Sister chromatid cohesion establishment during DNA replication termination.

Newly copied sister chromatids are tethered together by the cohesin complex, but how sister chromatid cohesion coordinates with DNA replication is poorly understood. Prevailing models suggest that cohesin complexes, bound to DNA before replication, remain behind the advancing replication fork to keep sister chromatids together. By visualizing single replication forks colliding with preloaded cohesin complexes, we find that the replisome instead pushes cohesin to where a converging replisome is met. Whereas the converging replisomes are removed during DNA replication termination, cohesin remains on nascent DNA and provides cohesion. Additionally, we show that CMG (CDC45-MCM2-7-GINS) helicase disassembly during replication termination is vital for proper cohesion in budding yeast. Together, our results support a model wherein sister chromatid cohesion is established during DNA replication termination.

Sister chromatid cohesion halts DNA loop expansion.

Eukaryotic genomes are folded into DNA loops mediated by structural maintenance of chromosomes (SMC) complexes such as cohesin, condensin, and Smc5/6. This organization regulates different DNA-related processes along the cell cycle, such as transcription, recombination, segregation, and DNA repair. During the G2 stage, SMC-mediated DNA loops coexist with cohesin complexes involved in sister chromatid cohesion (SCC). However, the articulation between the establishment of SCC and the formation of SMC-mediated DNA loops along the chromatin remains unknown. Here, we show that SCC is indeed a barrier to cohesin-mediated DNA loop expansion along G2/M Saccharomyces cerevisiae chromosomes.

Securin acetylation prevents precocious separase activation and premature sister chromatid separation.

Lysine acetylation of non-histone proteins plays crucial roles in many cellular processes. In this study, we examine the role of lysine acetylation during sister chromatid separation in mitosis. We investigate the acetylation of securin at K21 by cell-cycle-dependent acetylome analysis and uncover its role in separase-triggered chromosome segregation during mitosis. Prior to the onset of anaphase, the acetylated securin via TIP60 prevents its degradation by the APC/CCDC20-mediated ubiquitin-proteasome system. This, in turn, restrains precocious activation of separase and premature separation of sister chromatids. Additionally, the acetylation-dependent stability of securin is also enhanced by its dephosphorylation. As anaphase approaches, HDAC1-mediated deacetylation of securin promotes its degradation, allowing released separase to cleave centromeric cohesin. Blocking securin deacetylation leads to longer anaphase duration and errors in chromosome segregation. Thus, this study illustrates the emerging role of securin acetylation dynamics in mitotic progression and genetic stability.

Sister chromatid cohesion is mediated by individual cohesin complexes.

Eukaryotic genomes are organized by loop extrusion and sister chromatid cohesion, both mediated by the multimeric cohesin protein complex. Understanding how cohesin holds sister DNAs together, and how loss of cohesion causes age-related infertility in females, requires knowledge as to cohesin's stoichiometry in vivo. Using quantitative super-resolution imaging, we identified two discrete populations of chromatin-bound cohesin in postreplicative human cells. Whereas most complexes appear dimeric, cohesin that localized to sites of sister chromatid cohesion and associated with sororin was exclusively monomeric. The monomeric stoichiometry of sororin:cohesin complexes demonstrates that sister chromatid cohesion is conferred by individual cohesin rings, a key prediction of the proposal that cohesion arises from the co-entrapment of sister DNAs.

Role of chromosomal cohesion and separation in aneuploidy and tumorigenesis.

