Nucleotide Excision Repair Mechanism Research Articles

Detection of bulky DNA lesions: DDB2 at the interface of chromatin and DNA repair in eukaryotes

Bulky DNA damage is corrected by the nucleotide excision repair (NER) pathway. Although the core biochemical mechanism of NER is understood, details including lesion recognition and repair in the context of chromatin remain to be elucidated. As more data become available, the complexity of lesion recognition in chromatin is becoming clear. This review will discuss current knowledge of DNA damage recognition in the context of chromatin, with a focus on the roles of chromatin remodeling and the specific lesion recognition protein DDB2 (DNA damage-binding protein 2) in chromatin repair. Additionally, we propose a model that ubiquitination-mediated DDB2 dissociation from chromatin, not its degradation, is important for GG-NER progression.

Mutagenic repair of DNA interstrand crosslinks

Formation of DNA interstrand crosslinks (ICLs) in chromosomal DNA imposes acute obstruction of all essential DNA functions. For over 70 years bifunctional alkylators, also known as DNA crosslinkers, have been an important class of cancer chemotherapeutic regimens. The mechanisms of ICL repair remains largely elusive. Here, we review a eukaryotic mutagenic ICL repair pathway discovered by work from several laboratories. This repair pathway, alternatively termed recombination-independent ICL repair, involves the incision activities of the nucleotide excision repair (NER) mechanism and lesion bypass polymerase(s). Repair of the ICL is initiated by dual incisions flanking the ICL on one strand of the double helix; the resulting gap is filled in by lesion bypass polymerases. The remaining lesion is subsequently removed by a second round of NER reaction. The mutagenic repair of ICL likely interacts with other cellular mechanisms such as the Fanconi anemia pathway and recombinational repair of ICLs. These aspects will also be discussed.

Abstract 1875: Dietary grape seed proanthocyanidins induce rapid repair of DNA damage via nucleotide excision repair genes in preventing UV-induced immunosuppression

Abstract Ultraviolet (UV) radiation-induced immunosuppression has been implicated in the development of skin cancers, including melanoma and non-melanoma. UV-induced DNA damage, predominantly the formation of cyclobutane pyrimidine dimers (CPDs), has been recognized as an important molecular trigger for the initiation of UVB-induced immunosuppression and skin carcinogenesis. We have shown earlier that administration of dietary grape (Vitis vinifera) seed proanthocyanidins (GSPs) supplemented with AIN76A control diet inhibit photocarcinogenesis in mice; however, the molecular mechanism of chemopreventive effects of GSPs are not clearly understood. To investigate the possible mechanism of action of GSPs we used genetically modified human cells and animal model. We observed that administration of dietary GSPs (0.2 and 0.5%, w/w) with supplementation of AIN76A control diet inhibited (52-65%, P<0.001) UV-induced suppression of contact hypersensitivity response to a contact sensitizer, 2,4-dinitrofluorobenzene, in a contact hypersensitivity mouse (C3H/HeN) model. Dietary GSPs repaired UV-induced DNA damage (CPD-positive cells) faster in the skin of mice as demonstrated by reduced number of CPD-positive cells (59%, p<0.001), and reduced the migration of CPD-positive cells from the skin to draining lymph nodes compared to non-GSPs-fed control mice, which was associated with the elevated levels of nucleotide excision repair (NER) genes. GSPs did not prevent UV-induced immunosuppression in NER-deficient mice but significantly prevented in NER-proficient mice (p<0.001) concomitantly repaired UV-induced DNA damage in NER-proficient mice (p<0.01) but did not repair in NER-deficient mice as indicated by immunohistochemical analysis of CPD-positive cells. Further, southwestern dot-blot analysis revealed that GSPs repaired UV-induced CPDs in xeroderma pigmentosum complementation group A (XPA)-proficient cells obtained from healthy person but did not repair in XPA-deficient cells obtained from the patients suffering from xeroderma pigmentosum disease, indicating that NER mechanism is involved in DNA repair. Together, these data identify a novel mechanism by which dietary GSPs prevent UV-induced immunosuppression which is mediated through rapid repair of damaged DNA, and that these effects lead to the prevention of UV radiation-induced skin tumors in mice. 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 1875.

