Subcellular localization of the Bacillus subtilis structural maintenance of chromosomes (SMC) protein

The Bacillus subtilis structural maintenance of chromosomes (SMC) protein is a member of a large family of proteins involved in chromosome organization. We found that SMC is a moderately abundant protein ( approximately 1000 dimers per cell). In vivo cross-linking and immunoprecipitation assays revealed that SMC binds to many regions on the chromosome. Visualization of SMC in live cells using a fusion to the green fluorescent protein (GFP) and in fixed cells using immunofluorescence microscopy indicated that a portion of SMC localizes as discrete foci in positions similar to that of the DNA replication machinery (replisome). When visualized simultaneously, SMC and the replisome were often in similar regions of the cell but did not always co-localize. Persistence of SMC foci did not depend on ongoing replication, but did depend on ScpA and ScpB, two proteins thought to interact with SMC. Our results indicate that SMC is bound to many sites on the chromosome and a concentration of SMC is localized near replication forks, perhaps there to bind and organize newly replicated DNA.     

SMC proteins in bacteria: condensation motors for chromosome segregation?

SMC proteins are a ubiquitous protein family, present in almost all organisms so far analysed except for a few bacteria. They function in chromosome condensation, segregation, cohesion, and DNA recombination repair in eukaryotes, and can introduce positive writhe into DNA in vitro. SMC proteins and the structurally homologous MukB protein are unusual ATPases that form antiparallel dimers, with long coiled coil segments separating globular ends capable of binding DNA. Recently, SMC proteins have been shown to be essential for chromosome condensation, segregation and cell cycle progression in bacteria. Identification of a suppressor mutation for MukB in topoisomerase I in Escherichia coli suggests that SMC proteins are involved in negative DNA supercoiling in vivo, and by this means organize and compact chromosomes. A model is discussed in which bacterial SMC proteins act after an initial separation of replicated chromosome origins into the future daughter cell, separating sister chromatids by condensing replicated DNA strands within both cell halves. This would be analogous to a pulling of DNA strands into opposite cell halves by a condensation mechanism exerted at two specialised subregions in the cell.     

Cloning and characterization of mammalian SMC1 and SMC3 genes and proteins, components of the DNA recombination complexes RC-1

Members of the evolutionary conserved Structural Maintenance of Chromosomes (SMC) protein family are involved in chromosome condensation and gene dosage compensation with the SMC2 and SMC4 subtypes, and sister chromatid cohesion with the SMC1 and SMC3 subtypes. The bovine recombination protein complex RC-1, which catalyzes DNA transfer reactions, contains two heterodimeric SMC polypeptides, the genes of which have now been cloned, sequenced, and classified as bovine (b)SMC1 and bSMC3. Both proteins display all the characteristic features of the SMC family. FISH analysis localized the mouse SMC3 gene to chromosome 19D2-D3. Mono- and polyclonal antibodies specific for either subtype detected high levels of protein expression in lymphoid tissues, lung, testis and ovary. No change in levels of bSMC1 and bSMC3 proteins occurred after X-ray or UV-light irradiation of various cell lines or primary cells, and the amounts of individual proteins and the heterodimer are roughly constant throughout the cell cycle. Immunofluorescence of mouse cells detected the SMC1 protein in foci associated with the chromatin. These foci dissolve and the SMC protein dissociates from the chromatin during M phase.     

SMC1: an essential yeast gene encoding a putative head-rod-tail protein is required for nuclear division and defines a new ubiquitous protein family

The smc1-1 mutant was identified initially as a mutant of Saccharomyces cerevisiae that had an elevated rate of minichromosome nondisjunction. We have cloned the wild-type SMC1 gene. The sequence of the SMC1 gene predicts that its product (Smc1p) is a 141-kD protein, and antibodies against Smc1 protein detect a protein with mobility of 165 kD. Analysis of the primary and putative secondary structure of Smc1p suggests that it contains two central coiled-coil regions flanked by an amino- terminal nucleoside triphosphate (NTP)-binding head and a conserved carboxy-terminal tail. These analyses also indicate that Smc1p is an evolutionary conserved protein and is a member of a new family of proteins ubiquitous among prokaryotes and eukaryotes. The SMC1 gene is essential for viability. Several phenotypic characteristics of the mutant alleles of smc1 gene indicate that its product is involved in some aspects of nuclear metabolism, most likely in chromosome segregation. The smc1-1 and smc1-2 mutants have a dramatic increase in mitotic loss of a chromosome fragment and chromosome III, respectively, but have no increase in mitotic recombination. Depletion of SMC1 function in the ts mutant, smc1-2, causes a dramatic mitosis-related lethality. Smc1p-depleted cells have a defect in nuclear division as evidenced by the absence of anaphase cells. This phenotype of the smc1- 2 mutant is not RAD9 dependent. Based upon the facts that Smc1p is a member of a ubiquitous family, and it is essential for yeast nuclear division, we propose that Smc1p and Smc1p-like proteins function in a fundamental aspect of prokaryotic and eukaryotic cell division.     

