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• W1998499862 abstract "DNA-damage signaling utilizes a multitude of posttranslational modifiers as molecular switches to regulate cell-cycle checkpoints, DNA repair, cellular senescence, and apoptosis. Here we show that RNF8, a FHA/RING domain-containing protein, plays a critical role in the early DNA-damage response. We have solved the X-ray crystal structure of the FHA domain structure at 1.35 Å. We have shown that RNF8 facilitates the accumulation of checkpoint mediator proteins BRCA1 and 53BP1 to the damaged chromatin, on one hand through the phospho-dependent FHA domain-mediated binding of RNF8 to MDC1, on the other hand via its role in ubiquitylating H2AX and possibly other substrates at damage sites. Moreover, RNF8-depleted cells displayed a defective G2/M checkpoint and increased IR sensitivity. Together, our study implicates RNF8 as a novel DNA-damage-responsive protein that integrates protein phosphorylation and ubiquitylation signaling and plays a critical role in the cellular response to genotoxic stress. DNA-damage signaling utilizes a multitude of posttranslational modifiers as molecular switches to regulate cell-cycle checkpoints, DNA repair, cellular senescence, and apoptosis. Here we show that RNF8, a FHA/RING domain-containing protein, plays a critical role in the early DNA-damage response. We have solved the X-ray crystal structure of the FHA domain structure at 1.35 Å. We have shown that RNF8 facilitates the accumulation of checkpoint mediator proteins BRCA1 and 53BP1 to the damaged chromatin, on one hand through the phospho-dependent FHA domain-mediated binding of RNF8 to MDC1, on the other hand via its role in ubiquitylating H2AX and possibly other substrates at damage sites. Moreover, RNF8-depleted cells displayed a defective G2/M checkpoint and increased IR sensitivity. Together, our study implicates RNF8 as a novel DNA-damage-responsive protein that integrates protein phosphorylation and ubiquitylation signaling and plays a critical role in the cellular response to genotoxic stress. Faithful duplication and segregation of DNA is essential to maintain genomic integrity during cell division. DNA lesions elicit a DNA-damage response, which collectively includes DNA repair, activation of cell-cycle checkpoints, chromatin remodeling, cellular senescence, and apoptosis. Mutations in a variety of components involved in these cellular processes directly contribute to tumorigenesis (Bartkova et al., 2005Bartkova J. Horejsi Z. Koed K. Kramer A. Tort F. Zieger K. Guldberg P. Sehested M. Nesland J.M. Lukas C. et al.DNA damage response as a candidate anti-cancer barrier in early human tumorigenesis.Nature. 2005; 434: 864-870Crossref PubMed Scopus (2121) Google Scholar, Gorgoulis et al., 2005Gorgoulis V.G. Vassiliou L.V. Karakaidos P. Zacharatos P. Kotsinas A. Liloglou T. Venere M. Ditullio Jr., R.A. Kastrinakis N.G. Levy B. et al.Activation of the DNA damage checkpoint and genomic instability in human precancerous lesions.Nature. 2005; 434: 907-913Crossref PubMed Scopus (1623) Google Scholar), highlighting the importance of these damage-induced signaling cascades in tumor suppression. Accumulating evidence suggests that the ATM/ATR-dependent phosphorylation of histone variant H2AX to create γ-H2AX is the initial signal for subsequent accumulation of various mediators/repair proteins to DNA lesions (Bassing et al., 2003Bassing C.H. Suh H. Ferguson D.O. Chua K.F. Manis J. Eckersdorff M. Gleason M. Bronson R. Lee C. Alt F.W. Histone H2AX: a dosage-dependent suppressor of oncogenic translocations and tumors.Cell. 2003; 114: 359-370Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar, Celeste et al., 2003Celeste A. Difilippantonio S. Difilippantonio M.J. Fernandez-Capetillo O. Pilch