Nucleotide Excision Repair Pathway Research Articles

UV-B-induced DNA damage and repair pathways in polar Pseudogymnoascus sp. from the Arctic and Antarctic regions and their effects on growth, pigmentation and conidiogenesis.

Solar radiation regulates most biological activities on Earth. Prolonged exposure to solar UV radiation can cause deleterious effects by inducing two major types of DNA damage, namely, cyclobutane pyrimidine dimers (CPDs) and pyrimidine 6-4 pyrimidone photoproducts. These lesions may be repaired by the photoreactivation (Phr) and nucleotide excision repair (NER) pathways; however, the principal UV-induced DNA repair pathway is not known in the fungal genus Pseudogymnoascus. In this study, we demonstrated that an unweighted UV-B dosage of 1.6kJ m-2 d-1 significantly reduced fungal growth rates (by between 22% and 35%) and inhibited conidia production in a 10 d exposure. The comparison of two DNA repair conditions, light or dark, which respectively induced photoreactivation (Phr) and NER, showed that the UV-B-induced CPDs were repaired significantly more rapidly in light than in dark conditions. The expression levels of two DNA repair genes, RAD2 and PHR1 (encoding a protein in NER and Phr respectively), demonstrated that NER rather than Phr was primarily activated for repairing UV-B-induced DNA damage in these Pseudogymnoascus strains. In contrast, Phr was inhibited after exposure to UV-B radiation, suggesting that PHR1 may have other functional roles. We present the first study to examine the capability of the Arctic and Antarctic Pseudogymnoascus sp. to perform photoreactivation and/or NER via RT-qPCR approaches, and also clarify the effects of light on UV-B-induced DNA damage repair in vivo by quantifying cyclobutene pyrimidine dimers and pyrimidine 6-4 pyrimidone photoproducts. Physiological response data, including relative growth rate, pigmentation and conidia production in these Pseudogymnoascus isolates exposed to UV-B radiation are also presented.

Reduced DNA Repair Capacity in Prostate Cancer Patients: A Phenotypic Approach Using the CometChip.

Simple SummaryProstate cancer (PCa) is the most commonly diagnosed cancer type in Hispanic men in the US. Among Hispanics, Puerto Rican (PR) men show the highest PCa-specific mortality. Various studies have shown that having low DNA repair capacity (DRC) is a significant risk factor for cancer development. The aim of this study was to evaluate variations in DRC, through the nucleotide excision repair (NER) pathway, in PR men with PCa using the CometChip. Overall, PCa cases had lower DRC than controls. When PCa cases were stratified into aggressive and indolent, controls had higher DRC than both groups. The contributions of additional factors (i.e., age and prostate-specific antigen levels) to DRC were also considered. Our data suggest that DRC levels may have the potential to discriminate between aggressive and indolent cases. Our results represent an innovative step in the development of a blood-based screening test for PCa based on DRC levels.Prostate cancer (PCa) accounts for 22% of the new cases diagnosed in Hispanic men in the US. Among Hispanics, Puerto Rican (PR) men show the highest PCa-specific mortality. Epidemiological studies using functional assays in lymphocytes have demonstrated that having low DRC is a significant risk factor for cancer development. The aim of this study was to evaluate variations in DRC in PR men with PCa. Lymphocytes were isolated from blood samples from PCa cases (n = 41) and controls (n = 14) recruited at a hospital setting. DRC levels through the nucleotide excision repair (NER) pathway were measured with the CometChip using UVC as a NER inductor. The mean DRC for controls and PCa cases were 20.66% (±7.96) and 8.41 (±4.88), respectively (p < 0.001). The relationship between DRC and tumor aggressiveness was also evaluated. Additional comparisons were performed to evaluate the contributions of age, anthropometric measurements, and prostate-specific antigen levels to the DRC. This is the first study to apply the CometChip in a clinical cancer study. Our results represent an innovative step in the development of a blood-based screening test for PCa based on DRC levels. Our data also suggest that DRC levels may have the potential to discriminate between aggressive and indolent cases.

