Review: SMCs in the world of chromosome biology- from prokaryotes to higher eukaryotes

The study of higher order chromosome structure and how it is modified through the course of the cell cycle has fascinated geneticists, biochemists, and cell biologists for decades. The results from many diverse technical avenues have converged in the discovery of a large superfamily of chromosome-associated proteins known as SMCs, for structural maintenance of chromosomes, which are predicted to have ATPase activity. Now found in all eukaryotes examined, and numerous prokaryotes as well, SMCs play crucial roles in chromatid cohesion, chromosome condensation, sex chromosome dosage compensation, and DNA recombination repair. In eukaryotes, SMCs exist in five subfamilies, which appear to associate with one another in particular pairs to perform their specific functions. In this review, we summarize current progress examining the roles these proteins, and the complexes they form, play in chromosome metabolism. We also present a twist in the SMC story, with the possibility of one SMC moonlighting in an unpredicted location.     

Structural maintenance of chromosomes (SMC) proteins, a family of conserved ATPases

The structural maintenance of chromosomes (SMC) proteins are essential for successful chromosome transmission during replication and segregation of the genome in all organisms. SMCs are generally present as single proteins in bacteria, and as at least six distinct proteins in eukaryotes. The proteins range in size from approximately 110 to 170 kDa, and each has five distinct domains: amino- and carboxy-terminal globular domains, which contain sequences characteristic of ATPases, two coiled-coil regions separating the terminal domains and a central flexible hinge. SMC proteins function together with other proteins in a range of chromosomal transactions, including chromosome condensation, sister-chromatid cohesion, recombination, DNA repair and epigenetic silencing of gene expression. Recent studies are beginning to decipher molecular details of how these processes are carried out.     

Prokaryotic structural maintenance of chromosomes (SMC) proteins: distribution, phylogeny, and comparison with MukBs and additional prokaryotic and eukaryotic coiled-coil proteins.

Structural maintenance of chromosomes (SMC) proteins are known to be essential for chromosome segregation in some prokaryotes and in eukaryotes. A systematic search for the distribution of SMC proteins in prokaryotes with fully or partially sequenced genomes showed that they form a larger family than previously anticipated and raised the number of known prokaryotic homologs to 54. Secondary structure predictions revealed that the length of the globular N-terminal and C-terminal domains is extremely well conserved in contrast to the hinge domain and coiled-coil domains which are considerably shorter in several bacterial species. SMC proteins are present in all gram-positive bacteria and in nearly all archaea while they were found in less than half of the gram-negative bacteria. Phylogenetic analyses indicate that the SMC tree roughly resembles the 16S rRNA tree, but that cyanobacteria and Aquifex aeolicus obtained smc genes by lateral transfer from archaea. Fourteen out of 22 smc genes located in fully sequenced genomes seem to be co-transcribed with a second gene out of six different gene families, indicating that the deduced gene products might be involved in similar functions. The SMC proteins were compared with other prokaryotic proteins with long coiled-coil domains. The lengths of different protein domains and signature sequences allowed to differentiate SMCs, MukBs, which were found to be confined to gamma proteobacteria, and two subfamilies of COG 0419 including the SbcC nuclease from E. coli. A phylogenetic analysis was performed including the prokaryotic coiled-coil proteins as well as SMCs and Rad18 proteins from selected eukaryotes.     

The evolution of SMC proteins: phylogenetic analysis and structural implications

The SMC proteins are found in nearly all living organisms examined, where they play crucial roles in mitotic chromosome dynamics, regulation of gene expression, and DNA repair. We have explored the phylogenetic relationships of SMC proteins from prokaryotes and eukaryotes, as well as their relationship to similar ABC ATPases, using maximum-likelihood analyses. We have also investigated the coevolution of different domains of eukaryotic SMC proteins and attempted to account for the evolutionary patterns we have observed in terms of available structural data. Based on our analyses, we propose that each of the six eukaryotic SMC subfamilies originated through a series of ancient gene duplication events, with the condensins evolving more rapidly than the cohesins. In addition, we show that the SMC5 and SMC6 subfamily members have evolved comparatively rapidly and suggest that these proteins may perform redundant functions in higher eukaryotes. Finally, we propose a possible structure for the SMC5/SMC6 heterodimer based on patterns of coevolution.     

