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DNA repair mechanisms counteract the formation of deleterious DNA lesions and maintain genomic integrity. Nucleotide excision repair (NER) is an important DNA repair pathway because of its extraordinarily large substrate specificity. P53 protein regulates NER pathway in a transcription-dependent or transcription-independent manner. Inherited polymorphisms of NER pathway genes (XPC, HR23B, XPA, DDB2, XPB, XPD, ERCC1, XPF, and XPG) and TP53 gene may contribute to individual variations in genetic susceptibility to OSCC and correlate with the prognosis of 204 OSCC patients. We carried out a hospital-based case-control study to investigate the association of 25 various polymorphisms of nine NER pathway genes and TP53 gene with the risk for OSCC. There were 34 newly diagnosed OSCC patients and 135 frequency-matched controls without BQ chewing and smoking habit as well as 313 newly diagnosed OSCC patients with BQ chewing or smoking habit and 312 frequency-matched controls being recruited between November 2003 and July 2007 at Kaoshiung Veterans General Hospital. Genotyping was performed using the PCR-RFLP techniques or TaqMan real-time PCR method. The significant association between polymorphisms of NER pathway genes and OSCC risk was mainly found among subjects with BQ chewing or smoking habit. In the single locus analysis, GA and AA genotypes of ERCC1 G-641A (AOR, 0.64; 95% CI, 0.45-0.93 and AOR, 0.48; 95% CI, 0.29-0.79, respectively; p for trend, 0.002), CT genotype of XPF C-850T (AOR, 1.53; 95% CI, 1.08-2.18; p for trend, 0.014), as well as GG genotype of XPB A-1039G (AOR, 0.51; 95% CI, 0.26-0.98; p for trend 0.034) were significantly associated with the risk of OSCC. Furthermore, -641G/ -425T or -641G/ -425C haplotype of ERCC1 (AORs, 1.34; 95% CI, 1.02-1.77 and AOR, 1.56; 95% CI, 1.18-2.07, respectively; p for trend 0.002) as well as -850T/ -247T and -850T/ -247C haplotype of XPF (AOR, 1.45; 95% CI, 1.09-1.94 for; p for trend 0.016) were strongly associated with the risk of OSCC. A trend toward increased risk of OSCC was observed when people with the increasing number of at risk genotypes in the combined analyses of nine NER pathway genes with (p for trend, <0.001) or without (p for trend 0.001) TP53 gene. Finally, in the stratification analysis, the combined effects of nine NER pathway genes had a significantly increased risk of OSCC among younger group (¡Ø50 years old), Fukienece population, BQ chewers, light smokers, or light drinkers. Besides, in the prognosis analysis of 204 OSCC patients, HR23B A-823C, polymorphisms of XPA gene, XPD C-643G, XPG C787G, and the number of at risk genotypes of NER pathway genes were associated with pathologic stage, T classification, or N classification. The association between NER genetic polymorphisms and survival of patients was only found in XPA C-1778T polymorphism. These results suggested that the single polymorphism of XPB A-1039G, ERCC1 G-641A and XPF C-850T, the joint effect of genetic polymorphisms of NER pathway genes, and gene-environment combined effect were associated with the risk of OSCC. Furthermore, in the analysis of NER genetic polymorphisms and prognosis of OSCC, we found polymorphisms of XPA gene might be a prognostic factor for OSCC.

In response to DNA damage, mammalian cells activate various DNA repair pathways to remove DNA lesions and, meanwhile, halt cell cycle progressions to allow sufficient time for repair. The nucleotide excision repair (NER) and the ATR-dependent cell cycle checkpoint activation are two major cellular responses to DNA damage induced by UV irradiation. However, how these two processes are coordinated in the response is poorly understood. Here we showed that the essential NER factor XPA (xeroderma pigmentosum group A) underwent nuclear accumulation upon UV irradiation, and strikingly, such an event occurred in an ATR (Ataxia-Telangiectasia mutated and RAD3-related)-dependent manner. Either treatment of cells with ATR kinase inhibitors or transfection of cells with small interfering RNA targeting ATR compromised the UV-induced XPA nuclear translocation. Consistently, the ATR-deficient cells displayed no substantial XPA nuclear translocation while the translocation remained intact in ATM (Ataxia-Telangiectasia mutated)-deficient cells in response to UV irradiation. Moreover, we found that ATR is required for the UV-induced nuclear focus formation of XPA. Taken together, our results suggested that the ATR checkpoint pathway may modulate NER activity through the regulation of XPA redistribution in human cells upon UV irradiation.