D.R. Sedelnikova O.A. Eckhaus M. Ried T. Bonner W.M. Nussenzweig A. H2AX haploinsufficiency modifies genomic stability and tumor susceptibility.Cell. 2003; 114: 371-383Abstract Full Text Full Text PDF PubMed Scopus (463) Google Scholar). A positive feedback loop has been proposed in which ATM/ATR concentrates at γ-H2AX-containing double-strand breaks via MDC1 to further phosphorylate adjacent H2AX molecules and amplify the DNA-damage signal (Lou et al., 2006Lou Z. Minter-Dykhouse K. Franco S. Gostissa M. Rivera M.A. Celeste A. Manis J.P. van Deursen J. Nussenzweig A. Paull T.T. et al.MDC1 maintains genomic stability by participating in the amplification of ATM-dependent DNA damage signals.Mol. Cell. 2006; 21: 187-200Abstract Full Text Full Text PDF PubMed Scopus (462) Google Scholar, Stucki et al., 2005Stucki M. Clapperton J.A. Mohammad D. Yaffe M.B. Smerdon S.J. Jackson S.P. MDC1 directly binds phosphorylated histone H2AX to regulate cellular responses to DNA double-strand breaks.Cell. 2005; 123: 1213-1226Abstract Full Text Full Text PDF PubMed Scopus (765) Google Scholar). Through this signal amplification step, a number of mediator/repair proteins, including BRCA1 and 53BP1, concentrate to sites of DNA damage to facilitate downstream checkpoint activation. We and others have previously demonstrated that tandem BRCT domains serve as phosphopeptide binding motifs that mediate protein-protein interactions (Manke et al., 2003Manke I.A. Lowery D.M. Nguyen A. Yaffe M.B. BRCT repeats as phosphopeptide-binding modules involved in protein targeting.Science. 2003; 302: 636-639Crossref PubMed Scopus (520) Google Scholar, Yu et al., 2003Yu X. Chini C.C. He M. Mer G. Chen J. The BRCT domain is a phospho-protein binding domain.Science. 2003; 302: 639-642Crossref PubMed Scopus (648) Google Scholar). Specifically, a number of DNA-damage response proteins, including BRCA1 and MDC1 (Fernandez-Capetillo et al., 2002Fernandez-Capetillo O. Chen H.T. Celeste A. Ward I. Romanienko P.J. Morales J.C. Naka K. Xia Z. Camerini-Otero R.D. Motoyama N. et al.DNA damage-induced G2-M checkpoint activation by histone H2AX and 53BP1.Nat. Cell Biol. 2002; 4: 993-997Crossref PubMed Scopus (545) Google Scholar, Goldberg et al., 2003Goldberg M. Stucki M. Falck J. D'Amours D. Rahman D. Pappin D. Bartek J. Jackson S.P. MDC1 is required for the intra-S-phase DNA damage checkpoint.Nature. 2003; 421: 952-956Crossref PubMed Scopus (416) Google Scholar, Lou et al., 2003aLou Z. Chini C.C. Minter-Dykhouse K. Chen J. Mediator of DNA damage checkpoint protein 1 regulates BRCA1 localization and phosphorylation in DNA damage checkpoint control.J. Biol. Chem. 2003; 278: 13599-13602Crossref PubMed Scopus (109) Google Scholar, Lou et al., 2003bLou Z. Minter-Dykhouse K. Wu X. Chen J. MDC1 is coupled to activated CHK2 in mammalian DNA damage response pathways.Nature. 2003; 421: 957-961Crossref PubMed Scopus (276) Google Scholar, Stewart et al., 2003Stewart G.S. Wang B. Bignell C.R. Taylor A.M. Elledge S.J. MDC1 is a mediator of the mammalian DNA damage checkpoint.Nature. 2003; 421: 961-966Crossref PubMed Scopus (667) Google Scholar), harbor BRCT domains that mediate binding to their respective partners in a phosphorylation-dependent manner (Yu and Chen, 2004Yu X. Chen J. DNA damage-induced cell cycle checkpoint control requires CtIP, a phosphorylation-dependent binding partner of BRCA1 C-terminal domains.Mol. Cell. Biol. 2004; 24: 9478-9486Crossref PubMed Scopus (295) Google Scholar, Stucki et al., 2005Stucki M. Clapperton J.A. Mohammad D. Yaffe M.B. Smerdon S.J. Jackson S.P. MDC1 directly binds phosphorylated histone H2AX to regulate cellular responses to DNA double-strand breaks.Cell. 2005; 123: 1213-1226Abstract Full Text Full Text PDF PubMed Scopus (765) Google Scholar). In addition to tandem BRCT