Regulation of DNA Damage Responses by Ubiquitin and SUMO
2013; Elsevier BV; Volume: 49; Issue: 5 Linguagem: Inglês
10.1016/j.molcel.2013.01.017
ISSN1097-4164
AutoresStephen P. Jackson, Daniel Durocher,
Tópico(s)Mitochondrial Function and Pathology
ResumoUbiquitylation and sumoylation, the covalent attachment of the polypeptides ubiquitin and SUMO, respectively, to target proteins, are pervasive mechanisms for controlling cellular functions. Here, we summarize the key steps and enzymes involved in ubiquitin and SUMO conjugation and provide an overview of how they are crucial for maintaining genome stability. Specifically, we review research that has revealed how ubiquitylation and sumoylation regulate and coordinate various pathways of DNA damage recognition, signaling, and repair at the biochemical, cellular, and whole-organism levels. In addition to providing key insights into the control and importance of DNA repair and associated processes, such work has established paradigms for regulatory control that are likely to extend to other cellular processes and that may provide opportunities for better understanding and treatment of human disease. Ubiquitylation and sumoylation, the covalent attachment of the polypeptides ubiquitin and SUMO, respectively, to target proteins, are pervasive mechanisms for controlling cellular functions. Here, we summarize the key steps and enzymes involved in ubiquitin and SUMO conjugation and provide an overview of how they are crucial for maintaining genome stability. Specifically, we review research that has revealed how ubiquitylation and sumoylation regulate and coordinate various pathways of DNA damage recognition, signaling, and repair at the biochemical, cellular, and whole-organism levels. In addition to providing key insights into the control and importance of DNA repair and associated processes, such work has established paradigms for regulatory control that are likely to extend to other cellular processes and that may provide opportunities for better understanding and treatment of human disease. Although initially discovered as a mechanism targeting proteins for destruction by the proteasome, ubiquitylation—the covalent attachment of the 76 amino acid residue protein ubiquitin to other proteins—is now also known to regulate protein activity, localization, and interactions (Bergink and Jentsch, 2009Bergink S. Jentsch S. Principles of ubiquitin and SUMO modifications in DNA repair.Nature. 2009; 458: 461-467Crossref PubMed Scopus (261) Google Scholar; Komander and Rape, 2012Komander D. Rape M. The ubiquitin code.Annu. Rev. Biochem. 2012; 81: 203-229Crossref PubMed Scopus (238) Google Scholar). In addition to ubiquitin, there are several ubiquitin-like proteins (UBLs) that are structurally related to ubiquitin. Ubiquitin and most UBLs are attached via their C-terminal glycine residues to target proteins by enzymatic reactions mediated by E1, E2, and E3 ligases (Figure 1). The most widely characterized UBL is the ∼100 residue protein SUMO (small ubiquitin-related modifier). Eukaryotes usually contain a single type of ubiquitin that is encoded by multiple genes. By contrast, vertebrate cells possess two types of SUMO: SUMO-1 and the highly related proteins SUMO-2 and SUMO-3 (SUMO2/3) that appear to be functionally redundant. Simpler organisms such as Saccharomyces cerevisiae and Schizosaccharomyces pombe, however, contain a single SUMO (Smt3 and Pmt3, respectively). In mammals, ubiquitylation involves two E1s, over 35 E2s, and over 600 E3s, while sumoylation is mediated by a single heterodimeric E1, one E2 (UBC9/UBE2I), and