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• W2910565537 abstract "•Interaction of XRCC1 with PAR and DNA are both mediated by the central BRCT domain•Interaction with PAR and DNA occurs through non-overlapping binding surfaces•Mutational disruption of DNA binding to XRCC1 impairs recruitment to DNA damage•Disruption of DNA binding by XRCC1 impairs repair of DNA single-strand breaks XRCC1 accelerates repair of DNA single-strand breaks by acting as a scaffold protein for the recruitment of Polβ, LigIIIα, and end-processing factors, such as PNKP and APTX. XRCC1 itself is recruited to DNA damage through interaction of its central BRCT domain with poly(ADP-ribose) chains generated by PARP1 or PARP2. XRCC1 is believed to interact directly with DNA at sites of damage, but the molecular basis for this interaction within XRCC1 remains unclear. We now show that the central BRCT domain simultaneously mediates interaction of XRCC1 with poly(ADP-ribose) and DNA, through separate and non-overlapping binding sites on opposite faces of the domain. Mutation of residues within the DNA binding site, which includes the site of a common disease-associated human polymorphism, affects DNA binding of this XRCC1 domain in vitro and impairs XRCC1 recruitment and retention at DNA damage and repair of single-strand breaks in vivo. XRCC1 accelerates repair of DNA single-strand breaks by acting as a scaffold protein for the recruitment of Polβ, LigIIIα, and end-processing factors, such as PNKP and APTX. XRCC1 itself is recruited to DNA damage through interaction of its central BRCT domain with poly(ADP-ribose) chains generated by PARP1 or PARP2. XRCC1 is believed to interact directly with DNA at sites of damage, but the molecular basis for this interaction within XRCC1 remains unclear. We now show that the central BRCT domain simultaneously mediates interaction of XRCC1 with poly(ADP-ribose) and DNA, through separate and non-overlapping binding sites on opposite faces of the domain. Mutation of residues within the DNA binding site, which includes the site of a common disease-associated human polymorphism, affects DNA binding of this XRCC1 domain in vitro and impairs XRCC1 recruitment and retention at DNA damage and repair of single-strand breaks in vivo. X-ray repair cross-complementing protein 1 (XRCC1) is a scaffold protein that coordinates the repair of DNA single-strand nicks and gaps (single strand breaks [SSBs]; Caldecott, 2003Caldecott K.W. XRCC1 and DNA strand break repair.DNA Repair (Amst.). 2003; 2: 955-969Crossref PubMed Scopus (510) Google Scholar). It constitutively associates with a DNA polymerase (Polβ) and a DNA ligase (Lig3α) to fill and ligate the broken strand (Caldecott et al., 1994Caldecott K.W. McKeown C.K. Tucker J.D. Ljungquist S. Thompson L.H. An interaction between the mammalian DNA repair protein XRCC1 and DNA ligase III.Mol. Cell. Biol. 1994; 14: 68-76Crossref PubMed Google Scholar, Caldecott et al., 1996Caldecott K.W. Aoufouchi S. Johnson P. Shall S. XRCC1 polypeptide interacts with DNA polymerase beta and possibly poly (ADP-ribose) polymerase, and DNA ligase III is a novel molecular ‘nick-sensor’ in vitro.Nucleic Acids Res. 1996; 24: 4387-4394Crossref PubMed Scopus (548) Google Scholar, Kubota et al., 1996Kubota Y. Nash R.A. Klungland A. Schär P. Barnes D.E. Lindahl T. Reconstitution of DNA base excision-repair with purified human proteins: interaction between DNA polymerase beta and the XRCC1 protein.EMBO J. 1996; 15: 6662-6670Crossref PubMed Scopus (690) Google Scholar, Nash et al., 1997Nash R.A. Caldecott K.W. Barnes D.E. Lindahl T. XRCC1 protein interacts with one of two distinct forms of DNA ligase III.Biochemistry. 