RESUMO
SMC (structural maintenance of chromosomes) protein complexes are an evolutionarily conserved family of motor proteins that hold sister chromatids together and fold genomes throughout the cell cycle by DNA loop extrusion. These complexes play a key role in a variety of functions in the packaging and regulation of chromosomes, and they have been intensely studied in recent years. Despite their importance, the detailed molecular mechanism for DNA loop extrusion by SMC complexes remains unresolved. Here, we describe the roles of SMCs in chromosome biology and particularly review in vitro single-molecule studies that have recently advanced our understanding of SMC proteins. We describe the mechanistic biophysical aspects of loop extrusion that govern genome organization and its consequences.
Assuntos
Proteínas Cromossômicas não Histona , Complexos Multiproteicos , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Complexos Multiproteicos/química , Cromossomos/genética , Cromossomos/metabolismo , DNA/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismoRESUMO
Structural maintenance of chromosomes (SMC) complexes organize genome topology in all kingdoms of life and have been proposed to perform this function by DNA loop extrusion. How this process works is unknown. Here, we have analyzed how loop extrusion is mediated by human cohesin-NIPBL complexes, which enable chromatin folding in interphase cells. We have identified DNA binding sites and large-scale conformational changes that are required for loop extrusion and have determined how these are coordinated. Our results suggest that DNA is translocated by a spontaneous 50 nm-swing of cohesin's hinge, which hands DNA over to the ATPase head of SMC3, where upon binding of ATP, DNA is clamped by NIPBL. During this process, NIPBL "jumps ship" from the hinge toward the SMC3 head and might thereby couple the spontaneous hinge swing to ATP-dependent DNA clamping. These results reveal mechanistic principles of how cohesin-NIPBL and possibly other SMC complexes mediate loop extrusion.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , DNA/química , Conformação de Ácido Nucleico , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Proteínas de Ciclo Celular/química , DNA/metabolismo , Transferência Ressonante de Energia de Fluorescência , Células HeLa , Humanos , Hidrólise , Cinética , Microscopia de Força Atômica , Modelos Moleculares , Proteínas Nucleares/metabolismo , Conformação Proteica , CoesinasRESUMO
The ring-shaped structural maintenance of chromosome (SMC) complexes are multi-subunit ATPases that topologically encircle DNA. SMC rings make vital contributions to numerous chromosomal functions, including mitotic chromosome condensation, sister chromatid cohesion, DNA repair, and transcriptional regulation. They are thought to do so by establishing interactions between more than one DNA. Here, we demonstrate DNA-DNA tethering by the purified fission yeast cohesin complex. DNA-bound cohesin efficiently and topologically captures a second DNA, but only if that is single-stranded DNA (ssDNA). Like initial double-stranded DNA (dsDNA) embrace, second ssDNA capture is ATP-dependent, and it strictly requires the cohesin loader complex. Second-ssDNA capture is relatively labile but is converted into stable dsDNA-dsDNA cohesion through DNA synthesis. Our study illustrates second-DNA capture by an SMC complex and provides a molecular model for the establishment of sister chromatid cohesion.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Cromátides/genética , Proteínas Cromossômicas não Histona/metabolismo , DNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Trifosfato de Adenosina/metabolismo , Cromátides/metabolismo , Replicação do DNA , Saccharomyces cerevisiae , Schizosaccharomyces , CoesinasRESUMO
DNA loop-extruding SMC complexes play crucial roles in chromosome folding and DNA immunity. Prokaryotic SMC Wadjet (JET) complexes limit the spread of plasmids through DNA cleavage, yet the mechanisms for plasmid recognition are unresolved. We show that artificial DNA circularization renders linear DNA susceptible to JET nuclease cleavage. Unlike free DNA, JET cleaves immobilized plasmid DNA at a specific site, the plasmid-anchoring point, showing that the anchor hinders DNA extrusion but not DNA cleavage. Structures of plasmid-bound JetABC reveal two presumably stalled SMC motor units that are drastically rearranged from the resting state, together entrapping a U-shaped DNA segment, which is further converted to kinked V-shaped cleavage substrate by JetD nuclease binding. Our findings uncover mechanical bending of residual unextruded DNA as molecular signature for plasmid recognition and non-self DNA elimination. We moreover elucidate key elements of SMC loop extrusion, including the motor direction and the structure of a DNA-holding state.
