SUSAN M. GASSER CHROMOSOME STRUCTURE SUSAN M. GASSER CHROMOSOME STRUCTURE Coiling up chromosomes The mechanism by which eukaryotic chromosomes condense as cells enter mitosis has long been inaccessible to molecular biologists. An important clue has now been provided by a ubiquitous , the SMCs.

The study of mitotic chromosome condensation has been coiled-coil structure, interrupted by a spacer of unknown handicapped by the fact that very few proteins were function. Finally, all SMC family members end in a glob- known to be directly involved in the process. Topoisom- ular tail domain of 100-200 amino acids, also containing erase II has long been suspected to play a crucial role, but a highly conserved motif, the DA box [2], which has a although necessary, it is clearly not sufficient to catalyze predicted helix-loop-helix structure. The DA box, the compaction of DNA into mitotic chromosomes [1]. named for several recurring aspartic acid and alanine Recently, however, five independent lines of research have residues, also shows homology to the 'Walker B' motif converged on a novel family of factors [2]. Each of the [8], which correlates with NTP hydrolysis, and is found studies presents unique evidence implicating its family in a number of RNA helicases and nucleotide-dependent member in chromosome condensation and/or mainten- transporter proteins [4]. The presence of these two motifs ance of a condensed state [2-6]. Sequence alignment may indicate that the heads and tails of SMC proteins identifies related factors in species ranging from halo- interact at some point to form an NTP-hydrolyzing bacteria to mouse, including two distantly related yeasts, a entity, although genetic proof for this is lacking. nematode, Xenopus and chicken; all contain several highly characteristic primary and secondary structural elements. The three-domain structure of the SMC family members, along with their highly conserved internal This group of proteins has been christened SMC, after motifs, is reminiscent of mechanochemical motor pro- the budding yeast () gene SMC1 teins like [9]. Closer comparison of primary ('stability of minichromosomes'), the first member of the sequences among the family members allows the defini- family for which a function was defined [2]. Since then, a tion of two subfamilies, defined by the degree of identity second budding yeast gene, SMC2, has been reported within the conserved head and tail domains. For instance, (cited in [4]). Similarly, in the fission yeast Schizo- SMC2, SC-II, XCAP-E and cutl4p share greater than saccharomyces pombe, two genes encoding SMC proteins 70 % amino-acid identity in their NTP-binding sites and were identified, called cut3+ and cutl4+, for 'chromo- DA boxes. The DPY-27, SMC1 and XCAP-C proteins somes untimely torn' [3]. The chicken SMC protein cor- also have a high degree of sequence identity, although responds to an abundant, non-histone protein that was between the two subfamilies only 30-50 % of the amino recovered from mitotic chromosomes and called SC-II acids are perfectly conserved in these characteristic (scaffolding protein II) [4], and the two gene products regions. The coiled-coil structure is ubiquitously present, from Xenopus, XCAP-C and XCAP-E (Xenopus chromo- but has low overall sequence conservation. some-associated polypeptides C and E), were isolated as abundant non-histone proteins that bound tightly to Deletion studies in both fission and budding yeast show chromosomes condensed in vitro using cytoplasmic that both head and tail domains are required for full extracts of Xenopus eggs [5]. Finally, dpy-27 is a function [2,3], and, in DPY-27, point mutations in the Caenorhabditiselegans gene required for the down-regula- amino-terminal NTP-binding motif render the protein tion of transcription from the entire X chromosome non-functional [6]. Evidence from the Xenopus system during dosage compensation in an XX hermaphrodite [6]. In each of these cases, the SMC family member is invariably associated with a mitotic or condensed- chromosomal state. Genes encoding highly related proteins were also cloned from mycoplasma [7], the purple bacterium Rhodospirillum rubrino and a mouse cDNA library [2], although no functional studies have yet been reported for these.

All of the deduced SMC family proteins share striking structural similarity (Fig. 1). They are large proteins (1 000-1 500 amino acids), containing a globular head domain with a conserved 57-residue region that includes Fig. 1. The relevant SMC family motifs and overall organization. a nucleotide-binding motif. This is followed by two Note that domains are not drawn to scale; only a partial consen- extended stretches of oa helix that are likely to form a sus for the 36 amino-acid DA box is given [2,4].

