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Review |
The ubiquitin landscape at DNA double-strand breaks
Correspondence to Roger A. Greenberg: rogergr{at}mail.med.upenn.edu
The intimate relationship between DNA double-strand break (DSB) repair and cancer susceptibility has sparked profound interest in how transactions on DNA and chromatin surrounding DNA damage influence genome integrity. Recent evidence implicates a substantial commitment of the cellular DNA damage response machinery to the synthesis, recognition, and hydrolysis of ubiquitin chains at DNA damage sites. In this review, we propose that, in order to accommodate parallel processes involved in DSB repair and checkpoint signaling, DSB-associated ubiquitin structures must be nonuniform, using different linkages for distinct functional outputs. We highlight recent advances in the study of nondegradative ubiquitin signaling at DSBs, and discuss how recognition of different ubiquitin structures may influence DNA damage responses.
Abbreviations used in this paper: ATM, ataxia telangiectasia mutated; ATR, ataxia telangiectasia and Rad3 related; CtIP, CtBP-interacting protein; DSB, double-strand break; DUB, deubiquitinating enzyme; IRIF, ionizing radiation–induced foci; MIU, motif interacting with ubiquitin; PIKK, phosphatidylinositol-3-kinase-related kinase; UIM, ubiquitin interaction motif.
© 2009 Messick and Greenberg
This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
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Introduction |
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Principles of ubiquitin
Numerous cellular processes are regulated by the posttranslational mark ubiquitin, including protein degradation, cell cycle regulation, DNA repair, transcription, and endocytosis. Specifically, the highly conserved 76–amino acid protein can alter the activity of its target in a variety of ways, from changing its localization or enzymatic activity to targeting it for degradation. Ubiquitylation, the process that involves the covalent attachment ubiquitin to the target protein, creates a covalent isopeptide linkage in a variety of different topologies to affect these diverse processes.
Ubiquitylation is a highly regulated process involving a specific cascade of activities performed by the E1, E2, and E3 series of enzymes (Hershko et al., 2000; Pickart, 2001). E1, or ubiquitin-activating enzyme, activates ubiquitin by forming a thiol ester link between the carboxy terminus of ubiquitin and the active site cysteine of E1 in an ATP-requiring step. The activated ubiquitin is then transferred to an E2 ubiquitin-conjugating enzyme, also through a thiol ester bond between ubiquitin and the active site cysteine of E2. E2, together with E3 or ubiquitin ligase, transfers the ubiquitin to its target, forming a covalent isopeptide linkage between the carboxyl terminus Gly-76 of ubiquitin to a primary amine (usually the 
-amino group of lysine) of the target protein.
Ubiquitylation is among the more unique forms of posttranslational modification in that a single ubiquitin monomer can be further ubiquitylated (polyubiquitylated) through one of seven lysines or through the amino terminus to create polyubiquitin chains (Fig. 1 A). Remarkably, different ubiquitin topologies or linkages between ubiquitin moieties can lead to vastly different biological outcomes. For example, the canonical lysine-48 (K48)-linked polyubiquitin targets the substrate protein for proteasomal degradation (Pickart and Cohen, 2004), whereas lysine-63 (K63)-linked polyubiquitin is often involved in localization or signaling events (Chen and Sun, 2009). Polyubiquitin can be linked through one residue to create a homogeneous chain, or through multiple residues, forming branched ubiquitin chains (Kim et al., 2007b).
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-helical structures that recognize the hydrophobic patch centered around Ile44 of ubiquitin (Harper and Schulman, 2006; Grabbe and Dikic, 2009). Though the affinities of individually isolated ubiquitin-binding domains are low to moderate, this affinity may increase in vivo due to multiple ubiquitin-binding members in a complex recognizing polyubiquitin chains in a synergistic fashion. The hydrolysis of the isopeptide bond connecting ubiquitin to a substrate protein is performed by deubiquitinating enzymes (DUBs). DUBs act in a variety of regulatory processes, from "rescuing" previously targeted proteins from degradation to attenuating signaling pathways (Reyes-Turcu et al., 2009). Given the tight regulatory control of ubiquitin and the possibility for many different linkages between ubiquitin moieties, it is perhaps not surprising that many DUBs exhibit specificity for one type of ubiquitin chain isoform. There are generally two classes of DUBs that are distinguished on the basis of catalytic mechanism (Reyes-Turcu et al., 2009). The first utilizes an active site cysteine nucleophile to hydrolyze the ubiquitin isopeptide bond, which is remarkably similar to the papain family of cysteine proteases. The second class of DUB enzymes is the Jab1/MPN/Mov34 (JAMM) family of zinc-binding metalloproteases. JAMM domains function by binding a highly electrophilic metal (usually zinc) that delivers a hydroxide ion for nucleophilic attack on the isopeptide bond, performing isopeptide bond hydrolysis in a similar mechanism to the Zn2+-containing protease thermolysin.