Green Tea Polyphenols Prevent UV-Induced Immunosuppression by Rapid Repair of DNA Damage and Enhancement of Nucleotide Excision Repair Genes

UV radiation-induced immunosuppression has been implicated in the development of skin cancers. Green tea polyphenols (GTP) in drinking water prevent photocarcinogenesis in the skin of mice. We studied whether GTPs in drinking water (0.1-0.5%, w/v) prevent UV-induced immunosuppression and (if so) potential mechanisms of this effect in mice. GTPs (0.2% and 0.5%, w/v) reduced UV-induced suppression of contact hypersensitivity (CHS) in response to a contact sensitizer in local (58-62% reductions; P < 0.001) and systemic (51-55% reductions; P < 0.005) models of CHS. Compared with untreated mice, GTP-treated mice (0.2%, w/v) had a reduced number of cyclobutane pyrimidine dimer-positive (CPD(+)) cells (59%; P < 0.001) in the skin, showing faster repair of UV-induced DNA damage, and had a reduced (2-fold) migration of CPD(+) cells from the skin to draining lymph nodes, which was associated with elevated levels of nucleotide excision repair (NER) genes. GTPs did not prevent UV-induced immunosuppression in NER-deficient mice but significantly prevented it in NER-proficient mice (P < 0.001); immunohistochemical analysis of CPD(+) cells indicated that GTPs reduced the numbers of UV-induced CPD(+) cells in NER-proficient mice (P < 0.001) but not in NER-deficient mice. Southwestern dot-blot analysis revealed that GTPs repaired UV-induced CPDs in xeroderma pigmentosum complementation group A (XPA)-proficient cells of a healthy person but did not in XPA-deficient cells obtained from XPA patients, indicating that a NER mechanism is involved in DNA repair. This study is the first to show a novel NER mechanism by which drinking GTPs prevents UV-induced immunosuppression and that inhibiting UV-induced immunosuppression may underlie the chemopreventive activity of GTPs against photocarcinogenesis.

DNA Polymerase POLN Participates in Cross-Link Repair and Homologous Recombination

All cells rely on DNA polymerases to duplicate their genetic material and to repair or bypass DNA lesions. In humans, 16 polymerases have been identified, and each bears specific functions in genome maintenance. We identified here the recently discovered polymerase POLN to be involved in repair of DNA cross-links. Such DNA lesions are highly toxic and are believed to be repaired by the sequential activity of nucleotide excision repair, translesion synthesis, and homologous recombination mechanisms. By functionally assaying its role in these processes, we unraveled an unexpected involvement of POLN in homologous recombination. Moreover, we obtained evidence for physical and functional interaction of POLN with factors belonging to the Fanconi anemia pathway, a master regulator of cross-link repair. Finally, we show that POLN interacts and cooperates in DNA repair with the helicase HEL308, which shares a common origin with POLN in the Drosophila mus308 gene. Our data indicate that this novel polymerase-helicase complex participates in homologous recombination repair and is essential for cellular protection against DNA cross-links.

Abstract A228: Noninvasive magnetic resonance spectroscopic PD markers of a minor-groove interstrand cross-linking agent (BN2629) in human colon carcinoma and melanoma xenografts

