Camptothecin compromises transcription recovery and cell survival against cisplatin and ultraviolet irradiation regardless of transcription-coupled nucleotide excision repair.

DNA-damaging anti-cancer drugs are used clinically to induce cell death by causing DNA strand breaks or DNA replication stress. Camptothecin (CPT) and cisplatin are commonly used anti-cancer drugs, and their combined use enhances the anti-tumour effects. However, the mechanism underlying this enhanced effect has not been well studied. In this study, we analysed the combined effect of CPT and cisplatin or ultraviolet (UV) and found that CPT suppresses transcription recovery after UV damage and induces the disappearance of the Cockayne syndrome group B (CSB) protein, a transcription-coupled nucleotide excision repair (TC-NER) factor. This CPT-induced disappearance of CSB expression was suppressed by proteasome and transcription inhibitors. Moreover, CSB ubiquitination was detected after CPT treatment in a transcription-dependent manner, suggesting that the transcription stress caused by CPT induces CSB ubiquitination, resulting in CSB undetectability. However, Cockayne syndrome group A (CSA) and CUL4A were not involved in the CPT-induced CSB undetectability, suggesting that CSB ubiquitination caused by CPT is regulated differently from the UV response. However, cisplatin or UV sensitivity was enhanced by CPT even in CSB- or CSA-knockout cells. Furthermore, the excessive CSB expression, which suppressed CSB ubiquitination, did not cancel the combined effect of CPT. These results suggest that CPT blocks the repair of cisplatin or UV-induced DNA damage regardless of TC-NER status. CPT possibly compromised the alternative repair pathways other than TC-NER, leading to the suppression of transcription recovery and enhancement of cell killing.

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.

Structural basis of ubiquitin recognition by the winged-helix domain of Cockayne syndrome group B protein.

Cockayne syndrome group B (CSB, also known as ERCC6) protein is involved in many DNA repair processes and essential for transcription-coupled repair (TCR). The central region of CSB has the helicase motif, whereas the C-terminal region contains important regulatory elements for repair of UV- and oxidative stress-induced damages and double-strand breaks (DSBs). A previous study suggested that a small part (∼30 residues) within this region was responsible for binding to ubiquitin (Ub). Here, we show that the Ub-binding of CSB requires a larger part of CSB, which was previously identified as a winged-helix domain (WHD) and is involved in the recruitment of CSB to DSBs. We also present the crystal structure of CSB WHD in complex with Ub. CSB WHD folds as a single globular domain, defining a class of Ub-binding domains (UBDs) different from 23 UBD classes identified so far. The second α-helix and C-terminal extremity of CSB WHD interact with Ub. Together with structure-guided mutational analysis, we identified the residues critical for the binding to Ub. CSB mutants defective in the Ub binding reduced repair of UV-induced damage. This study supports the notion that DSB repair and TCR may be associated with the Ub-binding of CSB.

Analysis of the conserved NER helicases (XPB and XPD) and UV-induced DNA damage in Hydra.

BACKGROUND: Nucleotide excision repair (NER) pathway is an evolutionarily conserved mechanism of genome maintenance. It detects and repairs distortions in DNA double helix. Xeroderma Pigmentosum group B (XPB) and group D (XPD) are important helicases in NER and are also critical subunits of TFIIH complex. We have studied XPB and XPD for the first time from the basal metazoan Hydra which exhibits lack of organismal senescence. METHODS: In silico analysis of proteins was performed using MEGA 6.0, Clustal Omega, Swiss Model, etc. Gene expression was studied by in situ hybridization and qRT-PCR. Repair of CPDs was studied by DNA blot assay. Interactions between proteins were determined by co- immunoprecipitation. HyXPB and HyXPD were cloned in pET28b, overexpressed and helicase activity of purified proteins was checked. RESULTS: In silico analysis revealed presence of seven classical helicase motifs in HyXPB and HyXPD. Both proteins revealed polarity-dependent helicase activity. Hydra repairs most of the thymine dimers induced by UVC (500 J/m2) by 72 h post-UV exposure. HyXPB and HyXPD transcripts, localized all over the body column, remained unaltered post-UV exposure indicating their constitutive expression. In spite of high levels of sequence conservation, XPB and XPD failed to rescue defects in human XPB- and XPD-deficient cell lines. This was due to their inability to get incorporated into the TFIIH multiprotein complex. CONCLUSIONS: Present results along with our earlier work on DNA repair proteins in Hydra bring out the utility of Hydra as model system to study evolution of DNA repair mechanisms in metazoans.