Abstract 3747: RPS3 positively regulates nucleotide excision repair with respect to XP-D and skin cancers

Abstract Ribosomal protein S3 (rpS3) is a multifunctional protein working in translation, DNA repair, cancer, apoptosis etc. UV induced DNA damage is repaired by the nucleotide excision repair (NER) pathways; defects in these pathways lead to a genetic instability known as xeroderma pigmentosum (XP) eventually developing skin cancers. We discovered that rpS3 associates with transcription factor IIH (TFIIH) via an interaction with the xeroderma pigmentosum complementation group D (XPD) protein and complements its function in the NER pathway. Here, we showed that XP-D cells overexpressing rpS3 showed markedly increased resistance to UV through XPD and rpS3 interaction thus augmenting the helicase activity of XPD protein. Additionally, the knockdown of rpS3 caused reduced NER efficiency and the subsequent addition of rpS3 restored the helicase activity of the TFIIH complex in cells. We present data suggesting that rpS3 is involved in post-excision processing in NER, assisting TFIIH in expediting the repair process by increasing its turnover rate when DNA is damaged. We also propose that rpS3 is a novel accessory protein of the NER pathway and its recruitment to the repair machinery upon UV damage augments repair efficiency by enhancing XPD helicase function and increasing its turnover rate, thus facilitating DNA repair. In addition to this, manipulations of rpS3 gene could complement XP-D phenotype and prevent UV damage and skin cancer occurrence. Animal studies strongly indicate that these result could be applied not only for the development of cancer drugs but also for cosmeceuticals. [1] FASEB J. (2020) 34: 8102 [2] Cell &amp; Mol Life Sci (2021) 78: 3591 Citation Format: Joon Kim, Yong-Joon Park, Tae Sung Kim, Woo Sung Ahn, Hag Dong Kim. RPS3 positively regulates nucleotide excision repair with respect to XP-D and skin cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3747.

Abstract 2598: AZD1390 radio-sensitizes p53-mutant GBM via disrupting homology directed DNA repair

Abstract The ATM inhibitor AZD1390 disrupts cellular responses to ionizing radiation (IR) and is a potent radiosensitizer being tested in clinical trials. Here, we evaluated, the effects of AZD1390 on radiation sensitivity and DNA damage repair pathways in glioblastoma (GBM) cells and patient derived xenografts (PDXs). AZD1390 (30 nM and higher) suppressed IR (5 Gy)-induced phosphorylation of ATM-Serine1981 and downstream phosphorylation sites on Kap1, Chk2 and H2AX in U251 cells and multiple PDXs. Consistent with enhanced DNA damage, AZD1390 increased IR induced G2/M arrest in U251 (80.6% with AZD1390/IR vs. 64.6% with IR, p= 0.01), GBM43 (61.9% vs. 25.7%, p= 0.01) and GBM39 (40.9% vs. 25.4%, p= 0.01). Moreover, in a clonogenic survival assay, AZD1390 sensitized U251 cells to 5 Gy IR (0.24% survival with AZD1390/IR vs. 2.3% with IR alone, p=0.01). In a reporter-based analysis of DNA repair capacity, ATM inhibition resulted in a 40 to 60% reduction in homologous recombination (HR) and modest but significant decrease in micro-homology mediated end joining (MMEJ) and gap fill-in synthesis in U251 cells but had no effect on non-homologous end joining, translesion synthesis, nucleotide or base excision repair pathways. Comparing effects of AZD1390 on repair in GBM14 (TP53-wt) and GBM43 (TP53-mutant), similar results were observed except that decreased MMEJ was seen only in GBM43 (0.03± 0.01% vs. 0.07± 0.01% in control, p=0.002). Intriguingly, RAD51 knockdown (to disrupt HR) sensitized U251 and GBM43 but not GBM14. The efficacy of AZD1390 ± IR was studied in vivo in 10 PDXs. IR was delivered to orthotopic tumors using opposed lateral 225 kVp beams. AZD1390 (20 mg/kg PO) was given just prior to each radiation dose (2 Gy x 5 fractions). AZD1390 monotherapy was mostly ineffective, IR alone was reasonably efficacious with an average 1.8 ± 0.1-fold-increase in survival relative to sham radiation (survival ratio) across all 10 models. IR/AZD1390 treatment resulted in significant survival extension relative to IR alone in 6 of 10 models. Analysis of the survival benefit of combination therapy compared to IR alone across the entire cohort of PDXs was statistically marginal (average survival ratio 2.3± 0.3 vs. 1.8 ± 0.1 with IR, p=0.08). However, when stratified by TP53 status, combination therapy was significantly more effective than IR (mean survival ratio 2.3 ± 0.1 vs. 1.6 ± 0.2 with IR alone, p=0.02) in TP53-mutant PDXs, where all 5 models benefited. In contrast, TP53-wt group had no benefit (mean survival ratio 2.2 ± 0.5 vs. 2.0 ± 0.2, p=0.61), GBM39 was only TP53-wt PDX that benefited from the combination. In conclusion, AZD1390 is an effective radio-sensitizer that causes disruption in HR and potentially other DNA repair pathways. Interestingly, in vivo radiosensitizing effects are mostly restricted to TP53-mutant GBM PDXs. Understandingmechanism of resistance in the context of different TP53 backgrounds remains an important future direction. Citation Format: Shiv K. Gupta, Jiajia Chen, Daniel J. Laverty, Surabhi Talele, Brett L. Carlson, Ann C. Mladek Tuma, Danielle Burgenske, Gaspar J. Kitange, Petra Hamerlik, Zachary D. Nagel, Stephen T. Durant, William F. Elmquist, Jann N. Sarkaria. AZD1390 radio-sensitizes p53-mutant GBM via disrupting homology directed DNA repair [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2598.