Abstract BN2629 (also known as SJG-136) is a novel pyrrolobenzodiazepine dimer analogue that binds covalently in the minor groove of DNA, inducing little or no distortion of the double helix. Hence, it evades the p53-mediated detection and up-regulation of nucleotide excision repair mechanisms but induces apoptosis. It has just completed Phase 1 clinical trials in the USA (NCI) and UK (CR-UK), and has been approved by the NCI for Phase II evaluation. The aim of this work was to develop a robust and non-invasive surrogate marker for tumor response following BN2629 treatment. We carried out an in vivo 31phosphorus magnetic resonance spectroscopy (31P MRS) study of BN2629 (80 g/kg via ip for 3 days) and vehicle in HT29 human colon carcinoma and LOX IMVI melanoma xenograft models. In vitro 31P MRS were performed on tumor extracts. Significant growth delays (p&amp;lt;0.0001) were observed in both BN2629-treated HT29 and LOX IMVI xenografts when compared with controls. In vivo31P MRS of the HT29 xenografts showed an increase in the phosphomonoester/total phosphorus signals (PME/TotP) (p=0.04) and PME/ - NTP ratios (p&amp;lt;0.02) after 3 days of BN2629 treatment. No significant changes were observed in the control group. In vitro 31P MRS of extracts from BN2629-treated HT29 tumors showed significant increases in phosphoethanolamine (PE; p&amp;lt;0.01), glycerophosphocholine (GPC; p&amp;lt;0.01) and glycerophosphoethanolamine (GPE; p&amp;lt;0.03) when compared with controls. In vivo 31P MRS of the LOX IMVI xenografts showed an increase in - NTP/TotP ratio ( -NTP/TotP) (p&amp;lt;0.01) after 3 days of BN2629 treatment. No significant changes were observed in the control group. In vitro 31P MRS of extracts from BN2629-treated LOX IMVI tumors showed significant increases in GPC (p&amp;lt;0.001), GPE (p&amp;lt;0.01) and ATP (p=0.04) when compared with controls. Treatment with BN2629 resulted in tumor growth delay and altered bioenergetic and phospholipid metabolism in vivo, which are consistent with metabolic changes observed following treatment with agents such as 5-FU [1] and cyclophosphamide [2]. The phospholipid changes may be associated with alteration in membrane turnover following treatment, and these changes may have potential as surrogate non-invasive markers for determining tumor response following treatment with BN2629. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A228.

Differential survival of Escherichia coli uvrA, uvrB, and uvrC mutants to psoralen plus UV-A (PUVA): Evidence for uncoupled action of nucleotide excision repair to process DNA adducts

The nucleotide excision repair mechanism (NER) of Escherichia coli is responsible for the recognition and elimination of more than twenty different DNA lesions. Herein, we evaluated the in vivo role of NER in the repair of DNA adducts generated by psoralens (mono- or bi-functional) and UV-A light (PUVA) in E. coli. Cultures of wild-type E. coli K12 and mutants for uvrA, uvrB, uvrC or uvrAC genes were treated with PUVA and cell survival was determined. In parallel, kinetics of DNA repair was also evaluated by the comparison of DNA sedimentation profiles in all the strains after PUVA treatment. The uvrB mutant was more sensitive to PUVA treatment than all the other uvr mutant strains. Wild-type strain, and uvrA and uvrC mutants were able to repair PUVA-induced lesions, as seen by DNA sedimentation profiles, while the uvrB mutant was unable to repair the lesions. In addition, a quadruple fpg nth xth nfo mutant was unable to nick PUVA-treated DNA when the crude cell-free extract was used to perform plasmid nicking. These data suggest that DNA repair of PUVA-induced lesions may require base excision repair functions, despite proficient UvrABC activity. These results point to a specific role for UvrB protein in the repair of psoralen adducts, which appear to be independent of UvrA or UvrC proteins, as described for the classical UvrABC endonuclease mechanism.

Two-Step Recognition of DNA Damage for Mammalian Nucleotide Excision Repair: Directional Binding of the XPC Complex and DNA Strand Scanning

For mammalian nucleotide excision repair (NER), DNA lesions are recognized in at least two steps involving detection of unpaired bases by the XPC protein complex and the subsequent verification of injured bases. Although lesion verification is important to ensure high damage discrimination and the accuracy of the repair system, it has been unclear how this is accomplished. Here, we show that damage verification involves scanning of a DNA strand from the site where XPC is initially bound. Translocation by the NER machinery exhibits a 5'-to-3' directionality, strongly suggesting involvement of the XPD helicase, a component of TFIIH. Furthermore, the initial orientation of XPC binding is crucial in that only one DNA strand is selected to search for the presence of lesions. Our results dissect the intricate molecular mechanism of NER and provide insights into a strategy for mammalian cells to survey large genomes to detect DNA damage.