The nucleotide excision repair pathway (NER) has been shown to efficiently remove bulky base lesions from the DNA, including those induced by solar light. It has been suggested that the NER pathway may be involved also in removing smaller oxidative base lesions from the DNA. Oxidative damage in the cell is caused by cellular aerobic respiration, with base damage to the nucleotides of the DNA being the most biologically relevant. One of the most common oxidative base lesions in the genome is the 7 ,8-dihydro-8-oxoguanine (8-oxoG). This lesion is pre-mutagenic since it can base pair with equal efficiency to the correct cytosine base, or the incorrect adenine base during DNA replication. Oxidative damage, including 8-oxoG, is repaired primarily by the base excision repair (BER) pathway, which is a multi-step, multi-protein pathway similar to NER. One key protein involved in both BER and NER is the p53 protein, which can act as a transcription factor and protein regulator to influence DNA repair. We have used a recombinant non-replicating human adenovirus, Ad5HCMVlacZ, which expresses the ~-galactosidase (~-gal) reporter gene, to examine the role of several NER proteins and the p53 protein in the BER of oxidative damage in human cells. Methylene blue (MB) acts as a photosenstizer, and after irradiation by visible light (VL) produces reactive oxygen species that cause 8-0xoG in the DNA. By infecting several normal, NER deficient and p53 deficient -tumor, primary and transformed fibroblast cell lines with a MB+VL-treated Ad5HCMVlacZ reporter construct, we were able to determine the host cell reactivation (HCR) of the oxidatively damaged reporter. Results indicate that the HCR of the MB+VL-treated reporter and the expression of p53 are enhanced by UVC pretreatment in normal human fibroblasts, suggesting that p53 may be involved in inducible BER. In addition, increased expression of p53 facilitated by pre-infection of normal cells with p53 expressing Ad5p53wt similarly enhanced HCR in the normal fibroblasts, giving further evidence that increased expression of p53 alone enhances BER. In contrast, although UVC pretreatment of p53 compromised cells resulted in enhanced HCR, the enhanced HCR did not correlate with enhanced p53 expression, suggesting that enhancement in BER can result from both p53 dependent and p53 independent mechanisms. We report also that HCR of the MB+VL-treated reporter gene was substantially reduced in SV40-transformed XP-C cells, with little or no reduction in SV40-transformed XPA, XPD, XPF, XPG and CSB cells, suggesting a role for the XPC protein in the BER ofMB+VL-induced DNA damage. In particular, the XPC protein appears to be involved in both the constitutive and inducible aspects of BER, as the HCR of the MB + VL-treated reporter was reduced in 3 UVC pretreated as well as untreated XP-C primary human fibroblast strains. In addition, pre-infection of cells with Ad5p53wt, resulted in an enhanced HCR of normal but not XP-C deficient fibroblasts consistent with a p53 dependent involvement of the XPC protein in BER of MB+VL-treated DNA. Additional studies were also conducted to determine the cell sensitivity of normal and NER deficient SV40-transformed cell lines to MB and MB+VL. The results show that MB alone and MC+VL are toxic to cells, and that cells deficient in NER are not more sensitive to MC or MB+VL compared to NER proficient normal cells. In fact, the NER deficient cell lines were more resistant to MB alone compared to NER proficient normal cells. In particular, although the SV40- transformed XP-C cell line showed a significant reduction in HCR of the MB-Vl-treated reporter gene, suggesting a deficiency in the repair of MB+VL-induced DNA damage, the SV40-transformed XP-C cells were not more sensitive to MB or MB+VL. This suggests that the toxicity of human cells to MB and MB+VL results primarily from damage to cellular components other than DNA such as membrane structures including the mitochondria and lysozomes as has been reported for other photosensitizers. / Thesis / Master of Science (MSc)

The stability of DNA is a critical factor for several diseases, the most prevalent of which is cancer. Several neurodegenerative and accelerated aging diseases are also characterized by genomic instability. The number and complexity of DNA repair pathways that human cells possess underscores the importance of genomic stability. These pathways ensure that damaged DNA is repaired and that a cells complement of DNA remains stable upon cell division. How one particular type of DNA alteration, a DNA loop, is processed in human cells was the focus of this study. We have employed an in vitro system to study defined DNA loop substrates by human nuclear extracts. The influence of either a 5 or 3 nick, the range of loop sizes processed, and the role of DNA mismatch repair, DNA nucleotide excision repair, and