Global Genome Nucleotide Excision Repair Research Articles

Interplay of the Tfb1 pleckstrin homology domain with Rad2 and Rad4 in transcription coupled and global genomic nucleotide excision repair.

Transcription-coupled repair (TCR) and global genomic repair (GGR) are two subpathways of nucleotide excision repair (NER). The TFIIH subunit Tfb1 contains a Pleckstrin homology domain (PHD), which was shown to interact with one PHD-binding segment (PB) of Rad4 and two PHD-binding segments (PB1 and PB2) of Rad2 in vitro. Whether and how the different Rad2 and Rad4 PBs interact with the same Tfb1 PHD, and whether and how they affect TCR and GGR within the cell remain mysterious. We found that Rad4 PB constitutively interacts with Tfb1 PHD, and the two proteins may function within one module for damage recognition in TCR and GGR. Rad2 PB1 protects Tfb1 from degradation and interacts with Tfb1 PHD at a basal level, presumably within transcription preinitiation complexes when NER is inactive. During a late step of NER, the interaction between Rad2 PB1 and Tfb1 PHD augments, enabling efficient TCR and GGR. Rather than interacting with Tfb1 PHD, Rad2 PB2 constrains the basal interaction between Rad2 PB1 and Tfb1 PHD, thereby weakening the protection of Tfb1 from degradation and enabling rapid augmentation of their interactions within TCR and GGR complexes. Our results shed new light on NER mechanisms.

Global genome and transcription-coupled nucleotide excision repair pathway in prokaryotes

Global genome and transcription-coupled nucleotide excision repair pathway in prokaryotes

Detection of oxaliplatin- and cisplatin-DNA lesions requires different global genome repair mechanisms that affect their clinical efficacy.

The therapeutic efficacy of cisplatin and oxaliplatin depends on the balance between the DNA damage induction and the DNA damage response of tumor cells. Based on clinical evidence, oxaliplatin is administered to cisplatin-unresponsive cancers, but the underlying molecular causes for this tumor specificity are not clear. Hence, stratification of patients based on DNA repair profiling is not sufficiently utilized for treatment selection. Using a combination of genetic, transcriptomics and imaging approaches, we identified factors that promote global genome nucleotide excision repair (GG-NER) of DNA-platinum adducts induced by oxaliplatin, but not by cisplatin. We show that oxaliplatin-DNA lesions are a poor substrate for GG-NER initiating factor XPC and that DDB2 and HMGA2 are required for efficient binding of XPC to oxaliplatin lesions and subsequent GG-NER initiation. Loss of DDB2 and HMGA2 therefore leads to hypersensitivity to oxaliplatin but not to cisplatin. As a result, low DDB2 levels in different colon cancer cells are associated with GG-NER deficiency and oxaliplatin hypersensitivity. Finally, we show that colon cancer patients with low DDB2 levels have a better prognosis after oxaliplatin treatment than patients with high DDB2 expression. We therefore propose that DDB2 is a promising predictive marker of oxaliplatin treatment efficiency in colon cancer.

NAT10 regulates the repair of UVB-induced DNA damage and tumorigenicity

Chemical modifications in messenger RNA (mRNA) regulate gene expression and play critical roles in stress responses and diseases. Recently we have shown that N6-methyladenosine (m6A), the most abundant mRNA modification, promotes the repair of UVB-induced DNA damage by regulating global genome nucleotide excision repair (GG-NER). However, the roles of other mRNA modifications in the UVB-induced damage response remain understudied. N4-acetylcytidine (ac4C) is deposited in mRNA by the RNA-binding acetyltransferase NAT10. This NAT10-mediated ac4C in mRNA has been reported to increase both mRNA stability and translation. However, the role of ac4C and NAT10 in the UVB-induced DNA damage response remains poorly understood. Here we show that NAT10 plays a critical role in the repair of UVB-induced DNA damage lesions through regulating the expression of the key GG-NER gene DDB2. We found that knockdown of NAT10 enhanced the repair of UVB-induced DNA damage lesions by promoting the mRNA stability of DDB2. Our findings are in contrast to the previously reported role of NAT10-mediated ac4C deposition in promoting mRNA stability and may represent a novel mechanism for ac4C in the UVB damage response. Furthermore, NAT10 knockdown in skin cancer cells decreased skin cancer cell proliferation in vitro and tumorigenicity in vivo. Chronic UVB irradiation increases NAT10 protein levels in mouse skin. Taken together, our findings demonstrate a novel role for NAT10 in the repair of UVB-induced DNA damage products by decreasing the mRNA stability of DDB2 and suggest that NAT10 is a potential novel target for preventing and treating skin cancer.