Adult-Onset Neurodegeneration in Nucleotide Excision Repair Disorders (NERDND ): Time to Move Beyond the Skin.

Variants in genes of the nucleotide excision repair (NER) pathway have been associated with heterogeneous clinical presentations ranging from xeroderma pigmentosum to Cockayne syndrome and trichothiodystrophy. NER deficiencies manifest with photosensitivity and skin cancer, but also developmental delay and early-onset neurological degeneration. Adult-onset neurological features have been reported in only a few xeroderma pigmentosum cases, all showing at least mild skin manifestations. The aim of this multicenter study was to investigate the frequency and clinical features of patients with biallelic variants in NER genes who are predominantly presenting with neurological signs. In-house exome and genome datasets of 14,303 patients, including 3543 neurological cases, were screened for deleterious variants in NER-related genes. Clinical workup included in-depth neurological and dermatological assessments. We identified 13 patients with variants in ERCC4 (n=8), ERCC2 (n=4), or XPA (n=1), mostly proven biallelic, including five different recurrent and six novel variants. All individuals had adult-onset progressive neurological deterioration with ataxia, dementia, and frequently chorea, neuropathy, and spasticity. Brain magnetic resonance imaging showed profound global brain atrophy in all patients. Dermatological examination did not show any skin cancer or pronounced ultraviolet damage. We introduce NERDND as adult-onset neurodegeneration (ND ) within the spectrum of autosomal recessive NER disorders (NERD). Our study demonstrates that NERDND is probably an underdiagnosed cause of neurodegeneration in adulthood and should be considered in patients with overlapping cognitive and movement abnormalities. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Analysis of Omics Data Reveals Nucleotide Excision Repair-Related Genes Signature in Highly-Grade Serous Ovarian Cancer to Predict Prognosis.

Most of the high-grade serous ovarian cancers (HGSOC) are accompanied by P53 mutations, which are related to the nucleotide excision repair (NER) pathway. This study aims to construct a risk signature based on NER-related genes that could effectively predict the prognosis for advanced patients with HGSOC. In our study, we found that two clusters of HGSOC with significantly different overall survival (OS) were identified by consensus clustering and principal component analysis (PCA). Then, a 7-gene risk signature (DDB2, POLR2D, CCNH, XPC, ERCC2, ERCC4, and RPA2) for OS prediction was developed subsequently based on TCGA cohort, and the risk score-based signature was identified as an independent prognostic indicator for HGSOC. According to the risk score, HGSOC patients were divided into high-risk group and low-risk group, in which the distinct OS and the predictive power were also successfully verified in the GEO validation sets. Then we constructed a nomogram, including the risk signature and clinical-related risk factors (age and treatment response) that predicted an individual’s risk of OS, which can be validated by assessing calibration curves. Furthermore, GSEA showed that the genes in the high-risk group were significantly enriched in cancer-related pathways, such as “MAPK signaling pathway”, “mTOR signaling pathway”, “VEGF signaling pathway” and so on. In conclusion, our study has developed a robust NER-related genes-based molecular signature for prognosis prediction, and the nomogram could be used as a convenient tool for OS evaluation and guidance of therapeutic strategies in advanced patients with HGSOC.

Genetic Variations in Nucleotide Excision Repair Pathway Genes and Risk of Allergic Rhinitis