the Werner Syndrome helicase proteins were variables tested. The results indicate tha t DNA loops containing between 5 to 12 nucleotides are processed in a strand - specific manner when either a 5 or 3 nick is present , with repair being targeted solely to the nicked strand . This repair occurs by both mismatch repair dependent and independent pathways. The processing of DNA loops containing 30 nucleotides in length is directed either by a 5 nick, or by the loop itself, but not by a 3 nick. The nick independent pathway results solely in loop removal. The large loop pathway is independent of mismatch repair, nucleotide excision repair, and the WRN helicase/exonuclease protein. Both of the 5 nick directed pathways occur by excision that initiates at the pre- existing nick and proceeds towards the loop along the shortest path between the nick and loop. DNA resynthesis occurs using either DNA polymerase , , or and also initiates at the pre-existing 5 nick. The 3 nick directed intermediate loop repair pathway proceeds in a similar fashion, likely after a nick is made 5 to the loop region on the strand that contained the pre-existing nick. DNA synthesis inhibition has only a minor affect on the nick independent loop removal pathway as only a short tract of DNA surrounding the loop site is processed. In total, the results point to at least 3 novel pathways that process DNA loops that likely contribute to total genomic stability.

Nucleotide excision repair (NER) is one of the major repair systems for removal of DNA lesions. The NER pathway has evolved mainly to repair UV-induced DNA damage and is also active against a broad range of endogenously generated oxidative lesions. Defects in NER result in the human inherited disorder xeroderma pigmentosum (XP), which is characterised by UV hypersensitivity and a 1000-fold increased risk of skin cancer. ERCC1 is essential for the NER pathway where it acts in a complex with the XPF protein to make the incision 5' to the DNA lesion. The normal 1.1kb Ercc1 transcript is expressed in all tissues. Our group has discovered a second larger 1.5 kb transcript, which initiates from an alternative promoter, and is the most abundant Ercc1 transcript in mouse skin. The aims of this project were: 1, To investigate the role of ERCC1 and of the 1.5kb skin specific Ercc1 transcript in protecting the skin against UV-induced DNA damage. 2, To study the importance of ERCC1 in melanoma skin cancer and investigate ERCC1as a possible target for therapy against melanoma. Using a panel of Ercc1 wild-type and deficient cells, we established a quantitative western blotting system to study the expression of ERCC1 in a range of mouse tissues and mouse and human cell types. Although the skin-specific Ercc1 transcript was found to be present at much higher levels in the skin of albino compared to pigmented mouse strains, this did not result in an elevated level of ERCC1 protein. We were also unable to demonstrate that UV-irradiation, or other stress-inducing treatments resulted in increased levels of ERCC1 protein in cultured mouse keratinocytes. We investigated the DNA methylation status of the normal Ercc1 promoter and that of two potential upstream promoter regions that were candidates for the source of the 1.5kb skin-specific Ercc1 transcript. We found no evidence that they were the source and, instead, used 5' RACE analysis to locate the skin-specific promoter to a polymorphic region 500bp upstream of the normal initiation site. In albino strains this region contains a SINE element, which we hypothesize could be involved in the production of the skin-specific Ercc1 transcript. We also investigated the protein level of ERCC1 and other DNA repair proteins, including XPF, MSH2, MSH6 and MLH1 in human melanoma cells and ovarian tumour cells. Significantly elevated protein levels of ERCC1 and XPF, as well as the mismatch repair protein MLH1 were found in melanoma cells. This could possibly contribute to the higher resistance to chemotherapy in melanoma, although the melanoma cell lines we tested did not show increased resistance to UV and cisplatin compared to the ovarian cancer cells tested. When Ercc1 proficient mouse melanoma cells were xenografted into nude mice the xenografts grew rapidly. Cisplatin treatment caused an initial shrinkage of the tumours, but re-growth rapidly followed. Cells re-isolated into culture from cisplatin treated xenografts had significantly higher levels of ERCC1 protein than either input cells, or cells re-isolated from untreated xenografts. An isogenic Ercc1 deficient derivative of the Ercc1 proficient mouse melanoma cell line grew as rapidly as the parent line in vitro, but grew much more slowly as xenografts. In addition, the xenografts shrank completely following cisplatin treatment and did not recover. This suggests that ERCC1 could be a drug target for melanoma therapy.

