Ubiquitin and SUMO in DNA repair at a glance

The task of preserving the stability of the genetic information of a cell is characterised by several important requirements (Hoeijmakers, 2009). First, genome maintenance is a largely cell-autonomous function, as each cell within an organism is obliged to prevent or repair injury of its own genome. Second, insults that endanger genome stability require a rapid response because damage to the genetic material quickly affects essential cellular functions by interfering with gene expression. Third, any response needs to be reversibly tuned to the physiological state of the cell, in particular its stage within the cell cycle. Last, but not least, mechanisms for the protection of genome integrity have to react flexibly to a large variety of damaging agents to which a cell may be exposed.Considering these characteristics of DNA repair, it is not surprising that post-translational modifications with members of the ubiquitin family, such as ubiquitin itself and small ubiquitin-related modifier (SUMO), have been identified as key contributors to genome maintenance. The notion that ubiquitin and SUMO can rapidly and reversibly change the properties, stability or localisation of their target proteins without the need for de novo protein synthesis makes them ideal regulators for fine-tuning DNA repair and damage response pathways. Their general action as modulators of protein function goes far beyond their contribution to regulated proteolysis, for which ubiquitin first became famous (Glickman and Ciechanover, 2002).In this Cell Science at a Glance article and the accompanying poster, I will summarise the contributions of ubiquitin and SUMO to the major pathways of genome maintenance. Several excellent reviews on this topic have been published over the past years, which give insight into the relevant mechanisms involved (Al-Hakim et al., 2010; Bergink and Jentsch, 2009; Huang and D'Andrea, 2006; Ulrich and Walden, 2010). Here, I will place more emphasis on the crosstalk between individual repair pathways and the specific contributions of selected proteins involved in ubiquitin and SUMO conjugation to the processes involved. For information about the general principles of protein ubiquitylation and SUMOylation, the reader is referred to pertinent reviews in this field (Glickman and Ciechanover, 2002; Kerscher et al., 2006).DNA double-strand breaks (DSBs) represent perhaps the most dangerous type of DNA lesion because they have dramatic effects on all DNA transactions, including proper segregation of chromosomes during cell division (Hoeijmakers, 2009). In order to initiate repair, a series of phosphorylation events, namely the phosphorylation of the histone variant H2AX and mediator of DNA-damage checkpoint 1 (MDC1) by the checkpoint kinase ataxia telangiectasia mutated (ATM), leads to the sequential recruitment of several E3 ubiquitin ligases in higher eukaryotes, as detailed below (Bekker-Jensen and Mailand, 2011; Panier and Durocher, 2009; Tang and Greenberg, 2010). These enzymes promote extensive ubiquitylation of histone H2A and other, yet unknown, chromatin-associated proteins in a cascade that eventually results in the localisation of another RING-finger ubiquitin ligase, BRCA1 (for breast cancer 1, early onset), as well as the checkpoint protein TP53BP1 (for tumor protein p53 binding protein 1), to the break. Whereas BRCA1 is essential for initiating DSB repair by homologous recombination, TP53BP1 has been associated with repair by non-homologous end-joining (Hiom, 2010).The mechanistic details of TP53BP1 recruitment to DSBs are poorly understood. The protein binds to methylated histones, and it is unclear how the damage-induced ubiquitylation cascade elicits this signal for chromatin association of TP53BP1. By contrast, the signalling pathway leading to BRCA1 recruitment has now been roughly elucidated. Although there are indications that the pathway is not entirely linear, the prevailing concept is based on the recognition of post-translational modifications, such as phosphate or ubiquitin moieties, by dedicated domains in the respective effector proteins, which themselves trigger the recruitment of additional enzymes for modification of other chromatin components. Specifically, phosphorylated H2AX (γ-H2AX) is recognised by tandem BRCA1 C-terminal (BRCT) motifs on MDC1. Following phosphorylation by ATM, MDC1 is then able to interact with the forkhead-associated (FHA) domains of the RING-finger E3 