Trabectedin: an anticancer drug from the sea

Background: Trabectedin (ET-743) is an anticancer agent originally isolated from Ecteinascidia turbinata, a marine organism. Objective: The goal of this review is to describe the chemical characteristics, mechanism of action and the results of clinical trials with this compound. The toxicities are described, as well as the pharmacokinetics. The regulatory affairs and marketing strategies for this compound are also discussed. Methods: Medline and meeting proceedings were searched to accomplish our objectives. Results/conclusions: Trabectedin is a unique alkylating agent. It affects a variety of transcription factors, cell proliferation, and nucleotide excision repair mechanism. In addition, it inhibits the MDR-1 gene, which is responsible for the resistance of cancer cells to chemotherapeutic agents. The main toxicities of this agent are transaminitis and myelosuppression, both reversible and noncumulative. At present, trabectedin is approved in Europe for the treatment of sarcoma. The combination of this compound with other chemotherapeutics has been tested in Phase I studies in sarcomas and is feasible. This drug in combination with doxil has shown to improve outcome in relapsed ovarian cancer, compared with doxil alone. The results of this study may lead to an FDA approval of the combination in the USA, for the treatment of relapsed ovarian cancer.

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.

DNA repair in mammalian cells : Nucleotide excision repair: variations on versatility.

Nucleotide excision repair (NER) is one of the most versatile DNA repair systems. It can be subdivided into several, differentially regulated, subpathways: global genome repair (GGR), transcription-coupled repair (TCR), and transcription domain-associated repair (DAR). This review begins with a brief overview of the numerous types of DNA lesions handled by NER, and proceeds to describe in detail the molecular mechanisms of NER. It then addresses heterogeneities in NER activity in physiological situations (e. g. in differentiated cells) and explores the underlying regulatory mechanism. It then reviews several inherited diseases associated with NER deficiencies: xeroderma pigmentosum, Cockayne syndrome, trichothiodystrophy, UV-sensitive syndrome. It concludes by discussing several currently unresolved issues, relating either to the cause of the above diseases or to the mechanistic details of the various NER subpathways and of their regulation. (Part of a Multi-author Review).

Crosslinking of the NER damage recognition proteins XPC-HR23B, XPA and RPA to photoreactive probes that mimic DNA damages

A new assay to probe the mechanism of mammalian nucleotide excision repair (NER) was developed. Photoreactive arylazido analogues of dNMP in DNA were shown to be substrates for the human NER system. Oligonucleotides carrying photoreactive “damages” were prepared using the multi-stage protocol including one-nucleotide gap filling by DNA polymerase β using photoreactive dCTP or dUTP analogues followed by ligation of the resulting nick. Photoreactive 60-mers were annealed with single-stranded pBluescript II SK (+) and subsequently primer extension reactions were performed. Incubation of HeLa extracts with the plasmids containing photoreactive moieties resulted in an excision pattern typical of NER. DNA duplexes containing photoreactive analogues were used to analyze the interaction of XPC-HR23B, RPA, and XPA with damaged DNA using the photocrosslinking assay. Crosslinking of the XPC-HR23B complex with photoreactive 60-mers resulted in modification of its XPC subunit. RPA crosslinked to ssDNA or mismatched dsDNA more efficiently than to dsDNA, whereas XPA did not show a preference for any of the DNA species. XPC and XPA photocrosslinking to DNA decreased in the presence of Mg 2+ whereas RPA crosslinking to DNA was not sensitive to this cofactor. Our data establish a photocrosslinking assay for the investigation of the damage recognition step in human nucleotide excision repair.

Replication-coupled repair of crotonaldehyde/acetaldehyde-induced guanine-guanine interstrand cross-links and their mutagenicity.