© Current Biology 1995, Vol 5 No 4 357 358 Current Biology 1995, Vol 5 No 4

suggests that XCAP-C and XCAP-E are likely to form Still more convincing is the fact that the pattern of heterodimers [5]. As more and more pairs of SMC pro- proteins associated with chromosomes condensed in teins are identified, it is reasonable to expect that any Xenopus egg extracts closely resembles the pattern of non- given species will have at least two family members and histone proteins recovered with highly purified mitotic that these may form both homodimers and heterodimers. chromosomes [5,10]. Among the relatively few proteins If the helix-loop-helix domain mediates DNA binding tightly associated with chromosomes condensed in vitro, (a possibility that remains to be proven), we might expect Hirano and Mitchison [5,12] identified topoisomerase II each heterodimer to be targeted to distinct sites in the and two SMC family members, XCAP-C and XCAP-E. chromosome depending on the particular partners All three seem to be essential for the condensation process. involved in the dimer. Whereas the depletion of topoisomerase II arrests conden- sation at a fairly early stage [1], the addition of anti-XCAP What evidence implicates these proteins in chromosome antibodies allows partial compaction, arresting when the condensation? The yeast system provides revealing chromosomes are long and extended [5]. As a depletion of mutant phenotypes: the fission yeast cut3ts and cutl4ts XCAPs from the extract was not performed, it is difficult temperature-sensitive mutants arrest with non-condensed to pinpoint their execution point in relation to that of chromosomes and show aberrant mitotic segregation; topoisomerase II. Remarkably, however, the addition of viability is rapidly lost upon shift to the non-permissive anti-XCAP antibodies after chromosome condensation temperature [3]. With various smcl mutant alleles in has been completed also destabilizes the structure that has budding yeast, the stability at mitosis of both natural been assembled in vitro, suggesting that XCAP activity chromosomes and minichromosomes drops [2], and in (perhaps NTP hydrolysis) is necessary for the maintenance situ hybridization studies in mutant cells show that of the condensed chromosome. XCAPs C and E seem to chromosomes are incompletely condensed at the mitotic function as a heterodimer and to form a highly insoluble arrest point (A. Strunnikov and D Koshland, personal complex with DNA in the conditions of the condensation communication). For both cut and smcl mutants, leth- assay [5]. Under these conditions (high 3-glycerol phos- ality is evident only in metaphase, and viability is not phate and EGTA), topoisomerase II no longer binds lost if cells are maintained in interphase at the non- tightly to chromosomes [12,13], suggesting that the permissive temperature. Conditional top2 (topoisomer-. XCAP complex can help generate a structure that, at least ase II) mutants also show a metaphase-specific lethality in vitro, no longer requires topoisomerase II [5]. and a high frequency of the cut or 'torn chromosome' phenotype. Intriguingly, the cut 14 t mutation shows The protein that implicates the SMC family in yet larger synthetic-lethality in combination with a conditional realms of chromosome function was cloned by Barbara mutation in top2, perhaps indicating an interaction Meyer's group [6] in its study of genes required for between the two proteins [3]. Moreover, the cut3 dosage compensation in C. elegans. In XX animals, tran- phenotype is partially suppressed by overexpression of scription of genes on the X chromosome is reduced topoisomerase I [3]. These observations implicate the cut globally, to a level roughly equivalent to that observed in gene products in functions that overlap those ascribed XO animals. Five dpy genes are implicated in this to topoisomerases. process, of which only dpy-27 is known (so far) to encode a member of the SMC family. Antibodies raised The isolation of metaphase chromosomal scaffolds was a to the DPY-27 protein bind specifically to the X method developed by Laemmli (for example, see [10]) in chromosome, but only in XX animals. In mutants in order to differentiate between readily extractable chro- which DPY-27 inappropriately binds to the X chromo- mosomal proteins and others that remain tightly bound. some in XO animals, the level of transcription of X- As loops of DNA were seen emanating from the residual linked genes is reduced and results in death [6]. Thus, 'scaffolding', the tightly bound non-histone scaffold pro- unlike the other members of the SMC family, this pro- teins were considered to be candidates for organizing tein shows a chromosome-specific association which is mitotic chromosomes. Years ago, topoisomerase II was maintained in interphase and which can reduce tran- identified as one of two major scaffold proteins, and only scriptional activity. Repression of X-linked transcription recently the gene for the second, SC-II, was cloned [4]. is presumed to function through modulation of higher- With antibodies raised against the recombinant chick SC- order structure. A second C. elegans SMC II protein, Earnshaw and colleagues observed an axial family member still more closely related to SMC1 has staining pattern in swollen human or chicken chromo- been reported (cited in [6]), leaving open the search for somes, very similar to that observed for topoisomerase II an SMC2-like nematode gene. The possibility of target- [4]. Furthermore, the two proteins were shown to co- ing an SMC protein to specific chromosomal domains, purify in a putative transcriptional regulatory complex perhaps to mediate specific patterns of condensation, called UB2 [11]. Although ascribing a function to suggests a link between mitotic chromosome conden- proteins on the basis of their the presence in a scaffold sation and chromatin-mediated gene repression which fraction is only 'guilt by association', taken with the yeast merits further study. and Xenopus results, it does suggest that the chromosomal scaffold extraction procedure is a valid means of How do SMC family members function? Here we can enriching for proteins involved in chromosomal structure. only speculate. Early stages of mitotic chromosome DISPATCH 359