domains, the FHA domain constitutes a separate class of phosphopeptide binding modules (Durocher et al., 2000Durocher D. Taylor I.A. Sarbassova D. Haire L.F. Westcott S.L. Jackson S.P. Smerdon S.J. Yaffe M.B. The molecular basis of FHA domain:phosphopeptide binding specificity and implications for phosphodependent signaling mechanisms.Mol. Cell. 2000; 6: 1169-1182Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar). Many FHA domain-containing proteins have been reported to play a role in DNA repair, cell-cycle arrest, and pre-mRNA processing (Sun et al., 1998Sun Z. Hsiao J. Fay D.S. Stern D.F. Rad53 FHA domain associated with phosphorylated Rad9 in the DNA damage checkpoint.Science. 1998; 281: 272-274Crossref PubMed Scopus (327) Google Scholar, Li et al., 2000Li J. Lee G.I. Van Doren S.R. Walker J.C. The FHA domain mediates phosphoprotein interactions.J. Cell Sci. 2000; 113: 4143-4149Crossref PubMed Google Scholar). The reversibility and sequence selectivity of ligand binding afforded by these and other phosphopeptide binding domain-containing proteins allows individual protein-protein interactions that control downstream responses to be tightly regulated in a stimulus-dependent manner. Recent studies have provided additional insight into the phosphorylation-dependent regulation of the DNA-damage signaling network. However, the detailed mechanisms by which the initial γ-H2AX signal at DNA lesions becomes propagated, amplified, and modified to concentrate checkpoint mediator proteins to these sites remain obscure. Here we report our study of an FHA and RING domain-containing protein, RNF8, which serves as the molecular linker for communication between the protein phosphorylation and protein ubiquitylation pathways that are crucial for the activation and maintenance of the DNA-damage response. We have previously studied the role of the FHA domain and RING domain-containing protein Chfr in mitosis (Yu et al., 2005Yu X. Minter-Dykhouse K. Malureanu L. Zhao W.M. Zhang D. Merkle C.J. Ward I.M. Saya H. Fang G. van Deursen J. et al.Chfr is required for tumor suppression and Aurora A regulation.Nat. Genet. 2005; 37: 401-406Crossref PubMed Scopus (168) Google Scholar). In the course of these studies we used a protein named RNF8 as a control because it is the only other known mammalian protein that shares a similar domain organization with Chfr (Figure S1A). RNF8 was initially reported to interact with class III human ubiquitin-conjugating enzymes (E2s) through its RING domain (Ito et al., 2001Ito K. Adachi S. Iwakami R. Yasuda H. Muto Y. Seki N. Okano Y. N-terminally extended human ubiquitin-conjugating enzymes (E2s) mediate the ubiquitination of RING-finger proteins, ARA54 and RNF8.Eur. J. Biochem. 2001; 268: 2725-2732Crossref PubMed Scopus (82) Google Scholar). RNF8 was later shown to bind to the Retinoid X Receptor and regulate its transcriptional activity (Takano et al., 2004Takano Y. Adachi S. Okuno M. Muto Y. Yoshioka T. Matsushima-Nishiwaki R. Tsurumi H. Ito K. Friedman S.L. Moriwaki H. et al.The RING finger protein, RNF8, interacts with retinoid X receptor alpha and enhances its transcription-stimulating activity.J. Biol. Chem. 2004; 279: 18926-18934Crossref PubMed Scopus (31) Google Scholar). Because several FHA domain-containing proteins are known to play a role in DNA-damage signaling, we investigated whether RNF8 or Chfr might participate in the DNA-damage response. Cells stably expressing tagged RNF8 or Chfr were irradiated. Interestingly and surprisingly, RNF8 foci can be readily observed after DNA damage, and these foci colocalized with the DNA-damage marker γ-H2AX (Figure S1A). Despite the resemblance of RNF8 and Chfr (Figure S1A), we did not observe any Chfr focus formation following DNA damage (Figure 1A), indicating that these two