approximately ten E3s. Ubiquitin and SUMO are usually attached to substrates via isopeptide linkages between their C termini and the εNH2 group of Lys residues on target proteins. In some cases, the target protein has a single ubiquitin or SUMO attached, while in others, several can be individually attached to multiple Lys residues on the target. Furthermore, because ubiquitin and some SUMOs possess modifiable lysine residues, conjugation cycles can often be repeated to produce polymeric chains (Bergink and Jentsch, 2009Bergink S. Jentsch S. Principles of ubiquitin and SUMO modifications in DNA repair.Nature. 2009; 458: 461-467Crossref PubMed Scopus (261) Google Scholar). In the case of ubiquitin, seven Lys residues can be used (Lys6, Lys11, Lys27, Lys29, Lys33, Lys48, and Lys63) along with the amino group of the N-terminal Met. SUMO2/3 but not SUMO1 bear internal sumoylatable Lys residues that can be used to form chains, while SUMO1 can be conjugated as a chain-terminator. Consistent with different ubiquitin and SUMO chains having different structures and physical properties, they have distinct functions. For example, while Lys48-, Lys29-, and Lys11-linked ubiquitin chains promote target-protein degradation by the proteasome, Lys63 chains generally regulate protein-protein interactions. There is also evidence for ubiquitin chains with mixtures of linkages (Komander and Rape, 2012Komander D. Rape M. The ubiquitin code.Annu. Rev. Biochem. 2012; 81: 203-229Crossref PubMed Scopus (238) Google Scholar), as well as chains containing both ubiquitin and SUMO (Praefcke et al., 2012Praefcke G.J. Hofmann K. Dohmen R.J. SUMO playing tag with ubiquitin.Trends Biochem. Sci. 2012; 37: 23-31Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Like other posttranslational modifications, sumoylation and ubiquitylation are reversible. While SUMO-protein isopeptide bonds are cleaved by a small family of peptidases (SENP1–SENP3 and SENP5–SENP7), there are ∼100 deubiquitylating enzymes (also known as deubiquitylases or DUBs). DUBs are grouped into five families: ubiquitin C-terminal hydrolases (UCHs), ubiquitin-specific proteases (USPs) and ovarian tumor proteases (OTUs), the Josephins, and the Jab1/MPN/Mov34 family (JAMM/MPN+). The first four families are Cys proteases, whereas the latter comprises Zn2+-dependent metalloproteases (Nijman et al., 2005Nijman S.M. Luna-Vargas M.P. Velds A. Brummelkamp T.R. Dirac A.M. Sixma T.K. Bernards R. A genomic and functional inventory of deubiquitinating enzymes.Cell. 2005; 123: 773-786Abstract Full Text Full Text PDF PubMed Scopus (710) Google Scholar). In addition to opposing ubiquitin/SUMO ligase activities, certain DUBs and SENPs process ubiquitin and SUMO precursors, and some DUBs are intrinsic components of the proteasome. Genome integrity is continuously undermined by exogenous and endogenously generated DNA-damaging chemicals, ionizing radiation (IR) and ultraviolet (UV) radiation, and by errors in DNA replication. To mitigate this, cells possess highly effective mechanisms—collectively called the DNA damage response (DDR)—to detect, signal, and repair DNA lesions. These processes have profound impacts on normal cell and organism physiology, with their deregulation or loss causing genome instability syndromes that are associated with cancer, stem cell exhaustion, developmental defects, infertility, immune deficiency, neurodegenerative disease, and premature aging (Jackson and Bartek, 2009Jackson S.P. Bartek J. The DNA-damage response in human biology and disease.Nature. 