Nucleotide Excision DNA Repair Pathway Research Articles

High-Throughput Flow Cytometry Combined with Genetic Analysis Brings New Insights into the Understanding of Chromatin Regulation of Cellular Quiescence.

Cellular quiescence is a reversible differentiation state when cells are changing the gene expression program to reduce metabolic functions and adapt to a new cellular environment. When fission yeast cells are deprived of nitrogen in the absence of any mating partner, cells can reversibly arrest in a differentiated G0-like cellular state, called quiescence. This change is accompanied by a marked alteration of nuclear organization and a global reduction of transcription. Using high-throughput flow cytometry combined with genetic analysis, we describe the results of a comprehensive screen for genes encoding chromatin components and regulators that are required for the entry and the maintenance of cellular quiescence. We show that the histone acetylase and deacetylase complexes, SAGA and Rpd3, have key roles both for G0 entry and survival during quiescence. We reveal a novel function for the Ino80 nucleosome remodeling complex in cellular quiescence. Finally, we demonstrate that components of the MRN complex, Rad3, the nonhomologous end-joining, and nucleotide excision DNA repair pathways are essential for viability in G0.

Design and Structure-Guided Development of Novel Inhibitors of the Xeroderma Pigmentosum Group A (XPA) Protein-DNA Interaction.

XPA is a unique and essential protein required for the nucleotide excision DNA repair pathway and represents a therapeutic target in oncology. Herein, we are the first to develop novel inhibitors of the XPA-DNA interaction through structure-guided drug design efforts. Ester derivatives of the compounds 1 (X80), 22, and 24 displayed excellent inhibitory activity (IC50 of 0.82 ± 0.18 μM and 1.3 ± 0.22 μM, respectively) but poor solubility. We have synthesized novel amide derivatives that retain potency and have much improved solubility. Furthermore, compound 1 analogs exhibited good specificity for XPA over RPA (replication protein A), another DNA-binding protein that participates in the nucleotide excision repair (NER) pathway. Importantly, there were no significant interactions observed by the X80 class of compounds directly with DNA. Molecular docking studies revealed a mechanistic model for the interaction, and these studies could serve as the basis for continued analysis of structure-activity relationships and drug development efforts of this novel target.

Mechanisms of Chromium and Uranium Toxicity in Pseudomonas stutzeri RCH2 Grown under Anaerobic Nitrate-Reducing Conditions.

Chromium and uranium are highly toxic metals that contaminate many natural environments. We investigated their mechanisms of toxicity under anaerobic conditions using nitrate-reducing Pseudomonas stutzeri RCH2, which was originally isolated from a chromium-contaminated aquifer. A random barcode transposon site sequencing library of RCH2 was grown in the presence of the chromate oxyanion (Cr[VI]) or uranyl oxycation (U[VI]). Strains lacking genes required for a functional nitrate reductase had decreased fitness as both metals interacted with heme-containing enzymes required for the later steps in the denitrification pathway after nitrate is reduced to nitrite. Cr[VI]-resistance also required genes in the homologous recombination and nucleotide excision DNA repair pathways, showing that DNA is a target of Cr[VI] even under anaerobic conditions. The reduced thiol pool was also identified as a target of Cr[VI] toxicity and psest_2088, a gene of previously unknown function, was shown to have a role in the reduction of sulfite to sulfide. U[VI] resistance mechanisms involved exopolysaccharide synthesis and the universal stress protein UspA. As the first genome-wide fitness analysis of Cr[VI] and U[VI] toxicity under anaerobic conditions, this study provides new insight into the impact of Cr[VI] and U[VI] on an environmental isolate from a chromium contaminated site, as well as into the role of a ubiquitous protein, Psest_2088.

Epidermal Pigmentation, Nucleotide Excision Repair and Risk of Skin Cancer

As a group, malignancies of the skin are the most commonly diagnosed cancers, with over a million cases each year identified in the United States alone. While the keratinocyte malignancies – basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) – represent the majority of clinical cases of skin cancer, malignant melanoma is the most deadly form of skin cancer. Each of these skin malignancies is clearly linked to UV radiation, with chronic cumulative UV exposure being most relevant for keratinocyte malignancies and intense, blistering sunburns most relevant for melanoma. In this review, we describe the epidemiology of skin cancer, the link with UV radiation, and the innate defenses used to resist UV damage with particular attention to the nucleotide excision DNA repair pathway. We also focus on the role of skin pigmentation and the molecular events that control melanization of the skin, since these signaling pathways appear to be major determinants of skin cancer risk. We are particularly interested in the melanocortin 1 receptor (MC1R) signaling pathway which influences basal skin pigmentation, the ability to tan and the efficiency by which UV-induced photolesions are repaired in melanocytes after UV exposure.