Cell division is a crucial process, and one of its essential steps involves copying the genetic material, which is organized into structures called chromosomes. Before a cell can divide into two, it needs to ensure that each newly copied chromosome is paired tightly with its identical twin. This pairing is maintained by a protein complex known as cohesin, which is conserved in various organisms, from single-celled ones to humans. Cohesin essentially encircles the DNA, creating a ring-like structure to handcuff, to keep the newly synthesized sister chromosomes together in pairs. Therefore, chromosomal cohesion and separation are fundamental processes governing the attachment and segregation of sister chromatids during cell division. Metaphase-to-anaphase transition requires dissolution of cohesins by the enzyme Separase. The tight regulation of these processes is vital for safeguarding genomic stability. Dysregulation in chromosomal cohesion and separation resulting in aneuploidy, a condition characterized by an abnormal chromosome count in a cell, is strongly associated with cancer. Aneuploidy is a recurring hallmark in many cancer types, and abnormalities in chromosomal cohesion and separation have been identified as significant contributors to various cancers, such as acute myeloid leukemia, myelodysplastic syndrome, colorectal, bladder, and other solid cancers. Mutations within the cohesin complex have been associated with these cancers, as they interfere with chromosomal segregation, genome organization, and gene expression, promoting aneuploidy and contributing to the initiation of malignancy. In summary, chromosomal cohesion and separation processes play a pivotal role in preserving genomic stability, and aberrations in these mechanisms can lead to aneuploidy and cancer. Gaining a deeper understanding of the molecular intricacies of chromosomal cohesion and separation offers promising prospects for the development of innovative therapeutic approaches in the battle against cancer.

Coordination of cohesin and DNA replication observed with purified proteins.

Two newly duplicated copies of genomic DNA are held together by the ring-shaped cohesin complex to ensure faithful inheritance of the genome during cell division1-3. Cohesin mediates sister chromatid cohesion by topologically entrapping two sister DNAs during DNA replication4,5, but how cohesion is established at the replication fork is poorly understood. Here, we studied the interplay between cohesin and replication by reconstituting a functional replisome using purified proteins. Once DNA is encircled before replication, the cohesin ring accommodates replication in its entirety, from initiation to termination, leading to topological capture of newly synthesized DNA. This suggests that topological cohesin loading is a critical molecular prerequisite to cope with replication. Paradoxically, topological loading per se is highly rate limiting and hardly occurs under the replication-competent physiological salt concentration. This inconsistency is resolved by the replisome-associated cohesion establishment factors Chl1 helicase and Ctf4 (refs. 6,7), which promote cohesin loading specifically during continuing replication. Accordingly, we found that bubble DNA, which mimics the state of DNA unwinding, induces topological cohesin loading and this is further promoted by Chl1. Thus, we propose that cohesin converts the initial electrostatic DNA-binding mode to a topological embrace when it encounters unwound DNA structures driven by enzymatic activities including replication. Together, our results show how cohesin initially responds to replication, and provide a molecular model for the establishment of sister chromatid cohesion.

Mechanical disengagement of the cohesin ring.

Cohesin forms a proteinaceous ring that is thought to link sister chromatids by entrapping DNA and counteracting the forces generated by the mitotic spindle. Whether individual cohesins encircle both sister DNAs and how cohesin opposes spindle-generated forces remains unknown. Here we perform force measurements on individual yeast cohesin complexes either bound to DNA or holding together two DNAs. By covalently closing the hinge and Smc3Psm3-kleisin interfaces we find that the mechanical stability of the cohesin ring entrapping DNA is determined by the hinge domain. Forces of ~20 pN disengage cohesin at the hinge and release DNA, indicating that ~40 cohesin molecules are sufficient to counteract known spindle forces. Our findings provide a mechanical framework for understanding how cohesin interacts with sister chromatids and opposes the spindle-generated tension during mitosis, with implications for other force-generating chromosomal processes including transcription and DNA replication.

Age-dependent loss of cohesion protection in human oocytes.