of one population from the mitotic chromosome as cells progress through metaphase [14]. Subpopulations of topoisomerase II might also have different conforma- tions; indeed, it has been shown that topoisomerase II can adopt both a closed-clamp configuration around two DNA helices, from which topologically closed circles can not be released [15], and an open clamp, created by the hydrolysis of ATP. If, at entry into mito- sis, a subclass of topoisomerase II molecules form the closed clamp structure and tag certain sites along the chromosome, then perhaps it is this subset that also attracts, or associates with, SMC proteins (see Fig. 2). These proteins could form cross-bridges that bring together distant stretches of DNA when ATP is hydro- lyzed by the SMC dimer. Thus, topoisomerase II and SMCs could act together to 'reel in' the chromosome: the closed-clamp form of topoisomerase II might form a hook around certain sites (possibly at AT-rich scaffold- associated regions, SARs [16]), and the motor-like SMC complex might hydrolyze ATP to coil up the chromatid. Once the bases of the loops are coiled, and coinci- dentally decatenated, topoisomerase II may no longer be necessary. It may flip back open, perhaps at the metaphase-anaphase transition.

Many alternative models are possible, as the experimental evidence to restrict our imagination is sparse. Whatever the mechanism may be, the presence of such proteins in bacteria and mycoplasma raises the exciting possibility that SMC proteins mediate an ancient function of coiling up the long thread of life, keeping replicated copies orga- nized in order to ensure their correct segregation. Perhaps SMCs arose early in evolution to allow genome growth beyond the size of a single loop of DNA.

Acknouledgements: I apologize to anyone whose paper has been overlooked, and I thank Emma Roberts, Francesca Palla- dino, Moira Cockell and Hubert Renauld for comments and discussions.

References 1. Adachi Y, Luke M, Laemmli UK: Chromosome assembly in vitro: topoisomerase II is required for condensation. Cell 1991, 64:137-148. 2. Strunnikov AV, Larionov VL, Koshland D: SMCI: an essential yeast gene encoding a putative head-rod-tail protein is required for nuclear division and defines a new ubiquitous protein family. J Cell Biol 1993, 123:1635-1648. Fig. 2. A highly speculative model for eukaryotic (and possibly 3. Saka Y, Sutani T, Yamashita Y, Saitoh S, Takeuchi M, Nakaseko Y, prokaryotic) chromosome condensation, in which SMC dimers Yanagida M: Fission yeast cut3 and cut14, members of a ubiquitous interact with a subset of topoisomerase molecules. Potential protein family, are required for chromosome condensation and seg- changes as the cells progress through metaphase are shown. regation in mitosis. EMBO J 1994, 13:4938-4952. 4. Saito N, Goldberg IG, Wood ER, Earnshaw WC: Scll: an abundant chromosome scaffold protein is a member of a family of putative with an unusual predicted tertiary structure. J Cell Biol condensation require the enzymatic activity of 1994, 127:303-318. 5. Hirano T, Mitchison TJ: A heterodimeric coiled-coil protein topoisomerase II, presumably to decatenate intertwined required for mitotic chromosome condensation in vitro. Cell 1994, strands of replicated sister chromatids. Whether this 79:449-458. has a structural role or simply releases catenated 6. Chuang P-T, Albertson DG, Meyer BJ: DPY-27: a chromosome con- densation protein homolog that regulates C. elegans dosage com- loops is still unclear. However, as topoisomerase II is pensation through association with the X chromosome. Cell 1994, modified at metaphase - it is the target of numerous .79:459-474. kinases - one can imagine that one form of topoiso- 7. Notarincola S, Mclntash M, Wise K: A Mycoplasma hyorhinis pro- tein with sequence similarities to nucleotide-binding . merase II is bound to specific sites while another is freely Gene 1991, 97:77-85. diffusing, and this might be reflected in the selective loss 8. Walker JE, Sarste M, Runswick MJ, Gay NJ: Distantly related 360 Current Biology 1995, Vol 5 No 4

