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A new SUMO ligase in the DNA damage response
DNA Repair 5 (2006) 138–141
Hot Topics in DNA Repair No. 17
A new SUMO ligase in the DNA damage response Karen M. Lee, Matthew J. O’Connell ∗ Accepted 8 August 2005 Available online 27 September 2005
Abstract SUMO is a small ubiquitin-like protein that is attached to target proteins, altering their localization and function. The condensin and cohesinrelated Smc5/6 complex has been linked to DNA repair and checkpoint responses, but details of its molecular function have remained obscure. Recent reports show one subunit of the complex is a SUMO ligase, providing another link between protein sumoylation and DNA damage responses. © 2005 Elsevier B.V. All rights reserved. Keywords: SUMO ligase; DNA damage response; SMC
1. Introduction All eukaryotic cells contain three multi-protein complexes that include members of the structural maintenance of chromosomes (SMC) proteins, together with several non-SMC subunits [1]. A large body of data has led to suggested mechanistic roles for the cohesin and condensin complexes, which include Smc1/3 and Smc2/4 heterodimers, respectively. Molecular details regarding the function of the third complex, currently known as the Smc5/6 complex, are far less understood, though genetic analysis points to an essential role in genome integrity, most likely involving homologous recombination [2]. Recently, two reports have described SUMO ligase activity for this complex in fission and budding yeasts, which resides within one of the non-SMC subunits, Nse2/Mms21 [3,4]. Do these observations account for the postulated role for SUMO modification of proteins to regulate DNA repair? 2. SUMO modification of target proteins Small ubiquitin-like modifier (SUMO) is a small protein distantly related to ubiquitin. Like ubiquitin, SUMO is covalently conjugated to lysines on target proteins using the combined activities of an E1 SUMO activator and an E2 SUMO conjugating enzyme, generally referred to as Ubc9. E3 SUMO ligases have also been described, but SUMO conjugation may not be absolutely E3-dependent, and there are a limited number of these enzymes compared to the very large family of E3 ubiquitin ligases. Modification by SUMO does not lead to protein degradation, and on the contrary can compete for the same 1568-7864/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.dnarep.2005.08.003
lysines otherwise modified by ubiquitin. Other diverse effects of protein sumoylation have been described, primarily involving nuclear proteins in signaling pathways, transcriptional activities and the regulation of sub-cellular localization. Also as seen with ubiquitylation, SUMO modification can be reversed by one of several SUMO isopeptidases. Whilst the yeasts contain a single SUMO isoform, higher eukaryotes have three distinct SUMO genes/polypeptides, providing further complexity to the regulation of protein function by this reversible modification [5]. 3. SUMO and DNA repair Many sumoylated proteins have been identified by either directed or proteomic approaches, and several observations have implicated protein sumoylation in DNA repair. Mutations in the E1 (rad31) [6] and E2 (hus5) [7] enzymes of fission yeast lead to a hypersensitivity to DNA damaging agents. Indeed, the SUMOencoding gene in this organism, pmt3, is not essential, and its deletion also leads to similar sensitivity to a range of genotoxins [8]. In each case there are also defects in chromosome segregation, which are also observed in cells lacking the SUMO ligase Pli1 [9]. Further, several proteins intimately involved in DNA repair have been reported to be sumoylated, or at least associate with SUMO, in various systems. These include the homologous recombination proteins Rad51 and Rad52. Human homologs of both proteins interact with SUMO in yeast two-hybrid assays [10,11]. Fission yeast Rad52, which is known as Rad22, is sumoylated in cells [12], and its interaction with Rad51 may be the cause of the two-hybrid interaction. Both types I and II topoisomerases (Top1 and Top2) have been shown to be sumoy-
K.M. Lee, M.J. O’Connell / DNA Repair 5 (2006) 138–141
lated in yeast and metazoans. For Top1, the enzyme becomes sumoylated when inhibited by camptothecin [13], and mutations in Ubc9 enhance the efficacy of camptothecin in Top1 inhibition [14]. The sumoylation of Top1 affects its sub-nuclear localization [15]. Similarly, Top2 isoforms are sumoylated, and whilst this occurs predominately during mitosis, it is also up-regulated in response to Top2 inhibition by teniposide or ICRF193 [16,17], two inhibitors that lock Top2 in different configurations. The modification of these enzymes may reflect the conformational change induced by the inhibitors, or may be a result of the DNA damage inflicted by the inhibited topoisomerases during decatenation. The human base excision repair enzyme thymine DNA glycosylase, which generates abasic sites in DNA, is also regulated by sumoylation. In this case, SUMO acts to regulate the conformation of the enzyme and in turn its interaction with templates [18–20]. Finally, in budding yeast post-replication repair, PCNA sumoylation likely antagonizes recombination promoted by PCNA ubiquitylation by the Rad18/Rad6 and Ubc13/Mms2/Rad5 ubiquitin ligases [21]. Srs2 helicase, which antagonizes recombination, is recruited to sumoylated PCNA and through this may antagonize PCNA in recombination rather than its ubiquitylation per se [22,23]. Proteomic studies in the budding yeast have also identified a large number of sumoylated proteins [24–28], and it will take an enormous effort to individually dissect the effects of SUMO on the function of these proteins. 4. SUMO ligase activity in the Smc5/6 complex Two protein complexes involved in chromosome dynamics have also been implicated in DNA repair. Cohesin, which consists of an Smc1/3 dimer together with Scc1 and Scc3, is required to maintain cohesion between replicated sister chromatids. Not surprisingly, defects in cohesin lead to defects in DNA repair, as close proximity to a sister chromatid allows for homologous recombination. Condensin, which consists of an Smc2/4 dimer and three non-SMC subunits, is required for mitotic chromosome condensation, and specific mutants in fission yeast have also uncovered a role in DNA repair during interphase [29]. Interestingly, both Smc and non-Smc subunits of these complexes are also subject to sumoylation [27,28,30]. The third and related Smc5/6 complex consists of an Smc5/6 dimer and four non-Smc subunits, Nse1–Nse4. The founding member of this complex was the Smc6 subunit in fission yeast [31], encoded by the rad18 gene, which has been renamed smc6 to avoid confusion with the post-replication repair protein referred to as Rad18 in most systems. All components of the Smc5/6 complex are essential for viability, and hypomorphic and conditional alleles manifest defects in DNA repair and maintenance of DNA damage checkpoints. The molecular events controlled by the complex are not known, though epistasis experiments and studies of suppression of smc6 alleles by the BRCT domain protein Brc1 point towards a role in establishing recombination [31,32]. One model pertaining to Smc protein function is that they facilitate the interaction of the non-Smc subunits with specific chromosomal domains or structures. Therefore, the dissection of the molecular functions for the Smc5/6 complex is likely to