RNF8 (Huen et al., 2007; Kolas et al., 2007; Mailand et al., 2007). The ubiquitin moieties conjugated by RNF8 and its cognate ubiquitin-conjugating enzyme (E2) UBC13 (officially known as ubiquitin-conjugating enzyme E2N, UBE2N) to histones H2A and H2AX, and possibly other chromatin-bound proteins, are thought to be recognised by a second RING-finger E3, RNF168, through its ‘motifs interacting with ubiquitin’ (MIU) domains (Doil et al., 2009; Stewart et al., 2009). RNF168, together with UBC13, catalyses the formation of polymeric chains, linked through lysine (K) 63 of ubiquitin, that in turn recruit the adapter protein RAP80 (receptor associated protein 80, also known as UIMC1) through its ubiquitin-interacting motifs (UIM). The dimeric BRCA1–BARD1 complex is eventually recruited through the interaction of the BRCA1 tandem BRCT motifs with the phosphorylated abraxas protein (also known as FAM175A), which is itself part of a multisubunit scaffold complex (Kim et al., 2007; Liu et al., 2007; Sobhian et al., 2007; Wang et al., 2007). Although it is clear that BRCA1 is an important mediator of DNA DSB repair through homologous recombination, its relevant substrate proteins and cognate E2 enzyme(s), the structure of the resulting ubiquitin conjugates and the physiological consequences of these modifications are not yet fully understood (Hiom, 2010).In addition to BRCA1, several other ubiquitin E3s participate in the cascade, although their roles are less well defined. On one hand, RAD18, a RING-finger E3 best known for its function in DNA damage bypass (see below), acts downstream of RNF8, presumably by being recruited to chromatin by means of a ubiquitin-binding zinc finger (UBZ) domain (Huang et al., 2009). Intriguingly, the binding of RAD18, but not its E3 ligase activity, is required for proper homologous recombination. On the other hand, HERC2, a large HECT domain family E3 protein, associates with the FHA domain of RNF8 and seems to promote interaction with UBC13 in an as yet unknown way (Bekker-Jensen et al., 2010). Finally, a number of polycomb group proteins, which mediate transcriptional repression through modulation of chromatin structure, have recently been shown to contribute to the recruitment of both BRCA1 and TP53BP1 through H2A and H2AX ubiquitylation (Gieni et al., 2011). These include the RING-finger proteins BMI1 and RNF2 (also called RING1B or RING2), which form a heterodimeric ubiquitin E3. Although the mechanism by which the complex is initially recruited is a matter of debate – it might involve the MRN (MRE11–RAD50–NBS1) damage recognition complex and/or poly-(ADP)-ribosylation (Chou et al., 2010; Ismail et al., 2010) – sustained localisation at the site of DSBs seems to require signalling by the ATM or ataxia-telangiectasia-related (ATR) kinases, H2AX phosphorylation and its ubiquitylation by RNF8 (Ginjala et al., 2011).Negative regulation of ubiquitylation in the context of DSB repair is exerted by a number of deubiquitylating enzymes (DUBs) (Al-Hakim et al., 2010), namely BRCC36 (for BRCA1/BRCA2-containing complex, subunit 3), ubiquitin specific peptidases (USP) 3 and 16 as well as OTUB1 (for OTU domain, ubiquitin aldehyde binding 1). BRCC36 is part of the RAP80 complex and exhibits a preference for K63-polyubiquitin chains (Sobhian et al., 2007). USP3 and USP16 have both been shown to act on histone H2A and seem to downregulate the pathway at the stage of RNF8 (Cai et al., 1999; Doil et al., 2009; Joo et al., 2007; Nicassio et al., 2007), whereas OTUB1 acts further downstream and might inhibit the action of UBC13 in a non-catalytic manner (Nakada et al., 2010). The negative influence of DUBs on the formation of damage-induced BRCA1 and TP53BP1 foci indicates that cells maintain a fine balance between ubiquitylation and deubiquitylation to regulate DSB repair.In addition to ubiquitylation, the post-translational modifications at DSBs also involve SUMOylation, which is mediated by the SUMO E3 PIAS1 and PIAS4 proteins (for protein inhibitor of activated STAT), which both associate with the single SUMO E2 UBC9 (also known as UBE2I) (Galanty et al., 2009; Morris et al., 2009). One of the substrates appears to be BRCA1 itself, whose catalytic activity is boosted by SUMO modification. Hence, SUMOylation of the ubiquitin E3 BRCA1 represents an interesting example of crosstalk between the two modifiers. In addition, the polycomb protein Pc2 (officially known as CBX4), which acts as a SUMO ligase on a number of substrates, including