sequences in the a and [i subunits of ATPsynthase, , kinases 13. Vassetzky Y, Dang Q, Benedetti P, Gasser SM: Topoisomerase II and other ATP-requiring enzymes and a common nucleotide bind- forms multimers in vitro: effects of metals, p-glycerophosphate and ing fold. EMBO11982, 1:945-951. phosphorylation of its C-terminal domain. Mol Cell Biol 1994, 9. Walker RA, Sheetz MP: Cytoplasmic microtubule-associated 14:6962-6974. motors. Annu Rev Biochem 1993, 62:429-451. 14. Swedlow JR, Sedat J, Agard DA: Multiple populations of topoiso- 10. Lewis CD, Laemmli UK: Higher-order metaphase chromosome merase II detected in vivo by time-lapse, three-dimensional wide- structure: evidence for metalloprotein interactions. Cell 1982, field microscopy. Cell 1993, 73:97-108. 29:1 71-181 15. Roca J, Wang JC: The capture of a DNA double helix by an ATP- 11. Ma X, Saitoh N, Curtis PJ: Purification and characterisation of a dependent protein clamp: a key step in DNA transport by type II nuclear DNA-binding factor complex containing topoisomerase II DNA topoisomerases. Cell 1992, 71:833-840. and chromosome scaffold protein-2. J Biol Chem 1993, 16. Gasser SM, Laemmli UK: A glimpse of chromosomal order. Trends 268:6182-6188. Genet 1987, 3:16-21. 12. Hirano T, Mitchison TJ: Topoisomerase II does not play a scaffolding role in the organization of mitotic chromosomes assembled in Xenopus egg extracts. I Cell Biol 1993, 120: Susan M. Gasser, Swiss Institute for Experimental Cancer 601-612. Research (ISREC), CH-1066 Epalinges, Switzerland.

THE APRIL 1995 ISSUE (VOL. 5, NO. 2) OF CURRENT OPINION IN GENETICS AND DEVELOPMENT

includes the following reviews, edited by Winship Herr and Robert Kingston, on Chromosomes and Expression Mechanisms:

On the nature of replication origins in higher eukaryotes by Joyce L. Hamlin and Pieter A. Dijkwel Eukaryotic replicators and associated protein complexes by Stephen P. Bell Chromatin-mediated transcriptional repression in yeast by Sharon Y. Roth Chromatin multiprotein complexes involved in the maintenance of transcription patterns by Valerio Orlando and Renato Paro Structure and function of DNA-binding proteins by Hillary C.M. Nelson Structure and function of transcriptional activation domains by Steven J. Triezenberg The RNA polymerase x subunit: structure and function by Richard H. Ebright and Steve Busby The RNA polymerase II carboxy-terminal domain: links to a bigger and better 'holoenzyme'? by Andrew Emili and C. James Ingles Regulation of transcriptional elongation by RNA polymerase II by David L. Bentley TFIIH: a link between transcription, DNA repair and cell cycle regulation by Thierry Seroz, Jae Ryoung Hwang, Vincent Moncollin and Jean Marc Egly A complex protein assembly catalyzes polyadenylation of mRNA precursors by James L. Manley The ins and outs of RNA nucleocytoplasmic transport by Maria L. Zapp DNA methylation in eukaryotes by Robert A. Martienssen and Eric J. Richards Sister chromatid separation in vivo and in vitro by Sandra L. Holloway Telomeres and telomerase in aging and cancer by Calvin B. Harley and Bryant Villeponteau