Background Allergic rhinitis (AR) is the most frequent inflammatory disorder in the nasal mucosa that remains unclear etiology. Mounting studies suggested that genetic instability could trigger and worsen the inflammatory response. The nucleotide excision repair (NER) system is an important pathway in maintaining the stability of the genome. Therefore, the genetic variations in NER pathway genes may have potential effects on AR risk. Methods We evaluated the correlation between 19 candidate single nucleotide polymorphisms (SNPs) in NER pathway genes and AR susceptibility by a case-control study in a Chinese population, which contains 508 AR cases and 526 controls. Results Three independent SNPs were identified as significantly associated with AR susceptibility, including ERCC1 rs2298881 C > A (recessive model: adjusted odds ratios (OR) = 0.30, 95%confidence interval (CI) = 0.18–0.50, P < 0.0001), ERCC1 rs11615 G > A (dominant model: adjusted OR = 1.44, 95%CI = 1.04–2.01, P = 0.030), and XPC rs2228001 A > C (dominant model: adjusted OR = 0.68, 95%CI = 0.49–0.95, P = 0.024). Stratified analysis showed that ERCC1 rs2298881 AA genotype was correlated with a lower risk of AR among all the subgroups compared with rs2298881 CC/CA genotype. XPC rs2228001 AC/CC genotype reduced AR risk among the following subgroups: age > 60 months, clinical stage I and III. Conclusion Our finding showed that genetic variations in NER pathway genes: ERCC1 and XPC may affect the risk of AR, which will provide new insights into the genetics of AR from the perspective of DNA damage repair.

Effects of ERCC5 rs751402 Polymorphism on Oxidative Stress and the Impact of Curcumin on Catalase Activity in Breast Carcinogenesis.

Background:Breast cancer is a complex multifactorial disease and polymorphisms in nucleotide excision repair pathway are associated with the potential risk of breast cancer. Pathological processes linked to breast cancer are associated with oxidative stress. Catalase plays an essential role in cell defense against oxidative stress. Curcumin has antioxidant activity that can significantly reduce oxidative stress levels. The aims of this study were to determine ERCC5 rs751402 polymorphism was associated with oxidative stress in breast carcinogenesis. The impact of curcumin on catalase activity for inhibiting breast cancer progression was also studied. Methods:The effect of ERCC5 rs751402 polymorphism on oxidative stress was studied with different H2O2 concentrations in HCC 1937 cell line for 24 h and then analysed by MTT assay. The impact of curcumin on catalase activity was studied in MCF-7 cell line treated with different curcumin concentrations for 24 h and then analysed by trypan blue exclusion assay and catalase activity assay. Results:It showed that this polymorphism involved in oxidative stress (p < 0.05) and curcumin caused the antiproliferative effect by the catalase activity increase (p < 0.05). Conclusion:Our study indicated that ERCC5 rs751402 polymorphism may contribute to the etiology of breast carcinogenesis about the failure of oxidative stress protection and lead to breast carcinogenesis. The antiproliferative effect of curcumin may be associated with catalase activity and protect breast carcinogenesis.

Pan-sarcoma analysis of DNA damage response pathway alterations and deficiency.

11548 Background: Alterations in DNA damage response (DDR) pathways contribute to genomic instability and malignant progression and have been shown to be of clinical significance in several carcinomas and solid tumors. While some studies have identified ostensibly pathogenic variations in known and novel cancer genes with implications for sarcoma risk and treatment opportunities, there is limited information regarding the role of DDR pathway alterations in sarcoma. We identified a gene alteration in ERCC2, a gene that codes for a DNA helicase in the nucleotide excision repair pathway, in a patient with multiply relapsed epithelioid sarcoma (ES), prompting an investigation of DDR pathway alterations in sarcoma samples using a global next-generation sequencing (NGS) platform. Methods: Sarcoma patient samples (N = 5310), representing 38 pediatric and adult histologic subtypes, underwent NGS of DNA (592 gene panel or whole exome) and RNA (whole transcriptome sequencing, N = 3612) at a CLIA-certified laboratory (Caris Life Sciences, Phoenix, AZ). A threshold of 10 mut/Mb was used to identify high tumor mutational burden (TMB-H). IHC was performed for PD-L1 (SP142; 2+|5% = positive). Homologous recombination deficiency (HRD) scores were calculated as a composite of loss of heterozygosity, telomeric allelic imbalance, and large-scale transitions, using a positive threshold of 42 (N = 2138). HRD score association with biomarker status was evaluated overall and in sarcoma subtypes. Results: A pathogenic DDR pathway mutation was noted in 842 (15.9%) of the total samples. ATRX was by far the most commonly altered DDR gene (10% of all samples), with mutations observed across 25 sarcoma subtypes (11 subtypes with &gt; 10% mutation rate: leiomyosarcoma [LMS], perivascular epithelioid cell tumor [PEComa], pleomorphic sarcoma [PLSARC], uterine sarcoma [OUSARC], osteosarcoma, spindle cell sarcoma, angiosarcoma, mesenchymal chondrosarcoma, sarcoma NOS, fibrosarcoma and ES). CHEK2, ATM, and MUTYH mutations were observed in 1-2% of sarcoma samples. More than 20 histologic subtypes showed distinct gene signatures with mutations occurring in &gt; 3% of the samples investigated. ERCC2 was mutated in 3% of ES and 6.5% in PEComa. Median HRD scores ranged between 20-58 across sarcoma subtypes. High rates of deficient HRD (HRDd ≥ 42) were observed in PLSARC (83.2%), OUSARC (73.7%), and dedifferentiated chondrosarcoma (71.4%), while low rates of HRDd were observed in Ewing sarcoma (0%) and clear cell sarcoma (10%). In the overall cohort, ERCC2, ATRX and BRCA2 were significantly associated with increased HRD scores (p = 0.01). Conclusions:DDR pathway alterations are present in numerous histologic subtypes of sarcoma. A more comprehensive analysis of individual histologic subtypes is in progress. Further research will evaluate the clinical implications of these known and novel mutations to guide risk stratification and potential therapeutic options.