Assuntos
DNA , Endonucleases , DNA/metabolismo , Plasmídeos/genética , Células Procarióticas , Proteínas de Ciclo Celular/metabolismoRESUMO
Condensin is a structural maintenance of chromosomes (SMC) complex family member thought to build mitotic chromosomes by DNA loop extrusion. However, condensin variants unable to extrude loops, yet proficient in chromosome formation, were recently described. Here, we explore how condensin might alternatively build chromosomes. Using bulk biochemical and single-molecule experiments with purified fission yeast condensin, we observe that individual condensins sequentially and topologically entrap two double-stranded DNAs (dsDNAs). Condensin loading transitions through a state requiring DNA bending, as proposed for the related cohesin complex. While cohesin then favors the capture of a second single-stranded DNA (ssDNA), second dsDNA capture emerges as a defining feature of condensin. We provide complementary in vivo evidence for DNA-DNA capture in the form of condensin-dependent chromatin contacts within, as well as between, chromosomes. Our results support a "diffusion capture" model in which condensin acts in mitotic chromosome formation by sequential dsDNA-dsDNA capture.
Assuntos
Proteínas de Ligação a DNA , Schizosaccharomyces , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/química , DNA/genética , Cromossomos , Proteínas de Ciclo Celular/genética , Schizosaccharomyces/genética , MitoseRESUMO
SMC protein complexes are molecular machines that provide structure to chromosomes. These complexes bridge DNA elements and by doing so build DNA loops in cis and hold together the sister chromatids in trans. We discuss how drastic conformational changes allow SMC complexes to build such intricate DNA structures. The tight regulation of these complexes controls fundamental chromosomal processes such as transcription, recombination, repair, and mitosis.
Assuntos
Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona , Proteínas de Ciclo Celular/metabolismo , Cromátides/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , DNA/genética , Mitose/genéticaRESUMO
Structural maintenance of chromosome (SMC) complexes fold DNA by loop extrusion to support chromosome segregation and genome maintenance. Wadjet systems (JetABCD/MksBEFG/EptABCD) are derivative SMC complexes with roles in bacterial immunity against selfish DNA. Here, we show that JetABCD restricts circular plasmids with an upper size limit of about 100 kb, whereas a linear plasmid evades restriction. Purified JetABCD complexes cleave circular DNA molecules, regardless of the DNA helical topology; cleavage is DNA sequence nonspecific and depends on the SMC ATPase. A cryo-EM structure reveals a distinct JetABC dimer-of-dimers geometry, with the two SMC dimers facing in opposite direction-rather than the same as observed with MukBEF. We hypothesize that JetABCD is a DNA-shape-specific endonuclease and propose the "total extrusion model" for DNA cleavage exclusively when extrusion of an entire plasmid has been completed by a JetABCD complex. Total extrusion cannot be achieved on the larger chromosome, explaining how self-DNA may evade processing.
Assuntos
Adenosina Trifosfatases , Clivagem do DNA , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Plasmídeos/genética , Cromossomos/metabolismo , DNA/genética , Proteínas de Ciclo Celular/genética , Cromossomos Bacterianos/genética , Cromossomos Bacterianos/metabolismoRESUMO
Mre11-Rad50-Xrs2 (MRX) is a highly conserved complex with key roles in various aspects of DNA repair. Here, we report a new function for MRX in limiting transcription in budding yeast. We show that MRX interacts physically and colocalizes on chromatin with the transcriptional co-regulator Mediator. MRX restricts transcription of coding and noncoding DNA by a mechanism that does not require the nuclease activity of Mre11. MRX is required to tether transcriptionally active loci to the nuclear pore complex (NPC), and it also promotes large-scale gene-NPC interactions. Moreover, MRX-mediated chromatin anchoring to the NPC contributes to chromosome folding and helps to control gene expression. Together, these findings indicate that MRX has a role in transcription and chromosome organization that is distinct from its known function in DNA repair.
Assuntos
Cromossomos Fúngicos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Cromossomos Fúngicos/genética , Proteínas de Ligação a DNA/genética , Endodesoxirribonucleases/genética , Exodesoxirribonucleases/genética , Complexos Multiproteicos/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The ring-shaped cohesin complex topologically entraps two DNA molecules to establish sister chromatid cohesion. Cohesin also shapes the interphase chromatin landscape with wide-ranging implications for gene regulation, and cohesin is thought to achieve this by actively extruding DNA loops without topologically entrapping DNA. The 'loop extrusion' hypothesis finds motivation from in vitro observations-whether this process underlies in vivo chromatin loop formation remains untested. Here, using the budding yeast S. cerevisiae, we generate cohesin variants that have lost their ability to extrude DNA loops but retain their ability to topologically entrap DNA. Analysis of these variants suggests that in vivo chromatin loops form independently of loop extrusion. Instead, we find that transcription promotes loop formation, and acts as an extrinsic motor that expands these loops and defines their ultimate positions. Our results necessitate a re-evaluation of the loop extrusion hypothesis. We propose that cohesin, akin to sister chromatid cohesion establishment at replication forks, forms chromatin loops by DNA-DNA capture at places of transcription, thus unifying cohesin's two roles in chromosome segregation and interphase genome organisation.