The repair of acetaldehyde/crotonaldehyde-induced guanine (N2)-guanine (N2) interstrand cross-links (ICLs), 3-(2-deoxyribos-1-yl)-5,6,7,8-(N2-deoxyguanosyl)-6(R or S)-methylpyrimido[1,2-alpha]purine-10(3H)-one, was studied using a shuttle plasmid bearing a site-specific ICL. Since the authentic ICLs can revert to monoadducts, a chemically stable model ICL, 1,3-bis(2'-deoxyguanos-N2-yl)butane derivative, was also employed to probe the ICL repair mechanism. Since the removal of ICL depends on the nucleotide excision repair (NER) mechanism in Escherichia coli, the plasmid bearing the model ICL failed to yield transformants in NER-deficient host cells, proving the stability of this ICL in cells. The authentic ICLs yielded transformants in the NER-deficient hosts; therefore, these transformants are produced by plasmid bearing spontaneously reverted monoadducts. In contrast, in NER-deficient human cells, the model ICL was removed by an NER-independent repair pathway, which is unique to higher eukaryotes. This repair did not associate with a transcriptional event, but with replication. The analysis of repaired molecules revealed that the authentic and model ICLs were repaired mostly (>94%) in an error-free manner in both hosts. The major mutations that were observed were G --> T transversions targeting the cross-linked dG located in the lagging strand template. These results support one of the current models for the mammalian NER-independent ICL repair mechanism, in which a DNA endonuclease(s) unhooks an ICL from the leading strand template at a stalled replication fork site by incising on both sides of the ICL and then translesion synthesis is conducted across the "half-excised" ICL attached to the lagging strand template to restore DNA synthesis.

Inhibition of nucleotide excision repair (NER) by microcystin-LR in CHO-K1 cells

Microcystin-LR (MC-LR), a potent inhibitor of PP1 and PP2A protein phosphatases, is related to tumor promotion and initiation. Although the genotoxic properties of this toxin have been extensively investigated with a variety of non-mammalian and mammalian test systems, the existing results are contradictory. Based on our previous results regarding the impact of MC-LR on the processes of DNA repair we decided to examine in greater detail its effect on the capacity of nucleotide excision repair (NER). CHO-K1 cells were pre-treated with increasing doses of MC-LR (1, 10 and 20 μg/ml) and then exposed to UV radiation (25 J/m 2). Apoptosis was analyzed to exclude the possibility of false positive results in the comet assay. The results suggest that MC-LR targets the nucleotide excision repair mechanisms by interference with the incision/excision phase as well as the rejoining phase of NER and leads to an increased level of UV-induced cytogenetic DNA damage in CHO-K1 cells.

(−)-Epigallocatechin-3-Gallate Prevents Photocarcinogenesis in Mice through Interleukin-12–Dependent DNA Repair

We have shown previously that topical application of (-)-epigallocatechin-3-gallate (EGCG), the major polyphenol of green tea, prevents photocarcinogenesis in mice. EGCG prevents UVB-induced immunosuppression by inducing interleukin-12 (IL-12). As immunosuppression is a risk factor for photocarcinogenesis, we investigated the possibility that EGCG also prevents UVB-induced photocarcinogenesis through an IL-12-dependent DNA repair mechanism. To investigate this possibility, we determined the effects of EGCG on photocarcinogenesis in IL-12 knockout (KO) mice using the formation of cyclobutane pyrimidine dimers (CPD) as an indicator of the extent of UVB-induced DNA damage. Topical application of EGCG (1 mg/cm(2) skin) prevented photocarcinogenesis in wild-type (C3H/HeN) mice in terms of tumor incidence and tumor multiplicity but did not prevent photocarcinogenesis in IL-12 KO mice. UVB-induced DNA damage, as determined by the formation of CPDs and the number of sunburn cells, was resolved more rapidly in the skin of wild-type mice treated with EGCG than untreated control mice. In contrast, the extent of UVB-induced DNA damage and the numbers of sunburn cells were not significantly different in the EGCG-treated IL-12 KO mice and untreated control mice. In addition, treatment of XPA-proficient human fibroblast cells with EGCG promoted repair of UVB-induced CPDs in a dose-dependent manner but not in an XPA-deficient cells, indicating that the nucleotide excision repair mechanism is involved in EGCG-mediated DNA repair. Taken together, these results indicate for the first time that EGCG can prevent photocarcinogenesis through an EGCG-induced IL-12-dependent DNA repair mechanism.