1997; 36: 5207-5211Crossref PubMed Scopus (229) Google Scholar) and recruits the end-processing enzymes polynucleotide kinase-3′-phosphatase (PNKP) and aprataxin (APTX), which ensure the presence of 3′-hydroxyl and 5′-phosphate groups at gap margins (Ahel et al., 2006Ahel I. Rass U. El-Khamisy S.F. Katyal S. Clements P.M. McKinnon P.J. Caldecott K.W. West S.C. The neurodegenerative disease protein aprataxin resolves abortive DNA ligation intermediates.Nature. 2006; 443: 713-716Crossref PubMed Scopus (293) Google Scholar, Jilani et al., 1999Jilani A. Ramotar D. Slack C. Ong C. Yang X.M. Scherer S.W. Lasko D.D. Molecular cloning of the human gene, PNKP, encoding a polynucleotide kinase 3′-phosphatase and evidence for its role in repair of DNA strand breaks caused by oxidative damage.J. Biol. Chem. 1999; 274: 24176-24186Crossref PubMed Scopus (237) Google Scholar, Loizou et al., 2004Loizou J.I. El-Khamisy S.F. Zlatanou A. Moore D.J. Chan D.W. Qin J. Sarno S. Meggio F. Pinna L.A. Caldecott K.W. The protein kinase CK2 facilitates repair of chromosomal DNA single-strand breaks.Cell. 2004; 117: 17-28Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar). Recruitment of XRCC1 complexes to sites of DNA damage is strongly dependent on activation of the DNA-damage-responsive poly(ADP-ribose) polymerases PARP1 and PARP2 (El-Khamisy et al., 2003El-Khamisy S.F. Masutani M. Suzuki H. Caldecott K.W. A requirement for PARP-1 for the assembly or stability of XRCC1 nuclear foci at sites of oxidative DNA damage.Nucleic Acids Res. 2003; 31: 5526-5533Crossref PubMed Scopus (522) Google Scholar, Hanzlikova et al., 2017Hanzlikova H. Gittens W. Krejcikova K. Zeng Z. Caldecott K.W. Overlapping roles for PARP1 and PARP2 in the recruitment of endogenous XRCC1 and PNKP into oxidized chromatin.Nucleic Acids Res. 2017; 45: 2546-2557PubMed Google Scholar, Mortusewicz et al., 2007Mortusewicz O. Amé J.-C. Schreiber V. Leonhardt H. Feedback-regulated poly(ADP-ribosyl)ation by PARP-1 is required for rapid response to DNA damage in living cells.Nucleic Acids Res. 2007; 35: 7665-7675Crossref PubMed Scopus (234) Google Scholar, Schreiber et al., 2002Schreiber V. Amé J.C. Dollé P. Schultz I. Rinaldi B. Fraulob V. Ménissier-de Murcia J. de Murcia G. Poly(ADP-ribose) polymerase-2 (PARP-2) is required for efficient base excision DNA repair in association with PARP-1 and XRCC1.J. Biol. Chem. 2002; 277: 23028-23036Crossref PubMed Scopus (576) Google Scholar). PARP-dependent recruitment of XRCC1 requires the central BRCT domain (BRCT1), which conserves components of a pocket similar to the phosphopeptide-binding BRCT domains in proteins such as TOPBP1 (Rappas et al., 2011Rappas M. Oliver A.W. Pearl L.H. Structure and function of the Rad9-binding region of the DNA-damage checkpoint adaptor TopBP1.Nucleic Acids Res. 2011; 39: 313-324Crossref PubMed Scopus (58) Google Scholar, Wardlaw et al., 2014Wardlaw C.P. Carr A.M. Oliver A.W. TopBP1: A BRCT-scaffold protein functioning in multiple cellular pathways.DNA Repair (Amst.). 2014; 22: 165-174Crossref PubMed Scopus (75) Google Scholar). However, rather than interacting with phosphorylated proteins, the phosphate-binding pocket in XRCC1-BRCT1 has been shown to mediate interaction with the poly(ADP-ribose) (PAR) chains generated by PARP1 or PARP2 (Breslin et al., 2015Breslin C. Hornyak P. Ridley A. Rulten S.L. Hanzlikova H. Oliver A.W. Caldecott K.W. The XRCC1 phosphate-binding pocket binds poly (ADP-ribose) and is required for XRCC1 function.Nucleic Acids Res. 2015; 43: 6934-6944Crossref PubMed Scopus (69) Google Scholar, Li et al., 2013Li M. Lu L.Y. Yang C.Y. Wang S. Yu X. The FHA and BRCT domains recognize ADP-ribosylation during DNA damage response.Genes Dev. 2013; 27: 1752-1768Crossref PubMed Scopus (116) Google Scholar). Although an interaction with PAR plays a major role in recruiting XRCC1 to sites of DNA damage, several studies have suggested that XRCC1 is able to interact directly with DNA (Mani et al., 2004Mani R.S. Karimi-Busheri F. Fanta M. Caldecott K.W. Cass C.E. Weinfeld M. Biophysical characterization of human XRCC1 and its binding to damaged and undamaged DNA.Biochemistry. 