Persistent TFIIH binding to non-excised DNA damage causes cell and developmental failure.

Congenital nucleotide excision repair (NER) deficiency gives rise to several cancer-prone and/or progeroid disorders. It is not understood how defects in the same DNA repair pathway cause different disease features and severity. Here, we show that the absence of functional ERCC1-XPF or XPG endonucleases leads to stable and prolonged binding of the transcription/DNA repair factor TFIIH to DNA damage, which correlates with disease severity and induces senescence features in human cells. In vivo, in C. elegans, this prolonged TFIIH binding to non-excised DNA damage causes developmental arrest and neuronal dysfunction, in a manner dependent on transcription-coupled NER. NER factors XPA and TTDA both promote stable TFIIH DNA binding and their depletion therefore suppresses these severe phenotypical consequences. These results identify stalled NER intermediates as pathogenic to cell functionality and organismal development, which can in part explain why mutations in XPF or XPG cause different disease features than mutations in XPA or TTDA.

Relationship between XPA, XPB/ERCC3, XPF/ERCC4, and XPG/ERCC5 Polymorphisms and the Susceptibility to Head and Neck Carcinoma: A Systematic Review, Meta-Analysis, and Trial Sequential Analysis.

Background and Objectives: Nucleotide Excision Repair (NER), the most extensively researched DNA repair mechanism, is responsible for repairing a variety of DNA damages, and Xeroderma Pigmentosum (XP) genes participate in NER. Herein, we aimed to update the previous results with a meta-analysis evaluating the association of XPA, XPB/ERCC3, XPF/ERCC4, and XPG/ERCC5 polymorphisms with the susceptibility to HNC. Materials and Methods: PubMed/Medline, Web of Science, Scopus, and Cochrane Library databases were searched without any restrictions until 18 November 2023 to find relevant studies. The Review Manager 5.3 (RevMan 5.3) software was utilized to compute the effect sizes, which were expressed as the odds ratio (OR) with a 95% confidence interval (CI). Results: Nineteen articles were involved in the systematic review and meta-analysis that included thirty-nine studies involving ten polymorphisms. The results reported that the CC genotype of rs17655 polymorphism showed a significantly decreased risk of HNC in the recessive model (OR: 0.89; 95%CI: 0.81, 0.99; p-value is 0.03). In addition, the CT genotype (OR: 0.65; 95%CI: 0.48, 0.89; p-value is 0.008) of the rs751402 polymorphism was associated with a decreased risk, and the T allele (OR: 1.28; 95%CI: 1.05, 1.57; p-value is 0.02), the TT (OR: 1.74; 95%CI: 1.10, 2.74; p-value is 0.02), and the TT + CT (OR: 2.22; 95%CI: 1.04, 4.74; p-value is 0.04) genotypes were associated with an increased risk of HNC. Conclusions: The analysis identified two polymorphisms, rs17655 and rs751402, as being significantly associated with the risk of HNC. The study underscored the influence of various factors, such as the type of cancer, ethnicity, source of control, and sample size on these associations.

Generation of site-specifically labelled fluorescent human XPA to investigate DNA binding dynamics during nucleotide excision repair.

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.

Small Molecule Antagonists of the DNA Repair ERCC1/XPA Protein-Protein Interaction.

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.

Cross-species investigation into the requirement of XPA for nucleotide excision repair.

After reconstitution of nucleotide excision repair (excision repair) with XPA, RPA, XPC, TFIIH, XPF-ERCC1 and XPG, it was concluded that these six factors are the minimal essential components of the excision repair machinery. All six factors are highly conserved across diverse organisms spanning yeast to humans, yet no identifiable homolog of the XPA gene exists in many eukaryotes including green plants. Nevertheless, excision repair is reported to be robust in the XPA-lacking organism, Arabidopsis thaliana, which raises a fundamental question of whether excision repair could occur without XPA in other organisms. Here, we performed a phylogenetic analysis of XPA across all species with annotated genomes and then quantitatively measured excision repair in the absence of XPA using the sensitive whole-genome qXR-Seq method in human cell lines and two model organisms, Caenorhabditis elegans and Drosophila melanogaster. We find that although the absence of XPA results in inefficient excision repair and UV-sensitivity in humans, flies, and worms, excision repair of UV-induced DNA damage is detectable over background. These studies have yielded a significant discovery regarding the evolution of XPA protein and its mechanistic role in nucleotide excision repair.

A Significant Increasing Risk Association between Cigarette Smoking and XPA and XPC Genes Polymorphisms.

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.

A novel XPA splice-site mutation identified in a 4-year-old Filipino girl with xeroderma pigmentosum.

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.

A disease-associated XPA allele interferes with TFIIH binding and primarily affects transcription-coupled nucleotide excision repair.