Résumé : Les télomères sont des structures nucléoprotéiques spécialisées qui assurent la stabilité du génome en protégeant les extrémités chromosomiques. Afin d’empêcher des activités indésirables, la réparation des dommages à l’ADN doit être convenablement régulée au niveau des télomères. Pourtant, il existe peu d’études de la réparation des dommages induits par les ultraviolets (UVs) dans un contexte télomérique. Le mécanisme de réparation par excision de nucléotides (NER pour « Nucleotide Excision Repair ») permet d’éliminer les photoproduits. La NER est un mécanisme très bien conservé de la levure à l’humain. Elle est divisée en deux sous voies : une réparation globale du génome (GG-NER) et une réparation couplée à la transcription (TC-NER) plus rapide et plus efficace. Dans notre modèle d’étude, la levure Saccharomyces cerevisiae, une forme compactée de la chromatine nommée plus fréquemment « hétérochromatine » a été décrite. Cette structure particulière est présente entre autres, au niveau des régions sous-télomériques des extrémités chromosomiques. La formation de cette chromatine particulière implique quatre protéines nommées Sir (« Silent Information Regulator »). Elle présente différentes marques épigénétiques dont l’effet est de réprimer la transcription. L’accès aux dommages par la machinerie de réparation est-il limité par cette chromatine compacte ? Nous avons donc étudié la réparation des lésions induites par les UVs dans différentes régions associées aux télomères, en absence ou en présence de protéines Sir. Nos données ont démontré une modulation de la NER par la chromatine, dépendante des nucléosomes stabilisés par les Sir, dans les régions sous-télomériques. La NER était moins efficace dans les extrémités chromosomiques que dans les régions plus proches du centromère. Cet effet était dépendant du complexe YKu de la coiffe télomérique, mais pas dépendant des protéines Sir. La transcription télomériques pourrait aider la réparation des photoproduits, par l’intermédiaire de la sous-voie de TC-NER, prévenant ainsi la formation de mutations dans les extrémités chromosomiques. Des ARN non codants nommés TERRA sont produits mais leur rôle n’est pas encore clair. Par nos analyses, nous avons confirmé que la transcription des TERRA faciliterait la NER dans les différentes régions sous-télomériques. / Abstract : Telomeric DNA is made of short tandem repeats located at the ends of chromosomes and their maintenance is critical to prevent genome instability. DNA lesions constitute a serious risk to genome integrity. Thus, DNA repair mechanisms are required for continuous and unabridged cell divisions. The nucleotide excision repair (NER) pathway removes bulky DNA lesions such as UV-induced photoproducts, like the cyclobutane pyrimidine dimers (CPD). NER is divided in two sub-pathways: global genome repair (GGR) and the faster transcription-coupled repair (TCR), which only differ in how they recognize UV-induced lesions. In eukaryotes, NER must find and repair DNA lesions that are buried in nucleosomes. In the yeast S. cerevisiae, genes positioned close to telomeres are silenced by a heterochromatin-like structure that is formed by silent information regulator proteins (Sir). To determine if nucleosomes and chromatin in subtelomeric regions affect the efficiency of NER, we studied the repair of photoproducts in different telomere-associated regions in both, WT and SIR genes deleted cells (sirΔ). We found that NER efficiency was modulated by the presence of nucleosomes on the subtelomeric type X element. In addition, in absence of Sir proteins, NER efficiency increased and was not modulated by nucleosomes, indicating that nucleosome positioning was less defined in sirΔ cells. Remarkably, in telomeric restriction fragment, NER was less efficient at telomeres than in the subtelomere type Y’ element. We suggest that low NER efficiency at the very end of chromosomes results from attachment sites to the nuclear periphery. Our data indicate that NER in sub-telomeric chromatin is modulated by Sir proteins stabilized-nucleosomes, and that NER is inhibited in telomeric chromatin by the presence of YKu, independently from the presence of Sir proteins. It was recently shown that the chromosome ends are transcribed and a non-coding RNA, called TERRA, is produced. Currently the precise functions of TERRA are not understood. Our second goal is to help understand the function of TERRA. We think that transcription at the chromosome ends could facilitate the removal of DNA lesions from heterochromatin by TCR, which would prevent the formation of mutations and, ultimately, chromosome shortening. Our data showed that TC-NER is effective in Y’ element and the telomere. Without Sir proteins, TERRA transcription is found in a particular region at the end of the X element. The transcription of TERRA could improve the repair of UV-induced lesions.