Comprehensive pathway‐based interrogation of genetic variations in the nucleotide excision DNA repair pathway and risk of bladder cancer

Growing evidence suggests that single nucleotide polymorphisms (SNPs) in nucleotide excision repair (NER) pathway genes play an important role in bladder cancer etiology. However, only a limited number of genes and variations in this pathway have been evaluated to date. In this study, the authors applied a comprehensive pathway-based approach to assess the effects of 207 tagging and potentially functional SNPs in 26 NER genes on bladder cancer risk using a large case-control study that included 803 bladder cancer cases and 803 controls. In total, 17 SNPs were associated significantly with altered bladder cancer risk (P < .05), of which, 7 SNPs retained noteworthiness after they were assessed with a Bayesian approach for the probability of false discovery. The most noteworthy SNP was reference SNP 11132186 (rs11132186) in the inhibitor of growth family, member 2 (ING2) gene. Compared with the major allele-containing genotypes, the odds ratio was 0.52 (95% confidence interval, 0.32-0.83; P = .005) for the homozygous variant genotype. Three additional ING2 variants also exhibited significant associations with bladder cancer risk. Significant gene-smoking interactions were observed for 3 of the top 17 SNPs. Furthermore, through an exploratory classification and regression tree (CART) analysis, potential gene-gene interactions were identified. In this a large association study of the NER pathway and the risk of bladder cancer, several novel predisposition variants were identified along with potential gene-gene and gene-environment interactions in modulating bladder cancer risk. The results reinforce the importance of a comprehensive, pathway-focused, and tagging SNP-based candidate gene approach to identify low-penetrance cancer susceptibility loci.

Cis -acting genetic variation at an E2F1/YY1 response site and putative p53 site is associated with altered allele-specific expression of ERCC5 (XPG) transcript in normal human bronchial epithelium

ERCC5 (XPG) is a key component of the nucleotide excision DNA repair pathway. In two recent case-control studies, we determined that ERCC5 transcript expression pattern in grossly normal human bronchial epithelial cells (NBEC) was different in individuals diagnosed with lung cancer compared with non-lung cancer controls. In this study, we tested the hypothesis that variation at cis-acting sites contributed to observed variation in ERCC5 transcript expression in NBEC. Allele-specific expression (ASE) was measured at transcribed polymorphic site rs1047768 in exon 2 of ERCC5 in NBEC complementary DNA (cDNA) of 22 individuals using allele-specific competitive polymerase chain reaction. ASE at rs1047768 was then assessed for association with allelotype at polymorphic sites rs751402 (E2F1 and YY1 recognition and response site) and rs2296147 (putative P53 recognition site) in the proximal promoter and 5' untranslated region, respectively, of ERCC5. Interindividual variation in recombination between rs751402, rs2296147 and rs1047768 in poly-heterozygotes was controlled for by allele-specific sequencing. Measured rs1047768 T:C allelic ratio was (i) significantly higher in NBEC cDNA compared with genomic DNA controls (P < 0.001) among samples heterozygous at both rs751402 and rs2296147; (ii) less high (P = 0.02) for samples homozygous at rs751402 but heterozygous at rs2296147 and (iii) not significantly different (P = 0.18) for doubly homozygous individuals. Here, we demonstrate that rs751402 A allele and rs2296147 T allele are associated with higher ASE of ERCC5 T allele transcript at rs1047768 in NBEC.

Interacting partners of the Tfb2 subunit from yeast TFIIH

TFIIH is an evolutionary conserved eukaryotic multi-protein complex composed of ten subunits. It is involved in transcription, cell cycle regulation, RNA splicing and the nucleotide excision DNA repair pathway (NER). Depending on the process in which it is functioning, the composition of TFIIH varies and activities of its subunits are differentially regulated. Here we focused on interplay between the Ssl2, Tfb2 and Tfb5 subunits of TFIIH from Saccharomyces cerevisiae. We found that Tfb2 bridges the Ssl2 helicase and the NER-specific Tfb5 subunit. Moreover, the Tfb5-interacting domain of Tfb2 also binds nucleic acids (NA), although the addition of Tfb5 triggers dissociation of NA from Tfb2. In yeast cells, deletion of TFB5 is more detrimental to NER than loss of the Tfb5/NA-interacting domain of Tfb2, while combining these mutations resulted in suppression of the UV sensitivity of tfb5Δ. The implications of our findings in regards to TFIIH function and group A trichothiodystrophy, an inherited disease associated with mutations in the human TFB5 gene, are discussed.

Polymorphisms in nucleotide excision repair genes, smoking and intake of fruit and vegetables in relation to lung cancer

Polymorphisms in nucleotide excision repair genes have been associated with risk for lung cancer. We examined gene-environment interactions in relation to lung cancer in 430 cases and 790 comparison persons identified within a prospective cohort of 57,053 persons. We included polymorphisms in the XPC, XPA and XPD genes involved in the nucleotide excision DNA repair pathway and analysed possible interactions with smoking and dietary intake of fruit and vegetables in relation to risk for lung cancer. We found that intake of fruit was associated with lower risk for lung cancer only among carriers of the XPA A23G variant genotype. The incidence rate ratio for lung cancer was 0.60 (95% confidence interval: 0.43-0.84; p=0.003) per 50% increase in fruit intake. No convincing interactions were detected between the polymorphisms and smoking.

