Báo cáo khoa học: Meiosis and small ubiquitin-related modifier (SUMO)conjugating enzyme, Ubc9 potx

REVIEW ARTICLE Meiosis and small ubiquitin-related modifier (SUMO)- conjugating enzyme, Ubc9 Kengo Sakaguchi, Akiyo Koshiyama and Kazuki Iwabata Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, Chiba, Japan Introduction Small ubiquitin-related modifier (SUMO) modification, known as sumoylation is a post-translational protein modification like ubiquitination, and appears to play important roles in many diverse processes [1–13]. SUMO family proteins and ubiquitin are similar in terms of both structure and the enzymatic reactions Keywords DNA polymerase; Lim15 ⁄ Dmc1; meiosis; PCNA; Rad51; SUMO; sumoylation; topoisomerase II; Ubc9 Correspondence K. Sakaguchi, Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda-shi, Chiba-ken 278, Japan Fax: +81 471 23 9767 Tel: +81 471 24 1501 (ext. 3409) E-mail: kengo@rs.noda.tus.ac.jp (Received 16 March 2007, revised 22 May 2007, accepted 30 May 2007) doi:10.1111/j.1742-4658.2007.05905.x In this review, we describe the role of a small ubiquitin-like protein modi- fier (SUMO)-conjugating protein, Ubc9, in synaptonemal complex forma- tion during meiosis in a basidiomycete, Coprinus cinereus. Because its meiotic cell cycle is long and naturally synchronous, it is suitable for molecular biological, biochemical and genetic studies of meiotic prophase events. In yeast two-hybrid screening using the meiotic-specific cDNA lib- rary of C. cinereus, we found that the meiotic RecA homolog CcLim15 interacted with CcUbc9, CcTopII and CcPCNA. Moreover, both TopII and PCNA homologs were known as Ubc9 interactors and the targets of sumoylation. Immunocytochemistry demonstrates that CcUbc9, CcTopII and CcPCNA localize with CcLim15 in meiotic nuclei during leptotene to zygotene when synaptonemal complex is formed and when homologous chromosomes pair. We discuss the relationships between Lim15 ⁄ Dmc1 (CcLim15), TopII (CcTopII), PCNA (CcPCNA) and CcUbc9, and subse- quently, the role of sumoylation in the stages. We speculate that CcLim15 and CcTopII work in cohesion between homologous chromatins initially and then, in the process of the zygotene events, CcUbc9 works with factors including CcLim15 and CcTopII as an inhibitor of ubiquitin-mediated deg- radation and as a metabolic switch in the meiotic prophase cell cycle. After CcLim15–CcTopII dissociation, CcLim15 remains on the zygotene DNA and recruits CcUbc9, Rad54B, CcUbc9, Swi5-Sfr1, CcUbc9 and then CcPCNA in rotation on the C-terminus. Finally during zygotene, CcPCNA replaces CcLim15 on the DNA and the free-CcLim15 is probably ubiquiti- nated and disappears. CcPCNA may recruit the polymerase. The idea that CcUbc9 intervenes in every step by protecting CcLim15 and by switching several factors at the C-terminus of CcLim15 is likely. At the boundary of the zygotene and pachytene stages, CcPCNA would be sumoylated. CcUbc9 may also be involved with CcPCNA in the switch from the repli- cative polymerase being recruited at zygotene to the repair-type DNA polymerases being recruited at pachytene. Abbreviations DSB, double-strand break; SC, synaptonemal complex; SSB, single-stranded break; SUMO, small ubiquitin-related modifier. FEBS Journal 274 (2007) 3519–3531 ª 2007 The Authors Journal compilation ª 2007 FEBS 3519 underlying their conjugation [10,11]. Furthermore, the enzymes involved in SUMO conjugation have sequences with similarities to their counterparts that mediate ubiquitin conjugation [5]. However, both sum- oylation and ubiquitination have distinct nonoverlap- ping functions [1–13]. The functions of sumoylation depend on the target proteins. A comprehensive survey of sumoylated pro- teins was recently performed by Wykoff and O’Shea [14]. Utilizing a collection of epitope-tagged yeast strains and immunoprecipitation of a large fraction of the proteome, they developed a novel approach for the identification of sumoylated proteins. At least 82 pro- teins were found to be candidate SUMO targets, inclu- ding many of low abundance. Based on their results, it is noteworthy that it is not only control processes of chromosome segregation and cell division, DNA repli- cation and repair, nuclear protein import, protein targeting to and formation of certain subnuclear struc- tures that involve sumoylation, but processes involved in the mammalian inflammatory response and plant flowering time have also been described as involving this protein modification. Since the discovery of SUMO about 10 years ago many excellent reviews with detailed discussions of SUMO research have been published [1–13]. To bor- row Ju ¨ rgen Dohmen’s phrase [10], ‘these reviews attempt to summarize the current status of the rapidly increasing knowledge of the mechanisms and functions of SUMO systems in various eukaryotic model organ- isms with an emphasis on the enzymes mediating SUMO conjugation and deconjugation. A few insight- ful examples point to one mode in which sumoylation is antagonistic to ubiquitination for some substrates, and to another mode in which sumoylation is either required for protein interaction or inhibitory to it.’ On the whole, the situation is similar in 2007. Because the roles of sumoylation are so many, it is difficult to pre- sent a summary of the whole field in anything other than a partly chaotic manner. Therefore, we would like to summarize one aspect of the field, namely meiosis and sumoylation, because we recently found sumoyla- tion of a meiosis-specific RecA homolog, Lim15 ⁄ Dmc1, via interaction with the SUMO-conjugating enzyme Ubc9 at a particular stage of meiosis [15]. Lim15 ⁄ Dmc1 is a most important key protein in the meiotic cell cycle, particularly during the stages when homologous chromosomes pair and recombine. Meiosis In meiosis, as is well known, homologous chromo- somes are paired and recombined during meiotic pro- phase I (also called synapsis) and then segregated into tetrads [16]. Prophase I is divided into five stages, namely leptotene, zygotene, pachytene, diplotene and diakinesis. Chromosomes condense from the dispersed state typical of interphase during early meiotic pro- phase (leptotene) to form long thin threads and each acquires a proteinaceous axial core to which the two sister chromatids are attached. Then, homologous chromosomes become aligned during zygotene and form the synaptonemal complex (SC), a proteinaceous framework assembled between homologous chromo- somes, and required for the subsequent maintenance of synapses. SC polymerization ensures continuous and stable association along the homologous chromosomes throughout pachytene, during which time completion of reciprocal strand-exchange events takes place [16]. At pachytene, nonsister chromatids of the completely paired chromosomes recombine by forming chiasmata which become visible during diplotene. This is followed by two cell divisions, namely reductional segregation of homologous chromosomes and equational segrega- tion of sister chromatids, resulting in four gametes. In Saccharomyces cerevisiae, SC polymerization initi- ates at sites undergoing meiotic recombination and requires the activities of an enzyme induced by double- strand breaks (DSBs) and strand-exchange proteins [17]. It should also be noted that the zygotene and pachytene stages, which are the most important pro- phase stages when homologous chromosomes pair and recombine, tend to be intermixed. By contrast, in higher plants and mammals, the SC forms exactly at zygotene, and when this is finished recombination begins at pachytene, as ascertained by cytogenetic research in the 20th century. The end of SC formation must be an initiation signal of recombination [16]. Moreover, in studies of meiosis using higher plant (lily) and mouse spermatocytes, the initiation of pachytene DNA recombination was shown to be related to single-stranded breaks (SSBs) rather than DSBs [16,18–20]. Since the 1980s, there have been few new insights into the role of SSBs in pachytene DNA recombination. However, meiotic recombination by nicks and ⁄ or gaps in Schizosaccharomyces pombe has been reported [21]. It was proposed that meiotic recombination could be initiated by DSBs, as well as by non-DSB lesions, such as nicks and gaps. At pre- sent, Spo11 is identified as the protein that catalyses DSBs and is widely conserved in eukaryotes [22]. Prior to the DSB-repair model of Resnick [23], it was sug- gested that DNA nicks or gaps induced meiotic recom- bination [24]. These SSB recombination models lost favor after the publication of another DSB repair model by Szostack et al. [25], the observation of Meiosis and Ubc9 K. Sakaguchi et al. 3520 FEBS Journal 274 (2007) 3519–3531 ª 2007 The Authors Journal compilation ª 2007 FEBS meiosis-specific DSBs at a recombination hotspot in S. cerevisiae [26], and the identification of Spo11 as a DSB enzyme [27]. Despite such differences between yeast and other eukaryotes in the meiotic cell-cycle pattern, the molecular machinery of meiotic DNA recombination is likely to be conserved. Meiotic DNA recombination is composed of several steps. First, meiosis-specific DSBs or SSBs appear to be introduced and this is followed by formation of single-stranded DNA. The formed single-stranded ends then invade regions of homology in the other allele. After strand invasion and initial repair synthesis, the crossover and the noncrossover pathways diverge [28]. These reactions are mediated by the coordinated activ- ity of various proteins including RecA-like protein, an ATPase playing a central role in the strand-exchange reaction [29]. In eukaryotes, Lim15 ⁄ Dmc1 and RAD51 have been identified as RecA homologs. Whereas RAD51 is expressed in both meiotic and somatic cells and is required in the DNA repair reaction, Lim15 ⁄ Dmc1 expression is restricted to meiotic cells [29]. Lim15 ⁄ Dmc1 has a critical role in meiotic chro- mosome events, but its molecular functions and differ- ences from Rad51 are