A radiação ultravioleta (UV) pode ser absorvida por diferentes moléculas celulares, incluindo o DNA no qual provoca distorções estruturais. As lesões mais comuns induzidas pela radiação UV são o ciclobutano de pirimidina (CPD) e o fotoproduto (6-4)-pirimidina-pirimidona [(6-4)PPs]. Estas lesões podem ser reparadas pela fotorreativação, caracterizada por ter uma única proteína (fotoliase) que remove lesões empregando luz visível (320-500 nm) como fonte de energia. Foram identificados dois tipos de fotoliases que diferem por sua especificidade ao substrato: CPD-fotoliase e (6-4)-fotoliase. Um outro mecanismo de reparo é o reparo por excisão de nucleotídeos (NER), um mecanismo que envolve múltiplos passos e proteínas. Enquanto os efeitos genotóxicos da UVC e UVB já estão relativamente esclarecidos e bem aceitos, ainda existem controvérsias sobre a genotoxicidade da radiação UVA, devido ao fato de ser fracamente absorvida pelo DNA. Alguns autores acreditam que os seus principais efeitos são gerados de forma indireta pela produção de espécies reativas de oxigênio enquanto outros acreditam que a UVA pode gerar danos ao DNA de forma direta, provocando a formação de dímeros de pirimidina. O objetivo deste trabalho foi verificar os efeitos genotóxicos da radiação UVA em fibroblastos humanos deficientes e proficientes em NER utilizando adenovírus recombinantes contendo uma ou outra fotoliase para verificar se as lesões CPD e (6-4)PP são geradas pela UVA e se elas teriam alguma importância nas respostas verificadas após irradiação. Foi verificado que as células deficientes no gene XPA são mais sensíveis à radiação UVA quando comparadas às células selvagens. Por meio da detecção imunológica, confirmamos a geração das lesões CPD, (6-4)PP e Dewar, fotoisômero da lesão (6-4)PP, após irradiação com UVA no genoma de células humanas. Empregando vetores adenovirais para transdução de fotoliase específica para lesões tipo CPD ou (6-4)PP, confirmamos que de fato essas lesões são formadas em células humanas deficientes em reparo de DNA após irradiação com UVA. Além disso, esses vírus permitiram verificar a relevância biológica dessas lesões na indução de morte celular em células XP-A irradiadas. De fato, os dados indicam que para doses baixas de radiação UVA essas lesões desempenham um importante papel na indução de morte. Não podemos descartar, porém, que lesões indiretas (provavelmente geradas por estresse oxidativo) também tenham papel na indução de morte pela radiação UVA, o que parece ser mais importante a doses médias e altas dessa radiação. / Ultraviolet radiation (UV) is absorbed by different cellular molecules, including DNA in which induces structural distortions. The most common lesions induced by UV radiation are the cyclobutane pyrimidine (CPD) and the photoproduct (6-4)-pyrimidine-pyrimidone [(6-4)PP]. These lesions can be repaired by the photoreactivation, characterized by a single protein (photolyase) that removes lesions using visible light (320-500 nm) as energy source. Two types of photolyases had been identified that differ by their substrate specificity: CPD-photolyase and (6-4)-photolyase. Another repair mechanism is the nucleotide excision repair (NER), a mechanism that involves multiple steps and proteins. While the genotoxic effects of UVB and UVC are already relatively well-understood and accepted, there is still controversy about the genotoxicity of UVA radiation, due to its low absorption by DNA. Some authors believe that the major effects are generated indirectly by the production of reactive oxygen species, while others believe that UVA can cause damage to DNA directly, inducing the formation of pyrimidine dimers. The aim of this study was to assess the genotoxic effects of UVA radiation in human fibroblasts deficient and proficient in NER, using recombinant adenovirus expressing the photolyases to verify if CPDs and (6-4)PPs are generated by UVA and whether they had any importance in the responses observed after irradiation. It was found that cells deficient in the XPA gene are more sensitive to UV radiation compared to wild type cells. By immunological detection, we confirm the generation of CPD, (6-4)PP and Dewar, photoisomer of the (6-4)PP lesion, in the genome of human cells after irradiation with UVA. Using adenoviral vectors for the transduction of photolyases specific for CPD or (6-4)PP lesions, we confirm that in fact these lesions are generated in human cells deficient in DNA repair after irradiation with UVA. Moreover, these viruses allowed us to verify the biological relevance of these lesions in the induction of cell death in irradiated XP-A cells. In fact, our data indicates that for low doses of UVA radiation, these lesions play important roles in the induction of death. We cannot rule out, however, that indirect lesions (probably caused by oxidative stress) could also have a role in the induction of death by UVA radiation, which seems to be more important in intermediate and high doses of this radiation.