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benefit from the study of Nse1–Nse4. Nse1 contains a highly conserved variant RING domain (C4HC3), which is indicative of proteins with E3 ubiquitin ligase activity, though such an activity has not been reported for this protein [33,34]. Nse3 and Nse4 do not contain domains that are suggestive of function or enzymatic activity [2]. Nse2, which in budding yeast is also known as Mms21, contains a RING-like domain, referred to as a SP-RING (Siz/PIAS), which is characteristic of E3 SUMO ligases. It has now been shown that Nse2/Mms21 indeed posses SUMO ligase activity, and among its substrates are components of the Smc5/6 complex itself. Andrews et al. [3] observed sequence homology between fission yeast Nse2 and the budding yeast SUMO ligase Siz1, and that recombinant Nse2 possessed auto-sumoylation activity in vitro. Using in vitro translated substrates, they found that Nse2 could catalyze the sumoylation of Nse3 and Smc6, but not Nse1 or Smc5. Using co-precipitation studies, Smc6 was shown to be sumoylated in cells, and that this was up-regulated upon treatment with the alkylating agent MMS. Mutation of SP-RING motif abolished SUMO ligase activity in Nse2, and severely diminished the levels of sumoylated Smc6. Surprisingly, strains harboring the mutant nse2 allele (nse2-SA) were viable, yet were hypersensitive to MMS and hydroxyurea (both agents cause replication arrest), and were also mildly sensitivity to UV-C and ionizing radiation. The SUMO ligase activity of Nse2 was the first catalytic activity associated with the Smc5/6 complex. It raises the possibility that sumoylation of Smc6 may in turn regulate the complex, possibly through localization, though this has not yet been shown. As nse2 is an essential gene, and nse2-SA is only a hypomorph, then the SUMO ligase activity of Nse2 cannot be the essential function of the complex, though Nse2 must be present to form a functional complex. Zhao and Blobel [4] found an allele of budding yeast MMS21 (mms21-11) in a screen for synthetic lethals with the mlp1∆mlp2∆, a double mutant in genes encoding myosinlike proteins that are localized to the nucleoplasmic side of nuclear pore complexes other than those near the nucleolus. The desumoylating enzyme Ulp1 shares this localization, and indeed Ulp1 localization is via the Mlps. The mms21-11 mutant was also hypersensitive to genotoxins, and showed defects in the structure of the nucleoli and in the clustering of telomeres. Similar observations have recently been reported for alleles of SMC5 and SMC6 [35]. As with fission yeast Nse2, Mms21 was shown to posses SUMO ligase activity. Taking a candidate approach, Zhao and Blobel found that Smc5 was sumoylated in MMS treated cells, and that this was abolished in the mms21-11 mutant. Sumoylation of Smc5 (but not Smc6) has been also been observed in several proteomic screens for sumoylated proteins in budding yeast [27,28]. They also found that Ku70 sumoylation was somewhat diminished in the mms21-11 strain, though this may in part be due to a defective DNA damage response. However, Mms21-catalyzed sumoylation of Smc5 and Ku70 was observed in vitro, while PCNA sumoylation was not. Precisely why mms21-11 is synthetically lethal with mlp1∆mlp2∆, and with delocalized Ulp1 is not clear, but possibly delocalized Ulp1, leading to a net reduction in essential sumoylation, antagonizes sumoylation by Mms21 and possibly overlapping ligases, such
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as Siz1 and Siz2. Unlike the case in fission yeast, SUMO is essential for viability in budding yeast [36]. 5. Another disparity between the two yeasts? Both studies show SUMO ligase activity in the homologous proteins. As Nse2/Mms21 is anchored to the Smc5/6 complex, its repertoire of substrates may be significantly smaller than the monomeric ligases Pli1, Siz1 and Siz2. Nevertheless, in fission yeast Smc6 was identified as a substrate, and its sumoylation was increased by MMS treatment, a condition requiring Smc5/6 function for viability. Conversely, Smc5 but not Smc6 has been identified in screens for sumoylated proteins, which was confirmed by Zhao and Blobel, although again it was upregulated by MMS treatment. It should be noted that fission and budding yeasts are evolutionarily distant, though other explanations can be proposed to explain this apparent discrepancy. Andrews et al. did not report assays for Smc5 sumoylation from cell extracts, and the lack of sumoylation of in vitro translated Smc5 may be due to some other lacking modification. Further, sumoylation of Smc6 may be less robust than for Smc5 in budding yeast, and so may have been missed. It is perhaps noteworthy that cell cycle distribution is very different in these organisms, and these modifications may be cell cycle regulated. As Nse2/Mms21 is but one member of a six-protein complex, it will be important to biochemically dissect the SUMO ligase activities in the context of an intact complex. Finally, we do not have better descriptors to distinguish Smc5 from Smc6 other than overall sequence similarity—i.e. there are no functional assays that distinguish between the two thus far, and so maybe the two studies are actually assaying the same event. Ultimately, functional dissection of this will require mapping and mutation of the SUMO acceptor sites, as Nse2 is likely to have more than one substrate. 6. Summary The studies by Andrews et al., and Zhao and Blobel, clearly show Nse2-mediated sumoylation is an important event in surviving DNA damage, perhaps particularly during DNA replication. It will also be of interest to decipher the other activity(-ies) in the Smc5/6 complex, that may include Nse1-mediated ubiquitylation. Deciphering all the relevant target proteins in this response will be challenging, but nevertheless the work presented in these papers represents an important advance. Acknowledgments KML is supported by an NIH/NCI training grant T32 CA78207. MJO’C is a Scholar of the Leukemia and Lymphoma Society. Work in our laboratory is supported by grants from the NIH/NCI (CA100076) and the Peter J. Sharp Foundation for Melanoma Research. References [1] A. Losada, T. Hirano, Dynamic molecular linkers of the genome: the first decade of SMC proteins, Genes Dev. 19 (2005) 1269–1287.