Aneuploid human eggs (oocytes) are a major cause of infertility, miscarriage, and chromosomal disorders. Such aneuploidies increase greatly as women age, with defective linkages between sister chromatids (cohesion) in meiosis as a common cause. We found that loss of a specific pool of the cohesin protector protein, shugoshin 2 (SGO2), may contribute to this phenomenon. Our data indicate that SGO2 preserves sister chromatid cohesion in meiosis by protecting a "cohesin bridge" between sister chromatids. In human oocytes, SGO2 localizes to both sub-centromere cups and the pericentromeric bridge, which spans the sister chromatid junction. SGO2 normally colocalizes with cohesin; however, in meiosis II oocytes from older women, SGO2 is frequently lost from the pericentromeric bridge and sister chromatid cohesion is weakened. MPS1 and BUB1 kinase activities maintain SGO2 at sub-centromeres and the pericentromeric bridge. Removal of SGO2 throughout meiosis I by MPS1 inhibition reduces cohesion protection, increasing the incidence of single chromatids at meiosis II. Therefore, SGO2 deficiency in human oocytes can exacerbate the effects of maternal age by rendering residual cohesin at pericentromeres vulnerable to loss in anaphase I. Our data show that impaired SGO2 localization weakens cohesion integrity and may contribute to the increased incidence of aneuploidy observed in human oocytes with advanced maternal age.

Defining a core configuration for human centromeres during mitosis.

The centromere components cohesin, CENP-A, and centromeric DNA are essential for biorientation of sister chromatids on the mitotic spindle and accurate sister chromatid segregation. Insight into the 3D organization of centromere components would help resolve how centromeres function on the mitotic spindle. We use ChIP-seq and super-resolution microscopy with single particle averaging to examine the geometry of essential centromeric components on human chromosomes. Both modalities suggest cohesin is enriched at pericentromeric DNA. CENP-A localizes to a subset of the α-satellite DNA, with clusters separated by ~562 nm and a perpendicular intervening ~190 nM wide axis of cohesin in metaphase chromosomes. Differently sized α-satellite arrays achieve a similar core structure. Here we present a working model for a common core configuration of essential centromeric components that includes CENP-A nucleosomes, α-satellite DNA and pericentromeric cohesion. This configuration helps reconcile how centromeres function and serves as a foundation to add components of the chromosome segregation machinery.

What AlphaFold tells us about cohesin's retention on and release from chromosomes.

Cohesin is a trimeric complex containing a pair of SMC proteins (Smc1 and Smc3) whose ATPase domains at the end of long coiled coils (CC) are interconnected by Scc1. During interphase, it organizes chromosomal DNA topology by extruding loops in a manner dependent on Scc1's association with two large hook-shaped proteins called SA (yeast: Scc3) and Nipbl (Scc2). The latter's replacement by Pds5 recruits Wapl, which induces release from chromatin via a process requiring dissociation of Scc1's N-terminal domain (NTD) from Smc3. If blocked by Esco (Eco)-mediated Smc3 acetylation, cohesin containing Pds5 merely maintains pre-existing loops, but a third fate occurs during DNA replication, when Pds5-containing cohesin associates with Sororin and forms structures that hold sister DNAs together. How Wapl induces and Sororin blocks release has hitherto remained mysterious. In the 20 years since their discovery, not a single testable hypothesis has been proposed as to their role. Here, AlphaFold 2 (AF) three-dimensional protein structure predictions lead us to propose formation of a quarternary complex between Wapl, SA, Pds5, and Scc1's NTD, in which the latter is juxtaposed with (and subsequently sequestered by) a highly conserved cleft within Wapl's C-terminal domain. AF also reveals how Scc1's dissociation from Smc3 arises from a distortion of Smc3's CC induced by engagement of SMC ATPase domains, how Esco acetyl transferases are recruited to Smc3 by Pds5, and how Sororin prevents release by binding to the Smc3/Scc1 interface. Our hypotheses explain the phenotypes of numerous existing mutations and are highly testable.

Using Sister Chromatid Exchange Assay to Detect Homologous Recombination Deficiency in Epigenetically Deregulated Urothelial Carcinoma Cells.

