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Nucleotide excision repair (NER) is the most universal repair pathway, which removes a wide range of DNA helix-distorting lesions caused by chemical or physical agents. The final steps of this repair process are gap-filling repair synthesis and subsequent ligation. XPA is the central NER scaffolding protein factor and can be involved in post-incision NER stages. Replication machinery is loaded after the first incision of the damaged strand that is performed by the XPF-ERCC1 nuclease forming a damaged 5'-flap processed by the XPG endonuclease. Flap endonuclease I (FEN1) is a critical component of replication machinery and is absolutely indispensable for the maturation of newly synthesized strands. FEN1 also contributes to the long-patch pathway of base excision repair. Here, we use a set of DNA substrates containing a fluorescently labeled 5'-flap and different size gap to analyze possible repair factor-replication factor interactions. Ternary XPA-FEN1-DNA complexes with each tested DNA are detected. Furthermore, we demonstrate XPA-FEN1 complex formation in the absence of DNA due to protein-protein interaction. Functional assays reveal that XPA moderately inhibits FEN1 catalytic activity. Using fluorescently labeled XPA, formation of ternary RPA-XPA-FEN1 complex, where XPA accommodates FEN1 and RPA contacts simultaneously, can be proposed. We discuss possible functional roles of the XPA-FEN1 interaction in NER related DNA resynthesis and/or other DNA metabolic processes where XPA can be involved in the complex with FEN1.

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Nucleotide excision repair (NER) promotes genomic integrity by removing bulky DNA adducts introduced by external factors such as ultraviolet light. Defects in NER enzymes are associated with pathological conditions such as Xeroderma Pigmentosum, trichothiodystrophy, and Cockayne syndrome. A critical step in NER is the binding of the Xeroderma Pigmentosum group A protein (XPA) to the ss/ds DNA junction. To better capture the dynamics of XPA interactions with DNA during NER we have utilized the fluorescence enhancement through non-canonical amino acids (FEncAA) approach. 4-azido-L-phenylalanine (4AZP or pAzF) was incorporated at Arg-158 in human XPA and conjugated to Cy3 using strain-promoted azide-alkyne cycloaddition. The resulting fluorescent XPA protein (XPACy3) shows no loss in DNA binding activity and generates a robust change in fluorescence upon binding to DNA. Here we describe methods to generate XPACy3 and detail in vitro experimental conditions required to stably maintain the protein during biochemical and biophysical studies.

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The DNA excision repair protein ERCC1 and the DNA damage sensor protein, XPA are highly overexpressed in patient samples of cisplatin-resistant solid tumors including lung, bladder, ovarian, and testicular cancer. The repair of cisplatin-DNA crosslinks is dependent upon nucleotide excision repair (NER) that is modulated by protein-protein binding interactions of ERCC1, the endonuclease, XPF, and XPA. Thus, inhibition of their function is a potential therapeutic strategy for the selective sensitization of tumors to DNA-damaging platinum-based cancer therapy. Here, we report on new small-molecule antagonists of the ERCC1/XPA protein-protein interaction (PPI) discovered using a high-throughput competitive fluorescence polarization binding assay. We discovered a unique structural class of thiopyridine-3-carbonitrile PPI antagonists that block a truncated XPA polypeptide from binding to ERCC1. Preliminary hit-to-lead studies from compound 1 reveal structure-activity relationships (SAR) and identify lead compound 27 o with an EC50 of 4.7 µM. Furthermore, chemical shift perturbation mapping by NMR confirms that 1 binds within the same site as the truncated XPA67-80 peptide. These novel ERCC1 antagonists are useful chemical biology tools for investigating DNA damage repair pathways and provide a good starting point for medicinal chemistry optimization as therapeutics for sensitizing tumors to DNA damaging agents and overcoming resistance to platinum-based chemotherapy.