2004; 43: 16505-16514Crossref PubMed Scopus (51) Google Scholar, Nazarkina et al., 2007aNazarkina ZhK. Khodyreva S.N. Marsin S. Radicella J.P. Lavrik O.I. Study of interaction of XRCC1 with DNA and proteins of base excision repair by photoaffinity labeling technique.Biochemistry (Mosc.). 2007; 72: 878-886Crossref PubMed Scopus (14) Google Scholar, Nazarkina et al., 2007bNazarkina Z.K. Khodyreva S.N. Marsin S. Lavrik O.I. Radicella J.P. XRCC1 interactions with base excision repair DNA intermediates.DNA Repair (Amst.). 2007; 6: 254-264Crossref PubMed Scopus (81) Google Scholar, Ström et al., 2011Ström C.E. Mortusewicz O. Finch D. Parsons J.L. Lagerqvist A. Johansson F. Schultz N. Erixon K. Dianov G.L. Helleday T. CK2 phosphorylation of XRCC1 facilitates dissociation from DNA and single-strand break formation during base excision repair.DNA Repair (Amst.). 2011; 10: 961-969Crossref PubMed Scopus (30) Google Scholar) and that this plays a role in its DNA repair function (Berquist et al., 2010Berquist B.R. Singh D.K. Fan J. Kim D. Gillenwater E. Kulkarni A. Bohr V.A. Ackerman E.J. Tomkinson A.E. Wilson 3rd, D.M. Functional capacity of XRCC1 protein variants identified in DNA repair-deficient Chinese hamster ovary cell lines and the human population.Nucleic Acids Res. 2010; 38: 5023-5035Crossref PubMed Scopus (42) Google Scholar, Wei et al., 2013Wei L. Nakajima S. Hsieh C.L. Kanno S. Masutani M. Levine A.S. Yasui A. Lan L. Damage response of XRCC1 at sites of DNA single strand breaks is regulated by phosphorylation and ubiquitylation after degradation of poly(ADP-ribose).J. Cell Sci. 2013; 126: 4414-4423Crossref PubMed Scopus (52) Google Scholar). Previous NMR studies implicated the N-terminal domain of XRCC1 in high-affinity interactions with gapped DNA molecules (Marintchev et al., 1999Marintchev A. Mullen M.A. Maciejewski M.W. Pan B. Gryk M.R. Mullen G.P. Solution structure of the single-strand break repair protein XRCC1 N-terminal domain.Nat. Struct. Biol. 1999; 6: 884-893Crossref PubMed Scopus (167) Google Scholar), but subsequent work has cast doubt on this, and there is currently no coherent understanding of which part of XRCC1 is involved (London, 2015London R.E. The structural basis of XRCC1-mediated DNA repair.DNA Repair (Amst.). 2015; 30: 90-103Crossref PubMed Scopus (95) Google Scholar). We show here that both PAR and DNA interactions are mediated by non-overlapping binding sites on the first of the two BRCT domains in XRCC1 (BRCT1). Targeted mutations in the DNA-binding site, which contains a common human polymorphism, impair XRCC1 interaction with DNA in vitro and markedly affect the kinetics of XRCC1 recruitment, its retention on damaged chromatin, and the efficiency of DNA single-strand break repair in vivo. These data resolve a critical unanswered question in the field. Previous studies had suggested that the N-terminal domain (NTD) of XRCC1, which is required for association of Polβ with XRCC1, possesses an inherent affinity for DNA with single-strand nicks and short gaps (Marintchev et al., 1999Marintchev A. Mullen M.A. Maciejewski M.W. Pan B. Gryk M.R. Mullen G.P. Solution structure of the single-strand break repair protein XRCC1 N-terminal domain.Nat. Struct. Biol. 1999; 6: 884-893Crossref PubMed Scopus (167) Google Scholar). To discover whether other parts of XRCC1 might also be involved, we expressed and purified separate N-terminal (residues 1–223) and C-terminal (224–631) constructs of murine XRCC1 and examined