Structural maintenance of chromosomes protein of Bacillus subtilis affects supercoiling in vivo

Structural maintenance of chromosomes (SMC) proteins are found in nearly all organisms. Members of this protein family are involved in chromosome condensation and sister chromatid cohesion. Bacillus subtilis SMC protein (BsSMC) plays a role in chromosome organization and partitioning. To better understand the function of BsSMC, we studied the effects of an smc null mutation on DNA supercoiling in vivo. We found that an smc null mutant was hypersensitive to the DNA gyrase inhibitors coumermycin A1 and norfloxacin. Furthermore, depleting cells of topoisomerase I substantially suppressed the partitioning defect of an smc null mutant. Plasmid DNA isolated from an smc null mutant was more negatively supercoiled than that from wild-type cells. In vivo cross-linking experiments indicated that BsSMC was bound to the plasmid. Our results indicate that BsSMC affects supercoiling in vivo, most likely by constraining positive supercoils, an activity which contributes to chromosome compaction and organization.     

Differential arrangements of conserved building blocks among homologs of the Rad50/Mre11 DNA repair protein complex

Structural maintenance of chromosomes (SMC) proteins have diverse cellular functions including chromosome segregation, condensation and DNA repair. They are grouped based on a conserved set of distinct structural motifs. All SMC proteins are predicted to have a bipartite ATPase domain that is separated by a long region predicted to form a coiled coil. Recent structural data on a variety of SMC proteins shows them to be arranged as long intramolecular coiled coils with a globular ATPase at one end. SMC proteins function in pairs as heterodimers or as homodimers often in complexes with other proteins. We expect the arrangement of the SMC protein domains in complex assemblies to have important implications for their diverse functions. We used scanning force microscopy imaging to determine the architecture of human, Saccharomyces cerevisiae, and Pyrococcus furiosus Rad50/Mre11, Escherichia coli SbcCD, and S.cerevisiae SMC1/SMC3 cohesin SMC complexes. Two distinct architectural arrangements are described, based on the way their components were connected. The eukaryotic complexes were similar to each other and differed from their prokaryotic and archaeal homologs. These similarities and differences are discussed with respect to their diverse mechanistic roles in chromosome metabolism.     

SMC5 and MMS21 are required for chromosome cohesion and mitotic progression

Members of the structural maintenance of chromosome (SMC) protein family have essential functions during mitosis, ensuring chromosome condensation (SMC2/4) and cohesion (SMC1/3). The SMC5/6 complex has been implicated in a variety of DNA maintenance processes but unlike the other SMC proteins, SMC5/6 have not been attributed any role in mitosis. Here, we find that ablation of either SMC5 or the SUMO-ligase MMS21 leads to premature sister chromatid separation prior to anaphase. The failure of normal chromosome alignment activates the spindle assembly checkpoint and blocks mitotic progression. Interestingly, there is no similar mitotic response to ablation of SMC6. Further, we show that mitotic SMC5 co-elutes from column fractions that contain MMS21 but lack SMC6. Our results thus establish that SMC5 is crucial for mitotic progression and maintenance of sister chromatid cohesion during mitosis, and that this role of SMC5 seems to be independent of the SMC5/6 complex.     

Human Rad50/Mre11 is a flexible complex that can tether DNA ends.

The human Rad50 protein, classified as a structural maintenance of chromosomes (SMC) family member, is complexed with Mre11 (R/M) and has important functions in at least two distinct double-strand break repair pathways. To find out what the common function of R/M in these pathways might be, we investigated its architecture. Scanning force microscopy showed that the complex architecture is distinct from the described SMC family members. R/M consisted of two highly flexible intramolecular coiled coils emanating from a central globular DNA binding domain. DNA end-bound R/M oligomers could tether linear DNA molecules. These observations suggest that a unified role for R/M in multiple aspects of DNA repair and chromosome metabolism is to provide a flexible, possibly dynamic, link between DNA ends.     