Generation of splice switching oligonucleotides targeting the Cockayne syndrome group B gene product in order to change the diseased cell state.

Cockayne syndrome (CS) is a severe disorder with no effective treatment. The Cockayne syndrome group B (CSB) gene is one gene responsible for CS and also causes UV sensitive syndrome (UVSS), a disorder that causes mild symptoms. How the CSB gene determines a patient's fate is unknown, but one intriguing point is that in UVSS patient cell, there are nonsense mutations in both alleles at the same position in each upstream region of the PiggyBac transposable element derived 3 (PGBD3) inserted region. In contrast, in CS patient cells, there is at least one allele with several mutations downstream of the PGBD3 inserted region, or there are homozygous mutations in exon 1. Here, we designed and synthesized 24 splice switching oligonucleotides (SSOs) to skip exon 3 in CSB mRNA. Use of these SSOs induced a frame shift in order to generate an alternative stop codon at the upstream region of the PGBD3 invasion site. As a result, a reduction of mitochondrial membrane potential following H2O2 treatment in CS cell was recovered. It was demonstrated that up-regulation of several gene expression brought about by SSOs are related to mitochondrial dysfunction in CS cells.

Inhibition of UVSSA ubiquitination suppresses transcription-coupled nucleotide excision repair deficiency caused by dissociation from USP7.

Transcription-coupled nucleotide excision repair (TC-NER) is a subpathway of nucleotide excision repair that efficiently removes transcription-blocking DNA damage from the transcribed strands of active genes. UVSSA is a causative gene for UV-sensitive syndrome (UVS S), which is an autosomal recessive disorder characterized by hypersensitivity to UV light and deficiency in TC-NER. UV-stimulated scaffold protein A (UVSSA), the product of UVSSA, forms a complex with ubiquitin-specific peptidase 7 (USP7) and is stabilized by interaction with USP7. The central region of UVSSA, which contains the tumor necrosis factor receptor-associated factor (TRAF)-binding motif, is required for the interaction with the N-terminal TRAF domain of USP7. Here, we showed that UVSSA is mono-ubiquitinated in vitro and identified a lysine residue (Lys414 ) in UVSSA as the target of ubiquitination. The deubiquitination activity of USP7 was inhibited by the ubiquitin-conjugating enzyme UbcH6. Lys414 was also modified by poly-ubiquitin chains in vivo. UVSSA deficient in the interaction with USP7 is ubiquitinated and degraded by the proteasome, and the degradation leads to deficiency in TC-NER. The substitution of Lys414 by Arg of UVSSA inhibited its degradation and thereby suppressed the deficiency in TC-NER.

DGCR8 Mediates Repair of UV-Induced DNA Damage Independently of RNA Processing.

Ultraviolet (UV) radiation is a carcinogen that generates DNA lesions. Here, we demonstrate an unexpected role for DGCR8, an RNA binding protein that canonically functions with Drosha to mediate microRNA processing, in the repair of UV-induced DNA lesions. Treatment with UV induced phosphorylation on serine 153 (S153) of DGCR8 in both human and murine cells. S153 phosphorylation was critical for cellular resistance to UV, the removal of UV-induced DNA lesions, and the recovery of RNA synthesis after UV exposure but not for microRNA expression. The RNA-binding and Drosha-binding activities of DGCR8 were not critical for UV resistance. DGCR8 depletion was epistatic to defects in XPA, CSA, and CSB for UV sensitivity. DGCR8 physically interacted with CSB and RNA polymerase II. JNKs were involved in the UV-induced S153 phosphorylation. These findings suggest that UV-induced S153 phosphorylation mediates transcription-coupled nucleotide excision repair of UV-induced DNA lesions in a manner independent of microRNA processing.

Stabilization of Ultraviolet (UV)-stimulated Scaffold Protein A by Interaction with Ubiquitin-specific Peptidase 7 Is Essential for Transcription-coupled Nucleotide Excision Repair.