their ability to interact with a 39-base-pair DNA duplex containing a single-strand nick, in an electrophoretic mobility shift assay (EMSA) (see STAR Methods). Contrary to the published model, we were unable to detect any significant interaction in EMSAs with the construct containing the NTD domain. By contrast, the C-terminal construct lacking the putative DNA binding NTD produced robust EMSA band shifts (Figure 1A). The marked difference in behavior of the two parts of XRCC1 suggests that its inherent DNA-binding ability resides in the C-terminal region, which incorporates the two BRCT domains, rather than in the Polβ-binding N-terminal domain. As BRCT domains in other proteins have been implicated in binding to DNA (Leung and Glover, 2011Leung C.C. Glover J.N. BRCT domains: easy as one, two, three.Cell Cycle. 2011; 10: 2461-2470Crossref PubMed Scopus (113) Google Scholar and references therein), and as PAR and DNA have many structural and chemical features in common, we considered the notion that BRCT1, which mediates the interaction of XRCC1 with PAR (Breslin et al., 2015Breslin C. Hornyak P. Ridley A. Rulten S.L. Hanzlikova H. Oliver A.W. Caldecott K.W. The XRCC1 phosphate-binding pocket binds poly (ADP-ribose) and is required for XRCC1 function.Nucleic Acids Res. 2015; 43: 6934-6944Crossref PubMed Scopus (69) Google Scholar, Li et al., 2013Li M. Lu L.Y. Yang C.Y. Wang S. Yu X. The FHA and BRCT domains recognize ADP-ribosylation during DNA damage response.Genes Dev. 2013; 27: 1752-1768Crossref PubMed Scopus (116) Google Scholar), might also bind DNA. To address this, we expressed and purified the isolated BRCT1 domain of human XRCC1 and assessed its interaction with DNA using a fluorescence polarization assay (see STAR Methods). We observed robust interaction of XRCC1-BRCT1 with a blunt-ended double-stranded DNA (dsDNA) oligonucleotide and a variety of different “damaged” dsDNA molecules with Kd values in the range ∼0.2–0.4 μM (Figures 1B and S1). Oligonucleotides incorporating single-strand gaps bound slightly more tightly than the nicked or intact oligonucleotides, but the presence or absence of 5′-phosphate groups at the nick or gap had little effect on the affinity of the interaction. We previously showed that mutation of residues in XRCC1-BRCT1 that are topologically equivalent to phosphate-binding residues in other BRCT domains disrupted the interaction of XRCC1 with PAR (Breslin et al., 2015Breslin C. Hornyak P. Ridley A. Rulten S.L. Hanzlikova H. Oliver A.W. Caldecott K.W. The XRCC1 phosphate-binding pocket binds poly (ADP-ribose) and is required for XRCC1 function.Nucleic Acids Res. 2015; 43: 6934-6944Crossref PubMed Scopus (69) Google Scholar). To further characterize the PAR-binding site, we recorded two-dimensional (2D) 1H–15N heteronuclear single quantum coherence (HSQC) NMR spectra on isotopically labeled samples of human XRCC1-BRCT1 (see STAR Methods) and measured chemical shift perturbations in the presence of a purified PAR oligomer (PAR4) (see STAR Methods; Figures 2A, 2B, and S2). We observed significant chemical shift perturbations in residues within and proximal to the putative phosphate-binding pocket, including Arg 335 and Lys 369, whose mutation disrupts binding to PAR in vitro and XRCC1 recruitment to DNA damage in vivo (Breslin et al., 2015Breslin C. Hornyak P. Ridley A. Rulten S.L. Hanzlikova H. Oliver A.W. Caldecott K.W. The XRCC1 phosphate-binding pocket binds poly (ADP-ribose) and is required for XRCC1 function.Nucleic Acids Res. 2015; 43: 6934-6944Crossref PubMed Scopus (69) Google Scholar and see below), confirming our identification of this pocket as critical for PAR binding. The exchange behavior of the chemical shift perturbations observed were in the slow-exchange range, suggesting an affinity for PAR4 in the sub-micromolar range, consistent with previous observations (Kim et al., 2015Kim I.K. Stegeman R.A. Brosey C.A. Ellenberger T. A quantitative assay reveals ligand specificity of the DNA scaffold repair protein XRCC1 and efficient disassembly of complexes of XRCC1 and the poly(ADP-ribose) polymerase 1 by poly(ADP-ribose) glycohydrolase.J. Biol. Chem. 2015; 290: 3775-3783Crossref PubMed Scopus (41) Google Scholar). 