not well understood [30]. In this review, we would like to discuss CcLim15 in terms of its function and interactions. In particular, we would like to discuss the relationship with CcTopII [31], CcPCNA [32] and CcUbc9 [15] at the meiotic prophase of Coprinus cinereus and subsequently, the role of sumoylation at zygotene and pachytene. The role of sumoylation in meiosis is still largely unclear except for its involvements in the synaptonemal com- plex [33–35], chromosome segregation [36] and sperma- togenesis [37–40]. We propose CcUbc9-mediated sumoylation as a novel regulator of meiotic chromo- some paring and recombination. Biomaterials for meiotic studies In the biochemical study of meiosis, important consid- eration should be paid to the choice of biomaterials, because meiosis is a distinct part of sexual develop- ment which occurs only at a certain point in time. The meiotic cell cycle must be synchronous and usable over a year for such meiotic study. Lilium microsporocytes during the 1980s and before [16,41–47], and S. cerevisi- ae more recently [17], have mainly been used for the studies. The former system is not usable over a year and has a genome that is too large for modern genet- ics. The latter system is very convenient for genetic engineering but differs in the process of zygotene and pachytene, two crucial stages for pairing and recombi- nation, from the other eukaryotes. To avoid such problems, we have long used a basidi- omycete, C. cinereus, as a model organism in studies of sexual development and meiosis. Despite the rapid morphogenesis of its multicellular structure, its meiotic cell cycle is long [48–50] and meiotic cells develop syn- chronously after photoinduction. Each fruiting cap is extremely rich in meiotic cells at the same stage [48–50]. Moreover, as is the case for yeasts, the gen- ome project for C. cinereus has been completed and the genome is not so large. C. cinereus has been analyzed using forward genetics approaches because of the ease of mutagenesis by transformation of an asexual spore of the haploid mycelium known as oidium [51–55]. We have also suc- ceeded in performing gene repression by double-stran- ded RNA-mediated gene silencing as an alternative reverse genetics technique in C. cinereus [56]. Zolan et al. also reported molecular analyses of the C. cinere- us meiotic recombination process [57–62]. By taking advantage of the properties of this organ- ism as described above, we succeeded in establishing cDNA libraries from mRNAs from C. cinereus meiotic cells at leptotene, zygotene and pachytene and have studied 3R (DNA replication, repair, recombination) enzymes from each stage [31,63–69]. We found that transcripts of the 3R enzymes as described below are abundant at meiotic prophase I and we have previ- ously discussed the roles of the 3R enzymes during meiosis. The enzymes are PCNA (CcPCNA) [63], DNA ligase I [64], DNA ligase IV [65], Flap endonuc- lease-1 [66], Lim15 ⁄ Dmc1 (CcLim15) [67], Rad51 (CcRad51) [68,69] and DNA topoisomerase II (CcTop- II) [31]. We have also investigated the C. cinereus DNA polymerase group in the database, and know that the C. cinereus genome has genes homologous to DNA polymerases a, d, e, f, k and l at a minimum and lacks genes homologous to DNA polymerase b and g [70–72] (A. Sakamoto et al., unpublished results). Meiocytes at zygotene express at a minimum DNA polymerase a (CcPola), k (CcPolk) and l (CcPoll) and pachytene cells express CcPolk and CcPoll [72] (A. Sakamoto et al., unpublished results). With regard to DNA polymerase d, e and f, their expression has not been examined in C. cinereus meiotic prophase. According to biochemical studies of lily meiosis, a small amount of DNA replicates at zygotene (zygotene DNA synthesis) and repair synthesis of DNA occurs at pachytene (pachytene DNA synthesis) [16,41,45]. Zygotene and pachytene DNA syntheses are thought to be the molecular basis of SC formation and recom- bination repair, respectively, and play a role in the progression of meiosis [16,19,46]. As is well known, K. Sakaguchi et al. Meiosis and Ubc9 FEBS Journal 274 (2007) 3519–3531 ª 2007 The Authors Journal compilation ª 2007 FEBS 3521 Pola is involved in replication, and Polk and Poll are repair-type polymerases [73,74]. Taking the meiotic expression patterns of these DNA polymerases and the two sequential DNA syntheses into consideration, CcPola may contribute to zygotene DNA synthesis, and CcPolk and CcPoll may be involved in pachytene DNA synthesis in C. cinereus. Lim15 ⁄ Dmc1 and related factors in meiosis In a series of studies, we investigated RecA homologs. Two homologs of RecA-like protein, Lim15 ⁄ Dmc1 and Rad51 are known to be present in eukaryotes. According to Stassen et al. [58], phylogenetic analyses of eukaryotic RecA homologs reveal gene duplication early in eukaryotic evolution giving rise to two puta- tively monophyletic groups of RecA-like genes. Excep- tionally, higher plants possess one more additional RecA-like