related proteins have distinct cellular functions. To confirm the observed IR-induced focus localization of RNF8, we generated a polyclonal antibody specifically recognizing RNF8 (Figure S1B). IR-induced foci (IRIF) of endogenous RNF8 can be readily visualized (Figure 1B). The fact that RNF8 foci overlap with those of γ-H2AX prompted us to speculate that RNF8 might function in the DNA-damage response. We therefore examined the localization of RNF8 with several proteins known to be involved in this damage-induced signaling cascade. As expected, RNF8 colocalizes with MDC1, NBS1, 53BP1, BRCA1, pATM, and MCPH1, further lending credence to the potential role of RNF8 in the DNA-damage response (Figure S1C). The DNA-damage-induced focus formation of checkpoint proteins reflects their localization to chromatin structures at the vicinity of DNA breaks. Indeed, increased amount of RNF8 accumulated in the acid extractable fraction after IR treatment (Figure 1C). Moreover, the less-soluble fraction of RNF8 can be released by nuclease treatment (Figure 1D), suggesting that RNF8 accumulates at the chromatin upon DNA damage. Together, our studies suggest that RNF8 is a novel DNA-damage-responsive protein. It is generally accepted that the phosphorylation of histone variant H2AX is the initial signal upon DNA lesion detection. γ-H2AX is required for the sustained localization of a number of DNA-damage mediator/repair factors at chromatin regions at or near the sites of DNA damage (Paull et al., 2000Paull T.T. Rogakou E.P. Yamazaki V. Kirchgessner C.U. Gellert M. Bonner W.M. A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage.Curr. Biol. 2000; 10: 886-895Abstract Full Text Full Text PDF PubMed Scopus (1597) Google Scholar). To delineate where RNF8 fits in the established DNA-damage signaling cascade, we examined IRIF formation of RNF8 in a number of human or mouse cells with deficiencies in various DNA-damage checkpoint proteins. Our anti-RNF8 antibody could not detect endogenous RNF8 in mouse embryonic fibroblasts (MEFs), so we used retroviral particles containing a RNF8 expression construct to infect these cells (Figure 1E). In sharp contrast to the control wild-type (WT) MEFs, no IR-induced RNF8 focus formation was observed in H2AX-deficient MEFs or those reconstituted with the S139A phosphomutant (Figures 1E and S1D). Likewise, RNF8 focus formation was also abrogated in MDC1-deficient cells. On the other hand, RNF8 relocalization to γ-H2AX-containing foci is not noticeably affected in cells with BRCA1, 53BP1, or NBS1 deficiency (Figure 1E). These data suggest that RNF8 acts downstream of H2AX and MDC1 in the DNA-damage-responsive pathway. The FHA domain is a phosphoprotein-binding module (Durocher et al., 2000Durocher D. Taylor I.A. Sarbassova D. Haire L.F. Westcott S.L. Jackson S.P. Smerdon S.J. Yaffe M.B. The molecular basis of FHA domain:phosphopeptide binding specificity and implications for phosphodependent signaling mechanisms.Mol. Cell. 2000; 6: 1169-1182Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar, Li et al., 2000Li J. Lee G.I. Van Doren S.R. Walker J.C. The FHA domain mediates phosphoprotein interactions.J. Cell Sci. 2000; 113: 4143-4149Crossref PubMed Google Scholar). Figure 1F shows that tagged WT RNF8 formed foci that colocalize with γ-H2AX following IR treatment. Similarly, foci formation can also be observed for the delRING mutant. On the other hand, the FHA deletion mutant (i.e., delFHA) failed to localize to γ-H2AX-containing foci, suggesting that the FHA domain of RNF8 is important for targeting RNF8 to IR-induced DNA-damage sites (Figure S1E). FHA domains, like tandem BRCT domains, recognize amino acid sequences extending 3–4 residues around a central phosphorylated amino acid, with selection determined primarily by residues