2009; 461: 1071-1078Crossref PubMed Scopus (915) Google Scholar). Different DNA lesions are repaired by distinct systems. Thus, DNA double-strand breaks (DSBs) are repaired by nonhomologous end joining (NHEJ), alternative NHEJ, or homologous recombination (HR), UV-induced DNA lesions, and other bulky DNA adducts are repaired by nucleotide excision repair (NER), simpler base lesions are repaired by base-excision repair (BER) whose components and reactions overlap with those of single-strand break repair, and DNA base mismatches are corrected by mismatch repair (MMR), while the Fanconi anemia (FA) pathway repairs DNA crosslinks. Lesions are first recognized by proteins that trigger and coordinate the recruitment and activities of additional DNA repair components. DNA damage induction elicits cascades of posttranslational modifications, including phosphorylation, ubiquitylation, and sumoylation that orchestrate the aforementioned processes as well as additional aspects of the DDR, such as regulating deoxyribonucleotide supply and triggering of cell-cycle delays (cell-cycle checkpoints). These events are largely initiated by the apical DDR kinases ATM and ATR, whose importance in coordinating the DDR is illustrated by their mutation in Ataxia-telangiectasia (A-T) and Seckel syndrome, respectively (Durocher and Jackson, 2001Durocher D. Jackson S.P. DNA-PK, ATM and ATR as sensors of DNA damage: variations on a theme?.Curr. Opin. Cell Biol. 2001; 13: 225-231Crossref PubMed Scopus (327) Google Scholar; Kerzendorfer and O'Driscoll, 2009Kerzendorfer C. O'Driscoll M. Human DNA damage response and repair deficiency syndromes: linking genomic instability and cell cycle checkpoint proficiency.DNA Repair (Amst.). 2009; 8: 1139-1152Crossref PubMed Scopus (38) Google Scholar). In this review, we focus on the rapidly emerging functions of ubiquitin and SUMO in controlling various aspects of the DDR, with a particular emphasis on responses to DSBs, which are the most cytotoxic of all DNA lesions. The first association between DNA repair and ubiquitylation arose when yeast Rad6, which functions in postreplication repair (PRR), was shown to be a ubiquitin E2 (Jentsch et al., 1987Jentsch S. McGrath J.P. Varshavsky A. The yeast DNA repair gene RAD6 encodes a ubiquitin-conjugating enzyme.Nature. 1987; 329: 131-134Crossref PubMed Google Scholar). PRR is a DNA-damage-tolerance pathway that allows replication past bulky DNA lesions and is orchestrated by ubiquitylation and sumoylation of the DNA polymerase processivity factor PCNA (Figure 2). Two subpathways comprise eukaryotic PRR: translesion synthesis (TLS), and a template-switch mechanism associated with HR (Ulrich, 2011Ulrich H.D. Timing and spacing of ubiquitin-dependent DNA damage bypass.FEBS Lett. 2011; 585: 2861-2867Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). An early step in yeast PRR is PCNA monoubiquitylation by the RING-type E3, Rad18, in conjunction with the E2, Rad6 (Hoege et al., 2002Hoege C. Pfander B. Moldovan G.L. Pyrowolakis G. Jentsch S. RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO.Nature. 2002; 419: 135-141Crossref PubMed Scopus (1158) Google Scholar; Stelter and Ulrich, 2003Stelter P. Ulrich H.D. Control of spontaneous and damage-induced mutagenesis by SUMO and ubiquitin conjugation.Nature. 