Independent Mechanisms Target SMCHD1 to Trimethylated Histone H3 Lysine 9-Modified Chromatin and the Inactive X Chromosome

The chromosomal protein SMCHD1 plays an important role in epigenetic silencing at diverse loci, including the inactive X chromosome, imprinted genes, and the facioscapulohumeral muscular dystrophy locus. Although homology with canonical SMC family proteins suggests a role in chromosome organization, the mechanisms underlying SMCHD1 function and target site selection remain poorly understood. Here we show that SMCHD1 forms an active GHKL-ATPase homodimer, contrasting with canonical SMC complexes, which exist as tripartite ring structures. Electron microscopy analysis demonstrates that SMCHD1 homodimers structurally resemble prokaryotic condensins. We further show that the principal mechanism for chromatin loading of SMCHD1 involves an LRIF1-mediated interaction with HP1γ at trimethylated histone H3 lysine 9 (H3K9me3)-modified chromatin sites on the chromosome arms. A parallel pathway accounts for chromatin loading at a minority of sites, notably the inactive X chromosome. Together, our results provide key insights into SMCHD1 function and target site selection.     

A human condensin complex containing hCAP-C-hCAP-E and CNAP1, a homolog of Xenopus XCAP-D2, colocalizes with phosphorylated histone H3 during the early stage of mitotic chromosome condensation

Structural maintenance of chromosomes (SMC) family proteins play critical roles in structural changes of chromosomes. Previously, we identified two human SMC family proteins, hCAP-C and hCAP-E, which form a heterodimeric complex (hCAP-C-hCAP-E) in the cell. Based on the sequence conservation and mitotic chromosome localization, hCAP-C-hCAP-E was determined to be the human ortholog of the Xenopus SMC complex, XCAP-C-XCAP-E. XCAP-C-XCAP-E is a component of the multiprotein complex termed condensin, required for mitotic chromosome condensation in vitro. However, presence of such a complex has not been demonstrated in mammalian cells. Coimmunoprecipitation of the endogenous hCAP-C-hCAP-E complex from HeLa extracts identified a 155-kDa protein interacting with hCAP-C-hCAP-E, termed condensation-related SMC-associated protein 1 (CNAP1). CNAP1 associates with mitotic chromosomes and is homologous to Xenopus condensin component XCAP-D2, indicating the presence of a condensin complex in human cells. Chromosome association of human condensin is mitosis specific, and the majority of condensin dissociates from chromosomes and is sequestered in the cytoplasm throughout interphase. However, a subpopulation of the complex was found to remain on chromosomes as foci in the interphase nucleus. During late G(2)/early prophase, the larger nuclear condensin foci colocalize with phosphorylated histone H3 clusters on partially condensed regions of chromosomes. These results suggest that mitosis-specific function of human condensin may be regulated by cell cycle-specific subcellular localization of the complex, and the nuclear condensin that associates with interphase chromosomes is involved in the reinitiation of mitotic chromosome condensation in conjunction with phosphorylation of histone H3.     

SMC proteins and chromosome mechanics: from bacteria to humans

Chromosome cohesion and condensation are essential prerequisites of proper segregation of genomes during mitosis and meiosis, and are supported by two structurally related protein complexes, cohesin and condensin, respectively. At the core of the two complexes lie members of the structural maintenance of chromosomes (SMC) family of ATPases. SMC proteins are also found in most bacterial and archaeal species, implicating the existence of an evolutionarily conserved theme of higher-order chromosome organization and dynamics. SMC dimers adopt a two-armed structure with an ATP-binding cassette (ABC)-like domain at the distal end of each arm. This article reviews recent work on the bacterial and eukaryotic SMC protein complexes, and discusses current understanding of how these uniquely designed protein machines may work at a mechanistic level. It seems most likely that the action of SMC proteins is highly dynamic and plastic, possibly involving a diverse array of intramolecular and intermolecular protein-protein interactions.     

Disturbance in function and expression of condensin affects chromosome compaction in HeLa cells

Condensin, a major non-histone protein complex on chromosomes, is responsible for the formation of rod-shaped chromosome in mitosis. A heterodimer composed of SMC2 (structural maintenance of chromosomes) and SMC4 subunits constitutes the core part of condensin. Although extensive studies have been done in yeast, fruit fly and Xenopus to uncover the mechanisms and molecular nature of SMC proteins, little is known about the complex in mammalian cells. We have conducted a series of experiments to unveil the nature of condensin complex in human chromosome formation. The results show that overexpression of the C-terminal domain of SMC subunits disturbs chromosome condensation, leading to formation of swollen chromosomes, while knockdown of SMC subunits severely disturbs mitotic chromosome formation, resulting in chromatin bridges between daughter cells and multiple nuclei in single cells. The salt extraction assay indicates that a fraction of the condensin complex is bound to chromatin in interphase, but most of the condensin bind to chromatin at the onset of mitosis. Thus, disturbance in condensin function or expression affects chromosome condensation and influences mitotic progression.     