The role of Transcription Factor IIH complex in nucleotide excision repair.

DNA damage occurs throughout life from a variety of sources, and it is imperative to repair damage in a timely manner to maintain genome stability. Thus, DNA repair mechanisms are a fundamental part of life. Nucleotide excision repair (NER) plays an important role in the removal of bulky DNA adducts, such as cyclobutane pyrimidine dimers from ultraviolet light or DNA crosslinking damage from platinum-based chemotherapeutics, such as cisplatin. A main component for the NER pathway is transcription factor IIH (TFIIH), a multifunctional, 10-subunit protein complex with crucial roles in both transcription and NER. In transcription, TFIIH is a component of the pre-initiation complex and is important for promoter opening and the phosphorylation of RNA Polymerase II (RNA Pol II). During repair, TFIIH is important for DNA unwinding, recruitment of downstream repair factors, and verification of the bulky lesion. Several different disease states can arise from mutations within subunits of the TFIIH complex. Most strikingly are xeroderma pigmentosum (XP), XP combined with Cockayne syndrome (CS), and trichothiodystrophy (TTD). Here, we summarize the recruitment and functions of TFIIH in the two NER subpathways, global genomic (GG-NER) and transcription-coupled NER (TC-NER). We will also discuss how TFIIH's roles in the two subpathways lead to different genetic disorders.

ASH1L-MRG15 methyltransferase deposits H3K4me3 and FACT for damage verification in nucleotide excision repair

To recognize DNA adducts, nucleotide excision repair (NER) deploys the XPC sensor, which detects damage-induced helical distortions, followed by engagement of TFIIH for lesion verification. Accessory players ensure that this factor handover takes place in chromatin where DNA is tightly wrapped around histones. Here, we describe how the histone methyltransferase ASH1L, once activated by MRG15, helps XPC and TFIIH to navigate through chromatin and induce global-genome NER hotspots. Upon UV irradiation, ASH1L adds H3K4me3 all over the genome (except in active gene promoters), thus priming chromatin for XPC relocations from native to damaged DNA. The ASH1L-MRG15 complex further recruits the histone chaperone FACT to DNA lesions. In the absence of ASH1L, MRG15 or FACT, XPC is misplaced and persists on damaged DNA without being able to deliver the lesions to TFIIH. We conclude that ASH1L-MRG15 makes damage verifiable by the NER machinery through the sequential deposition of H3K4me3 and FACT.

Distinctive Participation of Transcription-Coupled and Global Genome Nucleotide Excision Repair of Pyrimidine Dimers in the Transcribed Strand of Yeast rRNA Genes.

UV light causes the formation of pyrimidine dimers (PDs). Transcription-coupled (TC) nucleotide excision repair (NER) and global genome (GG) NER remove PDs from the transcribed strand (TS) of active genes and the inactive genome, respectively. TC-NER is triggered by elongating RNA polymerases that are blocked at PDs. The yeast rRNA genes are densely loaded with RNA polymerase-I. After UV irradiation, their density increases at the 5'-end of the gene, which results from continuous transcription initiation, followed by elongation and pausing/release at the first encountered PD, from the transcription start site. RNA polymerase-I posed at downstream PDs are released from the TS and are replaced by nucleosomes. Consequently, discrete chromatin structures are formed in the damaged transcribed rRNA genes. Singular assignation of the two NER sub-pathways could therefore be required to eliminate PDs from the TS. To advance our understanding of NER in the dynamic structure of transcribed chromatin, we investigated the repair of PDs at nucleotide resolution in separate rRNA gene coding regions. In the TS, the TC-NER efficiency reflected the density of RNA polymerase-I, and PDs were removed faster in the 5'-end than in the 3'-end of the gene. GG-NER removed PDs from the TS where RNA polymerase-I was transiently replaced by a nucleosome. The two NER sub-pathways inversely participated to remove PDs from the TS. In the non-TS of both nucleosome and non-nucleosome rRNA gene coding regions, GG-NER was solely responsible to remove UV-induced DNA lesions.

Transcription-Coupled Nucleotide Excision Repair and the Transcriptional Response to UV-Induced DNA Damage.