1H–15N HSQC spectra recorded in the presence of a nicked dsDNA oligonucleotide with the internal 5′ end phosphorylated (see STAR Methods) instead of PAR also display clear chemical shift changes consistent with the sub-micromolar affinity of the nicked DNA for XRCC1-BRCT1 observed in the fluorescence polarization experiments (see above) and confirming an interaction between XRCC1-BRCT1 and DNA. However, most of the observed perturbations upon DNA binding occurred in residues that were not strongly affected by PAR (Figure 2C), suggesting that the DNA and PAR molecules were binding to distinct sites on XRCC1-BRCT1. We tested this by titrating in increasing amounts of nicked dsDNA into XRCC1-BRCT1 already saturated by PAR4 and observed a pattern of chemical shift perturbations that represented the superposition of perturbations observed for the separate additions of PAR and DNA to protein alone (Figure 2D). Mapped onto the NMR solution structure of XRCC1-BRCT1 (PDB: 2D8M), the sets of residues perturbed by binding of PAR or by binding of DNA define distinct non-overlapping patches on the solvent accessible surface of the domain (Figures 2E and 2F). The residues perturbed by PAR binding lie on the face of the domain formed by the C-terminal end of the central parallel β sheet and map in and around the phosphate-binding “pocket,” which is conserved in many BRCT domains that mediate interaction with phosphorylated peptide motifs (Leung and Glover, 2011Leung C.C. Glover J.N. BRCT domains: easy as one, two, three.Cell Cycle. 2011; 10: 2461-2470Crossref PubMed Scopus (113) Google Scholar). The residues perturbed by DNA binding localize to the opposite face of the domain within the N-terminal ends of the β strands and from a segment of polypeptide extending from the C-terminal α helix. Next, we sought to validate the results of the NMR experiments by exploring the effect of disruptive mutations in the predicted DNA-binding site on biochemical and functional assays. In the absence of a high-resolution structure for a complex, predicting a single point mutation that abrogates XRCC1-BRCT1 interaction with DNA, as we have been able to do with phosphopeptide interactions with other BRCT domains (Qu et al., 2013Qu M. Rappas M. Wardlaw C.P. Garcia V. Ren J.Y. Day M. Carr A.M. Oliver A.W. Du L.L. Pearl L.H. Phosphorylation-dependent assembly and coordination of the DNA damage checkpoint apparatus by Rad4(TopBP1).Mol. Cell. 2013; 51: 723-736Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, Rappas et al., 2011Rappas M. Oliver A.W. Pearl L.H. Structure and function of the Rad9-binding region of the DNA-damage checkpoint adaptor TopBP1.Nucleic Acids Res. 2011; 39: 313-324Crossref PubMed Scopus (58) Google Scholar), is challenging. However, the highly basic nature of the surface patch revealed by NMR titration experiments with DNA suggests that mutations altering the electrostatics should affect interaction of the XRCC1-BRCT1 domain with DNA (Figure 3A). We therefore mutated a number of residues in this region that were perturbed by DNA binding in the NMR studies and found that an XRCC1-BRCT1-R399D,R400Q double mutant, which would be expected to substantially disrupt the basic nature of the putative DNA-binding site without perturbing the structure of the domain, could be readily expressed and