Principles of DSB recognition
DSBs are potentially lethal lesions that can arise from endogenous and exogenous sources. To protect the genome, cells use a vast network of proteins to monitor DNA integrity and mount a response to DSBs. This DNA damage response usually includes cell cycle arrest and activation of the DNA repair pathways. In extreme cases, when cells are unable to properly repair DSBs, apoptosis or senescence pathways may be triggered.
Recognition and signaling at DSBs proceeds rapidly, with a distinct temporal and spatial order of association and dissociation of numerous DNA repair factors with the site of the break (Lisby et al., 2003). DNA repair protein recruitment and retention is conveniently visualized by fluorescence imaging of repair foci (often called ionizing radiation–induced foci [IRIF]). Foci are easily observed because many DNA repair proteins are retained at DSBs in suprastoichiometric ratios. It has been estimated that individual breaks contains at least 1,000 molecules of each DNA repair protein (Lisby et al., 2004), revealing that many repair proteins are concentrated at repair sites by a factor of 103. The reasons for this stoichiometry are unknown, although it is speculated that high local concentrations of repair factors are necessary to amplify signals for repair and checkpoint responses.
Robust foci formation occurs within minutes of DSB induction, which dictates that molecular recognition must rapidly develop at the site of DNA damage for repair protein recruitment. At the pinnacle of this chain of events is phosphorylation of the histone H2A variant H2AX. DNA damage activates cellular ataxia telangiectasia mutated (ATM) kinase and related PIKKs to phosphorylate H2AX at its C terminus on Ser139. Phospho H2AX (
-H2AX) formation occurs within minutes after damage, and extends for up to a megabase from the site of the break in mammalian cells, providing a platform for subsequent DNA repair protein recruitment and amplification at DSBs. Indeed, H2AX-null cells demonstrate strongly reduced repair protein focus formation, which is consistent with H2AX being a master regulator of the recruitment of DNA repair proteins to chromatin at DSBs (Celeste et al., 2002). These findings provided a basis to understand repair factor–DSB stoichiometry. Thousands of repair protein molecules associate along chromatin that contains -H2AX in cis to the DSB, which explains the suprastoichiometric relationship of repair proteins to DSBs.
Although -H2AX is a master regulator of visible foci formation, -H2AX deficiency does not eliminate all DNA repair protein recruitment to DSBs, which indicates that foci are just part of the DSB repair puzzle. During DSB repair, there is a clearing of -H2AX and nucleosomes directly adjacent to the DSB (Shroff et al., 2004; Berkovich et al., 2007). These dechromatinized regions are thought to be the site of the majority of DSB repair chemistry. For example, proteins that perform nonhomologous end joining do not form foci, presumably because they are present only at the DSB termini and not along chromatin that contains -H2AX. Conversely, proteins dedicated to homologous recombination, such as the breast cancer early onset gene product BRCA1, are present at both non-nucleosomal regions and at chromatin that contains -H2AX (Celeste et al., 2003; Bekker-Jensen et al., 2006). Thus, DSB recognition at different locales on both DNA and chromatin flanking the break is essential for repair and the maintenance of genome integrity.
Connections between PIKK activity and ubiquitylation
Ubiquitylation directs repair proteins to DSBs.
Several prominent DNA repair pathways use a paired process in which PIKK-mediated phosphorylation is coupled to substrate ubiquitylation (Table I). In each instance, ubiquitylation of a protein associated with DNA repair is essential for its recruitment to DSBs. Notably, mutation in each of these DNA damage–associated ubiquitylation pathways is responsible for a human cancer susceptibility syndrome (Scully and Livingston, 2000; Chenevix-Trench et al., 2002; Wang, 2007).