2003; 425: 188-191Crossref PubMed Scopus (488) Google Scholar). PCNA is primarily monoubiquitylated on Lys164, with this modification being recognized by specialized TLS polymerases such as Polη, Pol ι, Pol κ, and Polζ via their ubiquitin-binding domains (UBDs) of the UBM and UBZ families in conjunction with motifs such as PIP boxes that recognize other features of PCNA (Lehmann, 2011Lehmann A.R. Ubiquitin-family modifications in the replication of DNA damage.FEBS Lett. 2011; 585: 2772-2779Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). Unlike canonical high-fidelity polymerases, the catalytic sites of TLS polymerases can synthesize over and past DNA lesions but usually at the cost of fidelity, thus making them error prone. Yeast PCNA can also be polyubiquitylated on Lys164 by Rad5 (Hoege et al., 2002Hoege C. Pfander B. Moldovan G.L. Pyrowolakis G. Jentsch S. RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO.Nature. 2002; 419: 135-141Crossref PubMed Scopus (1158) Google Scholar), an E3 ligase that cooperates with a dimeric E2 Ubc13-Mms2. Rad5-Ubc13-Mms2 yields Lys63-linked ubiquitin chains on PCNA that promote the template-switching pathway that involves the newly synthesized sister chromatid and the HR machinery. While it is still unclear how PCNA ubiquitylation promotes template switching, the mammalian ZRANB3 translocase was recently identified as an effector of PCNA polyubiquitylation (Zeman and Cimprich, 2012Zeman M.K. Cimprich K.A. Finally, polyubiquitinated PCNA gets recognized.Mol. Cell. 2012; 47: 333-334Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar). While differences in PRR may exist between yeast and man, the pathway has been generally evolutionarily conserved, with mammals having counterparts of Rad6 (human HR6A/UBE2A and HR6B/UBE2B), Rad18 (RAD18), and Rad5 (SHPRH and HLTF). Knockout or depletion of RAD18 in a variety of species results in defective PRR, PCNA monoubiquitylation, and accumulation of TLS polymerases such as Polη at sites of replication fork blockage (Lee and Myung, 2008Lee K.Y. Myung K. PCNA modifications for regulation of post-replication repair pathways.Mol. Cells. 2008; 26: 5-11PubMed Google Scholar). Furthermore, small interfering RNA depletion studies suggest that human HLTF and SHPRH contribute to PCNA polyubiquitylation in response to fork-causing lesions (Motegi et al., 2008Motegi A. Liaw H.J. Lee K.Y. Roest H.P. Maas A. Wu X. Moinova H. Markowitz S.D. Ding H. Hoeijmakers J.H. Myung K. Polyubiquitination of proliferating cell nuclear antigen by HLTF and SHPRH prevents genomic instability from stalled replication forks.Proc. Natl. Acad. Sci. USA. 2008; 105: 12411-12416Crossref PubMed Scopus (77) Google Scholar) and in the suppression of mutagenesis (Lin et al., 2011Lin J.R. Zeman M.K. Chen J.Y. Yee M.C. Cimprich K.A. SHPRH and HLTF act in a damage-specific manner to coordinate different forms of postreplication repair and prevent mutagenesis.Mol. Cell. 2011; 42: 237-249Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), phenotypes consistent with functions for these E3 ligases in error-free PRR. In addition to being ubiquitylated, budding yeast PCNA is sumoylated on Lys164 (and to a lesser degree on Lys127) by the E3 Siz1 and the E2 Ubc9 (Pfander et al., 2005Pfander B. Moldovan G.L. Sacher M. Hoege C. Jentsch S. SUMO-modified PCNA recruits Srs2 to prevent recombination during S phase.Nature. 2005; 436: 428-433Crossref PubMed Scopus (0) Google Scholar; Stelter and Ulrich, 2003Stelter P. Ulrich H.D. Control of spontaneous and damage-induced mutagenesis by SUMO and ubiquitin conjugation.Nature. 2003; 425: 188-191Crossref PubMed Scopus (488) Google Scholar). PCNA sumoylation prevents unscheduled recombination during DNA replication by recruiting Srs2, a UvrD-type helicase that can strip the key HR protein Rad51 from chromatin (Krejci et al., 2003Krejci L. Van Komen S. Li Y. Villemain J. Reddy M.S. Klein H. Ellenberger T. Sung P. DNA helicase Srs2 disrupts the Rad51 presynaptic filament.Nature. 