itself (Wotton and Merrill, 2007), is also recruited to chromatin in a damage-dependent manner (Chou et al., 2010). There are clearly additional physiologically relevant targets, whose SUMOylation is important during DSB repair, but these remain to be identified.Apart from the direct involvement in the recruitment of repair factors, histone ubiquitylation is known to affect chromatin on a structural level, which is particularly important for the activation of transcription. In higher eukaryotes, this involves the relaxation of chromatin through monoubiquitylation of histone H2B by the heterodimeric RING finger E3 complex RNF20–RNF40 (Fierz et al., 2011; Weake and Workman, 2008). Very recently, it has been discovered that RNF20–RNF40-dependent H2B ubiquitylation is also required for efficient DSB repair, presumably by the same mechanism of chromatin decompaction (Moyal et al., 2011). As a consequence, inhibition of this modification causes defects in the recruitment of repair factors that are involved in both homologous recombination and non-homologous end-joining processes. Interestingly, this H2B-dependent contribution appears to be independent of the signalling pathway that involves RNF8-mediated H2A ubiquitylation.As discussed above, the ubiquitylation and SUMOylation cascades elicited at DSBs culminate in the recruitment of factors that initiate the two major pathways for the repair of such lesions (i.e. homologous recombination through recruitment of BRCA1 and non-homologous end-joining through TP53BP1). Whereas this particular signalling pathway is restricted to higher eukaryotes, SUMO also modifies several core recombination factors in both higher and lower eukaryotes. Among its prominent targets is RAD52, which promotes formation of the recombinogenic RAD51 filament. In budding yeast, SUMOylation by the E3 Siz2 stabilises Rad52 (Sacher et al., 2006) while at the same time also reducing the DNA-binding and single-strand annealing activities of this protein (Altmannova et al., 2010). SUMOylation of replication protein A (RPA), a single-stranded binding complex that is essential for recombination, replication and repair, has been detected in yeast and mammalian cells (Burgess et al., 2007; Dou et al., 2010). In mammals, this modification facilitates the recruitment of RAD51 and has been shown to be counteracted by the SUMO-specific isopeptidase SENP6 (Dou et al., 2010).As SUMOylation is often less dependent on specific ligases than ubiquitylation, the cognate E3s have not been well defined for all SUMO targets. On the basis of the phenotypes of the respective deletion mutants, it is clear that all three major SUMO E3s in budding yeast, Siz1, Siz2 and Mms21, participate in the modification of relevant substrates. Whereas the two yeast members of the PIAS family, Siz1 and Siz2, associate with DNA through SAP (for SAF-A/B, Acinus and PIAS) domains (Okubo et al., 2004), Mms21 is recruited to DNA through its association with the Smc5–Smc6 complex, a cohesin-like assembly with an important function in homologous recombination and replication fork restart in both yeast and humans (Potts and Yu, 2005; Zhao and Blobel, 2005). Although the importance of these SUMO ligases to genome maintenance by means of homologous recombination is undeniable, many of the relevant target proteins, as well as the mechanisms by which they affect the function of these targets, have yet to be elucidated.In addition to protein modification by SUMO, the regulation of DNA repair by homologous recombination also involves ubiquitin. In fission yeast, this modifier acts in its ‘classical’ way by inducing the degradation of the recombination factor Rad54 during the G1 phase of the cell cycle, which is consistent with a downregulation of recombination activity at this stage (Trickey et al., 2008). In this case, ubiquitin is attached by the anaphase promoting complex/cyclosome (APC/C), an E3 protein involved in cell cycle regulation that acts together with its G1-specific regulator Fzr (fizzy related, also referred to as Cdh1 and Hct1).An interesting crosstalk between ubiquitin and SUMO was uncovered by the identification of a set of ubiquitin ligases that recognise SUMOylated proteins as their substrates by means of SUMO-interaction motifs (SIMs) (Prudden et al., 2007; Sun et al., 2007; Uzunova et al., 2007; Xie et al., 2007). In yeast, one of these SUMO-targeted ubiquitin ligases, the heterodimeric Slx5–Slx8 complex in S. cerevisiae (Rfp1/2–Slx8 in S. pombe), acts