真核生物SMC基因家族中拷贝数目的长期稳定进化

Members of the Structural Maintenance of Chromosome (SMC) family have long been of interest to molecular and evolutionary biologists for their role in chromosome structural dynamics, particularly sister chromatid cohesion, condensation, and DNA repair. SMC and related proteins are found in all major groups of living organisms and share a common structure of conserved N and C globular domains separated from the conserved hinge domain by long coiled-coil regions. In eukaryotes there are six paralogous proteins that form three heterodimeric pairs, whereas in prokaryotes there is only one SMC protein that homodimerizes. From recently completed genome sequences, we have identified SMC genes from 34 eukaryotes that have not been described in previous reports. Our phylogenetic analysis of these and previously identified SMC genes supports an origin for the vertebrate meiotic SMC1 in the most recent common ancestor since the divergence from invertebrate animals. Additionally, we have identified duplicate copies due to segmental duplications for some of the SMC paralogs in plants and yeast, mainly SMC2 and SMC6, and detected evidence that duplicates of other paralogs were lost, suggesting differential evolution for these genes. Our analysis indicates that the SMC paralogs have been stably maintained at very low copy numbers, even after segmental (genome-wide) duplications. It is possible that such low copy numbers might be selected during eukaryotic evolution, although other possibilities are not ruled out.     

A role for Drosophila SMC4 in the resolution of sister chromatids in mitosis

BACKGROUND: Faithful segregation of the genome during mitosis requires interphase chromatin to be condensed into well-defined chromosomes. Chromosome condensation involves a multiprotein complex known as condensin that associates with chromatin early in prophase. Until now, genetic analysis of SMC subunits of the condensin complex in higher eukaryotic cells has not been performed, and consequently the detailed contribution of different subunits to the formation of mitotic chromosome morphology is poorly understood. RESULTS: We show that the SMC4 subunit of condensin is encoded by the essential gluon locus in Drosophila. DmSMC4 contains all the conserved domains present in other members of the structural-maintenance-of-chromosomes protein family. DmSMC4 is both nuclear and cytoplasmic during interphase, concentrates on chromatin during prophase, and localizes to the axial chromosome core at metaphase and anaphase. During decondensation in telophase, most of the DmSMC4 leaves the chromosomes. An examination of gluon mutations indicates that SMC4 is required for chromosome condensation and segregation during different developmental stages. A detailed analysis of mitotic chromosome structure in mutant cells indicates that although the longitudinal axis can be shortened normally, sister chromatid resolution is strikingly disrupted. This phenotype then leads to severe chromosome segregation defects, chromosome breakage, and apoptosis. CONCLUSIONS: Our results demonstrate that SMC4 is critically important for the resolution of sister chromatids during mitosis prior to anaphase onset.     

Discovery of two novel families of proteins that are proposed to interact with prokaryotic SMC proteins,and characterization of the Bacillus subtilis family members ScpA and ScpB

Structural maintenance of chromosomes (SMC) proteins are present in all eukaryotes and in many prokaryotes. Eukaryotic SMC proteins form complexes with various non-SMC subunits, which affect their function, whereas the prokaryotic homologues had no known non-SMC partners and were thought to act as simple homodimers. Here we describe two novel families of proteins, widespread in archaea and (Gram-positive) bacteria, which we denote 'segregation and condensation proteins' (Scps). ScpA genes are localized next to smc genes in nearly all SMC- containing archaea, suggesting that they belong to the same operon and are thus involved in a common process in the cell. The function of ScpA was studied in Bacillus subtilis, which also harbours a well characterized smc gene. Here we show that scpA mutants display characteristic phenotypes nearly identical to those of smc mutants, including temperature- sensitive growth, production of anucleate cells, formation of aberrant nucleoids, and chromosome splitting by the so-called guillotine effect. Thus, both SMC and ScpA are required for chromosome segregation and condensation. Interestingly, mutants of another B. subtilis gene, scpB, which is localized downstream from scpA, display the same phenotypes, which indicate that ScpB is also involved in these functions. ScpB is generally present in species that also encode ScpA. The physical interaction of ScpA and SMC was proven (i) by the use of the yeast two-hybrid system and (ii) by the isolation of a complex containing both proteins from cell extracts of B. subtilis. By extension, we speculate that interaction of orthologues of the two proteins is important for chromosome segregation in many archaea and bacteria, and propose that SMC proteins generally have non-SMC protein partners that affect their function not only in eukaryotes but also in prokaryotes.     

