covalently closed circular heteroduplexes or heteroduplexes containing a single-strand break 3' to the heterology, such structures are subject to a low level repair reaction that occurs with little strand bias. However, the presence of a single-strand break 5' to the insertion/deletion heterology greatly increases the efficiency of rectification and directs repair to the incised DNA strand. Because nick direction of repair is independent of the strand in which a particular heterology is placed, the observed strand bias is not due to asymmetry imposed on the heteroduplex by the extrahelical DNA segment. Strand-specific repair by this system requires ATP and the four dNTPs and is inhibited by aphidicolin. Repair is independent of the mismatch repair proteins MSH2, MSH6, MLH1, and PMS2 and occurs by a mechanism that is distinct from that of the conventional mismatch repair system. Large heterology repair in nuclear extracts of human cells is also independent of the XPF gene product, and extracts of Chinese hamster ovary cells deficient in the ERCC1 and ERCC4 gene products also support the reaction.     

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

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

8-OxoG retards the activity of the ligase III/XRCC1 complex during the repair of a single-strand break, when present within a clustered DNA damage site

Ionising radiation produces clustered DNA damage. Recent studies have established that the efficiency of excision of a lesion within clustered damage sites is reduced. This study presents evidence that the repair of clustered DNA damage is compromised, relative to that of the isolated lesions, since the lifetime of both lesions is extended by up to eight fold. Simple clustered damage sites, comprised of a single-strand break, one or five bases 3' or 5' to 8-oxoG on the opposite strand, were synthesised in oligonucleotides and repair carried out in XRS5 nuclear extracts. The rate of repair of the single-strand break within these clustered damage sites is reduced, mainly due to inhibition of the DNA ligase III/XRCC1 complex. The single-strand break, present as an isolated lesion, is repaired by short-patch base excision repair, however the mechanism of repair of the single-strand break within the clustered damage site is asymmetric. When the lesions are 5' to each other, the single-strand break is rejoined by short-patch repair whereas the rejoining of the single-strand break occurs by long-patch type repair when the lesions are 3' to one another. The retardation of DNA ligase III/XRCC1 complex, following addition of one base, is responsible for the initiation of long-patch base excision repair when the lesions are 3' to each other. The lesions within the cluster are processed sequentially, the single-strand break being repaired before excision of 8-oxoG, limiting the formation of double-strand breaks to <2%. Stalled processing of clustered DNA damage is suggested to have implications for mutation induction by radiation.     

Thermodynamic Cooperativity and Kinetic Proofreading in DNA Damage Recognition and Repair

In humans UV-induced cyclobutane thymine dimers are excised by the joint action of six repair factors, RPA, XPA, XPC, TFIIH, XPG, and XPF?ERCC1. Yet, in vitro assays show that none of these six factors is capable of detectably discriminating thymine dimer-containing DNA from undamaged DNA. We show how two elementary principles in macromolecular recognition, (1) cooperativity and (2) kinetic proofreading, are utilized to confer specificity to the repair system where none exists at the individual repair factor level and enable human cells to excise thymine dimers with a physiologically relevant specificity and at a biologically acceptable rate.