[2] A.R. Lehmann, The role of SMC proteins in the responses to DNA damage, DNA Repair (Amst) 4 (2005) 309–314. [3] E.A. Andrews, J. Palecek, J. Sergeant, E. Taylor, A.R. Lehmann, F.Z. Watts, Nse2, a component of the Smc5–6 complex, is a SUMO ligase required for the response to DNA damage, Mol. Cell Biol. 25 (2005) 185–196. [4] X. Zhao, G. Blobel, From the cover: a SUMO ligase is part of a nuclear multiprotein complex that affects DNA repair and chromosomal organization, Proc. Natl. Acad. Sci U.S.A. 102 (2005) 4777–4782. [5] E.S. Johnson, Protein modification by SUMO, Annu. Rev. Biochem. 73 (2004) 355–382. [6] M. Shayeghi, C.L. Doe, M. Tavassoli, F.Z. Watts, Characterisation of Schizosaccharomyces pombe rad31, a Uba-related gene required for DNA damage tolerance, Nucl. Acid Res. 25 (1997) 1162–1169. [7] F. Al-Khodairy, T. Enoch, I.M. Hagan, A.N. Carr, The Schizosaccharomyces pombe hus5 gene encodes a ubiquitin conjugating enzyme required for normal mitosis, J. Cell Sci. 108 (1995) 475–486. [8] K. Tanaka, J. Nishide, K. Okazaki, H. Kato, O. Niwa, T. Nakagawa, H. Matsuda, M. Kawamukai, Y. Murakami, Characterization of a fission yeast SUMO-1 homologue, Pmt3p, required for multiple nuclear events, including the control of telomere length and chromosome segregation, Mol. Cell Biol. 19 (1999) 8660–8672. [9] B. Xhemalce, J.S. Seeler, G. Thon, A. Dejean, B. Arcangioli, Role of the fission yeast SUMO E3 ligase Pli1p in centromere and telomere maintenance, EMBO J. 23 (2004) 3844–3853. [10] Z. Shen, P.E. Pardington-Purtymun, J.C. Comeaux, R.K. Moyzis, D.J. Chen, Associations of Ube2i with Rad52, Ubl1, p53, and Rad51 proteins in a yeast two-hybrid system, Genomics 37 (1996) 183– 186. [11] Z. Shen, P.E. Pardington-Purtymun, J.C. Comeaux, R.K. Moyzis, D.J. Chen, Ubl1, a human ubiquitin-like protein associating with human Rad51/Rad52 proteins, Genomics 36 (1996) 271–279. [12] J.C. Ho, N.J. Warr, H. Shimizu, F.Z. Watts, SUMO modification of Rad22, the Schizosaccharomyces pombe homologue of the recombination protein Rad52, Nucl. Acid Res. 29 (2001) 4179–4186. [13] Y. Mao, M. Sun, S.D. Desai, L.F. Liu, Sumo-1 conjugation to topoisomerase. I. A possible repair response to topoisomerase-mediated DNA damage, Proc. Natl. Acad. Sci. U.S.A. 97 (2000) 4046–4051. [14] H.R. Jacquiau, R.C. van Waardenburg, R.J. Reid, M.H. Woo, H. Guo, E.S. Johnson, M.A. Bjornsti, Defects in SUMO (small ubiquitin-related modifier) conjugation and deconjugation alter cell sensitivity to DNA topoisomerase I-induced DNA damage, J. Biol. Chem. 280 (2005) 23566–23575. [15] Y.Y. Mo, Y. Yu, Z. Shen, W.T. Beck, Nucleolar delocalization of human topoisomerase I in response to topotecan correlates with sumoylation of the protein, J. Biol. Chem. 277 (2002) 2958–2964. [16] S. Isik, K. Sano, K. Tsutsui, M. Seki, T. Enomoto, H. Saitoh, K. Tsutsui, The SUMO pathway is required for selective degradation of DNA topoisomerase IIbeta induced by a catalytic inhibitor ICRF-193(1), FEBS Lett. 546 (2003) 374–378. [17] Y. Mao, S.D. Desai, L.F. Liu, Sumo-1 conjugation to human DNA topoisomerase II isozymes, J. Biol. Chem. 275 (2000) 26066–26073. [18] R. Steinacher, P. Schar, Functionality of human thymine DNA glycosylase requires SUMO-regulated changes in protein conformation, Curr. Biol. 15 (2005) 616–623. [19] H. Takahashi, S. Hatakeyama, H. Saitoh, K.I. Nakayama, Noncovalent SUMO-1 binding activity of thymine DNA glycosylase (tdg) is required for its SUMO-1 modification and colocalization with the promyelocytic leukemia protein, J. Biol. Chem. 280 (2005) 5611–5621. [20] U. Hardeland, R. Steinacher, J. Jiricny, P. Schar, Modification of the human thymine-DNA glycosylase by ubiquitin-like proteins facilitates enzymatic turnover, EMBO J. 21 (2002) 1456–1464. [21] S. Prakash, R.E. Johnson, L. Prakash, Eukaryotic translesion synthesis DNA polymerases: specificity of structure and function, Annu. Rev. Biochem. 74 (2005) 317–353. [22] B. Pfander, G.L. Moldovan, M. Sacher, C. Hoege, S. Jentsch, Sumomodified PCNA recruits Srs2 to prevent recombination during S phase, Nature 436 (2005) 428–433.