in the third C-terminal (+3) position (Durocher et al., 2000Durocher D. Taylor I.A. Sarbassova D. Haire L.F. Westcott S.L. Jackson S.P. Smerdon S.J. Yaffe M.B. The molecular basis of FHA domain:phosphopeptide binding specificity and implications for phosphodependent signaling mechanisms.Mol. Cell. 2000; 6: 1169-1182Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar). However, in contrast to BRCT domains that recognize both pSer- and pThr-containing sequences, FHA domains appear only to recognize pThr-containing motifs. We determined the optimal phosphopeptide motifs recognized by the RNF8 FHA domain using pThr-oriented peptide library screening (Figure 2A). Intriguingly, the RNF8 FHA domain showed strong selection for Tyr and Phe in the +3 position. This selection for aromatic amino acids differs substantially from the acidic and aliphatic residue selection in the +3 position shown by all other FHA domains for which X-ray crystal structures are available (Durocher et al., 2000Durocher D. Taylor I.A. Sarbassova D. Haire L.F. Westcott S.L. Jackson S.P. Smerdon S.J. Yaffe M.B. The molecular basis of FHA domain:phosphopeptide binding specificity and implications for phosphodependent signaling mechanisms.Mol. Cell. 2000; 6: 1169-1182Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar, Li et al., 2002Li J. Williams B.L. Haire L.F. Goldberg M. Wilker E. Durocher D. Yaffe M.B. Jackson S.P. Smerdon S.J. Structural and functional versatility of the FHA domain in DNA-damage signaling by the tumor suppressor kinase Chk2.Mol. Cell. 2002; 9: 1045-1054Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). Instead, this selection for aromatic amino acids at the +3 position closely resembles the optimal phosphopeptide motifs recognized by the tandem BRCT domains of BRCA1 and MDC1 (Manke et al., 2003Manke I.A. Lowery D.M. Nguyen A. Yaffe M.B. BRCT repeats as phosphopeptide-binding modules involved in protein targeting.Science. 2003; 302: 636-639Crossref PubMed Scopus (520) Google Scholar, Stucki et al., 2005Stucki M. Clapperton J.A. Mohammad D. Yaffe M.B. Smerdon S.J. Jackson S.P. MDC1 directly binds phosphorylated histone H2AX to regulate cellular responses to DNA double-strand breaks.Cell. 2005; 123: 1213-1226Abstract Full Text Full Text PDF PubMed Scopus (765) Google Scholar, Yu et al., 2003Yu X. Chini C.C. He M. Mer G. Chen J. The BRCT domain is a phospho-protein binding domain.Science. 2003; 302: 639-642Crossref PubMed Scopus (648) Google Scholar). To investigate the structural basis for this unusual motif selection, we used limited proteolysis to map the boundaries of the FHA domain and solved the high-resolution structure of the RNF8 FHA:optimal phosphopeptide complex by X-ray crystallography at 1.35 Å (Table S1; the PDB code for the RNF8 FHA domain is 2PIE). The global fold of the RNF8 FHA domain is an 11-stranded β sandwich with the phosphopeptide-binding surface comprised of residues in loops that connect the β-strands at one end of the sandwich (Figure 2B), similar to what has been previously observed in the crystal structures of Rad53 and Chk2 FHA domains (Durocher et al., 2000Durocher D. Taylor I.A. Sarbassova D. Haire L.F. Westcott S.L. Jackson S.P. Smerdon S.J. Yaffe M.B. The molecular basis of FHA domain:phosphopeptide binding specificity and implications for phosphodependent signaling mechanisms.Mol. Cell. 2000; 6: 1169-1182Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar, Li et al., 2002Li J. Williams B.L. Haire L.F. Goldberg M. Wilker E. Durocher D. Yaffe M.B. Jackson S.P. Smerdon S.J. Structural and functional versatility of the FHA domain in DNA-damage signaling by the tumor suppressor kinase Chk2.Mol. Cell. 2002; 9: 1045-1054Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). The bound phosphopeptide is in an extended