protein, RadA [75]. As in other eukaryotes, with the exception of higher plants, we found Lim15 ⁄ Dmc1 and Rad51 homologs in C. cinereus. cDNA cloning and characterization of CcLim15 and CcRad51 have been performed and both have been shown to be expressed in meiotic prophase, especially late leptotene to early zygotene [67,69]. CcLim15 is transcribed only in meiosis [67], whereas CcRad51 is present in both somatic and meiotic cells [58]. In two- hybrid assays and in vitro protein–protein interaction assays, both CcLim15 and CcRad51 homotypically interact via their C-terminal domains [68]. As des- cribed previously [69], these two proteins exist in mei- otic nuclei predominantly during late leptotene to zygotene. According to Lilium microsporocyte studies, two different types of DNA synthesis occur at zygo- tene and pachytene [16,41,45]. Because these DNA synthetic processes appear to be for juxtaposing homologous DNAs at zygotene and for exchanging between homologous DNAs at pachytene, both proces- ses would independently require D-loop formation. If this is the case, neither CcLim15 nor CcRad51 would be involved in recombination between homologous chromosomes at pachytene but rather in strand arrangement (or SC formation) at zygotene. In order to understand the roles of these two RecA homologs in meiosis, meiotic protein factors that inter- act with them should be looked for. Recent studies imply that Rad51 interacts with various nuclear factors such as RPA [76,77], Rad52 [78–80], Rad54 [81–83], BRCA2 [84–87], the Rad55–Rad57 heterodimer [88] and others. By contrast, only a few proteins are known to interact with Lim15 ⁄ Dmc1. The Rad54 homolog proteins, Rdh54 ⁄ Tid1 in yeast and Rad54B in human, interact with Lim15 ⁄ Dmc1 as well as Rad51. In S. cerevisiae Rdh54 ⁄ Tid1 is involved in crossover inter- ference [89,90], while Rad54B in human enhances Lim15 ⁄ Dmc1-mediated DNA-strand exchange. The Mei5–Sae3 complex has also been identified as a new assembly factor for meiotic-specific Lim15 ⁄ Dmc1 in S. cerevisiae [91], while the Swi5–Sfr1 complex, the Mei5–Sae3 homolog in Schizosaccharomyces pombe, physically interacts with both RecA homologs [92]. According to biochemical studies of Swi5–Sfr1, the complex stimulates strand exchange mediated by Lim15 ⁄ Dmc1, which indicates that Swi5–Sfr1 also acts as a Lim15 ⁄ Dmc1 mediator [92]. In addition, the Hop2–Mnd1 complex functionally associates with both RecA homologs and stimulates D-loop formation and strand exchange in yeast and mammals [93–95]. Fur- ther screening for Lim15 ⁄ Dmc1 interactors would shed light on the machinery of meiotic chromosome paring and recombination. This concept prompted us to screen for such proteins. As a result, we were successful in finding novel CcLim15-interacting proteins, namely DNA topoisom- erase II (CcTopII) [31], PCNA (CcPCNA) [32] and Ubc9 (CcUbc9) [15], through yeast two-hybrid screen- ing using the meiotic stage-specific cDNA library of C. cinereus. This led us to find the possible involve- ment of sumoylation in meiosis. Ubc9 is the E2 type enzyme for SUMO conjugation to targets. In C. cine- reus, CcLim15 is a target protein of sumoylation both in vivo and in vitro, via interaction with CcUbc9. Inter- estingly, another RecA protein Rad51 was also repor- ted to associate with Ubc9, particularly in pachytene chromosomes in mouse spermatocytes [96] and was shown to be sumoylated in vitro [97]. Furthermore, both TopII and PCNA also interact with Ubc9 and are well known targets of sumoylation [98,99]. These properties add clarity to what is known about the con- trol of the meiotic chromosome events through post- translational modifications such as sumoylation. The role of Ubc9 in meiosis Ubc9 in mitosis Ubc9 is known as a SUMO-conjugating enzyme (E2), which receives activated SUMO (SUMO-GG) from the Uba2 subunit of SUMO-activating enzyme (E1) and forms a SUMO–Ubc9 intermediate in the sumoy- lation pathway [2,7,9–13]. Crystal structure analysis showed Ubc9 to have a domain similar to the core domain of ubiquitin-conjugating enzymes [100,101]. The surface of Ubc9, however, is positively charged by two sequence insertions, while the corresponding Meiosis and Ubc9 K. Sakaguchi et al. 3522 FEBS Journal 274 (2007) 3519–3531 ª 2007 The Authors Journal compilation ª 2007 FEBS regions in ubiquitin E2 enzymes have negative or neut- ral charge [102–104]. Thus, Ubc9 binds to SUMO but not ubiquitin. Furthermore, Ubc9 was reported pre- viously to interact with many sumoylation targets. Within the hydrophobic groove of Ubc9, Asp127 appears to engage in hydrogen bonding with a Lys residue within the sumoylation consensus motif -Y-K-X-E ⁄ D-, where Y is a large hydrophobic amino acid and X is any amino acid [103,105]. Therefore, complexes of Ubc9–Lim15 ⁄ Dmc1, Ubc9–TopII and Ubc9–PCNA may be intermediates in the production of SUMO–Lim15 ⁄ Dmc1, SUMO–TopII and SUMO– PCNA complexes, respectively. It is known that sumoylated proteins can escape from