as a regulator of homologous recombination, although its mechanism of action and its relevant substrate proteins remain to be identified. Bulk removal of high-molecular-mass SUMO targets might contribute to this DNA repair process (Uzunova et al., 2007).Homologous recombination plays a major role not only in DNA DSB repair but also in promoting the restart of stalled or collapsed replication forks. In this aspect, the SUMO system again appears to be particularly important. Although it is very likely that not all SUMOylated proteins that are relevant to this process have been identified, appropriate targets include members of the family of RECQ helicases: BLM (Bloom syndrome, RecQ helicase-like) and WRN (Werner syndrome, RecQ helicase-like) in higher eukaryotes, and Sgs1 in budding yeast (Branzei et al., 2006; Eladad et al., 2005; Kawabe et al., 2000). How SUMO affects the activities of these proteins and the subsequent restart of replication forks, however, has not been fully elucidated.Homologous recombination is just one way of dealing with DNA damage during replication. It applies to a variety of situations, including those where replication fork progression is impeded by strand breaks or interstrand crosslinks. Small lesions, such as adducts in the template DNA, which cannot be processed by the replicative polymerases, represent more subtle disturbances to fork progression and can be resolved by different processes (Friedberg, 2005). One solution to this problem, which results in error-free bypass, is template switching through a recombination event. Alternatively, specialised, error-prone DNA polymerases that can accommodate abnormal structures in their active sites can be employed in a process referred to as translesion synthesis (TLS) (Lehmann et al., 2007). Both strategies contribute to cellular damage resistance, but they also require strict regulation, as they might themselves induce genomic instability by means of larger genome rearrangments or point mutations, respectively.Control over both bypass pathways is mediated by ubiquitylation of the sliding clamp protein proliferating cell 2009). at by a complex the E3 and the E2 the of a series of polymerases for and facilitates et al., 2005; and of additional ubiquitin moieties by the RING-finger E3 and the heterodimeric E2 results in a which in yeast is a for the error-free pathway et al., In addition, the enzymes involved in have been in of et al., 2006; et al., 2008). Intriguingly, higher two of factor and histone RING which act on different of DNA damage et al., 2011). How template switching is not at all understood. There is about the downstream by the modification involve the set of homologous recombination factors or a fork might be to the stalled with the Interestingly, however, both and template switching can in manner that is from the replication within which the of fork as a means to initiate error-free damage bypass et al., 2010; and Jentsch, yeast is also by SUMO et al., In to ubiquitylation, this is not but during and also the of the clamp for its interaction Specifically, SUMOylation the binding of an which the formation of and the bypass pathway to act of DNA damage et al., 2005; et al., 2005). of SUMOylated by is mediated by a in the of the same SUMOylation the binding of which is involved in the of et al., and the recruitment of an clamp again through a number of which genome stability in a poorly defined way et al., 2010). Intriguingly, the of was to promote deubiquitylation of of SUMO, by a complex of the 1 and the factor 1 to et al., 2010). SUMOylation of was also in cells and in et al., 2006; and however, its functions in these system have not been and are to involve a Hence, the crosstalk with homologous recombination might be specific to budding of DNA interstrand is particularly important for genome stability and as these lesions strand not only for the of replication but also for transcription. In repair homologous recombination or a of repair and have a system for the which is a associated with defects in this process and monoubiquitylation of a heterodimeric complex of the (for proteins and whose modification their localisation to is to the pathway et al., et al., 2007; et al., 2007). is mediated by the core complex, whose is a RING-finger E3 that with the E2 A complex of other chromatin-associated proteins, the recognition complex, with as its DNA-binding functions in the core complex to the appropriate The downstream of ubiquitylation are not entirely