conformation with extensive contacts between the peptide backbone and side chain and main chain atoms from the RNF8 FHA domain (Figure 2C). Three structural features observed in the RNF8 FHA:phosphopeptide complex are distinct from those in other FHA domains: First, the RNF8 FHA domain contains two divergent loops and a C-terminal α-helical extension that cluster along one face of the domain, well removed from the phosphopeptide-interacting surface (Figure 2B, shaded red). This region is likely involved in phospho-independent interactions with potential substrates or with additional portions of the full-length RNF8 molecule. Second, the RNF8 FHA domain makes extensive direct contacts to the phosphate group, including a novel bidentate interaction involving Arg61 that is not observed in any other FHA domain:phosphopeptide crystal structure (Figure 2D). Third, the selectivity for Tyr/Phe in the +3 position results from its interaction with a flat, mostly nonpolar surface relatively enriched in sulfur-containing amino acids (Cys55, Met58, Val110, and Leu112). Interestingly, the general character of the interaction between the +3 Tyr and the surface of the RNF8 FHA domain is strikingly similar to that observed between the +3 Tyr residue of a γ-H2AX pSer-containing phosphopeptide and the surface of the tandem BRCT domains of MDC1 critical for MDC1 foci formation (Figures 2E and 2F). On the other hand, the RNF8 FHA domain +3 interacting surface bears little resemblance to the analogous surfaces in other FHA domains (Figures 2G and 2H). Thus, it appears that the pThr-binding FHA domain of RNF8 has evolved to bind to similar motifs as those recognized by the BRCT domains of the foci-forming proteins BRCA1 and MDC1. We directly investigated whether phospho-dependent binding was critical for IRIF formation of RNF8. We found that mutation of Arg61 to Gln reduced FHA domain:phosphopeptide binding by over 160-fold (Figures S2A and S2B). When the full-length RNF8 R61Q mutant protein was introduced into cells, no R61Q foci were observed after radiation damage (Figure S2C), indicating that phospho-dependent binding is required for interaction between the RNF8 FHA domain and its upstream binding partner. In an experiment with cell lysates, WT RNF8 could be pulled down with a phospho-Ser-containing peptide derived from γ-H2AX (Figure S2D) but not with the control unphosphorylated peptide. This interaction was totally abolished in experiments with the delFHA or R61Q mutant proteins. Furthermore, consistent with the previous observation that Chfr does not form IRIF, Chfr did not bind to the phosphorylated H2AX peptide (Figure S2E). Although RNF8 bound to phospho-H2AX peptides in a pulldown experiment, we failed to detect any direct binding between the RNF8 FHA domain and a phospho-H2AX peptide by isothermal titration calorimetry (data not shown), raising the possibility that the RNF8:γ-H2AX interaction observed was indirect. Because the BRCT domains of MDC1 mediate its direct binding to phospho-H2AX, and MDC1 is required for RNF8 IRIF, we examined whether RNF8 interacts with MDC1. Intriguingly, the optimal motif for phosphopeptide binding to the RNF8 FHA domain is pTXXY/F (Figure 2A), and inspection of the MDC1 sequence reveals four potential ATM/ATR phophorylation sites that match this motif (T699QCF, T719QAF, T752QPF, and T765QPF). One of these TQPF sites was recently reported to be phosphorylated following DNA damage in a large-scale proteomic study (Matsuoka et al., 2007Matsuoka S. Ballif B.A. Smogorzewska A. McDonald 3rd, E.R. Hurov K.E. Luo J. Bakalarski C.E. Zhao Z. Solimini N. Lerenthal Y. et al.ATM and ATR substrate analysis reveals extensive protein networks responsive to DNA damage.Science. 