purified as a soluble protein. Human populations have a common CAG → CGG polymorphism in codon 399 (allele frequency between 16%–35%), which results in a glutamine rather than an arginine in the expressed protein (Hu et al., 2005Hu Z. Ma H. Chen F. Wei Q. Shen H. XRCC1 polymorphisms and cancer risk: a meta-analysis of 38 case-control studies.Cancer Epidemiol. Biomarkers Prev. 2005; 14: 1810-1818Crossref PubMed Scopus (202) Google Scholar). Multiple studies have suggested association of the G/G and A/G genotypes with enhanced susceptibility to a broad range of cancer types (Casse et al., 2003Casse C. Hu Y.C. Ahrendt S.A. The XRCC1 codon 399 Gln allele is associated with adenine to guanine p53 mutations in non-small cell lung cancer.Mutat. Res. 2003; 528: 19-27Crossref PubMed Scopus (26) Google Scholar, Divine et al., 2001Divine K.K. Gilliland F.D. Crowell R.E. Stidley C.A. Bocklage T.J. Cook D.L. Belinsky S.A. The XRCC1 399 glutamine allele is a risk factor for adenocarcinoma of the lung.Mutat. Res. 2001; 461: 273-278Crossref PubMed Scopus (206) Google Scholar, Mateuca et al., 2008Mateuca R.A. Roelants M. Iarmarcovai G. Aka P.V. Godderis L. Tremp A. Bonassi S. Fenech M. Bergé-Lefranc J.L. Kirsch-Volders M. hOGG1(326), XRCC1(399) and XRCC3(241) polymorphisms influence micronucleus frequencies in human lymphocytes in vivo.Mutagenesis. 2008; 23: 35-41Crossref PubMed Scopus (59) Google Scholar, Mittal et al., 2008Mittal R.D. Singh R. Manchanda P.K. Ahirwar D. Gangwar R. Kesarwani P. Mandhani A. XRCC1 codon 399 mutant allele: a risk factor for recurrence of urothelial bladder carcinoma in patients on BCG immunotherapy.Cancer Biol. Ther. 2008; 7: 645-650Crossref PubMed Scopus (9) Google Scholar, Natukula et al., 2013Natukula K. Jamil K. Pingali U.R. Attili V.S. Madireddy U.R. The codon 399 Arg/Gln XRCC1 polymorphism is associated with lung cancer in Indians.Asian Pac. J. Cancer Prev. 2013; 14: 5275-5279Crossref PubMed Scopus (29) Google Scholar) and/or variable responses to chemotherapy (Deng et al., 2015Deng J.H. Deng J. Shi D.H. Ouyang X.N. Niu P.G. Clinical outcome of cisplatin-based chemotherapy is associated with the polymorphisms of GSTP1 and XRCC1 in advanced non-small cell lung cancer patients.Clin. Transl. Oncol. 2015; 17: 720-726Crossref PubMed Scopus (17) Google Scholar, Li and Li, 2013Li K. Li W. Association between polymorphisms of XRCC1 and ADPRT genes and ovarian cancer survival with platinum-based chemotherapy in Chinese population.Mol. Cell. Biochem. 2013; 372: 27-33Crossref PubMed Scopus (19) Google Scholar, Singh et al., 2017Singh A. Singh N. Behera D. Sharma S. Polymorphism in XRCC1 gene modulates survival and clinical outcomes of advanced North Indian lung cancer patients treated with platinum-based doublet chemotherapy.Med. Oncol. 2017; 34: 64Crossref PubMed Scopus (13) Google Scholar, Wu et al., 2012Wu J. Liu J. Zhou Y. Ying J. Zou H. Guo S. Wang L. Zhao N. Hu J. Lu D. Jin L. Li Q. Wang J.C. Predictive value of XRCC1 gene polymorphisms on platinum-based chemotherapy in advanced non-small cell lung cancer patients: a systematic review and meta-analysis.Clin. Cancer Res. 2012; 18: 3972-3981Crossref PubMed Scopus (42) Google Scholar). However, other studies and meta-analyses have failed to demonstrate such association, and the significance of this common polymorphism remains controversial (Jacobs and Bracken, 2012Jacobs D.I. Bracken M.B. Association between XRCC1 polymorphism 399 G->A and glioma among Caucasians: a systematic review and meta-analysis.BMC Med. Genet. 