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The hormonal form of vitamin D3, 1,25(OH)2D3, reduces UV-induced DNA damage. UV exposure initiates pre-vitamin D3 production in the skin, and continued UV exposure photoisomerizes pre-vitamin D3 to produce "over-irradiation products" such as lumisterol3 (L3). Cytochrome P450 side-chain cleavage enzyme (CYP11A1) in skin catalyzes the conversion of L3 to produce three main derivatives: 24-hydroxy-L3 [24(OH)L3], 22-hydroxy-L3 [22(OH)L3], and 20,22-dihydroxy-L3 [20,22(OH)L3]. The current study investigated the photoprotective properties of the major over-irradiation metabolite, 24(OH)L3, in human primary keratinocytes and human skin explants. The results indicated that treatment immediately after UV with either 24(OH)L3 or 1,25(OH)2D3 reduced UV-induced cyclobutane pyrimidine dimers and oxidative DNA damage, with similar concentration response curves in keratinocytes, although in skin explants, 1,25(OH)2D3 was more potent. The reductions in DNA damage by both compounds were, at least in part, the result of increased DNA repair through increased energy availability via increased glycolysis, as well as increased DNA damage recognition proteins in the nucleotide excision repair pathway. Reductions in UV-induced DNA photolesions by either compound occurred in the presence of lower reactive oxygen species. The results indicated that under in vitro and ex vivo conditions, 24(OH)L3 provided photoprotection against UV damage similar to that of 1,25(OH)2D3.

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Cigarette smoking (CS) is a major cause of various serious diseases due to tobacco chemicals. There is evidence suggesting that CS has been linked with the DNA damage repair system, as it can affect genomic stability, inducing genetic changes in the genes involved in the repair system, specifically the nucleotide excision repair (NER) pathway, affecting the function and/or regulation of these genes. Single nucleotide polymorphism (SNP), along with CS, can affect the work of the NER pathway and, therefore, could lead to different diseases. This study explored the association of four SNPs in both XPA and XPC genes with CS in the Saudi population. The Taq Man genotyping assay was used for 220 healthy non-smokers (control) and 201 healthy smokers to evaluate four SNPs in the XPA gene named rs10817938, rs1800975, rs3176751, and rs3176752 and four SNPs in the XPC gene called rs1870134, rs2228000, rs2228001, and rs2607775. In the XPA gene, SNP rs3176751 showed a high-risk association with CS-induced diseases with all clinical parameters, including CS duration, CS intensity, gender, and age of smokers. On the other hand, SNP rs1800975 showed a statistically significant low-risk association with all clinical parameters. In addition, rs10817938 showed a high-risk association only with long-term smokers and a low-risk association only with younger smokers. A low-risk association was found in SNP rs3176752 with older smokers. In the XPC gene, SNP rs2228001 showed a low-risk association only with female smokers. SNP rs2607775 revealed a statistically significant low-risk association with CS-induced diseases, concerning all parameters, except for male smokers. However, SNP rs2228000 and rs1870134 showed no association with CS. Overall, the study results demonstrated possible significant associations (effector/and protector) between CS and SNPs polymorphisms in DNA repair genes, such as XPA and XPC, except for rs2228000 and rs1870134 polymorphisms.

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Periodontitis is a chronic inflammatory disease that affects the supporting structures of teeth. In the literature, the association between the pathogenicity of bacteria and environmental factors in this regard have been extensively examined. In the present study, we will shed light on the potential role that epigenetic change can play on different facets of its process, more particularly the modifications concerning the genes involved in inflammation, defense, and immune systems. Since the 1960s, the role of genetic variants in the onset and severity of periodontal disease has been widely demonstrated. These make some people more susceptible to developing it than others. It has been documented that the wide variation in its frequency for various racial and ethnic populations is due primarily to the complex interplay among genetic factors with those affecting the environment and the demography. In molecular biology, epigenetic modifications are defined as any change in the promoter for the CpG islands, in the structure of the histone protein, as well as post-translational regulation by microRNAs (miRNAs), being known to contribute to the alteration in gene expression for complex multifactorial diseases such as periodontitis. The key role of epigenetic modification is to understand the mechanism involved in the gene-environment interaction, and the development of periodontitis is now the subject of more and more studies that attempt to identify which factors are stimulating it, but also affect the reduced response to therapy.