K.M. Lee, M.J. O’Connell / DNA Repair 5 (2006) 138–141 [23] E. Papouli, S. Chen, A.A. Davies, D. Huttner, L. Krejci, P. Sung, H.D. Ulrich, Crosstalk between SUMO and ubiquitin on PCNA is mediated by recruitment of the helicase Srs2p, Mol. Cell 19 (2005) 123–133. [24] W. Zhou, J.J. Ryan, H. Zhou, Global analyses of sumoylated proteins in Saccharomyces cerevisiae. Induction of protein sumoylation by cellular stresses, J. Biol. Chem. 279 (2004) 32262–32268. [25] V.G. Panse, U. Hardeland, T. Werner, B. Kuster, E. Hurt, A proteomewide approach identifies sumoylated substrate proteins in yeast, J. Biol. Chem. 279 (2004) 41346–41351. [26] G. Rosas-Acosta, W.K. Russell, A. Deyrieux, D.H. Russell, V.G. Wilson, A universal strategy for proteomic studies of SUMO and other ubiquitinlike modifiers, Mol. Cell Proteomics 4 (2005) 56–72. [27] C. Denison, A.D. Rudner, S.A. Gerber, C.E. Bakalarski, D. Moazed, S.P. Gygi, A proteomic strategy for gaining insights into protein sumoylation in yeast, Mol. Cell Proteomics 4 (2005) 246–254. [28] J.A. Wohlschlegel, E.S. Johnson, S.I. Reed, J.R. Yates III, Global analysis of protein sumoylation in Saccharomyces cerevisiae, J. Biol. Chem. 279 (2004) 45662–45668. [29] N. Aono, T. Sutani, T. Tomonaga, S. Mochida, M. Yanagida, Cnd2 has dual roles in mitotic condensation and interphase, Nature 417 (2002) 197–202. [30] D. D’Amours, F. Stegmeier, A. Amon, Cdc14 and condensin control the dissolution of cohesin-independent chromosome linkages at repeated DNA, Cell 117 (2004) 455–469. [31] A.R. Lehmann, M. Walicka, D.J.F. Grittiths, J.M. Murray, F.Z. Watts, S. McCready, A.M. Carr, The rad18 gene of Schizosaccharomyces pombe defines a new subgroup of the SMC superfamily involved in DNA repair, Mol. Cell Biol. 15 (1995) 7067–7080. [32] D.M. Sheedy, D. Dimitrova, J.K. Rankin, K.L. Bass, K.M. Lee, C. TapiaAlveal, S.H. Harvey, J.M. Murray, M.J. O’Connell. Brc1-mediated DNA repair and damage tolerance, Genetics 171 (2) (2005) in press. [33] Y. Fujioka, Y. Kimata, K. Nomaguchi, K. Watanabe, K. Kohno, Identification of a novel non-structural maintenance of chromosomes (SMC) component of the Smc5–Smc6 complex involved in DNA repair, J. Biol. Chem. 277 (2002) 21585–21591.
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[34] S.H. Harvey, D.M. Sheedy, A.R. Cuddihy, M.J. O’Connell, Coordination of DNA damage responses via the Smc5/Smc6 complex, Mol. Cell Biol. 24 (2004) 662–674. [35] J. Torres-Rosell, F. Machin, S. Farmer, A. Jarmuz, T. Eydmann, J.Z. Dalgaard, L. Aragon, Smc5 and smc6 genes are required for the segregation of repetitive chromosome regions, Nat. Cell Biol. 7 (2005) 412– 419. [36] G. Giaever, A.M. Chu, L. Ni, C. Connelly, L. Riles, S. Veronneau, S. Dow, A. Lucau-Danila, K. Anderson, B. Andre, A.P. Arkin, A. Astromoff, M. El-Bakkoury, R. Bangham, R. Benito, S. Brachat, S. Campanaro, M. Curtiss, K. Davis, A. Deutschbauer, K.D. Entian, P. Flaherty, F. Foury, D.J. Garfinkel, M. Gerstein, D. Gotte, U. Guldener, J.H. Hegemann, S. Hempel, Z. Herman, D.F. Jaramillo, D.E. Kelly, S.L. Kelly, P. Kotter, D. LaBonte, D.C. Lamb, N. Lan, H. Liang, H. Liao, L. Liu, C. Luo, M. Lussier, R. Mao, P. Menard, S.L. Ooi, J.L. Revuelta, C.J. Roberts, M. Rose, P. Ross-Macdonald, B. Scherens, G. Schimmack, B. Shafer, D.D. Shoemaker, S. Sookhai-Mahadeo, R.K. Storms, J.N. Strathern, G. Valle, M. Voet, G. Volckaert, C.Y. Wang, T.R. Ward, J. Wilhelmy, E.A. Winzeler, Y. Yang, G. Yen, E. Youngman, K. Yu, H. Bussey, J.D. Boeke, M. Snyder, P. Philippsen, R.W. Davis, M. Johnston, Functional profiling of the Saccharomyces cerevisiae genome, Nature 418 (2002) 387–391.