2012; 13: 97Crossref PubMed Scopus (26) Google Scholar, Taylor et al., 2002Taylor R.M. Thistlethwaite A. Caldecott K.W. Central role for the XRCC1 BRCT I domain in mammalian DNA single-strand break repair.Mol. Cell. Biol. 2002; 22: 2556-2563Crossref PubMed Scopus (150) Google Scholar, Yuan et al., 2010Yuan P. Liu L. Wu C. Zhong R. Yu D. Wu J. Xu Y. Nie S. Miao X. Sun Y. et al.No association between XRCC1 polymorphisms and survival in non-small-cell lung cancer patients treated with platinum-based chemotherapy.Cancer Biol. Ther. 2010; 10: 854-859Crossref PubMed Scopus (14) Google Scholar, Zeng et al., 2013Zeng X.Y. Huang J.M. Xu J.W. Xu Y. Yu H.P. Ji L. Qiu X.Q. Meta-analysis demonstrates lack of a relationship between XRCC1-399 gene polymorphisms and susceptibility to hepatocellular carcinoma.Genet. Mol. Res. 2013; 12: 1916-1923Crossref PubMed Scopus (6) Google Scholar). Because the participation of this polymorphic residue in DNA binding provides the first suggestion of a biochemical role, we compared Gln399 and Arg399 variants of the XRCC1-BRCT1 for functionality, alongside the R399D/R400Q double mutant. Using a previously described assay (Breslin et al., 2015Breslin C. Hornyak P. Ridley A. Rulten S.L. Hanzlikova H. Oliver A.W. Caldecott K.W. The XRCC1 phosphate-binding pocket binds poly (ADP-ribose) and is required for XRCC1 function.Nucleic Acids Res. 2015; 43: 6934-6944Crossref PubMed Scopus (69) Google Scholar), we tested the ability of the XRCC1-BRCT1 constructs to bind to PAR chains generated on histone H1 by PARP1 in the presence of NAD+ (see STAR Methods; Figure 3B). PAR binding by the DNA-binding site double mutant and the Gln399 variant were essentially identical to that of the Arg399 XRCC1-BRCT1 domain, whereas a construct with a previously described double mutation in the PAR-binding pocket (R335A, K369A; Breslin et al., 2015Breslin C. Hornyak P. Ridley A. Rulten S.L. Hanzlikova H. Oliver A.W. Caldecott K.W. The XRCC1 phosphate-binding pocket binds poly (ADP-ribose) and is required for XRCC1 function.Nucleic Acids Res. 2015; 43: 6934-6944Crossref PubMed Scopus (69) Google Scholar) failed to interact with PAR. These data demonstrate that the DNA-binding site identified by the NMR titration experiments does not contribute significantly to the interaction with PAR and confirms that neither the double mutation nor the polymorphic variation have any substantial effect on the three-dimensional structure and consequent functional integrity of the BRCT domain. By contrast, although both codon 399 variants and the PAR-binding pocket mutant protein displayed low or sub-micromolar affinity for 5′-phosphorylated or unphosphorylated nicked dsDNA in a fluorescence polarization assay (see STAR Methods), the R399D,R400Q double mutant failed to bind DNA, confirming the critical involvement of these residues in DNA binding by XRCC1-BRCT1 (Figures 3C and S3). To determine whether the ability of XRCC1-BRCT1 to bind DNA plays a role in its function as a DNA repair scaffold, we employed U2OS cells in which the XRCC1 gene was disrupted by CRISPR/Cas9-mediated gene editing and XRCC1 expression then restored in the edited cells by transfection with wild-type or mutant EGFP-XRCC1 fusion protein (see STAR Methods). We observed robust and rapid recruitment of both R399 and Q399 variants of the EGFP-XRCC1 fusion to DNA damage caused by laser micro-irradiation in these cell lines (see STAR Methods), whereas we failed to detect recruitment of the PAR-binding-defective R335A,K369A double mutant, as previously described (Breslin et al., 2015Breslin C. Hornyak P. Ridley A. Rulten S.L. Hanzlikova H. Oliver A.W. Caldecott K.W. The XRCC1 phosphate-binding pocket binds poly (ADP-ribose) and is required for XRCC1 function.Nucleic Acids Res. 2015; 43: 6934-6944Crossref PubMed Scopus (69) Google Scholar). The R399D,R400Q double mutant that is competent for PAR binding but defective in DNA binding (see above) was still recruited to DNA damage. However, this occurred with markedly