Genetic variants in XPD gene and glioma susceptibility in Chinese children: A multicenter case-control study.

Glioma is one of the central nervous system (CNS) tumors in children, accounting for 80% of malignant brain tumors. Nucleotide excision repair (NER) is a vital pathway during DNA damage repair progression. Xeroderma pigmentosum group D (XPD) or excision repair cross-complementing group 2 (ERCC2) is a critical factor in the NER pathway, playing an indispensable role in the DNA repair process. Therefore, the genetic variants in XPD may be associated with carcinogenesis induced by defects in DNA repair. We are the first to conduct a multi-center case-control study to investigate the correlation between XPD gene polymorphisms and pediatric glioma risk. We chose three single nucleotide polymorphisms and genotyped them using the TaqMan assay. Although there is no significant association of these genetic variations with glioma susceptibility, the stratified analysis revealed that in the subtype of astrocytic tumors, the rs13181 TG/GG genotype enhanced glioma risk than the TT genotype, and carriers with two to three genotypes also elevated the tumor risk than 0-1 genotypes. In conclusion, our findings provided an insight into the impact of XPD genetic variants on glioma risk.

BAP1 promotes the repair of UV-induced DNA damage via PARP1-mediated recruitment to damage sites and control of activity and stability.

BRCA1-associated protein-1 (BAP1) is a ubiquitin C-terminal hydrolase domain-containing deubiquitinase with tumor suppressor activity. The gene encoding BAP1 is mutated in various human cancers, with particularly high frequency in kidney and skin cancers, and BAP1 is involved in many cancer-related cellular functions, such as DNA repair and genome stability. Although BAP1 stimulates DNA double-strand break repair, whether it functions in nucleotide excision repair (NER) is unknown. Here, we show that BAP1 promotes the repair of ultraviolet (UV)-induced DNA damage via its deubiquitination activity in various cell types, including primary melanocytes. Poly(ADP-ribose) polymerase 1 (PARP1) interacts with and recruits BAP1 to damage sites, with BAP1 recruitment peaking after the DDB2 and XPC damage sensors. BAP1 recruitment also requires histone H2A monoubiquitinated at Lys119, which accumulates at damage sites. PARP1 transiently poly(ADP-ribosyl)ates (PARylates) BAP1 at multiple sites after UV damage and stimulates the deubiquitination activity of BAP1 both intrinsically and via PARylation. PARP1 also promotes BAP1 stability via crosstalk between PARylation and ubiquitination. Many PARylation sites in BAP1 are mutated in various human cancers, among which the glutamic acid (Glu) residue at position 31, with particularly frequent mutation in kidney cancer, plays a critical role in BAP1 stabilization and promotes UV-induced DNA damage repair. Glu31 also participates in reducing the viability of kidney cancer cells. This study therefore reveals that BAP1 functions in the NER pathway and that PARP1 plays a role as a novel factor that regulates BAP1 enzymatic activity, protein stability, and recruitment to damage sites. This activity of BAP1 in NER, along with its cancer cell viability-reducing activity, may account for its tumor suppressor function.

XPG in the Nucleotide Excision Repair and Beyond: a study on the different functional aspects of XPG and its associated diseases.