but to the ubiquitylation cascade at DSBs above, they in an activation of the pathway 2007). It has recently been shown that the 1 is recruited to by means of a domain, which might at in for the function of ubiquitylation et al., 2010; Liu et al., 2010; et al., 2010; et al., the pathway not only with the BRCA1 pathway that at DSBs but also exhibits crosstalk with damage On one hand, and are by the same (Huang et al., On the other hand, it has been recently that monoubiquitylation is a for ubiquitylation, by resulting in the recruitment and direct of by et al., 2010; et al., 2010; et al., 2010). Hence, activation of the pathway seems to be to as a to lesions during repair is of DNA replication on lesions that the and involves the of the the lesion as a single-stranded and of the resulting by A of called genome repair the genome. In damage recognition is mediated by (for group and – in the of – the protein complex, which and 2010). Whereas binds to the DNA, part of a E3 protein, the E3 and Whereas ubiquitylation results in that the modification et al., 2005). By contrast, the protein from budding yeast is and DNA damage et al., the details of how and ubiquitylation regulate remain a non-catalytic role for the in this process has been et al., an to damage recognition on the strand of can be by in a process called repair Although ubiquitin or SUMO have not been in this ubiquitylation and subsequent degradation of the large subunit of the itself has been recognised as an important way of that are stalled by DNA damage 2010). The budding yeast E3 its together with the and mediate monoubiquitylation of the et al., 2007; et al., can also chains on these to be to removal by the et al., 2009). ubiquitylation of the by a E3 complex results in chains, which induce degradation of the enzyme et al., 2009; et al., 2007). It has also been shown that a second is involved in deubiquitylation of the et al., and on the of single-stranded that from the of the Intriguingly, although these are presumably of lesions, monoubiquitylation and recruitment of the DNA have been to contribute to the efficient repair of these lesions in humans et al., 2010). In yeast, however, to be independent of by this of In addition, ubiquitylation of histone H2A by RNF8 and polycomb proteins have been in the et al., 2006; et al., in the signalling pathways at DSBs and during lesions, small adducts such as or damage are processed by the repair which is by a series of that The resulting site is processed further by of the DNA by removal of the and of the Although ubiquitylation has not been in this pathway a prominent example of SUMO modification how this modification of a protein can change the of its targets in of its acts on in DNA, but the enzyme is by binding to the In this SUMOylation of promotes catalytic by inducing the of the enzyme from DNA et al., is mediated by a large change in the enzyme and 2005). the structure of a domain a with DNA binding et al., 2005). SUMO binding might also contribute to the of catalytic et al., 2005). Finally, the modification might also the localisation of as well as the of the enzyme to the downstream the and 1 et al., the of ubiquitin and SUMO targets and conjugation factors involved in the maintenance of genome stability is far from the the of mechanisms by which members of the ubiquitin family are known to Both can initiate protein but they often function in a by downstream through dedicated recognition domains or by in the or catalytic activities of their target is the of crosstalk between the individual pathways of damage In addition to in the conjugation or factors or the modification targets, there are where one particular modification promotes or a subsequent is in the SUMO-targeted ubiquitin ligases, but also in the activation of the BRCA1 ubiquitin ligase by its own SUMOylation and in the activation of the ubiquitin ligase by the variety of and on DNA replication and repair very well the by which cells react to insults to their genetic and these processes the importance of the between the and DNA As most of the ubiquitylation and SUMOylation on The modifications in turn can induce in the of the target proteins for DNA, as with Rad52 or Alternatively, they might a direct influence on chromatin structure, such as with histone It has however, that mechanistic insight into the consequences of ubiquitin and SUMO modification for not most of their targets. are likely to the of principles of how the two contribute to genome maintenance.