UV-sensitive syndrome is an autosomal recessive disorder characterized by hypersensitivity to UV light and deficiency in transcription-coupled nucleotide excision repair (TC-NER), a subpathway of nucleotide excision repair that rapidly removes transcription-blocking DNA damage. UV-sensitive syndrome consists of three genetic complementation groups caused by mutations in the CSA, CSB, and UVSSA genes. UV-stimulated scaffold protein A (UVSSA), the product of UVSSA, which is required for stabilization of Cockayne syndrome group B (CSB) protein and reappearance of the hypophosphorylated form of RNA polymerase II after UV irradiation, forms a complex with ubiquitin-specific peptidase 7 (USP7). In this study, we demonstrated that the deubiquitination activity of USP7 is suppressed by its interaction with UVSSA. The interaction required the tumor necrosis factor receptor-associated factor domain of USP7 and the central region of UVSSA and was disrupted by an amino acid substitution in the tumor necrosis factor receptor-associated factor-binding motif of UVSSA. Cells expressing mutant UVSSA were highly sensitive to UV irradiation and defective in recovery of RNA synthesis after UV irradiation. These results indicate that the interaction between UVSSA and USP7 is important for TC-NER. Furthermore, the mutant UVSSA was rapidly degraded by the proteasome, and CSB was also degraded after UV irradiation as observed in UVSSA-deficient cells. Thus, stabilization of UVSSA by interaction with USP7 is essential for TC-NER.

The C-terminal Region and SUMOylation of Cockayne Syndrome Group B Protein Play Critical Roles in Transcription-coupled Nucleotide Excision Repair.

Cockayne syndrome (CS) is a recessive disorder that results in deficiencies in transcription-coupled nucleotide excision repair (TC-NER), a subpathway of nucleotide excision repair, and cells from CS patients exhibit hypersensitivity to UV light. CS group B protein (CSB), which is the gene product of one of the genes responsible for CS, belongs to the SWI2/SNF2 DNA-dependent ATPase family and has an ATPase domain and an ubiquitin-binding domain (UBD) in the central region and the C-terminal region, respectively. The C-terminal region containing the UBD is essential for the functions of CSB. In this study, we generated several CSB deletion mutants and analyzed the functions of the C-terminal region of CSB in TC-NER. Not only the UBD but also the C-terminal 30-amino acid residues were required for UV light resistance and TC-NER. This region was needed for the interaction of CSB with RNA polymerase II, the translocation of CS group A protein to the nuclear matrix, and the association of CSB with chromatin after UV irradiation. CSB was modified by small ubiquitin-like modifier 2/3 in a UV light-dependent manner. This modification was abolished in a CSB mutant lacking the C-terminal 30 amino acid residues. However, the substitution of lysine residues in this region with arginine did not affect SUMOylation or TC-NER. By contrast, substitution of a lysine residue in the N-terminal region with arginine decreased SUMOylation and resulted in cells with defects in TC-NER. These results indicate that both the most C-terminal region and SUMOylation are important for the functions of CSB in TC-NER.

Regulation of Transcription Elongation by the XPG-TFIIH Complex Is Implicated in Cockayne Syndrome.

XPG is a causative gene underlying the photosensitive disorder xeroderma pigmentosum group G (XP-G) and is involved in nucleotide excision repair. Here, we show that XPG knockdown represses epidermal growth factor (EGF)-induced FOS transcription at the level of transcription elongation with little effect on EGF signal transduction. XPG interacted with transcription elongation factors in concert with TFIIH, suggesting that the XPG-TFIIH complex serves as a transcription elongation factor. The XPG-TFIIH complex was recruited to promoter and coding regions of both EGF-induced (FOS) and housekeeping (EEF1A1) genes. Further, EGF-induced recruitment of RNA polymerase II and TFIIH to FOS was reduced by XPG knockdown. Importantly, EGF-induced FOS transcription was markedly lower in XP-G/Cockayne syndrome (CS) cells expressing truncated XPG than in control cells expressing wild-type (WT) XPG, with less significant decreases in XP-G cells with XPG nuclease domain mutations. In corroboration of this finding, both WT XPG and a missense XPG mutant from an XP-G patient were recruited to FOS upon EGF stimulation, but an XPG mutant mimicking a C-terminal truncation from an XP-G/CS patient was not. These results suggest that the XPG-TFIIH complex is involved in transcription elongation and that defects in this association may partly account for Cockayne syndrome in XP-G/CS patients.