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We herein report a case of a 4-year-old Filipino girl initially seen through online consultation from a general physician. She was born to a 22-year-old primigravid mother, with no birth complications nor a history of consanguinity in the family. During the 1st month of life, she developed hyperpigmented macules over the face, neck, upper back, and limbs, which were exacerbated by sun exposure. At 2 years old, she developed a solitary erythematous papule on the nasal area, which gradually enlarged within one year and developed into an exophytic ulcerating tumor extending to the right supra-alar crease. Xeroderma pigmentosum and squamous cell carcinoma were confirmed by whole-exome sequencing and skin biopsy, respectively.

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Nucleotide excision repair (NER) is a central DNA repair pathway responsible for removing a wide variety of DNA-distorting lesions from the genome. The highly choreographed cascade of core NER reactions requires more than 30 polypeptides. The xeroderma pigmentosum group A (XPA) protein plays an essential role in the NER process. XPA interacts with almost all NER participants and organizes the correct NER repair complex. In the absence of XPA's scaffolding function, no repair process occurs. In this review, we briefly summarize our current knowledge about the XPA protein structure and analyze the formation of contact with its protein partners during NER complex assembling. We focus on different ways of regulation of the XPA protein's activity and expression and pay special attention to the network of post-translational modifications. We also discuss the data that is not in line with the currently accepted hypothesis about the functioning of the XPA protein.

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Defects in DNA repair pathways have been associated with an improved response to immune checkpoint inhibition (ICI). In particular, patients with the nucleotide excision repair (NER) defect disease Xeroderma pigmentosum (XP) responded impressively well to ICI treatment. Recently, in melanoma patients, pretherapeutic XP gene expression was predictive for anti-programmed cell death-1 (PD-1) ICI response. The underlying mechanisms of this finding are still to be revealed. Therefore, we used CRISPR/Cas9 to disrupt XPA in A375 melanoma cells. The resulting subclonal cell lines were investigated by Sanger sequencing. Based on their genetic sequence, candidates from XPA exon 1 and 2 were selected and further analyzed by immunoblotting, immunofluorescence, HCR and MTT assays. In XPA exon 1, we established a homozygous (c.19delG; p.A7Lfs*8) and a compound heterozygous (c.19delG/c.19_20insG; p.A7Lfs*8/p.A7Gfs*55) cell line. In XPA exon 2, we generated a compound heterozygous mutated cell line (c.206_208delTTG/c.208_209delGA; p.I69_D70delinsN/p.D70Hfs*31). The better performance of the homozygous than the heterozygous mutated exon 1 cells in DNA damage repair (HCR) and post-UV-C cell survival (MTT), was associated with the expression of a novel XPA protein variant. The results of our study serve as the fundamental basis for the investigation of the immunological consequences of XPA disruption in melanoma.

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Non-melanoma skin cancer (NMSC) is mainly caused by ultraviolet (UV)-induced somatic mutations and is characterized by UV signature modifications. Xeroderma pigmentosum group A (Xpa) knockout mice exhibit extreme UV-induced photo-skin carcinogenesis, along with a photosensitive phenotype. We performed whole-exome sequencing (WES) of squamous cell carcinoma (SCC) samples after repetitive ultraviolet B (UVB) exposure to investigate the differences in the landscape of somatic mutations between Xpa knockout and wild-type mice. Although the tumors that developed in mice harboured UV signature mutations in a similar set of cancer-related genes, the pattern of transcriptional strand asymmetry was largely different; UV signature mutations in Xpa knockout and wild-type mice preferentially occurred in transcribed and non-transcribed strands, respectively, reflecting a deficiency in transcription-coupled nucleotide excision repair in Xpa knockout mice. Serial time point analyses of WES for a tumor induced by only a single UVB exposure showed pathogenic mutations in Kras, Fat1, and Kmt2c, which may be driver genes for the initiation and promotion of SCC in Xpa knockout mice. Furthermore, the inhibitory effects on tumor production in Xpa knockout mice by the anti-inflammatory CXCL1 monoclonal antibody affected the pattern of somatic mutations, wherein the transcriptional strand asymmetry was attenuated and the activated signal transduction was shifted from the RAS/RAF/MAPK to the PIK3CA pathway.