Fanconi anemia is a recessive monogenic disease in which patients display developmental abnormalities, bone marrow failure, and cancer predisposition phenotypes. Fanconi syndrome is comprised of 13 different genetic complementation groups, and at least three pathways (Wang, 2007). All Fanconi mutant cell lines display the common characteristic of sensitivity to DNA cross-linking agents. The classical Fanconi pathway culminates in monoubiquitylation of the FancD2 and FancI proteins at a single lysine residue on each protein (Garcia-Higuera et al., 2001; Sims et al., 2007; Smogorzewska et al., 2007; Wang, 2007). FancD2 ubiquitylation occurs in S phase in response to phosphorylation by the Rad3-related PIKK, ataxia telangiectasia and Rad3 related (ATR; Andreassen et al., 2004). Monoubiquitylation is critical for FancD2 and FancI localization to DSBs and chromatin association (Meetei et al., 2004; Wang et al., 2004; Sims et al., 2007; Smogorzewska et al., 2007). Though it is not clear how ubiquitylation controls Fanconi pathway activity, a carboxy-terminal FancD2-ubiquitin fusion protein strongly associated with DSBs and chromatin, whereas a fusion containing the Ile44Ala mutation in ubiquitin did not. Because most ubiquitin-binding domains require direct binding to Ile44 on ubiquitin, this result raises the possibility that a chromatin-bound ubiquitin receptor mediates ubiquitylated FancD2 and FancI localization (Matsushita et al., 2005).
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The identification of BRCA1-BARD1 E3 substrates will be critical to understand how BRCA1 ligase activity contributes to the DNA damage response. In this regard, BRCA1 mediated, DNA damage–inducible ubiquitylation of the BRCA1 carboxy-terminal interacting partner, CtBP-interacting protein (CtIP), was reported in human cells (Yu et al., 2006). Interestingly, BRCA1 RING mutations that disrupt E3 ligase activity or CtIP deficiency failed to support CHK1 phosphorylation via ATR, resulting in a defective ionizing radiation–induced G2 checkpoint. BRCA1 E3 activity and interaction with CtIP was necessary for CtIP ubiquitylation and IRIF formation. As with the case of FancD2 and FancI ubiquitylation, mechanisms responsible for CtIP-Ub foci formation are not presently understood.
Evidence for ubiquitin recognition at DSBs.
The concept of retention of DNA repair proteins at DSBs by ubiquitin receptors was finally validated in studies that revealed a molecular basis for BRCA1 DSB recruitment. BRCA1 is targeted to DSBs via an interaction with a five-component complex containing RAP80, a DNA repair protein that contains tandem ubiquitin interaction motifs (UIMs; Kim et al., 2007a; Sobhian et al., 2007; Wang et al., 2007; Yan et al., 2007). These UIM domains preferentially recognize K63-linked ubiquitin over K48-linked structures, which suggests that BRCA1 would be targeted to nondegradative ubiquitin signals (Sobhian et al., 2007). Indeed, K63-linked structures accumulate in DSB foci, whereas K48-linked chains do not (Sobhian et al., 2007; Doil et al., 2009; Stewart et al., 2009). RAP80 DSB localization requires -H2AX and MDC1, both of which are necessary for polyubiquitylation at DSBs. Interestingly, an in-frame deletion in RAP80 UIM1 is associated with breast cancer in northern Finnish populations (Nikkilä et al., 2009). This RAP80 variant, RAP80
E81, demonstrates reduced ubiquitin binding and DSB localization while maintaining all other protein interactions. Because of these properties, RAP80E81 functions as a dominant-negative allele by titrating BRCA1 away from DSBs.