Constructive rescue of TFIIH instability by an alternative isoform of XPD derived from a mutated XPD allele in mild but not severe XP-D/CS.

Mutations in XPD cause xeroderma pigmentosum (XP), XP and Cockayne syndrome (CS) crossover syndrome (XP/CS), trichothiodystrophy and cerebro-oculo-facio-skeletal syndrome (COFS). COFS represents the most severe end of the CS spectrum. This study reports two Japanese patients, COFS-05-135 and COFS-Chiba1, who died at ages of <1 year and exhibited typical COFS manifestations caused by XPD mutations p.[I619del];[R666W] and p.[G47R];[I619del], respectively. Two other cases of severe XP-D/CS (XP group D/CS), XP1JI (p.[G47R];[0]) and XPCS1PV (p.[R666W];[0]), died at ages <2 years. On the other hand, two cases of mild XP-D/CS, XP1NE (p.[G47R];[L461V;V716_R730del]) and XPCS118LV (p.[L461V;V716_R730del];[R666W]), lived beyond 37 years of age. p.I619Del and p.[L461V;V716_R730del] are functionally null; therefore, despite the differences in clinical manifestations, the functional protein in all of these patients was either p.G47R or p.R666W. To resolve the discrepancies in these XPD genotype-phenotype relationships, the p.[L461V;V716_R730del] allele was analyzed and we found that p.[L461V;A717G] was expressed from the same allele as p.[L461V;V716_R730del] by authentic splicing. Additionally, p.[L461V;A717G] could partially rescue the loss of XPD function, resulting in the milder manifestations observed in XP1NE and XPCS118LV.

The role of Cockayne syndrome group A (CSA) protein in transcription-coupled nucleotide excision repair.

Nucleotide excision repair (NER) removes a variety of DNA lesions, including ultraviolet-induced cyclobutane pyrimidine dimers. NER comprises two subpathways: transcription-coupled NER (TC-NER) and global genome NER. TC-NER efficiently removes lesions from the transcribed strands of active genes. Mutations in Cockayne syndrome groups A and B genes (CSA and CSB) result in defective TC-NER. In mammalian cells, TC-NER is presumably initiated by the arrest of RNA polymerase II at a lesion on the transcribed strand of an active gene, but the molecular mechanism underlying TC-NER remains unclear. The CSA protein has seven WD40 repeat motifs and beta-propeller architecture. A protein complex consisting of CSA, DDB1, cullin 4A, and Roc1 exhibits ubiquitin ligase activity. The role of CSA protein in TC-NER is described in this review.

Mutations in UVSSA cause UV-sensitive syndrome and destabilize ERCC6 in transcription-coupled DNA repair.

UV-sensitive syndrome (UV(S)S) is an autosomal recessive disorder characterized by photosensitivity and deficiency in transcription-coupled repair (TCR), a subpathway of nucleotide-excision repair that rapidly removes transcription-blocking DNA damage. Cockayne syndrome is a related disorder with defective TCR and consists of two complementation groups, Cockayne syndrome (CS)-A and CS-B, which are caused by mutations in ERCC8 (CSA) and ERCC6 (CSB), respectively. UV(S)S comprises three groups, UV(S)S/CS-A, UV(S)S/CS-B and UV(S)S-A, caused by mutations in ERCC8, ERCC6 and an unidentified gene, respectively. Here, we report the cloning of the gene mutated in UV(S)S-A by microcell-mediated chromosome transfer. The predicted human gene UVSSA (formerly known as KIAA1530)(7) corrects defective TCR in UV(S)S-A cells. We identify three nonsense and frameshift UVSSA mutations in individuals with UV(S)S-A, indicating that UVSSA is the causative gene for this syndrome. The UVSSA protein forms a complex with USP7 (ref. 8), stabilizes ERCC6 and restores the hypophosphorylated form of RNA polymerase II after UV irradiation.