Several proteins are involved in DNA repair mechanisms attempting to repair damages to the DNA continuously. One such protein is Xeroderma Pigmentosum Complementation Group G (XPG), a significant component in the Nucleotide Excision Repair (NER) pathway. XPG is accountable for making the 3' incision in the NER, while XPF-ERCC4 joins ERCC1 to form the XPF-ERCC1 complex. This complex makes a 5' incision to eliminate bulky DNA lesions. XPG is also known to function as a cofactor in the Base Excision Repair (BER) pathway by increasing hNth1 activity, apart from its crucial involvement in the NER. Reports suggest that XPG also plays a non-catalytic role in the Homologous Recombination Repair (HRR) pathway by forming higher-order complexes with BRCA1, BRCA2, Rad51, and PALB2, further influencing the activity of these molecules. Studies show that, apart from its vital role in repairing DNA damages, XPG is also responsible for R-loop formation, which facilitates exhibiting phenotypes of Werner Syndrome. Though XPG has a role in several DNA repair pathways and molecular mechanisms, it is primarily a NER protein. Unrepaired and prolonged DNA damage leads to genomic instability and facilitates neurological disorders, aging, pigmentation, and cancer susceptibility. This review explores the vital role of XPG in different DNA repair mechanisms which are continuously involved in repairing these damaged sites and its failure leading to XP-G, XP-G/CS complex phenotypes, and cancer progression.

Photorepair of Either CPD or 6-4PP DNA Lesions in Basal Keratinocytes Attenuates Ultraviolet-Induced Skin Effects in Nucleotide Excision Repair Deficient Mice

Ultraviolet (UV) radiation is one of the most genotoxic, universal agents present in the environment. UVB (280-315 nm) radiation directly damages DNA, producing cyclobutane pyrimidine dimers (CPDs) and pyrimidine 6-4 pyrimidone photoproducts (6-4PPs). These photolesions interfere with essential cellular processes by blocking transcription and replication polymerases, and may induce skin inflammation, hyperplasia and cell death eventually contributing to skin aging, effects mediated mainly by keratinocytes. Additionally, these lesions may also induce mutations and thereby cause skin cancer. Photolesions are repaired by the Nucleotide Excision Repair (NER) pathway, responsible for repairing bulky DNA lesions. Both types of photolesions can also be repaired by distinct (CPD- or 6-4PP-) photolyases, enzymes that specifically repair their respective photolesion by directly splitting each dimer through a light-dependent process termed photoreactivation. However, as photolyases are absent in placental mammals, these organisms depend solely on NER for the repair of DNA UV lesions. However, the individual contribution of each UV dimer in the skin effects, as well as the role of keratinocytes has remained elusive. In this study, we show that in NER-deficient mice, the transgenic expression and photorepair of CPD-photolyase in basal keratinocytes completely inhibited UVB-induced epidermal thickness and cell proliferation. On the other hand, photorepair by 6-4PP-photolyase in keratinocytes reduced but did not abrogate these UV-induced effects. The photolyase mediated removal of either CPDs or 6-4PPs from basal keratinocytes in the skin also reduced UVB-induced apoptosis, ICAM-1 expression, and myeloperoxidase activation. These findings indicate that, in NER-deficient rodents, both types of photolesions have causal roles in UVB-induced epidermal cell proliferation, hyperplasia, cell death and inflammation. Furthermore, these findings also support the notion that basal keratinocytes, instead of other skin cells, are the major cellular mediators of these UVB-induced effects.

Acetylation of H3K56 orchestrates UV-responsive chromatin events that generate DNA accessibility during Nucleotide Excision Repair

Histone modifications have long been related to DNA damage response. Nucleotide excision repair pathway that removes helix-distorting lesions necessitates DNA accessibility through chromatin modifications. Previous studies have linked H3 tail residue acetylation to UV-induced NER. Here we present evidences that acetylation of H3K56 is crucial for early phases of NER. Using H3K56 mutants K56Q and K56R, which mimic acetylated and unacetylated lysines respectively, we show that recruitment of the repair factor Rad16, a Swi/Snf family member is dependent on H3K56 acetylation. With constitutive H3K56 acetylation, Rad16 recruitment became UV-independent. Furthermore, H3K56 acetylation promoted UV-induced hyperacetylation of H3K9 and H3K14. Importantly, constitutive H3K56 acetylation prominently increased chromatin accessibility. During NER, lack of H3K56 acetylation that effectively aborted H3 tail residue acetylation and Rad16 recruitment, thus failed to impart essential chromatin modulations. The NER-responsive oscillation of chromatin structure observed in wild type, was distinctly eliminated in absence of H3K56 acetylation. In vitro assay with wild type and H3K56 mutant cell extracts further indicated that absence of H3K56 acetylation negatively affected DNA relaxation during NER. Overall, H3K56 acetylation regulates Rad16 redistribution and UV-induced H3 tail residue hyperacetylation, and the resultant modification code promotes chromatin accessibility and recruitment of subsequent repair factors during NER.