Karen M. Lee, Matthew J. O’Connell ∗ are in the Department of Oncological Sciences, Mount Sinai School of Medicine, 1 Gustave L. Levy Place, New York, NY 10029, USA ∗ Corresponding
author. Tel.: +1 212 659 5468; fax: +1 212 987 2240. E-mail address: [email protected] (M.J. O’Connell)
Hot Topics in DNA Repair No. 17
A new SUMO ligase in the DNA damage response Karen M. Lee, Matthew J. O’Connell ∗ Accepted 8 August 2005 Available online 27 September 2005
Abstract SUMO is a small ubiquitin-like protein that is attached to target proteins, altering their localization and function. The condensin and cohesinrelated Smc5/6 complex has been linked to DNA repair and checkpoint responses, but details of its molecular function have remained obscure. Recent reports show one subunit of the complex is a SUMO ligase, providing another link between protein sumoylation and DNA damage responses. © 2005 Elsevier B.V. All rights reserved. Keywords: SUMO ligase; DNA damage response; SMC
1. Introduction All eukaryotic cells contain three multi-protein complexes that include members of the structural maintenance of chromosomes (SMC) proteins, together with several non-SMC subunits [1]. A large body of data has led to suggested mechanistic roles for the cohesin and condensin complexes, which include Smc1/3 and Smc2/4 heterodimers, respectively. Molecular details regarding the function of the third complex, currently known as the Smc5/6 complex, are far less understood, though genetic analysis points to an essential role in genome integrity, most likely involving homologous recombination [2]. Recently, two reports have described SUMO ligase activity for this complex in fission and budding yeasts, which resides within one of the non-SMC subunits, Nse2/Mms21 [3,4]. Do these observations account for the postulated role for SUMO modification of proteins to regulate DNA repair? 2. SUMO modification of target proteins Small ubiquitin-like modifier (SUMO) is a small protein distantly related to ubiquitin. Like ubiquitin, SUMO is covalently conjugated to lysines on target proteins using the combined activities of an E1 SUMO activator and an E2 SUMO conjugating enzyme, generally referred to as Ubc9. E3 SUMO ligases have also been described, but SUMO conjugation may not be absolutely E3-dependent, and there are a limited number of these enzymes compared to the very large family of E3 ubiquitin ligases. Modification by SUMO does not lead to protein degradation, and on the contrary can compete for the same 1568-7864/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.dnarep.2005.08.003
lysines otherwise modified by ubiquitin. Other diverse effects of protein sumoylation have been described, primarily involving nuclear proteins in signaling pathways, transcriptional activities and the regulation of sub-cellular localization. Also as seen with ubiquitylation, SUMO modification can be reversed by one of several SUMO isopeptidases. Whilst the yeasts contain a single SUMO isoform, higher eukaryotes have three distinct SUMO genes/polypeptides, providing further complexity to the regulation of protein function by this reversible modification [5]. 3. SUMO and DNA repair Many sumoylated proteins have been identified by either directed or proteomic approaches, and several observations have implicated protein sumoylation in DNA repair. Mutations in the E1 (rad31) [6] and E2 (hus5) [7] enzymes of fission yeast lead to a hypersensitivity to DNA damaging agents. Indeed, the SUMOencoding gene in this organism, pmt3, is not essential, and its deletion also leads to similar sensitivity to a range of genotoxins [8]. In each case there are also defects in chromosome segregation, which are also observed in cells lacking the SUMO ligase Pli1 [9]. Further, several proteins intimately involved in DNA repair have been reported to be sumoylated, or at least associate with SUMO, in various systems. These include the homologous recombination proteins Rad51 and Rad52. Human homologs of both proteins interact with SUMO in yeast two-hybrid assays [10,11]. Fission yeast Rad52, which is known as Rad22, is sumoylated in cells [12], and its interaction with Rad51 may be the cause of the two-hybrid interaction. Both types I and II topoisomerases (Top1 and Top2) have been shown to be sumoy-
K.M. Lee, M.J. O’Connell / DNA Repair 5 (2006) 138–141
lated in yeast and metazoans. For Top1, the enzyme becomes sumoylated when inhibited by camptothecin [13], and mutations in Ubc9 enhance the efficacy of camptothecin in Top1 inhibition [14]. The sumoylation of Top1 affects its sub-nuclear localization [15]. Similarly, Top2 isoforms are sumoylated, and whilst this occurs predominately during mitosis, it is also up-regulated in response to Top2 inhibition by teniposide or ICRF193 [16,17], two inhibitors that lock Top2 in different configurations. The modification of these enzymes may reflect the conformational change induced by the inhibitors, or may be a result of the DNA damage inflicted by the inhibited topoisomerases during decatenation. The human base excision repair enzyme thymine DNA glycosylase, which generates abasic sites in DNA, is also regulated by sumoylation. In this case, SUMO acts to regulate the conformation of the enzyme and in turn its interaction with templates [18–20]. Finally, in budding yeast post-replication repair, PCNA sumoylation likely antagonizes recombination promoted by PCNA ubiquitylation by the Rad18/Rad6 and Ubc13/Mms2/Rad5 ubiquitin ligases [21]. Srs2 helicase, which antagonizes recombination, is recruited to sumoylated PCNA and through this may antagonize PCNA in recombination rather than its ubiquitylation per se [22,23]. Proteomic studies in the budding yeast have also identified a large number of sumoylated proteins [24–28], and it will take an enormous effort to individually dissect the effects of SUMO on the function of these proteins. 4. SUMO ligase activity in the Smc5/6 complex Two protein complexes involved in chromosome dynamics have also been implicated in DNA repair. Cohesin, which consists of an Smc1/3 dimer together with Scc1 and Scc3, is required to maintain cohesion between replicated sister chromatids. Not surprisingly, defects in cohesin lead to defects in DNA repair, as close proximity to a sister chromatid allows for homologous recombination. Condensin, which consists of an Smc2/4 dimer and three non-SMC subunits, is required for mitotic chromosome condensation, and specific mutants in fission yeast have also uncovered a role in DNA repair during interphase [29]. Interestingly, both Smc and non-Smc subunits of these complexes are also subject to sumoylation [27,28,30]. The third and related Smc5/6 complex consists of an Smc5/6 dimer and four non-Smc subunits, Nse1–Nse4. The founding member of this complex was the Smc6 subunit in fission yeast [31], encoded by the rad18 gene, which has been renamed smc6 to avoid confusion with the post-replication repair protein referred to as Rad18 in most systems. All components of the Smc5/6 complex are essential for viability, and hypomorphic and conditional alleles manifest defects in DNA repair and maintenance of DNA damage checkpoints. The molecular events controlled by the complex are not known, though epistasis experiments and studies of suppression of smc6 alleles by the BRCT domain protein Brc1 point towards a role in establishing recombination [31,32]. One model pertaining to Smc protein function is that they facilitate the interaction of the non-Smc subunits with specific chromosomal domains or structures. Therefore, the dissection of the molecular functions for the Smc5/6 complex is likely to