immediate ubiqu- itin-dependent degradation when both modifications target the same lysine within the substrate [1–13]. Taken together with the manner of the substrate recog- nition by Ubc9, the Ubc9-conjugated intermediates may also be protected from ubiquitination. However, in addition to a role in mediating sumoylation, another role of Ubc9 has been reported. Binding of Ubc9 to a nuclear-localization signal contributes to nuclear local- ization of the homeobox protein Vsx1 [106]. Ubc9 in meiosis In meiosis, a few roles of Ubc9 and sumoylation were known. Analysis of the Drosophila Ubc9 mutant less- wright has implicated SUMO modification in the dis- junction of homologous chromosomes in meiotic M1 [36]. Ubc9 was shown to localize on meiotic chromo- somes in S. cerevisiae and mice and bind to the consti- tutive proteins of the synaptonemal complex [33,96]. Recently, Cheng et al. clarified the relationship between SC formation and Ubc9-mediated sumoyla- tion [35]. In S. cerevisiae Zip3, a protein involved in the initiation of SC formation, is a SUMO E3 ligase [33,35]. In a Zip3-lacking mutant, a polycomplex was formed instead of the SC. Moreover, their results sug- gested that Zip1, a building block of the SC, binds to SUMO-conjugated proteins. These interactions may be important for homology sorting during early prophase, as well as in triggering extensive SC polymerization. As described, meiosis is a special cell cycle associ- ated with homologous chromosome pairing and recombination [16]. In mitosis, TopII is sumoylated in a cell-cycle-controlled manner indicating that SUMO modification serves to synchronize the function of many of its substrates with the mitotic cell cycle [107,108]. By contrast, sumoylated PCNA has been observed in the S phase but not in G 2 ⁄ M [99]. We found that CcLim15, CcRad51, CcTopII, CcPCNA and CcUbc9 are all present at meiotic prophase in C. cinereus and that each of CcLim15, CcTopII, CcPCNA and CcRad51 has the potential to interact with CcUbc9. Moreover, CcLim15 can also independ- ently interact with either CcTopII or CcPCNA at zygotene [31,32]. CcUbc9 is expressed from the premeiotic S phase through the tetrad stages, suggesting that CcUbc9 acts in concert with many of the meiotic events [15]. Expression of CcPCNA temporarily becomes most prominent at the transition between leptotene and zyg- otene, although small amounts of CcPCNA are con- stantly detected in nuclei from the premeiotic S phase through the tetrad stages [63]. In contrast, CcLim15 and CcRad51 are expressed from late leptotene to early zygotene with CcLim15 and CcRad51 proteins present at the same stages, then rapidly disappearing by early pachytene [67,69]. CcTopII transcripts begin to accu- mulate during late leptotene, slightly earlier than the CcLim15 transcript, becoming most abundant at early zygotene [31]. Thus, the interaction of CcLim15 with each of CcTopII, CcPCNA and CcUbc9 is always lim- ited around the transition between leptotene and zygo- tene, which is the point at which the homologous chromosomes pair (zygotene). Taking the localization during meiotic prophase I and interactions of these proteins into consideration, CcLim15, CcRad51, CcTopII and CcPCNA may be the meiotic target proteins of sumoylation. Because of the mechanism of Ubc9-mediated SUMO conjugation, analysis of the interaction between CcUbc9 and each of CcLim15, CcRad51, CcTopII and CcPCNA would give a clue to homologous chromosomes pairing in relation to sumoylation. For example, in late leptotene or early zygotene, which of CcPCNA or CcUbc9 inter- acts the earliest with CcLim15 or is it a simultaneous interaction? CcLim15–CcUbc9 complex in meiosis CcLim15 is distributed on the chromosomes in the nuclei at meiotic prophase, and becomes most pro- minent in late leptotene to zygotene [15,31,69]. A CcLim15-repressed strain shows defects in SC forma- tion and abnormal homologous chromosome pairing during meiosis [56]. CcLim15 is not detected after the late pachytene stages at all, whereas CcUbc9 is con- stant throughout meiosis, indicating that the CcLim15–CcUbc9 complex must occur and separate only for a limited period, namely during late leptotene to zygotene [15]. CcRad51 is also likely to behave in the same way, because its expression profile is the same as CcLim15 [69]. Therefore, the meiotic expression data for CcUbc9 indicates that chromosome paring, K. Sakaguchi et al. Meiosis and Ubc9 FEBS Journal 274 (2007) 3519–3531 ª 2007 The Authors Journal compilation ª 2007 FEBS 3523 which is closely related to the function of CcLim15 and ⁄ or CcRad51, may be partly controlled by SUMO- mediated regulation. Meanwhile, each of the CcUbc9 complexes may function independently in sumoylation. As described above, Ubc9 can conjugate to the lysine residue within a sumoylation consensus motif in the sumoylation pathway [105]. This conjugation may inhi- bit ubiquitin-mediated proteolysis. RecA homologs, Lim15 ⁄ Dmc1 and Rad51, promote strand exchange