Ultraviolet (UV) irradiation and other genotoxic stresses induce bulky DNA lesions, which threaten genome stability and cell viability. Cells have evolved two main repair pathways to remove such lesions: global genome nucleotide excision repair (GG-NER) and transcription-coupled nucleotide excision repair (TC-NER). The modes by which these subpathways recognize DNA lesions are distinct, but they converge onto the same downstream steps for DNA repair. Here, we first summarize the current understanding of these repair mechanisms, specifically focusing on the roles of stalled RNA polymerase II, Cockayne syndrome protein B (CSB), CSA and UV-stimulated scaffold protein A (UVSSA) in TC-NER. We also discuss the intriguing role of protein ubiquitylation in this process. Additionally, we highlight key aspects of the effect of UV irradiation on transcription and describe the role of signaling cascades in orchestrating this response. Finally, we describe the pathogenic mechanisms underlying xeroderma pigmentosum and Cockayne syndrome, the two main diseases linked to mutations in NER factors. Expected final online publication date for the Annual Review of Biochemistry, Volume 92 is June 2023. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Loss-of-Function of xpc Sensitizes Zebrafish to Ultraviolet Irradiation

Xeroderma pigmentosum complementation group C (XPC) protein recognizes bulky DNA adducts to initiate global genomic nucleotide excision repair (GG-NER). Humans carrying germline mutations in the XPC gene display strong susceptibility to skin and certain internal cancers. In addition to its role in NER, recent studies have indicated that XPC is also involved in other DNA damage repair pathways and transcription regulation. In this report, we generated a zebrafish xpc knockout mutant. Zebrafish xpc−/− mutant fish develop relative normally and are fertile. However, the mutant embryos were more sensitive to ultraviolet (UV) irradiation. Upon UV irradiation, compared with the wild type embryos, mutant embryos accumulated significantly higher levels of unrepaired DNA damages and apoptotic cells, which led to more severe abnormal development. Transcriptome analysis showed that the p53 signal pathway and apoptosis were enriched in the over upregulated genes in UV-irradiated mutant embryos, suggesting that high levels of unrepaired DNA lesions activated p53 to trigger apoptotic activity in mutant embryos. More interestingly, up to 972 genes in the untreated mutant embryos were differentially expressed, compared with those in the untreated WT. Among these differentially expressed genes (DEGs), 379 genes did not respond to UV irradiation, indicating that Xpc plays a role in addition of DNA damage repair. Our results demonstrate that Xpc is an evolutionally conserved factor in NER repair. Zebrafish xpc−/− mutant also provides a platform to study other functions of Xpc beyond the DNA damage repair.

UV-DDB stimulates the activity of SMUG1 during base excision repair of 5-hydroxymethyl-2'-deoxyuridine moieties.

UV-damaged DNA-binding protein (UV-DDB) is a heterodimeric protein, consisting of DDB1 and DDB2 subunits, that works to recognize DNA lesions induced by UV damage during global genome nucleotide excision repair (GG-NER). Our laboratory previously discovered a non-canonical role for UV-DDB in the processing of 8-oxoG, by stimulating 8-oxoG glycosylase, OGG1, activity 3-fold, MUTYH activity 4-5-fold, and APE1 (apurinic/apyrimidinic endonuclease 1) activity 8-fold. 5-hydroxymethyl-deoxyuridine (5-hmdU) is an important oxidation product of thymidine which is removed by single-strand selective monofunctional DNA glycosylase (SMUG1). Biochemical experiments with purified proteins indicated that UV-DDB stimulates the excision activity of SMUG1 on several substrates by 4-5-fold. Electrophoretic mobility shift assays indicated that UV-DDB displaced SMUG1 from abasic site products. Single-molecule analysis revealed that UV-DDB decreases the half-life of SMUG1 on DNA by ∼8-fold. Immunofluorescence experiments demonstrated that cellular treatment with 5-hmdU (5 μM for 15 min), which is incorporated into DNA during replication, produces discrete foci of DDB2-mCherry, which co-localize with SMUG1-GFP. Proximity ligation assays supported a transient interaction between SMUG1 and DDB2 in cells. Poly(ADP)-ribose accumulated after 5-hmdU treatment, which was abrogated with SMUG1 and DDB2 knockdown. These data support a novel role for UV-DDB in the processing of the oxidized base, 5-hmdU.

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.