2003; 423: 305-309Crossref PubMed Scopus (347) Google Scholar; Pfander et al., 2005Pfander B. Moldovan G.L. Sacher M. Hoege C. Jentsch S. SUMO-modified PCNA recruits Srs2 to prevent recombination during S phase.Nature. 2005; 436: 428-433Crossref PubMed Scopus (0) Google Scholar; Veaute et al., 2003Veaute X. 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NER repairs bulky DNA base adducts and ultraviolet light-induced lesions. Inherited defects in NER factors yield pathologies that include xeroderma pigmentosum (XP), Cockayne syndrome (CS), and trichothiodystrophy (TTD), which are characterized by sun hypersensitivity, skin cancer predisposition (in the case of XP), cognitive impairments, premature aging, or developmental defects (Hoeijmakers, 2009Hoeijmakers J.H. DNA damage, aging, and cancer.N. Engl. J. Med. 2009; 361: 1475-1485Crossref PubMed Scopus (401) Google Scholar). NER comprises two main pathways: global genome repair (GG-NER) that operates on all nuclear DNA, and transcription-coupled repair (TC-NER) that specifically targets the template strand of transcribed genes. During human GG-NER, DNA lesions can be detected independently by two complexes, DDB1-DDB2/XPE and XPC-RAD23 (Scrima et al., 2011Scrima A. Fischer E.S. Lingaraju G.M. Böhm K. Cavadini S. Thomä N.H. Detecting UV-lesions in the genome: The modular CRL4 ubiquitin ligase does it best!.FEBS Lett. 2011; 585: 2818-2825Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar) (Figure 3). However, DDB1-DDB2 plays a unique role in NER owing to the observation that DDB1-DDB2 is required for the effective recruitment of XPC to chromatin (Fitch et al., 2003Fitch M.E. Nakajima S. Yasui A. Ford J.M. In vivo recruitment of XPC to UV-induced cyclobutane pyrimidine dimers by the DDB2 gene product.J. Biol. Chem. 2003; 278: 46906-46910Crossref PubMed Scopus (117) Google Scholar). Mechanistically, DDB2-DDB1 forms an E3 ligase in association with CUL4A/B that mediates monoubiquitylation of histones and polyubiquitylation of DDB2 and XPC (Scrima et al., 2011Scrima A. Fischer E.S. Lingaraju G.M. Böhm K. Cavadini S. Thomä N.H. Detecting UV-lesions in the genome: The modular CRL4 ubiquitin ligase does it best!.FEBS Lett. 2011; 585: 2818-2825Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). While autoubiquitylated DDB2 is targeted for degradation, XPC is not because it is protected from proteasome action by RAD23, a proteasome-interacting protein (El-Mahdy et al., 2006El-Mahdy M.A. Zhu Q. Wang Q.E. Wani G. Praetorius-Ibba M. Wani A.A. Cullin 4A-mediated proteolysis of DDB2 protein at DNA damage sites regulates in vivo lesion recognition by XPC.J. Biol. Chem. 2006; 281: 13404-13411Crossref PubMed Scopus (75) Google Scholar; Sugasawa, 2006Sugasawa K. UV-induced ubiquitylation of XPC complex, the UV-DDB-ubiquitin ligase complex, and DNA repair.J. Mol. Histol. 2006; 37: 189-202Crossref PubMed Scopus (35) Google Scholar; and references therein). When RNA polymerase II (RNAP II) stalls upon encountering a DNA lesion, two independent cascades can be triggered. The first event is TC-NER, and the second is the ubiquitylation, extraction, and degradation of RNAP II from chromatin (Figure 3). TC-NER is dependent on CSB (ERCC6), a SWI/SNF family protein that associates with RNAP II (Gaillard and Aguilera, 2013Gaillard H. Aguilera A. Transcription coupled repair at the interface between transcription elongation and mRNP biogenesis.Biochim. Biophys. Acta. 2013; 1829: 141-150Crossref PubMed Scopus (12) Google Scholar). In addition to possessing chromatin-remodeling