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SMC protein complexes and higher-order chromosome dynamics Tatsuya Hirano The structural maintenance of chromosome (SMC) family of proteins represents an expanding group of chromosomal ATPases that are highly conserved among Bacteria, Archaea and Eukarya. During the past year, significant progress has been made towards understanding the cellular functions and molecular activities of this new class of proteins. Emerging evidence suggests that eukaryotic SMC proteins form large protein complexes with non-SMC subunits and act as key components for a wide variety of higher-order chromosome dynamics.
Addresses Cold Spring Harbor Laboratory, PO Box 100, Cold Spring Harbor, New York, USA; e-mail:
[email protected]
Current Opinionin Cell Biology 1998, 10:317-322 http://biomednet.com/elecref10955067401000317 © Current Biology Ltd ISSN 0955-0674
Abbreviations SMC structural maintenance of chromosomes XCAP Xenopus chromosome-associated polypeptides RC-1 recombination complex-1 DPY dumpy
Introduction T h e structural maintenance of chromosomes (SMC) family was first defined in 1993 [1] through genetic analysis of the yeast gene product Smclp: mutations in Smclp resulted in a defect in the proper segregation of mitotic chromosomes. Subsequent identification of SMC proteins from several model organisms revealed that they are involved not only in mitotic chromosome dynamics, but also in a wide variety of chromosomal events, including dosage compensation and recombinational repair [2,3,4"]. Dosage compensation is a chromosome-wide regulatory process that equalizes expression of X-linked genes between males and females, despite their two-fold difference in X chromosome dose (for example in Caenorhabditiselegans, hermaphrodites [XX] reduce the level of transcripts from each of their X chromosomes to equalise X-chromosome gene expression with that of males [XO]). Recombinational repair is a mode of DNA repair mediated by homologous recombination. During the past year, we have witnessed an exciting series of biochemical and genetic studies that enhances our understanding of this new class of proteins [5°]. It has become increasingly clear that eukaryotic SMC proteins function as components of large protein complexes: combinatorial association of different SMC and non-SMC subunits produces a variety of complexes with diverse functions. In this review, I describe the cellular and biochemical functions of these SMC protein complexes,
and discuss how they might have evolved and acquired their structural and functional diversity. Classification of e u k a r y o t i c S M C proteins Initial attempts to classify SMC proteins gave partially inaccurate results, primarily because only limited sequence information was available [2,3]. T h e completion of the Saccharomycescerevisiaegenome project allowed reclassification of the SMC protein family, and eukaryotic members have now been grouped into four subfamilies (from SMC1- to SMC4-type proteins; Table I; [4"]). Besides the four authentic SMC proteins, the yeast genome contains two open reading frames encoding distantly related 'SMC-like' proteins: Rhcl8p, implicated in DNA repair [6], and YOL034Wp of unknown function. All these proteins share common structural motifs, including an amino-terminal nucleotide-binding motif, two central coiled-coil motifs, and a carboxy-terminal conserved sequence termed the DA-box [2,3,4"]. Protein c o m p l e x e s c o n t a i n i n g S M C subunits Condensins, chromosome condensation protein complexes Among the four SMC subfamilies, the function of the SMC2-type and SMC4-type proteins is best understood from biochemical and genetic analyses in several model organisms. In Xenopus laevis, XCAP-C (SMC4-type) and XCAP-E (SMC2-type) were originally identified as two of the major components of mitotic chromosomes assembled in cell-free extracts [7] (XCAP stands for Xenopus chromosome-associated polypeptides). Independently, SclI, the second most abundant component of the vertebrate chromosome scaffold, was shown to be an SMC2-type protein [8]. Recent purification of SMC proteins has revealed that XCAP-C and XCAP-E associate with each other in Xenopus egg extracts, forming two complexes that have sedimentation coefficients of 8S and 13S [9°']. The 8S form (termed 8S condensin) is a heterodimer of XCAP-C and XCAP-E whereas the 13S form (13S condensin) contains three additional subunits (Figure 1). 13S condensin is absolutely required for chromosome condensation. Depletion of 13S condensin from cell-free extracts using specific antibodies (immunodepletion) results in defects in chromosome condensation. Re-addition of purified 13S condensin to the depleted extract (add-back or reconstitution experiment) restores its condensation activity. T h e role of 8S condensin is unclear. Specific and stoichiometric association of SMC2- and SMC4-type proteins has been confirmed in the fission yeast Cut3p-Cutl4p complex [10"]; mutations in this complex result in a defect in condensation [11]. Although it remain's to be determined if this 'pairing rule'
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Table 1 SMC and SMC-like proteins in eukaryotes. Subfamily
S. cerevisiae S. pombe A. nidulans C. elegans Drosophila Xenopus Chicken Bovine Human
SMC2
SMC4
SMC1
Smc2 Cut14 MIX-1 XCAP-E Scll -
Smc4 Cut3 F35G12.8 Gluon XCAP-C . -
Smcl AF0031136 XSMC1 . . bSMC1 Sb1.8
SMC3
SMC4 v
SMC-like 1
SMC-like 2
Smc3 cl 0F6 SudA
DPY-27 -
Rhcl 8 Radl 8 C23H4.6 -
YOL034W SPAC14C4.02 C27A2.1 -
-
-
Dcap XSMC3 . bSMC3 .