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benefit from the study of Nse1–Nse4. Nse1 contains a highly conserved variant RING domain (C4HC3), which is indicative of proteins with E3 ubiquitin ligase activity, though such an activity has not been reported for this protein [33,34]. Nse3 and Nse4 do not contain domains that are suggestive of function or enzymatic activity [2]. Nse2, which in budding yeast is also known as Mms21, contains a RING-like domain, referred to as a SP-RING (Siz/PIAS), which is characteristic of E3 SUMO ligases. It has now been shown that Nse2/Mms21 indeed posses SUMO ligase activity, and among its substrates are components of the Smc5/6 complex itself. Andrews et al. [3] observed sequence homology between fission yeast Nse2 and the budding yeast SUMO ligase Siz1, and that recombinant Nse2 possessed auto-sumoylation activity in vitro. Using in vitro translated substrates, they found that Nse2 could catalyze the sumoylation of Nse3 and Smc6, but not Nse1 or Smc5. Using co-precipitation studies, Smc6 was shown to be sumoylated in cells, and that this was up-regulated upon treatment with the alkylating agent MMS. Mutation of SP-RING motif abolished SUMO ligase activity in Nse2, and severely diminished the levels of sumoylated Smc6. Surprisingly, strains harboring the mutant nse2 allele (nse2-SA) were viable, yet were hypersensitive to MMS and hydroxyurea (both agents cause replication arrest), and were also mildly sensitivity to UV-C and ionizing radiation. The SUMO ligase activity of Nse2 was the first catalytic activity associated with the Smc5/6 complex. It raises the possibility that sumoylation of Smc6 may in turn regulate the complex, possibly through localization, though this has not yet been shown. As nse2 is an essential gene, and nse2-SA is only a hypomorph, then the SUMO ligase activity of Nse2 cannot be the essential function of the complex, though Nse2 must be present to form a functional complex. Zhao and Blobel [4] found an allele of budding yeast MMS21 (mms21-11) in a screen for synthetic lethals with the mlp1∆mlp2∆, a double mutant in genes encoding myosinlike proteins that are localized to the nucleoplasmic side of nuclear pore complexes other than those near the nucleolus. The desumoylating enzyme Ulp1 shares this localization, and indeed Ulp1 localization is via the Mlps. The mms21-11 mutant was also hypersensitive to genotoxins, and showed defects in the structure of the nucleoli and in the clustering of telomeres. Similar observations have recently been reported for alleles of SMC5 and SMC6 [35]. As with fission yeast Nse2, Mms21 was shown to posses SUMO ligase activity. Taking a candidate approach, Zhao and Blobel found that Smc5 was sumoylated in MMS treated cells, and that this was abolished in the mms21-11 mutant. Sumoylation of Smc5 (but not Smc6) has been also been observed in several proteomic screens for sumoylated proteins in budding yeast [27,28]. They also found that Ku70 sumoylation was somewhat diminished in the mms21-11 strain, though this may in part be due to a defective DNA damage response. However, Mms21-catalyzed sumoylation of Smc5 and Ku70 was observed in vitro, while PCNA sumoylation was not. Precisely why mms21-11 is synthetically lethal with mlp1∆mlp2∆, and with delocalized Ulp1 is not clear, but possibly delocalized Ulp1, leading to a net reduction in essential sumoylation, antagonizes sumoylation by Mms21 and possibly overlapping ligases, such
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as Siz1 and Siz2. Unlike the case in fission yeast, SUMO is essential for viability in budding yeast [36]. 5. Another disparity between the two yeasts? Both studies show SUMO ligase activity in the homologous proteins. As Nse2/Mms21 is anchored to the Smc5/6 complex, its repertoire of substrates may be significantly smaller than the monomeric ligases Pli1, Siz1 and Siz2. Nevertheless, in fission yeast Smc6 was identified as a substrate, and its sumoylation was increased by MMS treatment, a condition requiring Smc5/6 function for viability. Conversely, Smc5 but not Smc6 has been identified in screens for sumoylated proteins, which was confirmed by Zhao and Blobel, although again it was upregulated by MMS treatment. It should be noted that fission and budding yeasts are evolutionarily distant, though other explanations can be proposed to explain this apparent discrepancy. Andrews et al. did not report assays for Smc5 sumoylation from cell extracts, and the lack of sumoylation of in vitro translated Smc5 may be due to some other lacking modification. Further, sumoylation of Smc6 may be less robust than for Smc5 in budding yeast, and so may have been missed. It is perhaps noteworthy that cell cycle distribution is very different in these organisms, and these modifications may be cell cycle regulated. As Nse2/Mms21 is but one member of a six-protein complex, it will be important to biochemically dissect the SUMO ligase activities in the context of an intact complex. Finally, we do not have better descriptors to distinguish Smc5 from Smc6 other than overall sequence similarity—i.e. there are no functional assays that distinguish between the two thus far, and so maybe the two studies are actually assaying the same event. Ultimately, functional dissection of this will require mapping and mutation of the SUMO acceptor sites, as Nse2 is likely to have more than one substrate. 6. Summary The studies by Andrews et al., and Zhao and Blobel, clearly show Nse2-mediated sumoylation is an important event in surviving DNA damage, perhaps particularly during DNA replication. It will also be of interest to decipher the other activity(-ies) in the Smc5/6 complex, that may include Nse1-mediated ubiquitylation. Deciphering all the relevant target proteins in this response will be challenging, but nevertheless the work presented in these papers represents an important advance. Acknowledgments KML is supported by an NIH/NCI training grant T32 CA78207. MJO’C is a Scholar of the Leukemia and Lymphoma Society. Work in our laboratory is supported by grants from the NIH/NCI (CA100076) and the Peter J. Sharp Foundation for Melanoma Research. References [1] A. Losada, T. Hirano, Dynamic molecular linkers of the genome: the first decade of SMC proteins, Genes Dev. 19 (2005) 1269–1287.