RAP80 exists as a stable complex together with four other core components: BRCC36, BRCC45, Abraxas, and MERIT40/NBA1 (Feng et al., 2009; Shao et al., 2009b; Wang et al., 2009). Bioinformatic analysis suggests similarity of the RAP80 core complex to the lid domain within the 19S subunit of the 26S proteasome (Wang et al., 2009). BRCC36 is a member of the JAMM family of metalloprotease DUB enzymes, and mutation of the zinc-binding residues results in increased sensitivity to DSBs, an impaired G2 checkpoint, and elevated levels of conjugated ubiquitin at DSBs (Shao et al., 2009a,b). This JAMM domain DUB specifically deubiquitylates K63-Ub, which matches the ubiquitin-binding preference of the RAP80 UIM domains (Cooper et al., 2009; Shao et al., 2009a). Generally, knockdown of any of the members of the complex results in failure of the other constituents to form DSB-associated foci, and increased sensitivity to ionizing radiation. Although structural evidence is needed to determine the actual degree of similarity to the proteasome, it is intriguing that multiple ubiquitin-binding domains would be present within the RAP80 complex (Wang et al., 2009). Perhaps these domains regulate BRCC36 catalytic activity or, alternatively, modulate the avidity of the complex for ubiquitin chains at DSBs. In vitro reconstitution of this complex with recombinant proteins will be necessary to definitively address these possibilities.
PIKK activity has been linked to histone ubiquitylation, the putative DSB-associated ligand for the BRCA1–RAP80 complex (Huen et al., 2007; Kolas et al., 2007; Mailand et al., 2007; Zhao et al., 2007). As mentioned earlier, -H2AX serves as a signal to initiate recruitment of additional factors to DNA repair foci. Chief among them is the mediator of DNA damage checkpoint 1 (MDC1). ATM phosphorylates MDC1 at an SQ/TQ-rich region, which is subsequently recognized by the E3 ubiquitin ligase RNF8, beginning the transition from primarily a phosphorylation cascade at DSBs to a series of ubiquitin-dependent signaling events. RNF8 works in conjunction with Ubc13 to ubiquitylate histones H2A and -H2AX. The RIDDLE syndrome E3 ligase RNF168 recognizes ubiquitin chains at DSBs synthesized by RNF8 through its two motif interacting with ubiquitin (MIU) domains which represent an inverted UIM (Doil et al., 2009; Stewart et al., 2009). RNF168, like RNF8, associates with Ubc13 to direct ubiquitylated H2A and K63-Ub synthesis at DSBs. RNF8 or RNF168 deficiency abrogates BRCA1–RAP80 foci formation, providing additional supportive evidence that ubiquitin is a DSB-targeting mechanism for the BRCA1–RAP80 complex.
RAP80 is not the only DNA repair protein that requires the combined efforts of RNF8 and RNF168 together with Ubc13 for DSB localization (Huang et al., 2009). The chromatin-bound DNA repair protein, p53-binding protein 1 (53BP1), also depends on RNF8 and RNF168 DSB-associated ubiquitylation, yet no evidence exists that 53BP1 directly binds ubiquitin. Instead, it appears to recognize methylated histones at DSBs (Huyen et al., 2004; Sanders et al., 2004; Botuyan et al., 2006). These results suggest that DSB-associated ubiquitin influences chromatin structure in a manner necessary to unveil modified histone epitopes. The current data are therefore consistent with ubiquitylation of DSB-associated proteins providing direct and indirect routes to damage site recognition for DNA repair proteins.
Structural basis for RAP80 K63-Ub specificity: a paradigm for DSB-associated ubiquitin recognition
The structural basis accounting for the K63-Ub specificity of the RAP80 tandem UIM domains was delineated in two recent papers (Sato et al., 2009; Sims and Cohen, 2009). Sims and Cohen (2009) measured the binding affinity for ubiquitin of the UIM1 and UIM2 to be quite modest (230 µM and 470 µM, respectively). The binding affinity increased >10-fold (17–22 µM) when the tandem UIM domains were measured for the ability to bind K63-linked diubiquitin. Mutational analysis showed that the linker region between the two UIMs was important for binding K63-linked polyubiquitin. Sato et al., (2009) recently described the mouse RAP80 tandem UIM domain structure bound to K63-linked diubiquitin (Fig. 2 A). The structure reveals that these domains consists of a single 60-Å long -helix (Sato et al., 2009). Interestingly, the inter-UIM region adopts an -helical secondary structure, as predicted by Sims and Cohen (2009). Each UIM domain binds to the hydrophobic patch surrounding Ile44 of the respective ubiquitin. The torsional freedom of K63-linked diubiquitin about the carboxy-terminal axis of the distal ubiquitin is evident when one compares the RAP80 bound and unbound structures. In the unbound structures (Datta et al., 2009; Komander et al., 2009), the Ile44 hydrophobic patches are rotated 
100° from each other. Upon binding to RAP80, the hydrophobic patches of the two ubiquitins align along one face rotated just 10° from one another (Fig. 2 B; Sato et al., 2009). Combining the structural information with the mutational analysis reveals the inter-UIM region as more than just a linker between the two UIM domains. It serves as a molecular ruler measuring the distance between K63-linked ubiquitin and orients the hydrophobic regions to lie on one side. These findings led to a model of linkage-specific avidity, in which linker region orientation of the UIM domains can dictate specificity for different ubiquitin chain topologies (Sims and Cohen, 2009).