Assuntos
Proteínas de Ciclo Celular , Cromatina , Proteínas Cromossômicas não Histona , Coesinas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Cromatina/metabolismo , Cromatina/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , DNA Fúngico/metabolismo , DNA Fúngico/genética , Cromátides/metabolismo , Cromátides/genética , Transcrição GênicaRESUMO
Structural maintenance of chromosomes (SMC) complexes are essential for genome organization from bacteria to humans, but their mechanisms of action remain poorly understood. Here, we characterize human SMC complexes condensin I and II and unveil the architecture of the human condensin II complex, revealing two putative DNA-entrapment sites. Using single-molecule imaging, we demonstrate that both condensin I and II exhibit ATP-dependent motor activity and promote extensive and reversible compaction of double-stranded DNA. Nucleosomes are incorporated into DNA loops during compaction without being displaced from the DNA, indicating that condensin complexes can readily act upon nucleosome-bound DNA molecules. These observations shed light on critical processes involved in genome organization in human cells.
Assuntos
Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , DNA/química , DNA/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Nucleossomos/metabolismo , Adenosina Trifosfatases/genética , Proteínas de Ligação a DNA/genética , Humanos , Modelos Moleculares , Complexos Multiproteicos/genética , Ligação Proteica , Conformação Proteica , Imagem Individual de Molécula/métodosRESUMO
Despite key roles in sister chromatid cohesion and chromosome organization, the mechanism by which cohesin rings are loaded onto DNA is still unknown. Here we combine biochemical approaches and cryoelectron microscopy (cryo-EM) to visualize a cohesin loading intermediate in which DNA is locked between two gates that lead into the cohesin ring. Building on this structural framework, we design experiments to establish the order of events during cohesin loading. In an initial step, DNA traverses an N-terminal kleisin gate that is first opened upon ATP binding and then closed as the cohesin loader locks the DNA against the ATPase gate. ATP hydrolysis will lead to ATPase gate opening to complete DNA entry. Whether DNA loading is successful or results in loop extrusion might be dictated by a conserved kleisin N-terminal tail that guides the DNA through the kleisin gate. Our results establish the molecular basis for cohesin loading onto DNA.
Assuntos
Proteínas de Ciclo Celular/ultraestrutura , Cromátides/ultraestrutura , Proteínas Cromossômicas não Histona/ultraestrutura , DNA/ultraestrutura , Troca de Cromátide Irmã/genética , Adenosina Trifosfatases/genética , Proteínas de Ciclo Celular/genética , Cromátides/genética , Proteínas Cromossômicas não Histona/genética , Segregação de Cromossomos/genética , Microscopia Crioeletrônica , DNA/genética , Conformação de Ácido Nucleico , Conformação Proteica , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/ultraestrutura , CoesinasRESUMO
Structural maintenance of chromosomes (SMC) complexes are key organizers of chromosome architecture in all kingdoms of life. Despite seemingly divergent functions, such as chromosome segregation, chromosome maintenance, sister chromatid cohesion, and mitotic chromosome compaction, it appears that these complexes function via highly conserved mechanisms and that they represent a novel class of DNA translocases.
Assuntos
Cromátides , Cromossomos/metabolismo , DNA/química , DNA/metabolismo , Complexos Multiproteicos/metabolismo , Adenosina Trifosfatases/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Cromátides/química , Cromátides/genética , Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos , Cromossomos/química , Cromossomos/genética , Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos , Mitose , Complexos Multiproteicos/química , Regiões Promotoras Genéticas , Recombinação V(D)J , CoesinasRESUMO
Condensin is a conserved SMC complex that uses its ATPase machinery to structure genomes, but how it does so is largely unknown. We show that condensin's ATPase has a dual role in chromosome condensation. Mutation of one ATPase site impairs condensation, while mutating the second site results in hyperactive condensin that compacts DNA faster than wild-type, both in vivo and in vitro. Whereas one site drives loop formation, the second site is involved in the formation of more stable higher-order Z loop structures. Using hyperactive condensin I, we reveal that condensin II is not intrinsically needed for the shortening of mitotic chromosomes. Condensin II rather is required for a straight chromosomal axis and enables faithful chromosome segregation by counteracting the formation of ultrafine DNA bridges. SMC complexes with distinct roles for each ATPase site likely reflect a universal principle that enables these molecular machines to intricately control chromosome architecture.