[2] A.R. Lehmann, The role of SMC proteins in the responses to DNA damage, DNA Repair (Amst) 4 (2005) 309–314. [3] E.A. Andrews, J. Palecek, J. Sergeant, E. Taylor, A.R. Lehmann, F.Z. Watts, Nse2, a component of the Smc5–6 complex, is a SUMO ligase required for the response to DNA damage, Mol. Cell Biol. 25 (2005) 185–196. [4] X. Zhao, G. Blobel, From the cover: a SUMO ligase is part of a nuclear multiprotein complex that affects DNA repair and chromosomal organization, Proc. Natl. Acad. Sci U.S.A. 102 (2005) 4777–4782. [5] E.S. Johnson, Protein modification by SUMO, Annu. Rev. Biochem. 73 (2004) 355–382. [6] M. Shayeghi, C.L. Doe, M. Tavassoli, F.Z. Watts, Characterisation of Schizosaccharomyces pombe rad31, a Uba-related gene required for DNA damage tolerance, Nucl. Acid Res. 25 (1997) 1162–1169. [7] F. Al-Khodairy, T. Enoch, I.M. Hagan, A.N. Carr, The Schizosaccharomyces pombe hus5 gene encodes a ubiquitin conjugating enzyme required for normal mitosis, J. Cell Sci. 108 (1995) 475–486. [8] K. Tanaka, J. Nishide, K. Okazaki, H. Kato, O. Niwa, T. Nakagawa, H. Matsuda, M. Kawamukai, Y. Murakami, Characterization of a fission yeast SUMO-1 homologue, Pmt3p, required for multiple nuclear events, including the control of telomere length and chromosome segregation, Mol. Cell Biol. 19 (1999) 8660–8672. [9] B. Xhemalce, J.S. Seeler, G. Thon, A. Dejean, B. Arcangioli, Role of the fission yeast SUMO E3 ligase Pli1p in centromere and telomere maintenance, EMBO J. 23 (2004) 3844–3853. [10] Z. Shen, P.E. Pardington-Purtymun, J.C. Comeaux, R.K. Moyzis, D.J. Chen, Associations of Ube2i with Rad52, Ubl1, p53, and Rad51 proteins in a yeast two-hybrid system, Genomics 37 (1996) 183– 186. [11] Z. Shen, P.E. Pardington-Purtymun, J.C. Comeaux, R.K. Moyzis, D.J. Chen, Ubl1, a human ubiquitin-like protein associating with human Rad51/Rad52 proteins, Genomics 36 (1996) 271–279. [12] J.C. Ho, N.J. Warr, H. Shimizu, F.Z. Watts, SUMO modification of Rad22, the Schizosaccharomyces pombe homologue of the recombination protein Rad52, Nucl. Acid Res. 29 (2001) 4179–4186. [13] Y. Mao, M. Sun, S.D. Desai, L.F. Liu, Sumo-1 conjugation to topoisomerase. I. A possible repair response to topoisomerase-mediated DNA damage, Proc. Natl. Acad. Sci. U.S.A. 97 (2000) 4046–4051. [14] H.R. Jacquiau, R.C. van Waardenburg, R.J. Reid, M.H. Woo, H. Guo, E.S. Johnson, M.A. Bjornsti, Defects in SUMO (small ubiquitin-related modifier) conjugation and deconjugation alter cell sensitivity to DNA topoisomerase I-induced DNA damage, J. Biol. Chem. 280 (2005) 23566–23575. [15] Y.Y. Mo, Y. Yu, Z. Shen, W.T. Beck, Nucleolar delocalization of human topoisomerase I in response to topotecan correlates with sumoylation of the protein, J. Biol. Chem. 277 (2002) 2958–2964. [16] S. Isik, K. Sano, K. Tsutsui, M. Seki, T. Enomoto, H. Saitoh, K. Tsutsui, The SUMO pathway is required for selective degradation of DNA topoisomerase IIbeta induced by a catalytic inhibitor ICRF-193(1), FEBS Lett. 546 (2003) 374–378. [17] Y. Mao, S.D. Desai, L.F. Liu, Sumo-1 conjugation to human DNA topoisomerase II isozymes, J. Biol. Chem. 275 (2000) 26066–26073. [18] R. Steinacher, P. Schar, Functionality of human thymine DNA glycosylase requires SUMO-regulated changes in protein conformation, Curr. Biol. 15 (2005) 616–623. [19] H. Takahashi, S. Hatakeyama, H. Saitoh, K.I. Nakayama, Noncovalent SUMO-1 binding activity of thymine DNA glycosylase (tdg) is required for its SUMO-1 modification and colocalization with the promyelocytic leukemia protein, J. Biol. Chem. 280 (2005) 5611–5621. [20] U. Hardeland, R. Steinacher, J. Jiricny, P. Schar, Modification of the human thymine-DNA glycosylase by ubiquitin-like proteins facilitates enzymatic turnover, EMBO J. 21 (2002) 1456–1464. [21] S. Prakash, R.E. Johnson, L. Prakash, Eukaryotic translesion synthesis DNA polymerases: specificity of structure and function, Annu. Rev. Biochem. 74 (2005) 317–353. [22] B. Pfander, G.L. Moldovan, M. Sacher, C. Hoege, S. Jentsch, Sumomodified PCNA recruits Srs2 to prevent recombination during S phase, Nature 436 (2005) 428–433.