2007; 316: 1160-1166Crossref PubMed Scopus (2162) Google Scholar). We therefore synthesized peptides containing each of these four putative phosphorylation sites and measured their binding to the RNF8 FHA domain. Three of the four bound with affinities comparable to the optimal peptide (Figures S2F–S2I), while the fourth bound more weakly. We next generated a deletion mutant spanning residues 698–768 (Del) of MDC1 (Figure 3A) and showed that MDC1, but not Del, specifically bound to purified GST-RNF8 (Figures 3B and 3C). In addition, RNF8 coprecipitated with WT but not Del mutant of MDC1 in vivo (Figure 3D), further implying that these putative phosphorylation sites are required for the interaction between RNF8 and MDC1. A control experiment using the delFHA mutant of RNF8 confirms that this interaction also requires the FHA domain of RNF8 (Figure 3E). Similar results were obtained in reciprocal immunoprecipitation assays. Consistent with the role of MDC1 in mediating RNF8 accumulation at DNA-damage sites, an increased amount of MDC1 bound to RNF8 after IR, and this MDC1 was abolished with prior phosphatase treatment (Figure 3F). Likewise, WT but not Del mutant of MDC1 restored RNF8 IRIF in MDC1-depleted HeLa cells (Figures S3A–S3B). Collectively, these in vitro and in vivo results support a possible direct role of MDC1 in facilitating RNF8 localization, via a phospho-specific interaction conferred by the RNF8 FHA domain, to the chromatin following DNA damage. To further probe the role of RNF8 at DNA-damage sites in vivo, we depleted RNF8 with two different siRNAs and tested whether the IRIF of a number of mediator/repair proteins were RNF8 dependent. RNF8 knockdown did not affect γ-H2AX or MDC1 foci formation at DNA-damage sites (Figures 4A and S4A–S4C); however, localization of 53BP1 and BRCA1 to foci was abrogated (Figure 4A), suggesting that RNF8 lies upstream of these DNA-damage signaling mediator/effectors and facilitates the accumulation of these proteins to the sites of DNA damage. The MDC1/RNF8 interaction experiments presented in Figure 3 imply that MDC1 may interact with RNF8 and regulate RNF8-dependent events following DNA damage. MDC1 was previously shown to be required for IRIF formation of these checkpoint mediator proteins (Goldberg et al., 2003Goldberg M. Stucki M. Falck J. D'Amours D. Rahman D. Pappin D. Bartek J. Jackson S.P. MDC1 is required for the intra-S-phase DNA damage checkpoint.Nature. 2003; 421: 952-956Crossref PubMed Scopus (416) Google Scholar, Lou et al., 2003aLou Z. Chini C.C. Minter-Dykhouse K. Chen J. Mediator of DNA damage checkpoint protein 1 regulates BRCA1 localization and phosphorylation in DNA damage checkpoint control.J. Biol. Chem. 2003; 278: 13599-13602Crossref PubMed Scopus (109) Google Scholar, Stewart et al., 2003Stewart G.S. Wang B. Bignell C.R. Taylor A.M. Elledge S.J. MDC1 is a mediator of the mammalian DNA damage checkpoint.Nature. 2003; 421: 961-966Crossref PubMed Scopus (667) Google Scholar; also see Figure S4D). Here, we examined whether the MDC1/RNF8 interaction is crucial for these events in vivo. As expected, ectopically expressed MDC1 restored the accumulation of BRCA1 and 53BP1 in response to IR in MDC1−/− MEFs. The MDC1 deletion mutation, which abolishes its interaction with RNF8, did not affect its own focus localization following IR but failed to restore the RNF8-dependent concentration of BRCA1 and 53BP1 at the foci (Figure 4B), suggesting that the MDC1/RNF8 interaction is likely to be required for RNF8-dependent functions following DNA damage. In order to further explore roles of the RNF8 FHA and RING domains in targeting 53BP1 and BRCA1 to foci, we knocked down RNF8 in HeLa cells using siRNF8#2 (Figure S4E) and reintroduced full-length RNF8, delFHA, or delRING using constructs containing silent mutations within the RNF8 coding sequence that rendered the