XPA is a central scaffold protein that coordinates the assembly of repair complexes in the global genome (GG-NER) and transcription-coupled nucleotide excision repair (TC-NER) subpathways. Inactivating mutations in XPA cause xeroderma pigmentosum (XP), which is characterized by extreme UV sensitivity and a highly elevated skin cancer risk. Here, we describe two Dutch siblings in their late forties carrying a homozygous H244R substitution in the C-terminus of XPA. They present with mild cutaneous manifestations of XP without skin cancer but suffer from marked neurological features, including cerebellar ataxia. We show that the mutant XPA protein has a severely weakened interaction with the transcription factor IIH (TFIIH) complex leading to an impaired association of the mutant XPA and the downstream endonuclease ERCC1-XPF with NER complexes. Despite these defects, the patient-derived fibroblasts and reconstituted knockout cells carrying the XPA-H244R substitution show intermediate UV sensitivity and considerable levels of residual GG-NER (~50%), in line with the intrinsic properties and activities of the purified protein. By contrast, XPA-H244R cells are exquisitely sensitive to transcription-blocking DNA damage, show no detectable recovery of transcription after UV irradiation, and display a severe deficiency in TC-NER-associated unscheduled DNA synthesis. Our characterization of a new case of XPA deficiency that interferes with TFIIH binding and primarily affects the transcription-coupled subpathway of nucleotide excision repair, provides an explanation of the dominant neurological features in these patients, and reveals a specific role for the C-terminus of XPA in TC-NER.

The XPA Protein-Life under Precise Control.

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.

Homozygous CRISPR/Cas9 Knockout Generated a Novel Functionally Active Exon 1 Skipping XPA Variant in Melanoma Cells.

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.

Two interaction surfaces between XPA and RPA organize the preincision complex in nucleotide excision repair.

The xeroderma pigmentosum protein A (XPA) and replication protein A (RPA) proteins fulfill essential roles in the assembly of the preincision complex in the nucleotide excision repair (NER) pathway. We have previously characterized the two interaction sites, one between the XPA N-terminal (XPA-N) disordered domain and the RPA32 C-terminal domain (RPA32C), and the other with the XPA DNA binding domain (DBD) and the RPA70AB DBDs. Here, we show that XPA mutations that inhibit the physical interaction in either site reduce NER activity in biochemical and cellular systems. Combining mutations in the two sites leads to an additive inhibition of NER, implying that they fulfill distinct roles. Our data suggest a model in which the interaction between XPA-N and RPA32C is important for the initial association of XPA with NER complexes, while the interaction between XPA DBD and RPA70AB is needed for structural organization of the complex to license the dual incision reaction. Integrative structural models of complexes of XPA and RPA bound to single-stranded/double-stranded DNA (ss/dsDNA) junction substrates that mimic the NER bubble reveal key features of the architecture of XPA and RPA in the preincision complex. Most critical among these is that the shape of the NER bubble is far from colinear as depicted in current models, but rather the two strands of unwound DNA must assume a U-shape with the two ss/dsDNA junctions localized in close proximity. Our data suggest that the interaction between XPA and RPA70 is key for the organization of the NER preincision complex.

Revisiting the structural features of the xeroderma pigmentosum proteins: Focus on mutations and knowledge gaps.

The nucleotide excision repair pathway is a broadly studied DNA repair mechanism because impairments of its key players, the xeroderma pigmentosum proteins (XPA to XPG), are associated with multiple hereditary diseases. Due to the massive number of novel mutations reported for these proteins and new structural data published every year, proper categorization and discussion of relevant observations is needed to organize this extensive inflow of knowledge. This review aims to revisit the structural data of all XP proteins while updating it with the information developed in of the past six years. Discussions and interpretations of mutation outcomes, mechanisms of action, and knowledge gaps regarding their structures are provided, as well as new perspectives based on recent research.

The landscape of genetic alterations of UVB-induced skin tumors in DNA repair-deficient mice.

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.

XPA Enhances Temozolomide Resistance of Glioblastoma Cells by Promoting Nucleotide Excision Repair.

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.

Global and transcription-coupled repair of 8-oxoG is initiated by nucleotide excision repair proteins.

UV-DDB, consisting of subunits DDB1 and DDB2, recognizes UV-induced photoproducts during global genome nucleotide excision repair (GG-NER). We recently demonstrated a noncanonical role of UV-DDB in stimulating base excision repair (BER) which raised several questions about the timing of UV-DDB arrival at 8-oxoguanine (8-oxoG), and the dependency of UV-DDB on the recruitment of downstream BER and NER proteins. Using two different approaches to introduce 8-oxoG in cells, we show that DDB2 is recruited to 8-oxoG immediately after damage and colocalizes with 8-oxoG glycosylase (OGG1) at sites of repair. 8-oxoG removal and OGG1 recruitment is significantly reduced in the absence of DDB2. NER proteins, XPA and XPC, also accumulate at 8-oxoG. While XPC recruitment is dependent on DDB2, XPA recruitment is DDB2-independent and transcription-coupled. Finally, DDB2 accumulation at 8-oxoG induces local chromatin unfolding. We propose that DDB2-mediated chromatin decompaction facilitates the recruitment of downstream BER proteins to 8-oxoG lesions.