Structural modeling and analyses of genetic variations in the human XPC nucleotide excision repair protein

Xeroderma pigmentosum C (XPC) is a key initiator in the global genome nucleotide excision repair pathway in mammalian cells. Inherited mutations in the XPC gene can cause xeroderma pigmentosum (XP) cancer predisposition syndrome that dramatically increases the susceptibility to sunlight-induced cancers. Various genetic variants and mutations of the protein have been reported in cancer databases and literature. The current lack of a high-resolution 3-D structure of human XPC makes it difficult to assess the structural impact of the mutations/genetic variations. Using the available high-resolution crystal structure of its yeast ortholog, Rad4, we built a homology model of human XPC protein and compared it with a model generated by AlphaFold. The two models are largely consistent with each other in the structured domains. We have also assessed the degree of conservation for each residue using 966 sequences of XPC orthologs. Our structure- and sequence conservation-based assessments largely agree with the variant’s impact on the protein’s structural stability, computed by FoldX and SDM. Known XP missense mutations such as Y585C, W690S, and C771Y are consistently predicted to destabilize the protein’s structure. Our analyses also reveal several highly conserved hydrophobic regions that are surface-exposed, which may indicate novel intermolecular interfaces that are yet to be characterized.Communicated by Ramaswamy H. Sarma

Unscheduled DNA Synthesis at Sites of Local UV-induced DNA Damage to Quantify Global Genome Nucleotide Excision Repair Activity in Human Cells.

Nucleotide excision repair (NER) removes a wide variety of structurally unrelated lesions from the genome, including UV-induced photolesions such as 6-4 pyrimidine-pyrimidone photoproducts (6-4PPs) and cyclobutane pyrimidine dimers (CPDs). NER removes lesions by excising a short stretch of single-stranded DNA containing the damaged DNA, leaving a single-stranded gap that is resynthesized in a process called unscheduled DNA synthesis (UDS). Measuring UDS after UV irradiation in non-dividing cells provides a measure of the overall NER activity, of which approximately 90% is carried out by the global genome repair (GGR) sub pathway. Here, we present a protocol for the microscopy-based analysis and quantification of UDS as a measurement for GGR activity. Following local UV-C irradiation, serum-starved human cells are supplemented with the thymidine analogue 5-ethynyl-2'-deoxyuridine (EdU), which is incorporated into repair patches following NER-dependent dual incision. The incorporated nucleotide analogue is coupled to a fluorophore using Click-iT chemistry, followed by immunodetection of CPD photolesions to simultaneously visualize both signals by fluorescence microscopy. Accompanying this protocol is a custom-built ImageJ plug-in to analyze and quantify UDS signals at sites of CPD-marked local damage. The local UDS assay enables an effective and sensitive fluorescence-based quantification of GGR activity in single cells with application in basic research to better understand the regulatory mechanism in NER, as well as in diagnostics to characterize fibroblasts from individuals with NER-deficiency disorder. Graphical abstract.

267 Generation and Characterization of a CRISPR-Cas9-Mediated XPC gene Knockout in Human Skin Cells

Xeroderma pigmentosum group C (XPC) is a multifunctional DNA damage recognition protein involved in the global genome nucleotide excision repair (GG-NER) pathway. Mammalian cells employ this pathway to eliminate bulky DNA lesions that induce double helix distortion caused by mutagens such as UV radiation or cisplatin. Loss-of-function mutations in the XPC gene lead to various malignancies, including skin cancers. Photosensitivity and the buildup of DNA damage can both define the phenotype of XPC patients’ cells.

Identification of 5 Potential Predictive Biomarkers for Alzheimer's Disease by Integrating the Unified Test for Molecular Signatures and Weighted Gene Coexpression Network Analysis.

Previous transcriptome-wide association study (TWAS) has documented 21 genes associated with Alzheimer's disease (AD) risk, but the predictive biomarkers remain unexplored. TWAS leveraging the unified test for molecular signatures (UTMOST) was performed in 75,000 cases and 420,000 controls with 10 brain tissue gene expression references. Weighted gene coexpression network analysis (WGCNA) was conducted in GSE5281 and GSE48350 data sets containing 167 AD samples and 247 controls. Random forest (RF) analysis was applied to screen the potential predictive biomarkers based on overlapping genes identified by TWAS and WGCNA, followed by comprehensive bioinformatic analyses with differential gene expression, functional enrichment, and correlation with immune cells. A nomogram was established to verify the predictive power of the identified biomarkers. TWAS revealed 78 candidate genes (p < 2.89 × 10-6). In WGCNA turquoise module, 3 718 AD-related genes were screened. RF identified 5 predictive biomarkers (FAM71E1, DDB2, AP4M1, GPR4, DOC2A), which are enriched in the global genome nucleotide excision repair pathway and associated with immune cell designations "Natural.killer.T.cell," "Memory.B.cell," "T.follicular.helper.cell," "Neutrophil," and "MDSC." The nomogram based on the 5 markers showed a high predictive power. Five potential predictive biomarkers for AD were identified, providing new insights into the pathogenesis and etiology of AD.