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Glioblastoma is the most frequent, as well as aggressive kind of high-grade malignant glioma. Chemoresistance is posing a significant clinical barrier to the efficacy of temozolomide-based glioblastoma treatment. By suppressing xeroderma pigmentosum group A (XPA), a pivotal DNA damage recognition protein implicated in nucleotide excision repair (NER), we devised a novel method to enhance glioblastoma therapy and alleviate temozolomide resistance. On the basis of preliminary assessment, we found that XPA dramatically increased in glioblastoma compared with normal cells and contributed to temozolomide resistance. By constructing XPA stably knockdown cells, we illustrate that XPA protects glioma cells from temozolomide-triggered reproductive cell death, apoptosis, as well as DNA repair. Besides, XPA silencing remarkably enhances temozolomide efficacy in vivo. This study revealed a crucial function of XPA-dependent NER in the resistance of glioma cells to temozolomide.

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The association of multiple pilomatricomas with xeroderma pigmentosum has not been described. We report a case of a child with multiple pilomatricomas and photosensitivity who was found to have a pathogenic variant in exon 4 of XPA and a likely pathogenic variant in COL6A1.

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The xeroderma pigmentosum group A (XPA) protein plays an essential role in the removal of UV photoproducts and other bulky lesions from DNA as a component of the nucleotide excision repair (NER) machinery. Using cell lysates prepared from confluent cultures of human cells and from human skin epidermis, we observed an additional XPA antibody-reactive band on immunoblots that was approximately 3-4 kDa smaller than the native, full-length XPA protein. Biochemical studies revealed this smaller molecular weight XPA species to be due to proteolysis at the C-terminus of the protein, which negatively impacted the ability of XPA to interact with the NER protein TFIIH. Further work identified the endopeptidase cathepsin L, which is expressed at higher levels in quiescent cells, as the protease responsible for cleaving XPA during cell lysis. These results suggest that supplementation of lysis buffers with inhibitors of cathepsin L is important to prevent cleavage of XPA during lysis of confluent cells.

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Cockayne syndrome (CS) is a rare, autosomal genetic disorder characterized by premature aging-like features, such as cachectic dwarfism, retinal atrophy, and progressive neurodegeneration. The molecular defect in CS lies in genes associated with the transcription-coupled branch of the nucleotide excision DNA repair (NER) pathway, though it is not yet clear how DNA repair deficiency leads to the multiorgan dysfunction symptoms of CS. In this work, we used a mouse model of severe CS with complete loss of NER (Csa-/-/Xpa-/-), which recapitulates several CS-related phenotypes, resulting in premature death of these mice at approximately 20 weeks of age. Although this CS model exhibits a severe progeroid phenotype, we found no evidence of in vitro endothelial cell dysfunction, as assessed by measuring population doubling time, migration capacity, and ICAM-1 expression. Furthermore, aortas from CX mice did not exhibit early senescence nor reduced angiogenesis capacity. Despite these observations, CX mice presented blood brain barrier disruption and increased senescence of brain endothelial cells. This was accompanied by an upregulation of inflammatory markers in the brains of CX mice, such as ICAM-1, TNFα, p-p65, and glial cell activation. Inhibition of neovascularization did not exacerbate neither astro- nor microgliosis, suggesting that the pro-inflammatory phenotype is independent of the neurovascular dysfunction present in CX mice. These findings have implications for the etiology of this disease and could contribute to the study of novel therapeutic targets for treating Cockayne syndrome patients.