Prevention of Ultraviolet Radiation–Induced Immunosuppression by (−)-Epigallocatechin-3-Gallate in Mice Is Mediated through Interleukin 12–Dependent DNA Repair

Solar UV radiation-induced immunosuppression is considered to be a risk factor for melanoma and nonmelanoma skin cancers. We previously have shown that topical application of (-)-epigallocatechin-3-gallate (EGCG) prevents UV-induced immunosuppression in mice. We studied whether prevention of UV-induced immunosuppression by EGCG is mediated through interleukin 12 (IL-12)-dependent DNA repair. IL-12 knockout (KO) mice on C3H/HeN background and DNA repair-deficient cells from xeroderma pigmentosum complementation group A (XPA) patients were used in this study. The effect of EGCG was determined on UV-induced suppression of contact hypersensitivity and UV-induced DNA damage in the form of cyclobutane pyrimidine dimers (CPD) in mice and XPA-deficient cells using immunohistochemistry and dot-blot analysis. Topical treatment with EGCG prevented UV-induced suppression of the contact hypersensitivity in wild-type (WT) mice but did not prevent it in IL-12 KO mice. Injection of anti-IL-12 monoclonal antibody to WT mice blocked the preventive effect of EGCG on UV-induced immunosuppression. EGCG reduced or repaired UV-induced DNA damage in skin faster in WT mice as shown by reduced number of CPDs(+) cells and reduced the migration of CPD(+) antigen-presenting cells from the skin to draining lymph nodes. In contrast, this effect of EGCG was not seen in IL-12 KO mice. Further, EGCG was able to repair UV-induced CPDs in XPA-proficient cells obtained from healthy person but did not repair in XPA-deficient cells, indicating that nucleotide excision repair mechanism is involved in DNA repair. These data identify a new mechanism by which EGCG prevents UV-induced immunosuppression, and this may contribute to the chemopreventive activity of EGCG in prevention of photocarcinogenesis.

Repair of DNA interstrand cross-links: Interactions between homology-dependent and homology-independent pathways

DNA interstrand cross-links (ICLs) are complex DNA lesions generated by bifunctional alkylating agents, a class of compounds extensively used in cancer chemotherapy. Formation of an ICL covalently links the opposing strands of the double helix and results in severe disruptions of normal DNA functions, such as replication, transcription, and recombination. Because of the structural complexity, ICLs are most likely recognized by a variety of repair recognition proteins and processed through multiple mechanisms. To study the involvement of different repair pathways in ICL processing, we examined a variety of mammalian mutants with distinct DNA repair deficiencies. We found that the presence of ICLs induces frequent recombination between direct repeat sequences, suggesting that the single-strand annealing pathway may be an important mechanism for the removal of ICLs situated within direct repeats. Unlike recombination-independent ICL repair, ICL-induced single-strand annealing does not require the nucleotide excision repair (NER) mechanism. In cells defective in the mismatch repair protein Msh2, the level of recombination-independent ICL repair was significantly increased, suggesting that processing by the mismatch repair mechanism may lead to recombinational repair of ICLs. Our results suggest that removal of ICLs may involve two error-prone mechanisms depending on the sequence context of the cross-linked site.