K.M. Lee, M.J. O’Connell / DNA Repair 5 (2006) 138–141 [23] E. Papouli, S. Chen, A.A. Davies, D. Huttner, L. Krejci, P. Sung, H.D. Ulrich, Crosstalk between SUMO and ubiquitin on PCNA is mediated by recruitment of the helicase Srs2p, Mol. Cell 19 (2005) 123–133. [24] W. Zhou, J.J. Ryan, H. Zhou, Global analyses of sumoylated proteins in Saccharomyces cerevisiae. Induction of protein sumoylation by cellular stresses, J. Biol. Chem. 279 (2004) 32262–32268. [25] V.G. Panse, U. Hardeland, T. Werner, B. Kuster, E. Hurt, A proteomewide approach identifies sumoylated substrate proteins in yeast, J. Biol. Chem. 279 (2004) 41346–41351. [26] G. Rosas-Acosta, W.K. Russell, A. Deyrieux, D.H. Russell, V.G. Wilson, A universal strategy for proteomic studies of SUMO and other ubiquitinlike modifiers, Mol. Cell Proteomics 4 (2005) 56–72. [27] C. Denison, A.D. Rudner, S.A. Gerber, C.E. Bakalarski, D. Moazed, S.P. Gygi, A proteomic strategy for gaining insights into protein sumoylation in yeast, Mol. Cell Proteomics 4 (2005) 246–254. [28] J.A. Wohlschlegel, E.S. Johnson, S.I. Reed, J.R. Yates III, Global analysis of protein sumoylation in Saccharomyces cerevisiae, J. Biol. Chem. 279 (2004) 45662–45668. [29] N. Aono, T. Sutani, T. Tomonaga, S. Mochida, M. Yanagida, Cnd2 has dual roles in mitotic condensation and interphase, Nature 417 (2002) 197–202. [30] D. D’Amours, F. Stegmeier, A. Amon, Cdc14 and condensin control the dissolution of cohesin-independent chromosome linkages at repeated DNA, Cell 117 (2004) 455–469. [31] A.R. Lehmann, M. Walicka, D.J.F. Grittiths, J.M. Murray, F.Z. Watts, S. McCready, A.M. Carr, The rad18 gene of Schizosaccharomyces pombe defines a new subgroup of the SMC superfamily involved in DNA repair, Mol. Cell Biol. 15 (1995) 7067–7080. [32] D.M. Sheedy, D. Dimitrova, J.K. Rankin, K.L. Bass, K.M. Lee, C. TapiaAlveal, S.H. Harvey, J.M. Murray, M.J. O’Connell. Brc1-mediated DNA repair and damage tolerance, Genetics 171 (2) (2005) in press. [33] Y. Fujioka, Y. Kimata, K. Nomaguchi, K. Watanabe, K. Kohno, Identification of a novel non-structural maintenance of chromosomes (SMC) component of the Smc5–Smc6 complex involved in DNA repair, J. Biol. Chem. 277 (2002) 21585–21591.
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[34] S.H. Harvey, D.M. Sheedy, A.R. Cuddihy, M.J. O’Connell, Coordination of DNA damage responses via the Smc5/Smc6 complex, Mol. Cell Biol. 24 (2004) 662–674. [35] J. Torres-Rosell, F. Machin, S. Farmer, A. Jarmuz, T. Eydmann, J.Z. Dalgaard, L. Aragon, Smc5 and smc6 genes are required for the segregation of repetitive chromosome regions, Nat. Cell Biol. 7 (2005) 412– 419. [36] G. Giaever, A.M. Chu, L. Ni, C. Connelly, L. Riles, S. Veronneau, S. Dow, A. Lucau-Danila, K. Anderson, B. Andre, A.P. Arkin, A. Astromoff, M. El-Bakkoury, R. Bangham, R. Benito, S. Brachat, S. Campanaro, M. Curtiss, K. Davis, A. Deutschbauer, K.D. Entian, P. Flaherty, F. Foury, D.J. Garfinkel, M. Gerstein, D. Gotte, U. Guldener, J.H. Hegemann, S. Hempel, Z. Herman, D.F. Jaramillo, D.E. Kelly, S.L. Kelly, P. Kotter, D. LaBonte, D.C. Lamb, N. Lan, H. Liang, H. Liao, L. Liu, C. Luo, M. Lussier, R. Mao, P. Menard, S.L. Ooi, J.L. Revuelta, C.J. Roberts, M. Rose, P. Ross-Macdonald, B. Scherens, G. Schimmack, B. Shafer, D.D. Shoemaker, S. Sookhai-Mahadeo, R.K. Storms, J.N. Strathern, G. Valle, M. Voet, G. Volckaert, C.Y. Wang, T.R. Ward, J. Wilhelmy, E.A. Winzeler, Y. Yang, G. Yen, E. Youngman, K. Yu, H. Bussey, J.D. Boeke, M. Snyder, P. Philippsen, R.W. Davis, M. Johnston, Functional profiling of the Saccharomyces cerevisiae genome, Nature 418 (2002) 387–391.
Karen M. Lee, Matthew J. O’Connell ∗ are in the Department of Oncological Sciences, Mount Sinai School of Medicine, 1 Gustave L. Levy Place, New York, NY 10029, USA ∗ Corresponding
author. Tel.: +1 212 659 5468; fax: +1 212 987 2240. E-mail address: [email protected] (M.J. O’Connell)