activity, CSB recruits CSA (ERCC8) to sites of DNA damage, the latter forming an E3 with DDB1 and CUL4. The action of CSB and CSA may be to license the TC-NER process, which includes RNAP II backtracking and subsequent recruitment of the core NER machinery (Gaillard and Aguilera, 2013Gaillard H. Aguilera A. Transcription coupled repair at the interface between transcription elongation and mRNP biogenesis.Biochim. Biophys. Acta. 2013; 1829: 141-150Crossref PubMed Scopus (12) Google Scholar). The identity of the key ubiquitylation event that initiates TC-NER is still unknown, but RNAP II and CSB ubiquitylation are possibilities. In that regard, CSB possess a functionally important UBD, suggesting that CSB recognizes this key ubiquitylation event (Anindya et al., 2010Anindya R. Mari P.O. Kristensen U. Kool H. Giglia-Mari G. Mullenders L.H. Fousteri M. Vermeulen W. Egly J.M. Svejstrup J.Q. A ubiquitin-binding domain in Cockayne syndrome B required for transcription-coupled nucleotide excision repair.Mol. Cell. 2010; 38: 637-648Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Furthermore, the DUB USP7 is recruited to stalled polymerases by UVSSA, the product of a gene mutated in a CS-like UV-sensitivity syndrome (Cleaver, 2012Cleaver J.E. Photosensitivity syndrome brings to light a new transcription-coupled DNA repair cofactor.Nat. Genet. 2012; 44: 477-478Crossref PubMed Scopus (9) Google Scholar). UVSSA-USP7 interacts with RNAP II and delays the CSA-dependent degradation of CSB by the proteasome. The degradation of RNAP II by the proteasome can be seen as a last-resort measure and provides a unique case study for the role of ubiquitin chain editing. In yeast, these processes involve the Rsp5 E3 (NEDD4 in mammals), which catalyzes Lys63-linked ubiquitin chain formation on RNAP II (Anindya et al., 2007Anindya R. Aygün O. Svejstrup J.Q. Damage-induced ubiquitylation of human RNA polymerase II by the ubiquitin ligase Nedd4, but not Cockayne syndrome proteins or BRCA1.Mol. Cell. 2007; 28: 386-397Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar; Wilson et al., 2013Wilson M.D. Harreman M. Svejstrup J.Q. Ubiquitylation and degradation of elongating RNA polymerase II: The last resort.Biochim. Biophys. Acta. 2013; 1829: 151-157Crossref PubMed Scopus (19) Google Scholar). These chains are trimmed down by Ubp2, a DUB, resulting in monoubiquitylated RNAP II. Lys48-linked ubiquitin chains are then built from monoubiquitylated RNAP II by an Elongin/Cullin 3 complex, which can then promote RNAP II degradation after its extraction from chromatin with the Cdc48 segregase, the yeast VCP/p97 homolog (Harreman et al., 2009Harreman M. Taschner M. Sigurdsson S. Anindya R. Reid J. Somesh B. Kong S.E. Banks C.A. Conaway R.C. Conaway J.W. Svejstrup J.Q. Distinct ubiquitin ligases act sequentially for RNA polymerase II polyubiquitylation.Proc. Natl. Acad. Sci. USA. 2009; 106: 20705-20710Crossref PubMed Scopus (55) Google Scholar; Verma et al., 2011Verma R. Oania R. Fang R. Smith G.T. Deshaies R.J. Cdc48/p97 mediates UV-dependent turnover of RNA Pol II.Mol. Cell. 2011; 41: 82-92Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar; Wilson et al., 2013Wilson M.D. Harreman M. Svejstrup J.Q. Ubiquitylation and degradation of elongating RNA polymerase II: The last resort.Biochim. Biophys. Acta. 2013; 1829: 151-157Crossref PubMed Scopus (19) Google Scholar). An important paradigm for ubiquitin and SUMO acting in intracellular signaling is provided by the orchestrated recruitment of proteins such 53BP1 and the tumor suppressor BRCA1 onto chromatin surrounding DSB sites (Lukas