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Ubiquitin dynamics and DNA repair mechanism
There is considerable evidence that ubiquitylation influences DNA repair mechanism. DSBs are primarily repaired by either homologous or nonhomologous methods. Each of these employs distinct DNA processing events. A pivotal step distinguishing the two repair mechanisms is the 5'-to-3' end resection of DSB ends. Zhao et al. (2007) reported an interesting observation that Ubc13 is required for homologous recombination and that Ubc13-deficient cells lack wild-type levels of resection at a break, as detected by single-stranded binding replication protein A recruitment to laser-induced DSBs (Zhao et al., 2007). Ubc13 deficiency eliminated detectable ubiquitin DSB focus formation and conferred exquisite sensitivity to agents that induce DNA damage that requires homologous recombination for repair. These data implicate DSB-associated ubiquitin in the initial stages of repair.
Why Ubc13 is necessary for DSB end resection is not known; however, BRCA1 DSB association and E3 ligase activity were dramatically reduced in Ubc13-null cells (Zhao et al., 2007). This data raises the possibility of a connection between Ubc13 and BRCA1-dependent ubiquitylation and end resection. Central to the process of DSB end resection is the interaction of CtIP with BRCA1 and Mre11. The BRCA1–CtIP complex interacts with Mre11 in a DNA damage–inducible manner (Greenberg et al., 2006; Chen et al., 2008), and this supercomplex has been implicated in nucleolytic degradation of double-stranded termini (Fig. 3; Sartori et al., 2007; Yun and Hiom, 2009). As previously discussed, CtIP is polyubiquitylated after DNA damage in a manner dependent on its interaction with BRCA1 (Yu et al., 2006). CtIP ubiquitylation is correlated with its ability to form IRIF and to activate CHK1. Because CHK1 activation requires ATR activation on single-stranded DNA (Zou and Elledge, 2003), it is tempting to speculate that BRCA1 teams up with Ubc13 to ubiquitylate CtIP, thus activating CtIP–Mre11 complex nuclease activity for end resection. The creation of a clean, genetic system to investigate BRCA1 E3 activity has severely injured this hypothesis. BRCA1 RING domain I26A mutant knock-in embryonic stem cells have recently been made (Reid et al., 2008). This allele abrogates BRCA1 RING domain interaction with E2 enzymes (Brzovic et al., 2003; Christensen et al., 2007), strongly reducing BRCA1 E3 activity in vitro and autoubiquitylation in vivo (Reid et al., 2008). Surprisingly, BRCA1 I26A knock-in ES cells performed homologous recombination at similar levels to cells expressing wild-type BRCA1 (Reid et al., 2008). These findings are inconsistent with BRCA1 E3 activity being involved in end resection and instead are more supportive of Ubc13 working in conjunction with other E3 ligases (e.g., RNF8 and RNF168) to concentrate BRCA1–CtIP and other resection-promoting factors at DSBs. Perhaps CtIP ubiquitylation by BRCA1 is not essential for end resection and instead influences CHK1 phosphorylation by other means. An alternative explanation is that CtIP does not require BRCA1 in mouse ES cells for ubiquitylation and that a different E3 suffices in this cell type.
Concluding remarks
Nondegradative forms of ubiquitin have, quite literally, left their mark on the DNA damage response. A variety of repair protein substrates, E3 ligases, and DUBs, each with their own specificity for synthesizing, recognizing, or hydrolyzing ubiquitin chains, appear to make important contributions to DNA repair. These initial studies have created new opportunities to understand the DNA damage response, and perhaps additional pharmacologic opportunities for treating human disease.
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Acknowledgments |
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Submitted: 17 August 2009
Accepted: 9 October 2009
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References |
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