The Deinococcus radiodurans SMC protein is dispensable for cell viability yet plays a role in DNA folding

Deinococcus radiodurans contains a highly condensed nucleoid that remains to be unaltered following the exposure to high doses of γ-irradiation. Proteins belonging to the structural maintenance of chromosome protein (SMC) family are present in all organisms and were shown to be involved in chromosome condensation, pairing, and/or segregation. Here, we have inactivated the smc gene in the radioresistant bacterium D. radiodurans, and, unexpectedly, found that smc null mutants showed no discernible phenotype except an increased sensitivity to gyrase inhibitors suggesting a role of SMC in DNA folding. A defect in the SMC-like SbcC protein exacerbated the sensitivity to gyrase inhibitors of cells devoid of SMC. We also showed that the D. radiodurans SMC protein forms discrete foci at the periphery of the nucleoid suggesting that SMC could locally condense DNA. The phenotype of smc null mutant leads us to speculate that other, not yet identified, proteins drive the compact organization of the D. radiodurans nucleoid.     

Novel meiosis-specific isoform of mammalian SMC1

Structural maintenance of chromosomes (SMC) proteins fulfill pivotal roles in chromosome dynamics. In yeast, the SMC1-SMC3 heterodimer is required for meiotic sister chromatid cohesion and DNA recombination. Little is known, however, about mammalian SMC proteins in meiotic cells. We have identified a novel SMC protein (SMC1beta), which-except for a unique, basic, DNA binding C-terminal motif-is highly homologous to SMC1 (which may now be called SMC1alpha) and is not present in the yeast genome. SMC1beta is specifically expressed in testes and coimmunoprecipitates with SMC3 from testis nuclear extracts, but not from a variety of somatic cells. This establishes for mammalian cells the concept of cell-type- and tissue-specific SMC protein isoforms. Analysis of testis sections and chromosome spreads of various stages of meiosis revealed localization of SMC1beta along the axial elements of synaptonemal complexes in prophase I. Most SMC1beta dissociates from the chromosome arms in late-pachytene-diplotene cells. However, SMC1beta, but not SMC1alpha, remains chromatin associated at the centromeres up to metaphase II. Thus, SMC1beta and not SMC1alpha is likely involved in maintaining cohesion between sister centromeres until anaphase II.     

SMC protein-dependent chromosome condensation during aerial hyphal development in Streptomyces

Members of the SMC (structural maintenance of chromosomes) protein family play a central role in higher-order chromosome dynamics from bacteria to humans. So far, studies of bacterial SMC proteins have focused only on unicellular rod-shaped organisms that divide by binary fission. The conversion of multigenomic aerial hyphae of the mycelial organism Streptomyces coelicolor into chains of unigenomic spores requires the synchronous segregation of multiple chromosomes. Here we focus on the contribution of SMC proteins to sporulation-associated chromosome segregation in S. coelicolor. Deletion of the smc gene causes aberrant DNA condensation and missegregation of chromosomes (7.5% anucleate spores). In vegetative mycelium, immunostained SMC proteins were observed sporadically, while in aerial hyphae about to undergo sporulation they appeared as irregularly spaced foci which accompanied but did not colocalize with ParB complexes. Our data demonstrate that efficient chromosome segregation requires the joint action of SMC and ParB proteins. SMC proteins, similarly to ParAB and FtsZ, presumably belong to a larger group of proteins whose expression is highly induced in response to the requirement of aerial hyphal maturation.     

Functional interplay between cohesin and Smc5/6 complexes

Chromosomes are subjected to massive reengineering as they are replicated, transcribed, repaired, condensed, and segregated into daughter cells. Among the engineers are three large protein complexes collectively known as the structural maintenance of chromosome (SMC) complexes: cohesin, condensin, and Smc5/6. As their names suggest, cohesin controls sister chromatid cohesion, condensin controls chromosome condensation, and while precise functions for Smc5/6 have remained somewhat elusive, most reports have focused on the control of recombinational DNA repair. Here, we focus on cohesin and Smc5/6 function. It is becoming increasingly clear that the functional repertoires of these complexes are greater than sister chromatid cohesion and recombination. These SMC complexes are emerging as interrelated and cooperating factors that control chromosome dynamics throughout interphase. However, they also release their embrace of sister chromatids to enable their segregation at anaphase, resetting the dynamic cycle of SMC-chromosome interactions.