slower kinetics than the native variants (Figure 4A). Chromatin retention of the EGFP-XRCC1 fusion protein following DNA damage was also strongly affected by mutational disruption of the DNA-binding site in BRCT1, with the R399D,R400Q double mutant being as poorly retained as the PAR-binding defective R335A,K369A mutant (Figure 4B). Finally, we looked at the ability of the variant and mutant XRCC1 proteins to support DNA repair in U2OS cells following treatment with varying doses of methyl methanesulfonate (MMS), using an alkaline comet assay that reports on unrepaired DNA SSBs (Breslin et al., 2006Breslin C. Clements P.M. El-Khamisy S.F. Petermann E. Iles N. Caldecott K.W. Measurement of chromosomal DNA single-strand breaks and replication fork progression rates.Methods Enzymol. 2006; 409: 410-425Crossref PubMed Scopus (34) Google Scholar). Wild-type U2OS cells (which contain the R399 XRCC1 variant) in which the endogenous XRCC1 gene was disrupted by gene editing accumulated far higher levels of SSBs than did wild-type U2OS cells (Figures 4C and S4B–S4D). The SSB repair defect in these XRCC1 gene-edited cells was effectively rescued by expression of either of the residue 399 polymorphic variants of EGFP-XRCC1, but not by the PAR-binding defective R335A,K369A double mutant (Figure 4C). Expression of the PAR-binding competent but DNA-binding-defective R399D,R400Q mutant resulted in an intermediate level of SSB repair that was significantly reduced compared to wild-type U2OS cells. A direct consequence of the activation of PARP1 and/or PARP2 at DNA strand breaks is the rapid formation of PAR chains covalently anchored primarily to the PARP enzymes themselves (Caldecott, 2008Caldecott K.W. Single-strand break repair and genetic disease.Nat. Rev. Genet. 2008; 9: 619-631Crossref PubMed Scopus (698) Google Scholar, Daniels et al., 2015Daniels C.M. Ong S.E. Leung A.K. The promise of proteomics for the study of ADP-ribosylation.Mol. Cell. 2015; 58: 911-924Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). A primary function of these PAR chains in the context of DNA repair is the recruitment of the XRCC1 scaffold protein to sites of DNA damage (London, 2015London R.E. The structural basis of XRCC1-mediated DNA repair.DNA Repair (Amst.). 2015; 30: 90-103Crossref PubMed Scopus (95) Google Scholar, Li et al., 2013Li M. Lu L.Y. Yang C.Y. Wang S. Yu X. The FHA and BRCT domains recognize ADP-ribosylation during DNA damage response.Genes Dev. 2013; 27: 1752-1768Crossref PubMed Scopus (116) Google Scholar, Breslin et al., 2015Breslin C. Hornyak P. Ridley A. Rulten S.L. Hanzlikova H. Oliver A.W. Caldecott K.W. The XRCC1 phosphate-binding pocket binds poly (ADP-ribose) and is required for XRCC1 function.Nucleic Acids Res. 2015; 43: 6934-6944Crossref PubMed Scopus (69) Google Scholar, Hanzlikova et al., 2017Hanzlikova H. Gittens W. Krejcikova K. Zeng Z. Caldecott K.W. Overlapping roles for PARP1 and PARP2 in the recruitment of endogenous XRCC1 and PNKP into oxidized chromatin.Nucleic Acids Res. 2017; 45: 2546-2557PubMed Google Scholar). XRCC1-dependent repair of single-strand DNA breaks generated by oxidative damage, alkylation, or abortive topoisomerase 1 activity requires the catalytic activity of up to four associated DNA repair enzymes (Polβ, Lig3α, PNKP, and APTX), each of which requires access to the 5′ and/or 3′ termini at the margins of the DNA break to perform its particular reaction. To facilitate this, XRCC1 functions as a DNA-binding scaffold protein to he" @default.
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• W2910565537 title "Efficient Single-Strand Break Repair Requires Binding to Both Poly(ADP-Ribose) and DNA by the Central BRCT Domain of XRCC1" @default.
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