.
. .
.
.
DPY-2?, an SMC protein of Caenorhabditis elegans involved in dosage compensation, is closest to the SMC4-type proteins in its primary structure. However, given the facts that this protein is not required for mitosis and that the C. elegans genome has a more authentic SMC4 homolog, DPY-27 should be considered as a specialized SMC protein and classified into a distinct subfamily (SMC4-variant [SMC4V]). Gluon (H Bellen, personal communication); XSMC1 and XSMC3 (T Hirano, unpublished data); bSMC1 and bSMC3 ([25"]; R Jessberger, personal communication); Dcap [39]; Sb1.8 [40]. Other references are found in the text. Open reading frame names are indicated with unpublished sequences.
can be also applied to the budding yeast Smc2p and Smc4p, several lines of genetic evidence are consistent with this idea ([12]; also see below). T h e newly identified three subunits of 13S condensin are identical to previously uncharacterized XCAPs, XCAPD2, XCAP-G and XCAP-H, suggesting that the whole complex is targeted to chromosomes without loss of any subunits [9°°]. T h e three subunits are highly conserved from yeast to humans, but are not related to SMC proteins ([9°']; M Hirano, R Kobayashi, T Hirano, unpublished
data). No functional characterization has been described for these homologs with the exception of the Drosophila Barren protein [13"'], which is homologous to XCAP-H [9"']. T h e mutant phenotype and the chromosomal localization of Barren are completely consistent with the idea that Barren is a component of the Drosophila condensin. More recently, a Drosophila SMC4-type protein has been identified as the gluon gene product; a mutant ofgluon shows a phenotype similar to that of barren ([14]; H Bellen, personal communication). It remains to be
Figure 1
( a ) l 3S condensin
(b) Cohesion complex?
(c) RC-1
(d) Dosage compensation complex
@ ?
?
Chromosome condensation
Sister chromatid cohesion
Recombinational repair
Dosage compensation Current Opinion in Cell Biology
Eukaryotic SMC protein complexes with diverse functions. (a) 13S condensin is a five-subunit protein complex that plays a central role in chromosome condensation in Xenopus egg cell-free extracts [7,9"]. Two SMC subunits, XCAP-C (SMC4-type) and XCAP-E (SMC2-type), constitute the core of this complex. XCAP-H is homologous to the Drosophila Barren protein [13""]. (b) A putative cohesion complex is deduced from genetic and biochemical data currently available in yeast [15"',16",21 "]. Physical interactions between Smcl p, S c c l p/Mcdl p and Trf4p have been reported on the basis of coimmunoprecipitation experiments. (e) RC-1 is a recombination protein complex purified from calf thymus. It contains DNA polymerase ~;, ligase III, and two SMC (SMCl-type and SMC3-type) subunits [25"]. (d) The dosage compensation complex has been identified through genetic and biochemical studies in C. elegans [26,27"',28"',29"]. It is composed of at least four subunits, including MIX-1 (SMC2-type), DPY-27 (SMC4V-type), and DPY-26 (weakly homologous to the XCAP-H subunit of 13S condensin [9"]). DPY-28 is suspected to be a part of the complex. SMC subunits are shown by rectangles whereas non-SMC subunits are shown by ellipses.