Base excision DNA repair defect in Gadd45a-deficient cells

As one of a number of p53-regulated genes, Gadd45a (growth arrest and DNA damage inducible gene) has been shown to delay carcinogenesis and decrease mutation frequency. Gadd45a is known to regulate nucleotide excision DNA repair (NER) in response to UV radiation. Here, we report an emerging role for Gadd45a in base excision repair (BER). Gadd45a-null mouse embryo fibroblasts MEF and gadd45a-deficient human colon cancer cells exhibited slow BER after treatment with methyl methanesulfonate (MMS) a pure base-damaging agent. In addition, removal of AP sites by apurinic/apyrimidinic endonuclease 1/redox factor 1 (APE1/Ref1) was significantly delayed in gadd45a-null cells. Moreover, the localization of APE1/Ref1 within the nucleus was observed in gadd45a wild-type cells, whereas APE1 become mainly distributed in the cytoplasm, and there is a reduced interaction with proliferating cell nuclear antigen (PCNA) in Gadd45a-deficient cells. Inasmuch as p53 has been shown to regulate BER in addition to the NER pathway, our data suggest that p53-regulated gene Gadd45a contributes to the BER response by affecting the interaction of cellular APE1/Ref1 with PCNA. Gadd45a might be a key component gene of the p53 pathway involved in protection from carcinogenic base damage and maintenance of genomic stability, although the downstream mechanism including APE1/Ref1 will need further study.

Adaptive Stress Response in Segmental Progeria Resembles Long-Lived Dwarfism and Calorie Restriction in Mice

How congenital defects causing genome instability can result in the pleiotropic symptoms reminiscent of aging but in a segmental and accelerated fashion remains largely unknown. Most segmental progerias are associated with accelerated fibroblast senescence, suggesting that cellular senescence is a likely contributing mechanism. Contrary to expectations, neither accelerated senescence nor acute oxidative stress hypersensitivity was detected in primary fibroblast or erythroblast cultures from multiple progeroid mouse models for defects in the nucleotide excision DNA repair pathway, which share premature aging features including postnatal growth retardation, cerebellar ataxia, and death before weaning. Instead, we report a prominent phenotypic overlap with long-lived dwarfism and calorie restriction during postnatal development (2 wk of age), including reduced size, reduced body temperature, hypoglycemia, and perturbation of the growth hormone/insulin-like growth factor 1 neuroendocrine axis. These symptoms were also present at 2 wk of age in a novel progeroid nucleotide excision repair-deficient mouse model (XPDG602D/R722W/XPA−/−) that survived weaning with high penetrance. However, despite persistent cachectic dwarfism, blood glucose and serum insulin-like growth factor 1 levels returned to normal by 10 wk, with hypoglycemia reappearing near premature death at 5 mo of age. These data strongly suggest changes in energy metabolism as part of an adaptive response during the stressful period of postnatal growth. Interestingly, a similar perturbation of the postnatal growth axis was not detected in another progeroid mouse model, the double-strand DNA break repair deficient Ku80 −/− mouse. Specific (but not all) types of genome instability may thus engage a conserved response to stress that evolved to cope with environmental pressures such as food shortage.

Deletion of the nucleotide excision repair gene Ercc1 reduces immunoglobulin class switching and alters mutations near switch recombination junctions.

The structure-specific endonuclease ERCC1-XPF is an essential component of the nucleotide excision DNA repair pathway. ERCC1-XPF nicks double-stranded DNA immediately adjacent to 3′ single-strand regions. Substrates include DNA bubbles and flaps. Furthermore, ERCC1 interacts with Msh2, a mismatch repair (MMR) protein involved in class switch recombination (CSR). Therefore, ERCC1-XPF has abilities that might be useful for antibody CSR. We tested whether ERCC1 is involved in CSR and found that Ercc1 − / − splenic B cells show moderately reduced CSR in vitro, demonstrating that ERCC1-XPF participates in, but is not required for, CSR. To investigate the role of ERCC1 in CSR, the nucleotide sequences of switch (S) regions were determined. The mutation frequency in germline Sμ segments and recombined Sμ-Sγ3 segments cloned from Ercc1 − / − splenic B cells induced to switch in culture was identical to that of wild-type (WT) littermates. However, Ercc1 − / − cells show increased targeting of the mutations to G:C bp in RGYW/WRCY hotspots and mutations occur at sites more distant from the S–S junctions compared with WT mice. The results indicate that ERCC1 is not epistatic with MMR and suggest that ERCC1 might be involved in processing or repair of DNA lesions in S regions during CSR.