Probing Protein-DNA Conformational Dynamics in DNA Damage Recognition: Xeroderma Pigmentosum Group A Stabilizes the Damaged DNA-RPA14 Complex by Controlling Conformational Fluctuation Dynamics.

DNA damage inside biological systems may result in diseases like cancer. One of the major repairing mechanisms is the nucleotide excision repair (NER) that recognizes and repairs the damage caused by several internal and external exposures, such as DNA double-strand distortion due to the chemical modifications. Recognition of lesions is the initial stage of the DNA damage repair, which occurs with the help of several proteins like Replication Protein A (RPA) and Xeroderma Pigmentosum group A (XPA). The recognition process involves complex conformational dynamics of the proteins. Studying the dynamics of damage recognition by these proteins helps us to understand the mechanism and to develop therapeutics to increase the efficiency of recognition. Here, we use single-molecule fluorescence fluctuation measurements of a dye, labeled at a damaged position on DNA, to understand the interaction of the damage site with RPA14 and XPA. Our results suggest that interactive conformational dynamics of RPA14 with damaged DNA is inhomogeneous due to its low affinity for DNA, whereas binding of XPA with the already formed DNA-RPA14 complex may increase the specificity of damage recognition by controlling the conformational fluctuation dynamics of the complex.

Acetaldehyde induces NER repairable mutagenic DNA lesions.

Nucleotide excision repair (NER) is a repair mechanism that removes DNA lesions induced by UV radiation, environmental mutagens and carcinogens. There exists sufficient evidence against acetaldehyde suggesting it to cause a variety of DNA lesions and be carcinogenic to humans. Previously, we found that acetaldehyde induces reversible intra-strand GG crosslinks in DNA similar to those induced by cis-diammineplatinum(II) that is subsequently repaired by NER. In this study, we analysed the repairability by NER mechanism and the mutagenesis of acetaldehyde. In an in vitro reaction setup with NER-proficient and NER-deficient xeroderma pigmentosum group A (XPA) cell extracts, NER reactions were observed in the presence of XPA recombinant proteins in acetaldehyde-treated plasmids. Using an in vivo assay with living XPA cells and XPA-correcting XPA cells, the repair reactions were also observed. Additionally, it was observed that DNA polymerase eta inserted dATP opposite guanine in acetaldehyde-treated oligonucleotides, suggesting that acetaldehyde-induced GG-to-TT transversions. These findings show that acetaldehyde induces NER repairable mutagenic DNA lesions.

Neurovascular dysfunction and neuroinflammation in a Cockayne syndrome mouse model.

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.

Xeroderma pigmentosum A homolog from Hydra partially complements DNA repair defect in human XPA-deficient cells.

Nucleotide excision repair (NER) pathway is a DNA repair mechanism that rectifies a wide spectrum of DNA lesions. Xeroderma pigmentosum group of proteins (XPA through XPG) orchestrate the NER pathway in humans. We have earlier studied XPA homolog from Hydra (HyXPA) and found it to be similar to human XPA. Here, we examined if HyXPA can functionally complement human XPA-deficient cells and reduce their sensitivity to UV radiation. We found that HyXPA was able to partially rescue XPA-deficient human cells from UV by its binding to chromatin of UV-irradiated cells. However, HyXPA failed to bind replication protein A (RPA70), a key interacting partner of human XPA in NER pathway. This could be attributed to changes in certain amino acid residues that have occurred during evolution, leading to prevention of some interactions between Hydra and human proteins.

Autophagy induction on impaired spermatogenesis of xeroderma pigmentosum group A gene-deficient mice.

Xeroderma pigmentosum (XP) involves a defect in the initial step of nucleotide excision repair (NER) and consists of eight genetic complementation groups (groups A-G and a variant). XP group A (XPA) patients have a high incidence of UV-induced skin tumors, immature testicular development, and neurological symptoms. In an earlier study, we have shown that XP group A (Xpa) gene-knockout mice (Xpa-/- mice) were highly sensitive to UV-induced skin carcinogenesis with a defect in NER and were highly susceptibility to spontaneous tumorigenesis with impaired spermatogenesis. However, the pathology of impaired spermatogenesis in Xpa-/- mice is unknown. To unravel the underlying pathology, we made a concerted effort using the testis of 3-month-old Xpa-/- mice. We found many large vacuoles in the seminiferous tubules of 3-month old Xpa-/- mice, while there were no large vacuoles in that of Xpa+/+ mice. Immunohistochemistry of microtubule-associated protein 1 light chain 3 (LC3), an autophagosome marker, showed degenerating cells with intense signal of LC3 in the seminiferous tubules, and immunoblotting revealed induction of LC3-II in the 3-month-old Xpa-/- mice. The results of the present study suggest autophagy induction as the possible mechanism underlying the impaired spermatogenesis in Xpa-/- mice. Therefore, Xpa-/- mice could be a useful model for investigating aging and male infertility with low expression of XPA.