SMC protein complexes and higher-order chromosome dynamics Hirano
determined if Gluon and Barren function together in the same complex. 13S condensin is targeted to chromosomes in a mitosisspecific manner, providing additional evidence for its essential role in mitotic chromosome condensation [9°°]. The three non-SMC subunits of 13S condensin, XCAP-D2, XCAP-G and XCAP-H, are hyperphosphorylated in a mitosis-specific manner, and they are therefore likely to play a regulatory role in condensin functions. Recent data show that cdc2 kinase is able to phosphorylate XCAP-D2 and XCAP-H in vitro, suggesting a direct link between the major mitotic kinase and the chromosome condensation machinery (K Kimura, T Hirano, unpublished data). Sister chromatid cohesion complex
T h e specific role of the SMC1- and SMC3-type proteins in chromosome dynamics is less clear than that of the SMC2- and SMC4-type proteins. Genetic studies showed that Smclp is required for the proper segregation of chromosomes [ 1], but apparently not for condensation [ 12]. More recently, two genetic studies in S. cerevisiae have reported a requirement for Smclp and Smc3p in sister chromatid cohesion. In one study, a genetic screen for mutants exhibiting precocious sister chromatid separation identified smcl and smc3 [15°°]. Notably, neither smc2 nor smc4 was isolated in the same screen, suggesting a specific contribution of the SMC1- and SMC3-type proteins to this process. Two additional mutants, sccl and scc2, showed a similar phenotype. An independent study found genetic and physical interactions between Scclp (named Mcdlp by this group) and Smclp [16°°]. Scclp/Mcdlp is an evolutionarily conserved protein that is homologous to the Schizosaccharomyces pombe Rad21p implicated in DNA repair [17-19]. Taking all these results into consideration, it has been proposed that Smclp and Smc3p, along with Scclp/Mcdlp, might be directly involved in sister chromatid cohesion. In a striking agreement with these genetic studies in yeast, a biochemical approach has recently identified a large protein complex in Xenopus egg extracts that contains homologs of Smclp, Smc3p and Scclp/Mcdlp (A Losada, M Hirano, T Hirano, unpublished data). This complex appears to have additional subunits, candidates for which might include homologs of Scc2p [15°°], Trf4p and BimD. Trf4p was originally identified in a genetic screen for mutants that become lethal in combination with a topoisomerase I mutation [20], and subsequently shown to interact with Smclp physically and genetically [21°]. Interestingly, a trf4 mutant also exhibits a cohesion defect (M Christman, personal communication). BimD is a putative DNA-binding protein required for mitosis in Aspergillus nidulans [22], and has been shown to interact genetically with SudA, an SMC3-type gene product [23°]. RC-1, a recombination protein complex
Recombination complex-1 (RC-1), purified from calf thymus, catalyzes recombinational repair of double-stranded gaps and deletions in vitro [24]. This complex contains
31g
DNA polymerase e, DNA ligase III and two SMC subunits (Figure 1). On the basis of their limited amino-terminal sequences, the two SMC subunits of RC-1 were initially identified as SMC1- and SMC2-type proteins [25°°]. T h e availability of their complete sequences, however, reclassified them as SMC1- and SMC3-type proteins (R Jessberger, personal communication). Although this is in good agreement with the 'pairing rule' found in the putative sister chromatid cohesion complex, the two complexes contain different sets of non-SMC subunits and therefore appear to be distinct. Nevertheless, identification of two complexes sharing two identical SMC subunits suggests a potential link between recombinational repair and sister chromatid cohesion, two cellular processes thought previously to be unrelated. Radl8p of S. pombe and its S. cerevisiae homolog Rhcl8p, distant relatives to the SMC family (Table 1), are also implicated in DNA repair [6]. Although this protein still has to be characterized biochemically, it is tempting to speculate that Radl8p might interact with the other 'SMC-like' protein of S. pombe (SPAC14C4.02). Dosage compensation complex
Dosage compensation in the nematode C. elegans is regulated by a protein complex that associates specifically with the X chromosomes of hermaphrodites to reduce their gene expression [26,27*°]; (Figure 1). This dosage compensation complex consists of at least four subunits, including two SMC proteins: DPY-27 (SMC4-variant-type) and MIX-1 (SMC2-type). DPY-27 is not required for mitosis and its function is limited to dosage compensation [26] whereas MIX-1 has a dual function, being required for both dosage compensation in interphase and chromosome segregation in mitosis [28°°]. DPY-27 is most similar to the SMC4-type protein in its primary structure so the dosage compensation complex still obeys the pairing rule of SMC2-SMC4. T h e role of MIX-1 in mitosis must be performed in combination with a more authentic SMC4-type protein encoded in the C. elegans genome (Table 1). This SMC-4 type protein has yet to be characterized functionally. These results demonstrate flexible but restricted selectivity in SMC heterodimer formation, and suggest there is an intimate relationship between dosage compensation and chromosome condensation. The connection between the two processes is also supported by the finding that DPY-26, a non-SMC subunit of the dosage compensation complex, shares a limited similarity to the XCAP-H subunit of 13S condensin [9*°,29°]. Interestingly, in the germline in which DPY-27 is not expressed, DPY-26 associates with all meiotic chromosomes, being involved in their proper segregation [29*]. DPY-28 is suspected to be the fourth subunit of the dosage compensation complex on the basis of genetic evidence [27*°], but has not yet been cloned. M o l e c u l a r m e c h a n i s m of S M C a c t i o n
It has been proposed that SMC proteins have an energydependent activity that modulates higher-order chromatin