Role of the nucleotide excision repair pathway proteins (UvrB and UvrD2) in recycling UdgB, a base excision repair enzyme in Mycobacterium smegmatis

Cross-talks between DNA repair pathways are emerging as a crucial strategy in the maintenance of the genomic integrity. A double-stranded (ds) DNA specific DNA glycosylase, UdgB is known to excise uracil, hypoxanthine and ethenocytosine. We earlier showed that Mycobacterium smegmatis (Msm) UdgB stays back on the AP-sites it generates in the DNA upon excision of the damaged bases. Here, we show that in an Msm strain deleted for a nucleotide excision repair (NER) protein, UvrB (uvrB–), UdgB expression is toxic, and its deletion from the genome (udgB–) rescues the strain from the genotoxic stress. However, UdgB bound AP-site is not a direct substrate for NER in vitro. We show that UvrD2 and UvrB, known helicases with single-stranded (ss) DNA translocase activity, facilitate recycling of UdgB from AP-DNA. Our studies reveal that the helicases play an important role in exposing the AP-sites in DNA and make them available for further repair.

Nucleotide Excision Repair Pathway Activity Is Inhibited by Airborne Particulate Matter (PM10) through XPA Deregulation in Lung Epithelial Cells.

Airborne particulate matter with a diameter size of ≤10 µm (PM10) is a carcinogen that contains polycyclic aromatic hydrocarbons (PAH), which form PAH–DNA adducts. However, the way in which these adducts are managed by DNA repair pathways in cells exposed to PM10 has been partially described. We evaluated the effect of PM10 on nucleotide excision repair (NER) activity and on the levels of different proteins of this pathway that eliminate bulky DNA adducts. Our results showed that human lung epithelial cells (A549) exposed to 10 µg/cm2 of PM10 exhibited PAH–DNA adducts as well as an increase in RAD23 and XPD protein levels (first responders in NER). In addition, PM10 increased the levels of H4K20me2, a recruitment signal for XPA. However, we observed a decrease in total and phosphorylated XPA (Ser196) and an increase in phosphatase WIP1, aside from the absence of XPA–RPA complex, which participates in DNA-damage removal. Additionally, an NER activity assay demonstrated inhibition of the NER functionality in cells exposed to PM10, indicating that XPA alterations led to deficiencies in DNA repair. These results demonstrate that PM10 exposure induces an accumulation of DNA damage that is associated with NER inhibition, highlighting the role of PM10 as an important contributor to lung cancer.

In vitro reconstitution of an efficient nucleotide excision repair system using mesophilic enzymes from Deinococcus radiodurans

Nucleotide excision repair (NER) is a universal and versatile DNA repair pathway, capable of removing a very wide range of lesions, including UV-induced pyrimidine dimers and bulky adducts. In bacteria, NER involves the sequential action of the UvrA, UvrB and UvrC proteins to release a short 12- or 13-nucleotide DNA fragment containing the damaged site. Although bacterial NER has been the focus of numerous studies over the past 40 years, a number of key questions remain unanswered regarding the mechanisms underlying DNA damage recognition by UvrA, the handoff to UvrB and the site-specific incision by UvrC. In the present study, we have successfully reconstituted in vitro a robust NER system using the UvrABC proteins from the radiation resistant bacterium, Deinococcus radiodurans. We have investigated the influence of various parameters, including temperature, salt, protein and ATP concentrations, protein purity and metal cations, on the dual incision by UvrABC, so as to find the optimal conditions for the efficient release of the short lesion-containing oligonucleotide. This newly developed assay relying on the use of an original, doubly-labelled DNA substrate has allowed us to probe the kinetics of repair on different DNA substrates and to determine the order and precise sites of incisions on the 5′ and 3′ sides of the lesion. This new assay thus constitutes a valuable tool to further decipher the NER pathway in bacteria.

Characterization of the nucleotide excision repair pathway and evaluation of compounds for overcoming the cisplatin resistance of non‑small cell lung cancer cell lines.