et al., 2011Lukas J. Lukas C. Bartek J. More than just a focus: The chromatin response to DNA damage and its role in genome integrity maintenance.Nat. Cell Biol. 2011; 13: 1161-1169Crossref PubMed Scopus (173) Google Scholar) (Figure 4). These events are initiated by phosphorylation of the histone variant H2AX (yielding γH2AX), which is recognized by MDC1 (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 (489) Google Scholar). MDC1 is then phosphorylated by the DSB-responsive kinase ATM, with these phospho-sites being bound by the FHA domain of the RING-E3 ligase RNF8 (Huen et al., 2007Huen M.S. Grant R. Manke I. Minn K. Yu X. Yaffe M.B. Chen J. RNF8 transduces the DNA-damage signal via histone ubiquitylation and checkpoint protein assembly.Cell. 2007; 131: 901-914Abstract Full Text Full Text PDF PubMed Scopus (479) Google Scholar; Kolas et al., 2007Kolas N.K. Chapman J.R. Nakada S. Ylanko J. Chahwan R. Sweeney F.D. Panier S. Mendez M. Wildenhain J. Thomson T.M. et al.Orchestration of the DNA-damage response by the RNF8 ubiquitin ligase.Science. 2007; 318: 1637-1640Crossref PubMed Scopus (413) Google Scholar; Mailand et al., 2007Mailand N. Bekker-Jensen S. Faustrup H. Melander F. Bartek J. Lukas C. Lukas J. RNF8 ubiquitylates histones at DNA double-strand breaks and promotes assembly of repair proteins.Cell. 2007; 131: 887-900Abstract Full Text Full Text PDF PubMed Scopus (520) Google Scholar). RNF8 then mediates ubiquitylation of proteins at DSB sites in a manner promoted via interactions with the large HECT-type ligase HERC2 (Bekker-Jensen et al., 2010Bekker-Jensen S. Rendtlew Danielsen J. Fugger K. Gromova I. Nerstedt A. Lukas C. Bartek J. Lukas J. Mailand N. HERC2 coordinates ubiquitin-dependent assembly of DNA repair factors on damaged chromosomes.Nat. Cell Biol. 2010; 12 (sup pp. 1–12): 80-86Crossref PubMed Scopus (94) Google Scholar). The RING-E3 ligase RNF168 is then recruited by its UBDs, recognizing RNF8 ubiquitylation products and products of its own activity. The UBDs of RNF168 are not equivalent and are integrated in functional modules containing targeting motifs, the LRMs that are also present on RAD18 and RAP80 (Panier et al., 2012Panier S. Ichijima Y. Fradet-Turcotte A. Leung C.C. Kaustov L. Arrowsmith C.H. Durocher D. Tandem protein interaction modules organize the ubiquitin-dependent response to DNA double-strand breaks.Mol. Cell. 2012; 47: 383-395Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). The primary outcome of RNF8/RNF168-dependent ubiquitylation is recruitment and/or retention of DSB repair and signaling factors on chromatin surrounding the DNA lesion, which include 53BP1, RAD18, BRCA1, the RAP80 complex (also known as BRCA1-A), HERC2, BMI1, RIF1, RNF169, NPM1, FAAP20, and NIPBL (Lukas et al., 2011Lukas J. Lukas C. Bartek J. More than just a focus: The chromatin response to DNA damage and its role in genome integrity maintenance.Nat. Cell Biol. 2011; 13: 1161-1169Crossref PubMed Scopus (173) Google Scholar). At the functional level, RNF8/RNF168-dependent ubiquitylation promotes NHEJ during immunoglobulin class switching and dysfunctional telomere fusion (Kracker and Durandy, 2011Kracker S. Durandy A. Insights into the B cell specific process of immunoglobulin class switch recombination.Immunol. Lett. 2011; 138: 97-103Crossref PubMed Scopus (22) Google Scholar; Peuscher and Jacobs, 2011Peuscher M.H. Jacobs J.J. DNA-damage response and repair activities at uncapped telomeres depend on RNF8.Nat. Cell Biol. 2011; 13: 1139-1145Crossref PubMed Scopus (22) Google Scholar; Rai et al., 2011Rai R. Li J.M. Zheng H. Lok G.T. Deng