with a donor DNA in an ATP-dependent manner [29]. CcLim15 is abundantly distributed on the chromo- somes in late leptotene to zygotene. CcRad51 is also distributed in a similar way to CcLim15 (unpublished data). The roles of CcLim15 and CcRad51 are likely to overlap but be independent of each other. Both pro- teins are likely to be required at zygotene. It was sug- gested that the CcUbc9 complexes are protected from degradation by ubiquitination at zygotene until strand arrangement between the homologous chromatins is complete (Fig. 1). There are two potential consensus motifs within CcLim15, the sequences surrounding Lys78 (-AKVE-) and Lys223 (-DKDF-). Although it is not clear whe- ther Lys223 is the target site, the sumoylation target sites are in the C-terminal part of CcLim15 (amino acids 105–347), which contains the ATPase domain [15]. Intriguingly, the sumoylation target domain of CcLim15 coincides with the domain that binds to CcUbc9 [15]. This correlation suggests that CcLim15 may be protected from degradation in the form of a CcLim15–CcUbc9 intermediate before the regulation of the functional activity by sumoylation. CcLim15 and CcTopII Previously the only known role for TopII in meiosis was in untying the entangling between chromatins, mainly at M1 [109,110]. Immunocytochemistry of C. cinereus meiotic cells shows that CcTopII is locali- zed on chromosomes in nuclei during the premeiotic S phase and also throughout the meiotic divisions, and that CcTopII signal culminated from leptotene to pachytene [31]. Furthermore, CcTopII and CcLim15 colocalized during leptotene and zygotene, suggesting that the CcLim15–CcTopII complex may be related to specific events in early stages of meiosis [31]. As reported previously, CcLim15 and CcTopII influ- ence the activities of each other. CcLim15 can potently activate the relaxation ⁄ catenation activity of CcTopII in vitro, but CcTopII suppresses CcLim15-dependent strand-transfer activity [31]. CcLim15’s DNA-depend- ent ATP digestion potential was strongly enhanced by the CcTopII protein with ssDNA. The ATPase activity of DNA topoisomerase II is suppressed by using ssDNA as the cofactor. We also measured DNA- dependent ATPase activity of CcTopII using double- stranded M13 DNA as a cofactor. Although CcLim15 itself had subtle DNA-dependent ATPase activity in the presence of 1 mm Ca 2+ , the ATPase activity of CcTopII was significantly inhibited by addition of CcLim15 in the presence of 1 mm Ca 2+ . The interac- tion between CcLim15 and CcTopII could easily form during meiotic pairing between homologous chromo- somes at the boundary of leptotene to zygotene, i.e. at the beginning of SC formation [31]. Therefore, the Fig. 1. Model of the sequential molecular machinery involved in the meiotic chromo- some events from leptotene to zygotene. Several steps in meiotic prophase are shown schematically. Initially Lim15 inter- acts with TopII and homologous chromatins initiate pairing. After dissociation of TopII, Lim15 remains on DNA and recruits Ubc9, Rad54B, Swi5–Sfr1 and PCNA. After PCNA replaces Lim15 on the zygotene DNA, the free-Lim15 disappears via ubiquitin-mediated degradation. The zygotene DNA is synthes- ized by Pola. At the end of zygotene PCNA is sumoylated and recruits Poll or ⁄ and Polk. The pachytene DNA synthesis is occurred by Poll or ⁄ and Polk. Meiosis and Ubc9 K. Sakaguchi et al. 3524 FEBS Journal 274 (2007) 3519–3531 ª 2007 The Authors Journal compilation ª 2007 FEBS zygotene role of CcTopII may differ from the role in M1, when it appears to control chromosome disjunc- tion and pachytene chromosome segregation. Interestingly, the C-terminus (amino acids 1066– 1569) of CcTopII as well as CcUbc9 binds to the C-terminus (amino acids 104–345) of CcLim15, indica- ting that CcTopII and CcUbc9 share the binding domain within CcLim15 [15,31]. In early meiotic pro- phase, either CcTopII or CcUbc9 is likely to interact at a similar if not the same site at the C-terminus of CcLim15. The question is therefore raised as to which of CcTopII or CcUbc9, binds to CcLim15 earlier? It seems, although the evidence is weak (stage-dependent expression order), that the CcTopII–CcLim15 interac- tion occurs at late leptotene to early zygotene [31] while the CcLim15–CcUbc9 interaction appears to occur throughout the whole of zygotene [15]. Initially the C-terminus of CcTopII binds to the C-terminus of CcLim15, and then with progression through the stages within zygotene, CcUbc9 may replace CcTopII on the C-terminus of CcLim15 (Fig. 1). The released CcTopII molecules may also simultaneously be sumo- ylated by CcUbc9. In meiosis, whether TopII is sumoylated or not is as yet unclear. However, the sumoylation of CcTopII may occur through meiotic prophase and at M1, because of its coexistence with CcUbc9. Three roles of sumoylated CcTopII could be considered. First, sumoylation may contribute to the stability of CcTopII protein. Accord- ing to our studies, CcTopII would be involved