Assuntos
Adenosina Trifosfatases/metabolismo , Montagem e Desmontagem da Cromatina/fisiologia , Proteínas de Ligação a DNA/metabolismo , Complexos Multiproteicos/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/fisiologia , Trifosfato de Adenosina/química , Sítios de Ligação/genética , Sítios de Ligação/fisiologia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Cromatina/fisiologia , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos/metabolismo , Cromossomos/fisiologia , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Humanos , Complexos Multiproteicos/fisiologia , Ligação Proteica/fisiologia , Subunidades Proteicas/metabolismo , CoesinasRESUMO
Smc5 and Smc6, together with the kleisin Nse4, form the heart of the enigmatic and poorly understood Smc5/6 complex, which is frequently viewed as a cousin of cohesin and condensin with functions in DNA repair. As novel functions for cohesin and condensin complexes in the organization of long-range chromatin architecture have recently emerged, new unsuspected roles for Smc5/6 have also surfaced. Here, I aim to provide a comprehensive overview of our current knowledge of the Smc5/6 complex, including its long-established function in genome stability, its multiple roles in DNA repair, and its recently discovered connection to the transcription inhibition of hepatitis B virus genomes. In addition, I summarize new research that is beginning to tease out the molecular details of Smc5/6 structure and function, knowledge that will illuminate the nuclear activities of Smc5/6 in the stability and dynamics of eukaryotic genomes.
Assuntos
Proteínas de Ciclo Celular/genética , Complexos Multiproteicos/genética , Proteínas de Schizosaccharomyces pombe/genética , Relação Estrutura-Atividade , Proteínas de Transporte/genética , Núcleo Celular/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Eucariotos/genética , Humanos , Recombinação Genética/genética , Schizosaccharomyces/genéticaRESUMO
The most prominent representatives of multisubunit SMC complexes, cohesin and condensin, are best known as structural components of mitotic chromosomes. It turned out that these complexes, as well as their bacterial homologues, are molecular motors, the ATP-dependent movement of these complexes along DNA threads leads to the formation of DNA loops. In recent years, we have witnessed an avalanche-like accumulation of data on the process of SMC dependent DNA looping, also known as loop extrusion. This review briefly summarizes the current understanding of the place and role of cohesin-dependent extrusion in cell physiology and presents a number of models describing the potential molecular mechanism of extrusion in a most compelling way. We conclude the review with a discussion of how the capacity of cohesin to extrude DNA loops may be mechanistically linked to its involvement in sister chromatid cohesion.
Assuntos
Fenômenos Fisiológicos Celulares , Coesinas , Animais , Humanos , Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/química , Cromátides/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/química , Coesinas/metabolismo , DNA/metabolismo , DNA/química , Proteínas de Ligação a DNA/metabolismo , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/químicaRESUMO
Accurate duplication and separation of long linear genomic DNA molecules is associated with a number of purely mechanical problems. SMC complexes are key components of the cellular machinery that ensures decatenation of sister chromosomes and compaction of genomic DNA during division. Cohesin, one of the essential eukaryotic SMC complexes, has a typical ring structure with intersubunit pore through which DNA molecules can be threaded. Capacity of cohesin for such topological entrapment of DNA is crucial for the phenomenon of post-replicative association of sister chromatids better known as cohesion. Recently, it became apparent that cohesin and other SMC complexes are, in fact, motor proteins with a very peculiar movement pattern leading to formation of DNA loops. This specific process has been called loop extrusion. Extrusion underlies multiple functions of cohesin beyond cohesion, but molecular mechanism of the process remains a mystery. In this review, we summarized the data on molecular architecture of cohesin, effect of ATP hydrolysis cycle on this architecture, and known modes of cohesin-DNA interactions. Many of the seemingly disparate facts presented here will probably be incorporated in a unified mechanistic model of loop extrusion in the not-so-distant future.