Structure and DNA-binding activity of the Pyrococcus furiosus SMC protein hinge domain

Structural Maintenance of Chromosomes (SMC) proteins are essential for a wide range of processes including chromosome structure and dynamics, gene regulation, and DNA repair. While bacteria and archaea have one SMC protein that forms a homodimer, eukaryotes possess three distinct SMC complexes, consisting of heterodimeric pairs of six different SMC proteins. SMC holocomplexes additionally contain several specific regulatory subunits. The bacterial SMC complex is required for chromosome condensation and segregation. In eukaryotes, this function is carried out by the condensin (SMC2-SMC4) complex. SMC proteins consist of N-terminal and C-terminal domains that fold back onto each other to create an ATPase "head" domain, connected to a central "hinge" domain via a long coiled-coil region. The hinge domain mediates dimerization of SMC proteins and binds DNA. This activity implicates a direct involvement of the hinge domain in the action of SMC proteins on DNA. We studied the SMC hinge domain from the thermophilic archaeon Pyrococcus furiosus. Its crystal structure shows that the SMC hinge domain fold is largely conserved between archaea and bacteria as well as eukarya. Like the eukaryotic condensin hinge domain, the P. furiosus SMC hinge domain preferentially binds single-stranded DNA (ssDNA), but its affinity for DNA is weaker than that of its eukaryotic counterpart, and point mutations reveal that its DNA-binding surface is more confined. The ssDNA-binding activity of its hinge domain might play a role in the DNA-loading process of the prokaryotic SMC complex during replication.     

SMC (structural maintenance of chromosomes) structural protein family and their role in chromatin reorganization

Structural chromatin proteins of the SMC (Structural Maintenance of Chromosomes) family play an important role in structural DNA reorganization in pro- and eukaryotes. Eukaryotic SMC proteins are the core components of the cohesin and condensin complexes. The cohesin complex is responsible for sister chromatid and homolog cohesion in mitosis and meiosis. The condensin complex uses ATP energy to induce positive coiled-coils in DNA, which results in compaction of the latter and formation of mitotic chromosome scaffold. In addition, the SMC proteins constitute recombination and recombination repair complexes. In hermaphrodites of nematode Caenorhabditis elegans, the SMC protein-containing complex controls dosage compensation and inactivation of the X chromosome genes.     

Structural Proteins of the SMC (Structural Maintenance of Chromosomes) Family and Their Role in Chromatin Reorganization

Structural chromatin proteins of the SMC (Structural Maintenance of Chromosomes) family play an important role in structural DNA reorganization in pro- and eukaryotes. Eukaryotic SMC proteins are the core components of the cohesin and condensin complexes. The cohesin complex is responsible for sister chromatid and homolog cohesion in mitosis and meiosis. The condensin complex uses ATP energy to induce positive coiled-coils in DNA, which results in compaction of the latter and formation of mitotic chromosome scaffold. In addition, the SMC proteins constitute recombination and recombination repair complexes. In hermaphrodites of nematode Caenorhabditis elegans, the SMC protein-containing complex controls dosage compensation and inactivation of the X chromosome genes.     

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.     

Identification of a novel non-structural maintenance of chromosomes (SMC) component of the SMC5-SMC6 complex involved in DNA repair

Structural maintenance of chromosomes (SMC) proteins play central roles in chromosome organization and dynamics. They have been classified into six subtypes, termed SMC1 to SMC6, and function as heterodimer components of large protein complexes that also include several non-SMC proteins. The SMC2-SMC4 and SMC1-SMC3 complexes are also known as condensin and cohesin, respectively, but the recently identified SMC5 and SMC6 complex is less well characterized. Here, we report that NSE1 from Saccharomyces cerevisiae encodes a novel non-SMC component of the SMC5(Yol034wp)-SMC6(Rhc18p) complex corresponding to the 2-3-MDa molecular mass. Nse1p is essential for cell proliferation and localizes primarily in the nucleus. nse1 mutants are highly sensitive to DNA-damaging treatments and exhibit abnormal cellular morphologies, suggesting aberrant mitosis as a terminal morphological phenotype. These results are consistent with the reported features of the Schizosaccharomyces pombe SMC6 gene, rad18, which is thought to be involved in recombinational DNA repair. We conclude that Nse1p and the SMC5-SMC6 heterodimer together form a high molecular mass complex that is conserved in eukaryotes and required for both DNA repair and proliferation.     

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.     

MukE and MukF form two distinct high affinity complexes

The MukBFE complex is essential for chromosome segregation and condensation in Escherichia coli. MukB is functionally related to the structural maintenance of chromosomes (SMC) proteins. Similar to SMCs, MukB requires accessory proteins (MukE and MukF) to form a functional complex for DNA segregation. MukF is a member of the kleisin family, which includes proteins that commonly mediate the interaction between SMCs and other accessory proteins, suggesting that the similarities between the MukBFE and the SMC complexes extend beyond MukB. Although SMCs have been carefully studied, little is known about the roles of their accessory components. In the present work, we characterize the oligomeric states of MukE and MukF using size exclusion chromatography and analytical ultracentrifugation. MukE self-associates to form dimers (K(D) 18 +/- 3 mum), which in turn interact with the MukF dimer to form two distinct high affinity complexes having 2:2 and 2:4 stoichiometries (F:E). Intermediate complexes are not found, and thus we propose that the equilibrium between these two complexes determines the formation of a functional MukBFE with stoichiometry 2:2:2.     