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BACKGROUND: To explore the potential biological function of XPA (Xeroderma pigmentosum group A) in hepatic neoplasms and the underlying molecular mechanisms. METHODS: Liver cells were used as experimental models to establish HCC (hepatocellular carcinoma) in vitro. Protein extractions were subjected to Western blotting to detect the proteins expression. The lentivirus transfection efficiency was confirmed by Western blot and RT-qPCR, Tunnel staining was used to detect apoptosis, and Transwell assays were used to observe cell migration and invasion. Cell proliferation was detected with colony formation and CCK-8 (cell counting kit-8) assays. RESULTS: XPA expression was obviously lower in HCC tissue and liver cancer cell lines. XPA overexpression induced autophagy and apoptosis by increasing LC3B II/I, Beclin1, cleaved-caspase-3, and Bax expression and decreasing p62 and Bcl2 protein levels. XPA also suppressed HCC EMT (Epithelial-Mesenchymal Transition) by increasing E-cadherin and decreasing N-cadherin and vimentin protein expression. Cell proliferation, migration and invasion in vivo were significantly inhibited by the overexpression of XPA, and p-PI3K, p-Akt, and p-mTOR expression were decreased in LV-XPA cells. In general, XPA inhibited HCC by inducing autophagy and apoptosis and by modulating the expression of PI3K/Akt/mTOR proteins. CONCLUSIONS: XPA overexpression was found to suppress HCC by inducing autophagy and apoptosis and repressing EMT and proliferation. Each of these effects may be involved in modulating the PI3K/Akt/mTOR signaling pathway.

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Cisplatin is a chemotherapeutic drug used for the treatment of a number of cancers. The efficacy of cisplatin relies on its binding to DNA and the induction of cytotoxic DNA damage to kill cancer cells. Cisplatin-based therapy is best known for curing testicular cancer; however, treatment of other solid tumors with cisplatin has not been as successful. Pre-clinical and clinical studies have revealed nucleotide excision repair (NER) as a major resistance mechanism against cisplatin in tumor cells. NER is a versatile DNA repair system targeting a wide range of helix-distorting DNA damage. The NER pathway consists of multiple steps, including damage recognition, pre-incision complex assembly, dual incision, and repair synthesis. NER proteins can recognize cisplatin-induced DNA damage and remove the damage from the genome, thereby neutralizing the cytotoxicity of cisplatin and causing drug resistance. Here, we review the molecular mechanism by which NER repairs cisplatin damage, focusing on the recent development of genome-wide cisplatin damage mapping methods. We also discuss how the expression and somatic mutations of key NER genes affect the response of cancer cells to cisplatin. Finally, small molecules targeting NER factors provide important tools to manipulate NER capacity in cancer cells. The status of research on these inhibitors and their implications in cancer treatment will be discussed.

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DNA repair is a highly dynamic process in which the actual damage recognition process occurs through an amazing dance between the DNA duplex containing the lesion and the DNA repair proteins. Single molecule investigations have revealed that DNA repair proteins solve the speed-stability paradox, of rapid search versus stable complex formation, by conformational changes induced in both the damaged DNA and the repair proteins. Using Rad4, XPA, PARP1, APE1, OGG1 and UV-DDB as examples, we have discovered how these repair proteins limit their travel on DNA, once a lesion is encountered through a process of anomalous diffusion. We have also observed how PARP1 and APE1, as well as UV-DDB and OGG1 or APE1, co-localize dynamically at sites near DNA damage. This review highlights how our group has greatly benefited from our productive collaborations with Sam Wilson's research group.