XPC–PARP complexes engage the chromatin remodeler ALC1 to catalyze global genome DNA damage repair

Cells employ global genome nucleotide excision repair (GGR) to eliminate a broad spectrum of DNA lesions, including those induced by UV light. The lesion-recognition factor XPC initiates repair of helix-destabilizing DNA lesions, but binds poorly to lesions such as CPDs that do not destabilize DNA. How difficult-to-repair lesions are detected in chromatin is unknown. Here, we identify the poly-(ADP-ribose) polymerases PARP1 and PARP2 as constitutive interactors of XPC. Their interaction results in the XPC-stimulated synthesis of poly-(ADP-ribose) (PAR) by PARP1 at UV lesions, which in turn enables the recruitment and activation of the PAR-regulated chromatin remodeler ALC1. PARP2, on the other hand, modulates the retention of ALC1 at DNA damage sites. Notably, ALC1 mediates chromatin expansion at UV-induced DNA lesions, leading to the timely clearing of CPD lesions. Thus, we reveal how chromatin containing difficult-to-repair DNA lesions is primed for repair, providing insight into mechanisms of chromatin plasticity during GGR.

Xeroderma Pigmentosum Complementation Group C (XPC): Emerging Roles in Non-Dermatologic Malignancies.

Xeroderma pigmentosum complementation group C (XPC) is a DNA damage recognition protein essential for initiation of global-genomic nucleotide excision repair (GG-NER). Humans carrying germline mutations in the XPC gene exhibit strong susceptibility to skin cancer due to defective removal via GG-NER of genotoxic, solar UV-induced dipyrimidine photoproducts. However, XPC is increasingly recognized as important for protection against non-dermatologic cancers, not only through its role in GG-NER, but also by participating in other DNA repair pathways, in the DNA damage response and in transcriptional regulation. Additionally, XPC expression levels and polymorphisms likely impact development and may serve as predictive and therapeutic biomarkers in a number of these non-dermatologic cancers. Here we review the existing literature, focusing on the role of XPC in non-dermatologic cancer development, progression, and treatment response, and highlight possible future applications of XPC as a prognostic and therapeutic biomarker.

Histone deacetylation regulates nucleotide excision repair through an interaction with the XPC protein

SummaryThe XPC protein complex plays a central role in DNA lesion recognition for global genome nucleotide excision repair (GG-NER). Lesion recognition can be accomplished in either a UV-DDB-dependent or -independent manner; however, it is unclear how these sub-pathways are regulated in chromatin. Here, we show that histone deacetylases 1 and 2 facilitate UV-DDB-independent recruitment of XPC to DNA damage by inducing histone deacetylation. XPC localizes to hypoacetylated chromatin domains in a DNA damage-independent manner, mediated by its structurally disordered middle (M) region. The M region interacts directly with the N-terminal tail of histone H3, an interaction compromised by H3 acetylation. Although the M region is dispensable for in vitro NER, it promotes DNA damage removal by GG-NER in vivo, particularly in the absence of UV-DDB. We propose that histone deacetylation around DNA damage facilitates the recruitment of XPC through the M region, contributing to efficient lesion recognition and initiation of GG-NER.

Set2 histone methyltransferase regulates transcription coupled-nucleotide excision repair in yeast.

Helix-distorting DNA lesions, including ultraviolet (UV) light-induced damage, are repaired by the global genomic-nucleotide excision repair (GG-NER) and transcription coupled-nucleotide excision repair (TC-NER) pathways. Previous studies have shown that histone post-translational modifications (PTMs) such as histone acetylation and methylation can promote GG-NER in chromatin. Whether histone PTMs also regulate the repair of DNA lesions by the TC-NER pathway in transcribed DNA is unknown. Here, we report that histone H3 K36 methylation (H3K36me) by the Set2 histone methyltransferase in yeast regulates TC-NER. Mutations in Set2 or H3K36 result in UV sensitivity that is epistatic with Rad26, the primary TC-NER factor in yeast, and cause a defect in the repair of UV damage across the yeast genome. We further show that mutations in Set2 or H3K36 in a GG-NER deficient strain (i.e., rad16Δ) partially rescue its UV sensitivity. Our data indicate that deletion of SET2 rescues UV sensitivity in a GG-NER deficient strain by activating cryptic antisense transcription, so that the non-transcribed strand (NTS) of yeast genes is repaired by TC-NER. These findings indicate that Set2 methylation of H3K36 establishes transcriptional asymmetry in repair by promoting canonical TC-NER of the transcribed strand (TS) and suppressing cryptic TC-NER of the NTS.

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.