A Role for Polymerase η in the Cellular Tolerance to Cisplatin-Induced Damage

Mutation of the POLH gene encoding DNA polymerase eta (pol eta) causes the UV-sensitivity syndrome xeroderma pigmentosum-variant (XP-V) which is linked to the ability of pol eta to accurately bypass UV-induced cyclobutane pyrimidine dimers during a process termed translesion synthesis. Pol eta can also bypass other DNA damage adducts in vitro, including cisplatin-induced intrastrand adducts, although the physiological relevance of this is unknown. Here, we show that independent XP-V cell lines are dramatically more sensitive to cisplatin than the same cells complemented with functional pol eta. Similar results were obtained with the chemotherapeutic agents, carboplatin and oxaliplatin, thus revealing a general requirement for pol eta expression in providing tolerance to these platinum-based drugs. The level of sensitization observed was comparable to that of XP-A cells deficient in nucleotide excision repair, a recognized and important mechanism for repair of cisplatin adducts. However, unlike in XP-A cells, the absence of pol eta expression resulted in a reduced ability to overcome cisplatin-induced S phase arrest, suggesting that pol eta is involved in translesion synthesis past these replication-blocking adducts. Subcellular localization studies also highlighted an accumulation of nuclei with pol eta foci that correlated with the formation of monoubiquitinated proliferating cell nuclear antigen following treatment with cisplatin, reminiscent of the response to UV irradiation and further indicating a role for pol eta in dealing with cisplatin-induced damage. Together, these data show that pol eta represents an important determinant of cellular responses to cisplatin, which could have implications for acquired or intrinsic resistance to this key chemotherapeutic agent.

The uvrA gene is involved in oxidative and acid stress responses in Lactobacillus helveticus CNBL1156

The uvrA gene of Lactobacillus helveticus CNBL1156 coding for subunit A of the excinuclease ABC complex involved in the nucleotide excision repair mechanism was identified. Analysis of the uvrA locus revealed the presence of three open reading frames, merR, sat and uvrA, which coded respectively for a MerR-like regulatory protein, a putative protein with homology to streptothricin acetyl transferase and for a UvrA protein. RNA analysis by northern blotting and RT-PCR showed that sat and uvrA were transcriptionally coupled. UvrA from L. helveticus contained the conserved domains of bacterial excinuclease A, as well as the two ATP binding sites and the zinc binding domains. The transcriptional activity of uvrA indicated that this gene was activated by exposure to UV radiation and oxidative stress. In addition, we observed that the expression of uvrA was inducible by pH; moreover, the role of UvrA in protection against stress was confirmed by acid adaptation experiments. Pretreatment of cells at pH 5 conferred resistance to H 2O 2, suggesting a specific adaptive response to pH-induced DNA damage. The results from this study indicate that UvrA contributes to acid and oxidative tolerance in L. helveticus, and suggest that it plays a role in survival at low pH under normal conditions.

Participation of BER and NER pathways in the repair of DNA lesions induced at low N-nitrosodiethylamine concentrations

In the present work, we evaluated ( p < 0.05) the participation of base excision repair (BER) and nucleotide excision repair (NER) mechanisms in repairing DNA lesions induced by N-nitrosodiethylamine (NDEA) at 1.5 ng/mL–36.5 μg/mL, through cell survival, in different single and double Escherichia coli DNA repair mutants ( uvrA, uvrB, uvrC, fpg, nth, xthA, fpg/nth, uvrA/fpg, fpg/xthA, mutY, and fpg/mutY), using pre-incubation periods of 90 min. Mutant strains BH20 ( fpg) and AB1886 ( uvrA) showed microsomal enzyme (S9 mix) independent NDEA cytotoxicity. Cytotoxicity was also detected at lowest NDEA concentrations, in the presence of S9 mix, with strains BH980 ( mutY) and BH990 ( fpg/mutY). NDEA cytotoxicity, without S9 mix, was detected for mutant strains AB1884 ( uvrC) and AB1885 ( uvrB). Through SOS chromotest with 90 min of pre-incubation for uvrA and nth strains, only NER was shown to be required for repairing NDEA-induced lesions with or without metabolic activation. PQ37 and PQ66 strains, both uvrA mutants, showed different levels of NDEA sensitivity. The findings suggest that, under the used conditions, and at low concentrations, NDEA-induced lesions require both repair pathways.