Condensin and cohesin display different arm conformations with characteristic hinge angles

Structural maintenance of chromosomes (SMC) proteins play central roles in higher-order chromosome dynamics from bacteria to humans. In eukaryotes, two different SMC protein complexes, condensin and cohesin, regulate chromosome condensation and sister chromatid cohesion, respectively. Each of the complexes consists of a heterodimeric pair of SMC subunits and two or three non-SMC subunits. Previous studies have shown that a bacterial SMC homodimer has a symmetrical structure in which two long coiled-coil arms are connected by a flexible hinge. A catalytic domain with DNA- and ATP-binding activities is located at the distal end of each arm. We report here the visualization of vertebrate condensin and cohesin by electron microscopy. Both complexes display the two-armed structure characteristic of SMC proteins, but their conformations are remarkably different. The hinge of condensin is closed and the coiled-coil arms are placed close together. In contrast, the hinge of cohesin is wide open and the coiled-coils are spread apart from each other. The non-SMC subunits of both condensin and cohesin form a globular complex bound to the catalytic domains of the SMC heterodimers. We propose that the "closed" conformation of condensin and the "open" conformation of cohesin are important structural properties that contribute to their specialized biochemical and physiological functions.     

Anucleate and titan cell phenotypes caused by insertional inactivation of the structural maintenance of chromosomes (smc) gene in the archaeon Methanococcus voltae

SMC (structural maintenance of chromosomes) proteins are highly conserved and present in eukaryotes, bacteria and archaea. They function in chromosome condensation and segregation and in DNA repair. Using an insertion vector containing the pac gene for resistance to puromycin, we have created an insertion in the smc gene of Methanococcus voltae. We used epifluorescence microscopy to examine the cell and nucleoid morphology, DNA content and metabolic activity. This insertion causes gross defects in chromosome segregation and cell morphology. Approximately 20% of mutant cells contain little or no DNA, and a subset of cells ( approximately 2%) IS abnormally large (three to four times their normal diameter) titan cells. We believe that these titan cells indicate cell division arrest at a cell cycle checkpoint. The results confirm that SMC in archaea is an important player in chromosome dynamics (as it is in bacteria and eukaryotes).     

Break the Cycle: The Role of Cell-Cycle Modulation in the Prevention of Vasculoproliferative Diseases

Atherosclerosis is a major health problem in the western world, and the effectiveness of interventional therapeutic modalities for symptomatic atherosclerotic lesions is limited by vessel restenosis. Proliferation and migration of smooth muscle cells (SMCs) play a central role in post-interventional restenosis. Accordingly, many therapeutic approaches attempt to inhibit SMC proliferation. As the cell cycle is a final common pathway in SMC proliferation, proteins of the cell cycle have emerged as logical targets for the treatment and prevention of restenotic lesions. In this review we discuss current approaches that target the cell cycle in smooth muscle cells, and also describe recent and ongoing clinical trials that involve cell-cycle manipulation in the treatment of vasculoproliferative diseases.     

Break the cycle: the role of cell-cycle modulation in the prevention of vasculoproliferative diseases

Atherosclerosis is a major health problem in the western world, and the effectiveness of interventional therapeutic modalities for symptomatic atherosclerotic lesions is limited by vessel restenosis. Proliferation and migration of smooth muscle cells (SMCs) play a central role in post-interventional restenosis. Accordingly, many therapeutic approaches attempt to inhibit SMC proliferation. As the cell cycle is a final common pathway in SMC proliferation, proteins of the cell cycle have emerged as logical targets for the treatment and prevention of restenotic lesions. In this review we discuss current approaches that target the cell cycle in smooth muscle cells, and also describe recent and ongoing clinical trials that involve cell-cycle manipulation in the treatment of vasculoproliferative diseases.     

ATP-dependent aggregation of single-stranded DNA by a bacterial SMC homodimer.