Assuntos
Coesinas , DNA , Animais , Humanos , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/química , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/química , Cromátides/metabolismo , Cromátides/química , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/química , Coesinas/química , Coesinas/metabolismo , DNA/metabolismo , DNA/químicaRESUMO
The RecQ helicase Sgs1 plays critical roles during DNA repair by homologous recombination, from end resection to Holliday junction (HJ) dissolution. Sgs1 has both pro- and anti-recombinogenic roles, and therefore its activity must be tightly regulated. However, the controls involved in recruitment and activation of Sgs1 at damaged sites are unknown. Here we show a two-step role for Smc5/6 in recruiting and activating Sgs1 through SUMOylation. First, auto-SUMOylation of Smc5/6 subunits leads to recruitment of Sgs1 as part of the STR (Sgs1-Top3-Rmi1) complex, mediated by two SUMO-interacting motifs (SIMs) on Sgs1 that specifically recognize SUMOylated Smc5/6. Second, Smc5/6-dependent SUMOylation of Sgs1 and Top3 is required for the efficient function of STR. Sgs1 mutants impaired in recognition of SUMOylated Smc5/6 (sgs1-SIMΔ) or SUMO-dead alleles (sgs1-KR) exhibit unprocessed HJs at damaged replication forks, increased crossover frequencies during double-strand break repair, and severe impairment in DNA end resection. Smc5/6 is a key regulator of Sgs1's recombination functions.
Assuntos
Proteínas de Ciclo Celular/metabolismo , DNA Cruciforme/metabolismo , RecQ Helicases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Motivos de Aminoácidos , Troca Genética , Dano ao DNA/genética , Reparo do DNA por Junção de Extremidades/genética , Mutação , RecQ Helicases/genética , Recombinação Genética/genética , Proteína SUMO-1/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , SumoilaçãoRESUMO
SMC and SMC-like complexes promote chromosome folding and genome maintenance in all domains of life. Recently, they were also recognized as factors in cellular immunity against foreign DNA. In bacteria and archaea, Wadjet and Lamassu are anti-plasmid/phage defence systems, while Smc5/6 and Rad50 complexes play a role in anti-viral immunity in humans. This raises an intriguing paradox - how can the same, or closely related, complexes on one hand secure the integrity and maintenance of chromosomal DNA, while on the other recognize and restrict extrachromosomal DNA? In this minireview, we will briefly describe the latest understanding of each of these complexes in immunity including speculations on how principles of SMC(-like) function may explain how the systems recognize linear or circular forms of invading DNA.
Assuntos
Proteínas de Ciclo Celular , Cromossomos , Humanos , Proteínas de Ciclo Celular/genética , DNA , PlasmídeosRESUMO
Structural maintenance of chromosomes (SMC) complexes are essential proteins found in genomes of all cellular organisms. Essential functions of these proteins, such as mitotic chromosome formation and sister chromatid cohesion, were discovered a long time ago. Recent advances in chromatin biology showed that SMC proteins are involved in many other genomic processes, acting as active motors extruding DNA, which leads to the formation of chromatin loops. Some loops formed by SMC proteins are highly cell type and developmental stage specific, such as SMC-mediated DNA loops required for VDJ recombination in B-cell progenitors, or dosage compensation in Caenorhabditis elegans and X-chromosome inactivation in mice. In this review, we focus on the extrusion-based mechanisms that are common for multiple cell types and species. We will first describe an anatomy of SMC complexes and their accessory proteins. Next, we provide biochemical details of the extrusion process. We follow this by the sections describing the role of SMC complexes in gene regulation, DNA repair, and chromatin topology.
Assuntos
Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona , Animais , Camundongos , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromatina , DNA/química , Caenorhabditis elegans/metabolismoRESUMO
The cohesin complex plays an important role in the maintenance of genome stability. Cohesin is composed of four core subunits and a set of regulatory subunits that interact with the core subunits. Less is known about cohesin dynamics in live cells and on the contribution of individual subunits to the overall complex. Understanding the tethering mechanism of cohesin is still a challenge, especially because the proposed mechanisms are still not conclusive. Models proposed to describe tethering depend on either the monomeric cohesin ring or a cohesin dimer. Here, we investigate the role of cohesin dynamics and stoichiometry in live yeast cells at single-molecule resolution. We explore the effect of regulatory subunit deletion on cohesin mobility and found that depletion of different regulatory subunits has opposing effects. Finally, we show that cohesin exists mostly as a canonical monomer throughout the cell cycle, and its monomeric form is independent of its regulatory factors. Our results demonstrate that single-molecule tools have the potential to provide new insights into the cohesin mechanism of action in live cells.