Xeroderma pigmentosum group F protein binds to Eg5 and is required for proper mitosis: implications for XP-F and XFE.

The xeroderma pigmentosum group F-cross-complementing rodent repair deficiency group 1 (XPF-ERCC1) complex is a structure-specific endonuclease involved in nucleotide excision repair (NER) and interstrand cross-link (ICL) repair. Patients with XPF mutations may suffer from two forms of xeroderma pigmentosum (XP): XP-F patients show mild photosensitivity and proneness to skin cancer but rarely show any neurological abnormalities, whereas XFE patients display symptoms of severe XP symptoms, growth retardation and accelerated aging. Xpf knockout mice display accelerated aging and die before weaning. These results suggest that the XPF-ERCC1 complex has additional functions besides NER and ICL repair and is essential for development and growth. In this study, we show a partial colocalization of XPF with mitotic spindles and Eg5. XPF knockdown in cells led to an increase in the frequency of abnormal nuclear morphology and mitosis. Similarly, the frequency of abnormal nuclei and mitosis was increased in XP-F and XFE cells. In addition, we showed that Eg5 enhances the action of XPF-ERCC1 nuclease activity. Taken together, these results suggest that the interaction between XPF and Eg5 plays a role in mitosis and DNA repair and offer new insights into the pathogenesis of XP-F and XFE.

BRCA1 contributes to transcription-coupled repair of DNA damage through polyubiquitination and degradation of Cockayne syndrome B protein.

BRCA1 is an important gene involved in susceptibility to breast and ovarian cancer and its product regulates the cellular response to DNA double-strand breaks. Here, we present evidence that BRCA1 also contributes to the transcription-coupled repair (TCR) of ultraviolet (UV) light-induced DNA damage. BRCA1 immediately accumulates at the sites of UV irradiation-mediated damage in cell nuclei in a manner that is fully dependent on both Cockayne syndrome B (CSB) protein and active transcription. Suppression of BRCA1 expression inhibits the TCR of UV lesions and increases the UV sensitivity of cells proficient in TCR. BRCA1 physically interacts with CSB protein. BRCA1 polyubiquitinates CSB and this polyubiquitination and subsequent degradation of CSB occur following UV irradiation, even in the absence of Cockayne syndrome A (CSA) protein. The depletion of BRCA1 expression increases the UV sensitivity of CSA-deficient cells. These results indicate that BRCA1 is involved in TCR and that a BRCA1-dependent polyubiquitination pathway for CSB exists alongside the CSA-dependent pathway to yield more efficient excision repair of lesions on the transcribed DNA strand.

A Japanese trichothiodystrophy patient with XPD mutations.

Trichothiodystrophy (TTD) is a rare autosomal recessive disorder characterized by sulfur-deficient brittle hair complicated with ichthyosis, physical and mental retardation, and proneness to infections. Approximately half of TTD patients exhibit cutaneous photosensitivity because of the defect of nucleotide excision repair. Three genes, XPB, XPD and TTDA, have been identified as causative genes of photosensitive TTD. These three genes are components of basal transcription factor IIH. Most TTD cases have been reported in Europe and North America. We report a severely affected Japanese TTD patient with XPD mutations. Interestingly, his father has ichthyotic skin. The alteration in the paternal allele was a nucleotide substitution leading to Arg-722 to Trp (R722W), as previously reported in TTD patients. The other alteration in the maternal allele was a novel 3-bp deletion at nucleotides 67-69, resulting in the deletion of Ser-23, which is located upstream of helicase motif I and is the closest to the N-terminal end of XPD in reported mutations. The expression study showed that the two alterations were causative mutations for TTD. In Asia, it is likely that there are TTD patients who have not been diagnosed.

Mutant Cockayne syndrome group B protein inhibits repair of DNA topoisomerase I-DNA covalent complex.