reintroduced constructs resistant to the siRNA. Unlike the deletion mutants, reconstitution with full-length RNF8 restored 53BP1 and BRCA1 IRIF in cells depleted of endogenous RNF8 (Figures 4C and S4F). Thus, both the phosphopeptide-binding and the ubiquitin ligase activity of RNF8 are required for its function in mediating the accumulation of DNA-damage checkpoint proteins at the sites of DNA damage. Our observation that IRIF formation of BRCA1 and 53BP1 requires the RNF8 RING domain suggests that the accumulation of these checkpoint proteins is dependent on protein ubiquitylation at the site of the damaged chromatin. The finding that DNA-damage-associated ubiquitin conjugates can be visualized using the anti-ubiquitin FK2 antibody (Morris and Solomon, 2004Morris J.R. Solomon E. BRCA1: BARD1 induces the formation of conjugated ubiquitin structures, dependent on K6 of ubiquitin, in cells during DNA replication and repair.Hum. Mol. Genet. 2004; 13: 807-817Crossref PubMed Scopus (192) Google Scholar, Polanowska et al., 2006Polanowska J. Martin J.S. Garcia-Muse T. Petalcorin M.I. Boulton S.J. A conserved pathway to activate BRCA1-dependent ubiquitylation at DNA damage sites.EMBO J. 2006; 25: 2178-2188Crossref PubMed Scopus (121) Google Scholar) is consistent with this hypothesis. If RNF8 is involved in the ubiquitylation of proteins at the damaged chromatin, H2AX and MDC1 deficiencies, which abrogate RNF8 accumulation at IRIF, might be expected to disrupt damage-dependent FK2 focus formation. This is indeed the case (see Figures S5A–S5C). Recently, the E2 ubiquitin-conjugating enzyme UBC13 was also implicated in the ubiquitylation of protein(s) at the chromatin following DNA damage (Zhao et al., 2007Zhao G.Y. Sonoda E. Barber L.J. Oka H. Murakawa Y. Yamada K. Ikura T. Wang X. Kobayashi M. Yamamoto K. et al.A critical role for the ubiquitin-conjugating enzyme Ubc13 in initiating homologous recombination.Mol. Cell. 2007; 25: 663-675Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). However, the E3 ligase, which provides substrate specificity, has yet to be identified. That RNF8 was demonstrated to interact with UBC13 for substrate ubiquitylation (Plans et al., 2006Plans V. Scheper J. Soler M. Loukili N. Okano Y. Thomson T.M. The RING finger protein RNF8 recruits UBC13 for lysine 63-based self polyubiquitylation.J. Cell. Biochem. 2006; 97: 572-582Crossref PubMed Scopus (80) Google Scholar) prompted us to speculate that RNF8 and UBC13 might act in concert in the DNA-damage-signaling cascade. In support of this speculation, we found that damage-associated ubiquitin conjugates were absent in either RNF8-depleted or UBC13-depleted cells (Figures 5A and S5D). UBC13 depletion also suppressed the accumulation of 53BP1 and BRCA1 at IRIF but did not affect focus formation of phospho-H2AX and MDC1 (Figure S5E). In addition, RNF8 IRIF can be readily visualized in UBC13-depleted cells, indicating that the damage-dependent RNF8 localization precedes UBC13 function in the DNA-damage response. These data, together with previous reports, imply that RNF8 acts with UBC13 to exert a mediator role in the DNA-damage-signaling cascade. The ubiquit" @default.
• W1998499862 created "2016-06-24" @default.
• W1998499862 creator A5002688713 @default.
• W1998499862 creator A5029892501 @default.
• W1998499862 creator A5030374291 @default.
• W1998499862 creator A5036737144 @default.
• W1998499862 creator A5069046588 @default.
• W1998499862 creator A5073615852 @default.
• W1998499862 creator A5086583107 @default.
• W1998499862 date "2007-11-01" @default.
• W1998499862 modified "2023-10-13" @default.
• W1998499862 title "RNF8 Transduces the DNA-Damage Signal via Histone Ubiquitylation and Checkpoint Protein Assembly" @default.
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