SMC (structural maintenance of chromosomes) proteins are putative ATPases that are highly conserved among Bacteria, Archaea and Eucarya. Eukaryotic SMC proteins are implicated in a diverse range of chromosome dynamics including chromosome condensation, dosage compensation and recombinational repair. In eukaryotes, two different SMC proteins form a heterodimer, which in turn acts as the core component of a large protein complex. Despite recent progress, no ATP-dependent activity has been found in individual SMC subunits. We report here the first biochemical characterization of a bacterial SMC protein from Bacillus subtilis. Unlike eukaryotic versions, the B.subtilis SMC protein (BsSMC) is a simple homodimer with no associated subunits. It binds preferentially to single-stranded DNA (ssDNA) and has a ssDNA-stimulated ATPase activity. In the presence of ATP, BsSMC forms large nucleoprotein aggregates in a ssDNA-specific manner. Proteolytic cleavage of BsSMC is changed upon binding to ATP and ssDNA. The energy-dependent aggregation of ssDNA might represent a primitive type of chromosome condensation that occurs during segregation of bacterial chromosomes.     

Galectin-1 interacts with beta-1 subunit of integrin

Galectin-1, a beta-galactoside-binding dimeric lectin, is involved in adhesion, migration, and proliferation of vascular smooth muscle cells (SMC), the key steps in the development of atherosclerosis and restenosis. Here we investigated the molecular basis of the interactions between galectin-1 and SMCs. Galectin-1 modulated SMC attachment in a dose- and beta-galactoside-dependent manner. Direct binding of galectin-1 to beta1 integrin was detected by the immune precipitation of beta1 integrin after chemical cross-linking of 125I-labelled galectin-1 to the cell surface proteins. Galectin-1 transiently increased availability of beta1 integrins on the cell surface to antibodies against beta1 integrin. Incubation of SMCs with galectin-1 transiently increased the amount of the active form of beta1 integrin and tyrosine phosphorylation of two cytoskeleton-associated proteins; one of them coincided with focal adhesion kinase (FAK). Galectin-1 is likely to affect SMC adhesion by interacting with beta1 integrin on the cell surface of SMCs and inducing outside-in signalling.     

Adherens junction-associated protein distribution differs in smooth muscle tissue and acutely isolated cells

This study was designed to examine how smooth muscle (SM) cell (SMC) isolation affects the distribution of some adherens junction (AJ) complex-associated proteins. Immunofluorescence procedures for identifying protein distribution were used on gastrointestinal and tracheal SM tissues and freshly isolated SMCs from dogs and rabbits. As confirmed by force measurements, relaxation, Ca(2+) depletion, and cholinergic activation of SM tissues do not cause significant redistribution of the AJ-associated proteins vinculin, talin, or fibronectin away from the plasma membrane. Unlike SMCs in tissue, freshly isolated SMCs show a variable peripheral/cytoplasmic vinculin and talin distribution that is not altered by activation. Enzymatic treatment of SM tissues (as done for the first step of SMC isolation) results in loss of fibronectin immunoreactivity in SMCs still in the tissue but fails to cause redistribution of vinculin, talin, or caveolin away from the periphery. The loss of fibronectin immunofluorescence with enzymatic digestion correlates significantly with loss of tissue force production. These results confirm that the AJ-associated proteins vinculin and talin do not redistribute throughout SMCs in tissues when relaxed, when generating force, or after enzymatic digestion. In addition, in freshly isolated SMCs, the distribution of these proteins is significantly altered in approximately 50% of the SMCs. The cause of this redistribution is currently unknown, as is the impact on intracellular signaling and mechanics of these cells. Use of these two systems (SMCs in tissues vs. freshly isolated SMCs) provides an ideal situation for studying the role of the AJ in SMC signaling and mechanics.     

Structure and DNA binding activity of the mouse condensin hinge domain highlight common and diverse features of SMC proteins

Structural Maintenance of Chromosomes (SMC) proteins are vital for a wide range of processes including chromosome structure and dynamics, gene regulation and DNA repair. Eukaryotes have three SMC complexes, consisting of heterodimeric pairs of six different SMC proteins along with several specific regulatory subunits. In addition to their other functions, all three SMC complexes play distinct roles in DNA repair. Cohesin (SMC1–SMC3) is involved in DNA double-strand break repair, condensin (SMC2–SMC4) participates in single-strand break (SSB) repair, and the SMC5–SMC6 complex functions in various DNA repair pathways. SMC proteins consist of N- and C-terminal domains that fold back onto each other to create an ATPase ‘head’ domain, connected to a central ‘hinge’ domain via long coiled-coils. The hinge domain mediates dimerization of SMC proteins and binds DNA, but it is not clear to what purpose this activity serves. We studied the structure and function of the condensin hinge domain from mouse. While the SMC hinge domain structure is largely conserved from prokaryotes to eukaryotes, its function seems to have diversified throughout the course of evolution. The condensin hinge domain preferentially binds single-stranded DNA. We propose that this activity plays a role in the SSB repair function of the condensin complex.