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structure because they share a common nucleotide-binding motif in their amino-terminal domains [1,7]. T h e first evidence for this prediction was provided last year from a biochemical analysis of 13S condensin [30°°1. 13S condensin is able to introduce positive supercoils into a relaxed circular DNA in the presence of topoisomerase I. T h e supercoiling activity requires ATP hydrolysis, and, consistently, 13S condensin has a DNA-stimulated ATPase activity. On the basis of these findings, a model for chromosome condensation has been proposed in which superhelical tension of DNA induced by 13S condensin acts as a driving force for the compaction of chromatin fibers in mitosis. Although 13S condensin has a high-affinity for structured DNAs, such as cruciform DNA, it is unknown if 13S condensin binds to specific sequence motifs implicated in chromosome condensation (e.g. scaffold-attachment regions; [31]). Another activity, reannealing of complementary DNA strands (DNA renaturation), has been found in two different SMC heterodimers: bSMCI-bSMC3 (a subcomplex of bovine RC-1; [25°°]) and the S. pombe Cut3p-Cutl4p complex [10°°]. This renaturation activity is likely to be one of the basic activities associated with all SMC heterodimers. It does not require ATP, however, and therefore may not represent a 'full' activity of SMC proteins. The mechanistic relationship between the renaturation activity and the ATP-dependent positive supercoiling activity is not yet clear. We expect that additional biochemical activities of SMC proteins will be discovered in the future perhaps adding a surprising twist to the field of chromosome research. Evolution
of SMC
proteins
Virtually nothing is known about the cellular and biochemical functions of bacterial and archaeal SMC proteins. Despite the existence of multiple SMC members within single eukaryal species, each of the bacterial and archaeal genomes fully sequenced so far contains a single SMC gene [32-37]. An ancestral SMC protein might therefore function as a homodimer. From an evolutionary point of view, it is of great interest to consider how such a primitive SMC protein has evolved and acquired complex cellular functions, I hypothesize here that four major transitions have given rise to the diversity and flexibility of SMC structure and function (Figure 2): an ancestral homodimer is converted into a heterodimeric form; a second class of heterodimer is created; an SMC subunit exchanges its SMC partner, providing an additional chance to fine-tune its function; the heterodimers associate with different sets of accessory subunits, forming functional holocomplexes that now exist in eukaryotic cells. We can imagine that multiple rounds of gene duplication played a key role during the evolution of SMC proteins. It seems reasonable to speculate that all eukaryotic complexes still possess an 'intrinsic' SMC activity (e.g. DNA renaturation), but each of them acquires a unique and more sophisticated activity devoted to its specific cellular function (e.g. positive
supercoiling). Finally, it would be interesting to ask why some bacterial species lack SMC protein (Escherichia coli is a notable example; [38]). Figure 2
m
SMC homodimer
SMC heterodimer
~/~ ~ 4
4
SMC holocornplex
Current Opinion in Cell Biology
A speculative model for the evolution of SMC proteins. Four evolutionary transitions are hypothesized to account for the diversification of SMC structure and function. (1) A primitive SMC homodimer (currently found in Bacteria and Archaea) is transformed into a heterodimeric form. (2) A second class of SMC heterodimer is created (this figure is drawn for simplicity and by no means implies that S M C 1 - S M C 3 is the ancestral form of SMC2-SMC4). (3) An SMC protein exchanges its SMC partner and modifies its existing function (e.g. from chromosome condensation to dosage compensation). (4) Different SMC heterodimers associate with different sets of 'accessory' subunits, forming functional holocomplexes. The same SMC heterodimer is able to recruit different sets of subunits, providing additional flexibility to acquire new functions (e.g. sister chromatid cohesion and recombination).
Conclusions
SMC protein complexes function as key components in a wide variety of chromosomal events. Despite the rapid progress summarized in this review, a number of fundamental questions remain to be addressed. What is the intrinsic (possibly ATP-dependent) activity common to all SMC proteins? What determines the specificity of heterodimeric association of eukaryotic SMC proteins? The current list of SMC-interacting proteins is far from complete, and there is no doubt that it will expand rapidly along with the discovery of new SMC protein complexes that have novel cellular functions. We also know little about cell-cycle regulation and targeting mechanisms of eukaryotic SMC protein complexes. How are sister chromatid cohesion and condensation regulated precisely and coordinately during the cell cycle? Is there a common mechanism underlying the apparently different cellular processes that depend on SMCs? We anticipate that answers to these specific questions will provide deep insights into our understanding of higher-order chromosome dynamics.
SMC protein complexes end higher-order chromosome dynamics Hirano
N o t e a d d e d in p r o o f T h e data cited in the text as A Losada, M Hirano, T Hirano, unpublished data is now in press [41].
condensation and segregation in mitosis. EMBO J 1994, 13:4938-4952. 12.
Acknowledgements I thank H Bellen, R Jessberger, and M Christman for unpublished results, B Meyer for a preprint, and D Koshland, K Nasmyth, and M Yanagida for discussion. I also thank members of my laboratory for critically reading the manuscript. The work in the author's laboratory was supported by grants from the National Institutes of Health and the Pew Scholars Program in the Biomedical Sciences.
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