Two UV-sensitive syndrome patients who have mild photosensitivity without detectable somatic abnormalities lack detectable Cockayne syndrome group B (CSB) protein because of a homozygous null mutation in the CSB gene. In contrast, mutant CSB proteins are produced in CS-B patients with the severe somatic abnormalities of Cockayne syndrome and photosensitivity. It is known that the piggyBac transposable element derived 3 is integrated within the CSB intron 5, and that CSB-piggyBac transposable element derived 3 fusion (CPFP) mRNA is produced by alternative splicing. We found that CPFP or truncated CSB protein derived from CPFP mRNA was stably produced in CS-B patients, and that wild-type CSB, CPFP, and truncated CSB protein interacted with DNA topoisomerase I. We also found that CPFP inhibited repair of a camptothecin-induced topoisomerase I-DNA covalent complex. The inhibition was suppressed by the presence of wild-type CSB, consistent with the autosomal recessive inheritance of Cockayne syndrome. These results suggested that reduced repair of a DNA topoisomerase I-DNA covalent complex because of truncated CSB proteins is involved in the pathogenesis of CS-B.

Nucleotide excision repair by mutant xeroderma pigmentosum group A (XPA) proteins with deficiency in interaction with RPA.

The xeroderma pigmentosum group A protein (XPA) is a core component of nucleotide excision repair (NER). To coordinate early stage NER, XPA interacts with various proteins, including replication protein A (RPA), ERCC1, DDB2, and TFIIH, in addition to UV-damaged or chemical carcinogen-damaged DNA. In this study, we investigated the effects of mutations in the RPA binding regions of XPA on XPA function in NER. XPA binds through an N-terminal region to the middle subunit (RPA32) of the RPA heterotrimer and through a central region that overlaps with its damaged DNA binding region to the RPA70 subunit. In cell-free NER assays, an N-terminal deletion mutant of XPA showed loss of binding to RPA32 and reduced DNA repair activity, but it could still bind to UV-damaged DNA and RPA. In contrast, amino acid substitutions in the central region reduced incisions at the damaged site in the cell-free NER assay, and four of these mutants (K141A, T142A, K167A, and K179A) showed reduced binding to RPA70 but normal binding to damaged DNA. Furthermore, mutants that had one of the four aforementioned substitutions and an N-terminal deletion exhibited lower DNA incision activity and binding to RPA than XPA with only one of these substitutions or the deletion. Taken together, these results indicate that XPA interaction with both RPA32 and RPA70 is indispensable for NER reactions.

DDB2 complex-mediated ubiquitylation around DNA damage is oppositely regulated by XPC and Ku and contributes to the recruitment of XPA.

UV-damaged-DNA-binding protein (UV-DDB) is a heterodimer comprised of DDB1 and DDB2 and integrated in a complex that includes a ubiquitin ligase component, cullin 4A, and Roc1. Here we show that the ubiquitin ligase activity of the DDB2 complex is required for efficient global genome nucleotide excision repair (GG-NER) in chromatin. Mutant DDB2 proteins derived from xeroderma pigmentosum group E patients are not able to mediate ubiquitylation around damaged sites in chromatin. We also found that CSN, a negative regulator of cullin-based ubiquitin ligases, dissociates from the DDB2 complex when the complex binds to damaged DNA and that XPC and Ku oppositely regulate the ubiquitin ligase activity, especially around damaged sites. Furthermore, the DDB2 complex-mediated ubiquitylation plays a role in recruiting XPA to damaged sites. These findings shed some light on the early stages of GG-NER.

RPAP3 interacts with Reptin to regulate UV-induced phosphorylation of H2AX and DNA damage.

We have previously reported that Monad, a novel WD40 repeat protein, potentiates apoptosis induced by tumor necrosis factor-alpha and cycloheximide. By affinity purification and mass spectrometry, RNA polymerase II-associated protein 3 (RPAP3) was identified as a Monad binding protein and may function with Monad as a novel modulator of apoptosis pathways. Here we report that Reptin, a highly conserved AAA + ATPase that is part of various chromatin-remodeling complexes, is also involved in the association of RPAP3 by immunoprecipitation and confocal microscopic analysis. Overexpression of RPAP3 induced HEK293 cells to death after UV-irradiation. Loss of RPAP3 by RNAi improved HeLa cell survival after UV-induced DNA damage and attenuated the phosphorylation of H2AX. Depletion of Reptin reduced cell survival and facilitated the phosphorylation on H2AX. These results suggest that RPAP3 modulates UV-induced DNA damage by regulating H2AX phosphorylation.