DNA in cells is constantly damaged by both endogenous and exogenous genotoxic agents. DNA repair is a cellular machinery that counters DNA damage and thus preserve genome integrity. Nucleotide excision repair (NER) in Escherichia coli (E. coli) is one of the DNA repair systems that recognizes and removes a variety of DNA damage such as pyrimidine dimers, bulky chemical adducts, DNA intrastrand cross-links, etc. The genes responsible for E. coli NER incisions are UvrA, UvrB, and UvrC. As the first step of E. coli NER, DNA damage recognition is achieved through the UvrA2B complex. Purification of UvrA, UvrB, and UvrC is essential for research to understand the molecular mechanisms of NER and carcinogenesis. Although UvrA, a 115 kDa protein, has been successfully purified in our lab in the past, the experimental procedures were very time-consuming and technically challenging. In this study we employed IMPACT (Intein Mediated Purification with an Affinity Chitin-binding Tag) system to subclone the cDNA of UvrA and express and purify the recombinant UvrA protein by a single-column step using the cloned expression construct. Furthermore, the purified protein was found to be fully functional in the UvrABC incision assay in which the DNA adduct of FABP [N-(20-deoxyguanosin-8-yl)-4-fluoro-4-aminobiphenyl] was efficiently cleaved in a time course-dependent manner.

Lung cancer is a particularly devastating disease, accounting for the most deaths among all cancer types in the United States. Despite a reduction in the country’s smoking rates, cigarette smoking remains the number one risk factor for lung cancer. Additionally arsenic exposure, which occurs primarily through contaminated drinking water in the U.S., is associated with increased lung cancer incidence. The nucleotide excision repair (NER) pathway is critical for maintenance of genomic fidelity, removing DNA lesions that could otherwise promote DNA mutations and drive carcinogenesis. Tobacco smoking introduces significant amounts of DNA damage and produces characteristic DNA mutations found in lung cancers of smokers, and arsenic increases lung cancer risk in smokers beyond the risk of smoking along. The contributions of these chemicals to DNA damage and cancer have been well documented, but few studies have examined their effects on DNA repair pathways, particularly the nucleotide excision repair (NER) pathway. Arsenic, while not directly mutagenic, promotes the carcinogenicity of other compounds including agents that produce DNA damage that is repaired by the NER pathway. In this dissertation I investigated the effects of cigarette smoke condensate (CSC, a whole-smoke tobacco surrogate) and arsenic on NER. I observed that CSC or arsenic treatment inhibited NER as measured by a slot-blot assay using UV-induced photolesions as model substrates to measure NER. The abundance of Xeroderma Pigmentosum complementation group C (XPC), a critical NER protein, was significantly reduced in all lines treated with either chemical, while XPA protein was unaffected. CSC and arsenic also affected RNA levels of certain NER genes. Finally, proteasome-regulated XPC turnover was affected by CSC and arsenic treatment, suggesting a potential mechanism for XPC protein inhibition. The observed impairment of NER by CSC is critically important in tobacco cancer etiology – CSC introduces DNA damage, some of which is repaired exclusively by NER, and CSC inhibits the NER pathway as well, providing a two-sided assault on cellular genetic fidelity. I then adapted the NER assay to measure repair in lymphocytes isolated from human subjects of a study investigating the high incidence of lung cancer in Appalachian Kentucky. I observed an age-dependent decline in NER efficiency that was modulated by subject smoking status and a reduced NER efficiency among current smokers in the lung cancer patient population compared to control subjects in the youngest age group, suggesting individual DNA repair capacity measured with this repair assay may be a biomarker for lung cancer susceptibility.