in chro- mosome pairing by interacting with CcLim15. Even if CcTopII is released by CcLim15 and becomes unstable, it may be protected by the immediate sumoylation from ubiquitin-mediated degradation. Second, sumoylated CcTopII during the zygotene stage may be related to SC initiation. In S. cerevisiae, SC formation is controlled by sumoylation during assembling proteins and chro- mosomes [34]. TopII is a candidate for a SUMO-conju- gated protein which binds to Zip1, a building block of SC [33,35]. Sumoylated CcTopII may interact with the Zip1 homolog and form the foundation of SC. A third possibility is that sumoylation of CcTopII may be rela- ted to chromosome segregation at M1. In mitosis, TopII was found to be SUMO-modified and sumoylation of TopII inhibits its ability to promote centromeric cohe- sion [108]. Sister chromatid cohesion at the centromere is suggested to be specifically regulated by SUMO-1 modification of TopII. Meanwhile, the disjunction of homologous chromosomes in meiotic M1 occurred in the Drosophila ubc9 mutant lesswright [36]. Thus, as seen in mitosis, CcTopII itself is also involved in untying DNA entangling and may be inhibited in function by sumoylation at M1. From a series of biochemical studies, we propose a hypothesis about the sequential molecular machinery related to the meiotic chromosome events from lepto- tene to zygotene. Initially, CcLim15 finds and binds to CcTopII to bring homologous chromatins closer together. Then some of the CcLim15–CcTopII com- plex are replaced by CcUbc9 resulting in a division into CcLim15–CcUbc9 and CcTopII–CcUbc9 com- plexes. The SUMO-mediated CcTopII may prepare to form the SC. Furthermore, CcLim15–Ccubc9 may need to be protected from proteolysis for it to still function in the next step. In the zygotene process, Rad54B, a member of the Swi2⁄ Snf2 family of DNA translocases and homolog of yeast Rdh54 ⁄ Tid1, pos- sesses the ability to generate negative supercoils in duplex DNA, leads to the transient opening of the DNA strands in the duplex [111–113] and interacts with both Rad51 and Lim15 ⁄ Dmc1. The CcLim15– CcUbc9 complex may recruit Rad54B and CcUbc9 may be replaced by Rad54B in the complex. In recent studies, Rad54B bound to the terminus of the Lim15 ⁄ Dmc1–ssDNA complex and caused stimulation of Lim15 ⁄ Dmc1-mediated DNA-strand exchange [113]. The CcLim15–Rad54B complex may stabilize the CcLim15–ssDNA complex and begin to pair homolog- ous zygotene DNAs (Fig. 1). Shortly after, Rad54B is released from the complex by recruitment of CcUbc9, again, to protect CcLim15 on the DNA from ubi- quitin-mediated proteolysis (Fig. 1). Next, the new CcLim15–CcUbc9 complex recruits the pairing elonga- tion factors (Swi5–Sfr1) with replacement of CcUbc9, and the CcLim15–Swi5–Sfr1 complex elongates the SC (Fig. 1). Even if homologous chromosomes pair incor- rectly, CcLim15–Swi5–Sfr1 homology searching could contribute to correct pairing [92]. Biochemical studies using yeast have provided evidence that Swi5–Sfr1 sti- mulates the strand exchange activity of Lim15 ⁄ Dmc1 [92]. Finally at zygotene, the SC begins to dissociate (Fig. 1). It is well-known that purified Hop2–Mnd1 stimu- lates the strand invasion activity of Dmc1 in vitro in yeast, mouse and human [93,94,114]. However, Hop2– Mnd1 has strand-exchange activity itself [115] and is required at pachytene according to fluorescence in situ hybridization of spread chromosomes [116]. Although the interaction between Hop2 and Mnd1 in yeast and human was easily detected, they failed to detect any measurable interaction between Hop2–Mnd1 and Rad51 or Lim15 ⁄ Dmc1 [95,114,116]. Hop2–Mnd1 appears to be able to form a complex and localize to chromosomes independent of Lim15 ⁄ Dmc1, suggesting that it might be required for the strand invasion pro- cess at pachytene. K. Sakaguchi et al. Meiosis and Ubc9 FEBS Journal 274 (2007) 3519–3531 ª 2007 The Authors Journal compilation ª 2007 FEBS 3525 Therefore, one of the roles of CcUbc9 is to protect the C-terminus of CcLim15 on the zygotene DNA from ubiquitination, since the dissociation of each complex may lead to CcLim15 degradation triggered by a specific proteolytic pathway such as the ubiqu- itin–proteasome pathway (Fig. 1). CcUbc9 is unable to ubiquitinate this site. Another role for CcUbc9 maybe to serve to synchronize the zygotene cell cycle at each point (Fig. 1), as SUMO modification serves to syn- chronize the function of many substrates with the mitotic cell cycle [10]. CcPCNA–CcUbc9 complex in meiosis In our previous study, CcPCNA was indicated to inter- act with CcLim15. CcPCNA is detected in nuclei from the premeiotic S phase through the tetrad stages [32,63]. Importantly, a significant proportion of CcLim15 and CcPCNA colocalizes on chromosomes from leptotene to zygotene. Unlike CcTopII, however, no enhancement of CcLim15-dependent strand transfer or DNA-dependent ATPase activities by CcPCNA have been found [32]. We suggest that the strand-trans- fer reaction