Y. Huen M.S. Chen J. Jin J. Chang S. The E3 ubiquitin ligase Rnf8 stabilizes Tpp1 to promote telomere end protection.Nat. Struct. Mol. Biol. 2011; 18: 1400-1407Crossref PubMed Scopus (20) Google Scholar). In addition to NHEJ, the RNF8 pathway can also promote HR. These repair defects likely contribute to the clinical phenotypes observed in individuals with inactivating mutations in RNF168, which are afflicted with an immunodeficiency and cellular radiosensitivity syndrome, RIDDLE, that is related to A-T (Stewart et al., 2009Stewart G.S. Panier S. Townsend K. Al-Hakim A.K. Kolas N.K. Miller E.S. Nakada S. Ylanko J. Olivarius S. Mendez M. et al.The RIDDLE syndrome protein mediates a ubiquitin-dependent signaling cascade at sites of DNA damage.Cell. 2009; 136: 420-434Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar). Significantly, the RNF8 pathway is turned off during mitosis, perhaps because chromatin ubiquitylation is incompatible with mitotic progression (Giunta et al., 2010Giunta S. Belotserkovskaya R. Jackson S.P. DNA damage signaling in response to double-strand breaks during mitosis.J. Cell Biol. 2010; 190: 197-207Crossref PubMed Scopus (88) Google Scholar). In another striking example of regulation, it recently emerged that RNF168 is a limiting factor in the RNF8 pathway, with the E3 ubiquitin ligases TRIP12 and UBR5 collaborating to regulate RNF168 levels, thereby preventing excessive histone ubiquitylation at DSB sites (Gudjonsson et al., 2012Gudjonsson T. Altmeyer M. Savic V. Toledo L. Dinant C. Grøfte M. Bartkova J. Poulsen M. Oka Y. Bekker-Jensen S. et al.TRIP12 and UBR5 suppress spreading of chromatin ubiquitylation at damaged chromosomes.Cell. 2012; 150: 697-709Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). It will be interesting to determine how TRIP12 and UBR5 affect RNF168 levels and whether any physiological conditions affect this regulation. In this regard, TRIP12 contains a WWE domain, which in other proteins binds to poly(ADP) ribose, suggesting that RNF168 levels might be controlled by poly(ADP) ribosylation. Herpes simplex virus (HSV) infection also regulates the RNF8 pathway, with the HSV ICP0 protein—which is necessary for the transition between the viral latent and lytic phases—being an E3 that targets RNF8 and RNF168 for degradation (Chaurushiya et al., 2012Chaurushiya M.S. Lilley C.E. Aslanian A. Meisenhelder J. Scott D.C. Landry S. Ticau S. Boutell C. Yates 3rd, J.R. Schulman B.A. et al.Viral E3 ubiquitin ligase-mediated degradation of a cellular E3: viral mimicry of a cellular phosphorylation mark targets the RNF8 FHA domain.Mol. Cell. 2012; 46: 79-90Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar; Lilley et al., 2010Lilley C.E. Chaurushiya M.S. Boutell C. Landry S. Suh J. Panier S. Everett R.D. Stewart G.S. Durocher D. Weitzman M.D. A viral E3 ligase targets RNF8 and RNF168 to control histone ubiquitination and DNA damage responses.EMBO J. 2010; 29: 943-955Crossref PubMed Scopus (70) Google Scholar). Specifically, ICP0 targets RNF8 for degradation through a "reverse-degron" mechanism mediated by a phospho-dependent interaction between ICP0 and the RNF8 FHA domain (Chaurushiya et al., 2012Chaurushiya M.S. Lilley C.E. Aslanian A. Meisenhelder J. Scott D.C. Landry S. Ticau S. Boutell C. Yates 3rd, J.R. Schulman B.A. et al.Viral E3 ubiquitin ligase-mediated degradation of a cellular E3: viral mimicry of a cellular phosphorylation mark targets the RNF8 FHA domain.Mol. Cell. 2012; 46: 79-90Abstract Full Text Full Text PDF PubMed Scopus (20) Google Schol
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