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Xeroderma pigmentosum complementation group A (XPA), is defective in xeroderma pigmentosum patients, causing pre-disposition to skin cancer and neurological abnormalities, which is not well understood. Here, we analyzed the XPA-deficient cells transcriptional profile under oxidative stress. The imbalance in of ubiquitin-proteasome system (UPS) gene expression was observed in XPA-deficient cells and the involvement of nuclear factor erythroid 2-related factor-2 (NFE2L2) was indicated. Co-immunoprecipitation assays showed the interaction between XPA, apurinic-apyrimidinic endonuclease 1 (APE1) and NFE2L2 proteins. Decreased NFE2L2 protein expression and proteasome activity was also observed in XPA-deficient cells. The data suggest the involvement of the growth arrest and DNA-damage-inducible beta (GADD45ß) in NFE2L2 functions. Similar results were obtained in xpa-1 (RNAi) Caenorhabditis elegans suggesting the conservation of XPA and NFE2L2 interactions. In conclusion, stress response activation occurs in XPA-deficient cells under oxidative stress; however, these cells fail to activate the UPS cytoprotective response, which may contribute to XPA patient's phenotypes.

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BACKGROUND: The objective of the present study is to comprehensively evaluate the impact of the rs1800975 A/G polymorphism within the human xeroderma pigmentosum group A (XPA) gene on susceptibility to overall cancer by performing an integrative analysis of the current evidence. METHODS: We retrieved possible relevant publications from a total of six electronic databases (updated to April 2020) and selected eligible case-control studies for pooled assessment. P-values of association and odds ratio (OR) were calculated for the assessment of association effect. We also performed Begg's test and Egger's test, sensitivity analysis, false-positive report probability (FPRP) analysis, trial sequential analysis (TSA), and expression/splicing quantitative trait loci (eQTL/sQTL) analyses. RESULTS: In total, 71 case-control studies with 19,257 cases and 30,208 controls from 52 publications were included for pooling analysis. We observed an enhanced overall cancer susceptibility in cancer cases compared with negative controls in the Caucasian subgroup analysis for the genetic models of allelic G vs. A, carrier G vs. A, homozygotic GG vs AA, heterozygotic AG vs. AA, dominant AG + GG vs. AA and recessive GG vs. AA + AG (P < 0.05, OR > 1). A similar positive conclusion was also detected in the "skin cancer" or "skin basal cell carcinoma (BCC)" subgroup analysis of the Caucasian population. Our FPRP analysis and TSA results further confirmed the robustness of the conclusion. However, our eQTL/sQTL data did not support the strong links of rs1800975 with the gene expression or splicing changes of XPA in the skin tissue. In addition, even though we observed a decreased risk of lung cancer under the homozygotic, heterozygotic and dominant models (P < 0.05, OR < 1) and an enhanced risk of colorectal cancer under the allelic, homozygotic, heterozygotic, dominant (P < 0.05, OR > 1), our data from FPRP analysis and another pooling analysis with only the population-based controls in the Caucasian population did not support the strong links between the XPA rs1800975 A/G polymorphism and the risk of lung or colorectal cancer. CONCLUSIONS: Our findings provide evidence of the close relationship between the XPA rs1800975 A/G polymorphism and susceptibility to skin cancer in the Caucasian population. The potential effect of XPA rs1800975 on the risk of developing lung or colorectal cancer still merits the enrollment of larger well-scaled studies.

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The nucleotide excision repair (NER) pathway is activated in response to a broad spectrum of DNA lesions, including bulky lesions induced by platinum-based chemotherapeutic agents. Expression levels of NER factors and resistance to chemotherapy has been examined with some suggestion that NER plays a role in tumour resistance; however, there is a great degree of variability in these studies. Nevertheless, recent clinical studies have suggested Xeroderma Pigmentosum group A (XPA) protein, a key regulator of the NER pathway that is essential for the repair of DNA damage induced by platinum-based chemotherapeutics, as a potential prognostic and predictive biomarker for response to treatment. XPA functions in damage verification step in NER, as well as a molecular scaffold to assemble other NER core factors around the DNA damage site, mediated by protein-protein interactions. In this review, we focus on the interacting partners and mechanisms of regulation of the XPA protein. We summarize clinical oncology data related to this DNA repair factor, particularly its relationship with treatment outcome, and examine the potential of XPA as a target for small molecule inhibitors.

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