by CcLim15 and the association between CcLim15 and CcPCNA may be temporally separable events in vivo. Furthermore, CcLim15 binds to the N- terminus of CcPCNA and CcPCNA binds to the C-ter- minus of CcLim15 [32], suggesting that CcTopII and CcPCNA compete on the C-terminus of CcLim15. One possible hypothesis is that CcTopII and CcPCNA would alternately bind to the C-terminus of CcLim15 at late leptotene to early zygotene, and each complex would function for the cohesion (CcTopII–CcLim15) and chromosome rearrangement (CcPCNA–CcLim15) (Fig. 1). Because chromosome rearrangement is thought to accompany zygotene DNA replication [16], CcPCNA may be involved in recruiting the replication polymerase (Fig. 1). PCNA is known to interact with Ubc9 and is sumo- ylated or ubiquitinated at Lys164. Ubiquitination of PCNA is involved in the DNA-damage-tolerance path- way, although the function of sumoylated PCNA is as yet unclear [117]. There are a few interesting reports [118–120] that SUMO-modified PCNA may inhibit Rad51-mediated DNA recombination after recruiting SRS2, which then leads to gross chromosome rear- rangement. Genetic evidence also suggests that sumoy- lation of PCNA on Lys164 inhibits Rad52-dependent recombinational repair, which may reduce the risk of chromosome rearrangements during the S phase [121]. It has not yet been examined whether PCNA is sumo- ylated in meiosis. In C. cinereus, however, because CcPCNA and CcUbc9 exist together in meiotic nuclei, the interaction between CcPCNA and CcUbc9 and sumoylation may occur at a certain point of meiosis. It is suggested that sumoylation of CcPCNA may prevent premature chromosomal recombination from late lep- totene to early zygotene, until the end of the strand arrangement between homologous chromatins by CcLim15. At the beginning of zygotene, the possible role of the CcPCNA–CcLim15 interaction may be to recruit free CcPCNA onto the zygotene CcLim15–CcTopII cohesion region with CcPCNA replacing CcTopII (Fig. 1). Alternatively, through sumoylation immedi- ately after cohesion, CcTopII–CcLim15 may separate into CcUbc9–CcTopII and CcLim15–CcUbc9, with CcLim15–CcUbc9 left on the cohesion regions leading to the recruitment of CcPCNA into the regions (Fig. 1). In our model, initially CcLim15–CcTopII occurring at late leptotene is involved in the coherence of the homologous chromatins at the boundary and after dissociation, CcLim15 or CcLim15–CcUbc9 remain on the zygotene DNA to recruit CcPCNA at early zygotene, and finally, the nonmodified CcPCNA is left there (Fig. 1). Then, a replicative-type of DNA polymerase, for example CcPola, may be recruited in order to replicate the zygotene DNA sequence (Fig. 1). Of course, some CcLim15 must be left for binding to other factors as described above, and alternatively be used for various events at zygotene. With progression of the zygotene stage, CcTopII and CcPCNA on the complexes may replace CcUbc9 and be sumoylated. For the next related-event to occur, each of CcLim15, CcTopII and CcPCNA has to be kept from the ubi- quitin-mediated degradation for a while (Fig. 1). As is well known, PCNA is closely related to DNA polymerases. And the modification states of PCNA eli- cit different responses and select the types of DNA polymerases. Unmodified PCNA acts as a processivity clamp for replicative DNA polymerases d and e [122]. Monoubiquitination of PCNA is induced by DNA damage and activates DNA polymerases f and g for translesion synthesis [123]. From S phase studies, it has been proposed that SUMO-modified PCNA may recruit DNA polymerase f in order to overcome repli- cation fork blocks not caused by DNA damage. These suggest that PCNA may play a role as a switchboard to shift DNA polymerases. Taking these observations into consideration, we would like to discuss the relationship between sumoy- lation of CcPCNA and meiotic DNA synthesis. At zygotene, no repair-type DNA synthesis is observed, but replication-type does occur [41,46]. Although PCNA is not modified during this stage in our model, it is unclear whether DNA polymerases d and e, which Meiosis and Ubc9 K. Sakaguchi et al. 3526 FEBS Journal 274 (2007) 3519–3531 ª 2007 The Authors Journal compilation ª 2007 FEBS are closely related to PCNA, are present in meiotic prophase (A. Sakamoto et al., unpublished results). In C. cinereus meiocytes CcPola is expressed at zygotene and its primase-lacking form is mostly functional, sug- gesting that this polymerase replicates the zygotene DNA sequence [70–72]. At the end of zygotene, SUMO conjugation of CcPCNA should occur after dissolution of the CcLim15–CcPCNA complex. 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REVIEW ARTICLE Meiosis and small ubiquitin-related modifier (SUMO)- conjugating enzyme, Ubc9 Kengo Sakaguchi, Akiyo Koshiyama and Kazuki Iwabata Department. zygotene PCNA is sumoylated and recruits Poll or ⁄ and Polk. The pachytene DNA synthesis is occurred by Poll or ⁄ and Polk. Meiosis and Ubc9 K. Sakaguchi et al. 3524