Lung cancer has been reported to be the leading cause of cancer‑related mortality worldwide. Cisplatin combination chemotherapy is a standard therapeutic strategy for patients with non‑small cell lung cancer (NSCLC) lacking driver mutations. However, the development of cisplatin resistance is a major obstacle to effective cancer treatment. The cellular mechanisms underlying cisplatin resistance have been previously revealed to be multifunctional. Accordingly, mechanistic analysis and the development of novel therapeutic strategies for cisplatin‑resistant NSCLC are urgently required. The present study mainly focused on the DNA repair mechanisms in cisplatin‑resistant NSCLC cells. Additionally, the effects of an Ecteinascidin (Et) derivative on cisplatin‑resistant cell lines were examined, by using a cisplatin‑resistant NSCLC cell line subjected to nucleotide excision repair (NER) pathway alterations. The results revealed that xeroderma pigmentosum group F‑complementing protein (XPF) mRNA expression was strongly associated with cisplatin resistance in cisplatin‑resistant NSCLC cell lines. XPF silencing significantly restored the sensitivity of cisplatin‑resistant PC‑14/CDDP cells to the drug. A potent anticancer effect of Et was observed in the cisplatin‑resistant cell line (PC‑14/CDDP), in which the NER pathway was altered. On the whole, these findings revealed that the expression levels of NER pathway‑related genes, including XPF, may have potential as biomarkers of cisplatin resistance. It was also suggested that Et may be a very promising compound for the development of novel anticancer drugs for the treatment of cisplatin‑resistant lung cancer.

The Prognostic and Predictive Role of Xeroderma Pigmentosum Gene Expression in Melanoma.

BackgroundAssessment of immune-specific markers is a well-established approach for predicting the response to immune checkpoint inhibitors (ICIs). Promising candidates as ICI predictive biomarkers are the DNA damage response pathway genes. One of those pathways, which are mainly responsible for the repair of DNA damage caused by ultraviolet radiation, is the nucleotide excision repair (NER) pathway. Xeroderma pigmentosum (XP) is a hereditary disease caused by mutations of eight different genes of the NER pathway, or POLH, here together named the nine XP genes. Anecdotal evidence indicated that XP patients with melanoma or other skin tumors responded impressively well to anti-PD-1 ICIs. Hence, we analyzed the expression of the nine XP genes as prognostic and anti-PD-1 ICI predictive biomarkers in melanoma.MethodsWe assessed mRNA gene expression in the TCGA-SKCM dataset (n = 445) and two pooled clinical melanoma cohorts of anti-PD-1 ICI (n = 75). In TCGA-SKCM, we applied hierarchical clustering on XP genes to reveal clusters, further utilized as XP cluster scores. In addition, out of 18 predefined genes representative of a T cell inflamed tumor microenvironment, the TIS score was calculated. Besides these scores, the XP genes, immune-specific single genes (CD8A, CXCL9, CD274, and CXCL13) and tumor mutational burden (TMB) were cross-correlated. Survival analysis in TCGA-SKCM was conducted for the selected parameters. Lastly, the XP response prediction value was calculated for the two pooled anti-PD-1 cohorts by classification models.ResultsIn TCGA-SKCM, expression of the XP genes was divided into two clusters, inversely correlated with immune-specific markers. A higher ERCC3 expression was associated with improved survival, particularly in younger patients. The constructed models utilizing XP genes, and the XP cluster scores outperformed the immune-specific gene-based models in predicting response to anti-PD-1 ICI in the pooled clinical cohorts. However, the best prediction was achieved by combining the immune-specific gene CD274 with three XP genes from both clusters.ConclusionOur results suggest pre-therapeutic XP gene expression as a potential marker to improve the prediction of anti-PD-1 response in melanoma.

Role of Xeroderma pigmentosum D (XPD) protein in genome maintenance in human cells under oxidative stress

Xeroderma pigmentosum D (XPD) protein plays a pivotal role in the nucleotide excision repair pathway. XPD unwinds the local area of the damaged DNA by virtue of constituting transcription factor II H (TFIIH) and is important not only for repair but also for basal transcription. Although cells deficient in XPD have shown to be defective in oxidative base-lesion repair, the effects of the oxidative assault on primary fibroblasts from patients suffering from Xeroderma Pigmentosum D have not been fully explored. Therefore, we sought to investigate the role of XPD in oxidative DNA damage-repair by treating primary fibroblasts derived from a patient suffering from Xeroderma Pigmentosum D, with hydrogen peroxide. Our results show dose-dependent increase in genotoxicity with minimal effect on cytotoxicity with H2O2 in XPD deficient cells compared to control cells. XPD deficient cells displayed increased susceptibility and reduced repair capacity when subjected to DNA damage induced by oxidative stress. XPD deficient fibroblasts exhibited increased telomeric loss after H2O2 treatment. In addition, we demonstrated that chronic oxidative stress induced accelerated premature senescence characteristics. Gene expression profiling revealed alterations in genes involved in transcription and nucleotide metabolisms, as well as in cellular and cell cycle processes in a more significant way than in other pathways. This study highlights the role of XPD in the repair of oxidative stress and telomere maintenance. Lack of functional XPD seems to increase the susceptibility of oxidative stress-induced genotoxicity while retaining cell viability posing as a potential cancer risk factor of Xeroderma Pigmentosum D patients.