Sister chromatid exchange (SCE) is the process of exchanging regions between two sister chromatids during DNA replication. Exchanges between replicated chromatids and their sisters can be visualized in cells when DNA synthesis in one chromatid is labelled by 5-bromo-2'-deoxyuridine (BrdU). Homologous recombination (HR) is considered as the principal mechanism responsible for the sister chromatid exchange (SCE) upon replication fork collapse, and therefore SCE frequency upon genotoxic conditions reflects the capacity of HR repair to respond to replication stress. During tumorigenesis, inactivating mutations or altered transcriptome can affect a plethora of epigenetic factors that participate in DNA repair processes, and there are an increasing number of reports which demonstrate a link between epigenetic deregulation in cancer and homologous recombination deficiency (HRD). Therefore, the SCE assay can provide valuable information regarding the HR functionality in tumors with epigenetic deficiencies. In this chapter, we provide a method to visualize SCEs. The technique outlined below is characterized by high sensitivity and specificity and has been successfully applied to human bladder cancer cell lines. In this context, this technique could be used to characterize the dynamics of HR repair in tumors with deregulated epigenome.

A dual-function SNF2 protein drives chromatid resolution and nascent transcripts removal in mitosis.

Mitotic chromatin is largely assumed incompatible with transcription due to changes in the transcription machinery and chromosome architecture. However, the mechanisms of mitotic transcriptional inactivation and their interplay with chromosome assembly remain largely unknown. By monitoring ongoing transcription in Drosophila early embryos, we reveal that eviction of nascent mRNAs from mitotic chromatin occurs after substantial chromosome compaction and is not promoted by condensin I. Instead, we show that the timely removal of transcripts from mitotic chromatin is driven by the SNF2 helicase-like protein Lodestar (Lds), identified here as a modulator of sister chromatid cohesion defects. In addition to the eviction of nascent transcripts, we uncover that Lds cooperates with Topoisomerase 2 to ensure efficient sister chromatid resolution and mitotic fidelity. We conclude that the removal of nascent transcripts upon mitotic entry is not a passive consequence of cell cycle progression and/or chromosome compaction but occurs via dedicated mechanisms with functional parallelisms to sister chromatid resolution.

The histone methyltransferase NSD3 contributes to sister chromatid cohesion and to cohesin loading at mitotic exit.

Sister chromatid cohesion is a multi-step process implemented throughout the cell cycle to ensure the correct transmission of chromosomes to daughter cells. Although cohesion establishment and mitotic cohesion dissolution have been extensively explored, the regulation of cohesin loading is still poorly understood. Here, we report that the methyltransferase NSD3 is essential for mitotic sister chromatid cohesion before mitosis entry. NSD3 interacts with the cohesin loader complex kollerin (composed of NIPBL and MAU2) and promotes the chromatin recruitment of MAU2 and cohesin at mitotic exit. We also show that NSD3 associates with chromatin in early anaphase, prior to the recruitment of MAU2 and RAD21, and dissociates from chromatin when prophase begins. Among the two NSD3 isoforms present in somatic cells, the long isoform is responsible for regulating kollerin and cohesin chromatin-loading, and its methyltransferase activity is required for efficient sister chromatid cohesion. Based on these observations, we propose that NSD3-dependent methylation contributes to sister chromatid cohesion by ensuring proper kollerin recruitment and thus cohesin loading.

Cohesin-mediated DNA loop extrusion resolves sister chromatids in G2 phase.

Genetic information is stored in linear DNA molecules, which are highly folded inside cells. DNA replication along the folded template path yields two sister chromatids that initially occupy the same nuclear region in an intertwined arrangement. Dividing cells must disentangle and condense the sister chromatids into separate bodies such that a microtubule-based spindle can move them to opposite poles. While the spindle-mediated transport of sister chromatids has been studied in detail, the chromosome-intrinsic mechanics presegregating sister chromatids have remained elusive. Here, we show that human sister chromatids resolve extensively already during interphase, in a process dependent on the loop-extruding activity of cohesin, but not that of condensins. Increasing cohesin's looping capability increases sister DNA resolution in interphase nuclei to an extent normally seen only during mitosis, despite the presence of abundant arm cohesion. That cohesin can resolve sister chromatids so extensively in the absence of mitosis-specific activities indicates that DNA loop extrusion is a generic mechanism for segregating replicated genomes, shared across different Structural Maintenance of Chromosomes (SMC) protein complexes in all kingdoms of life.

FIRRM/C1orf112 is synthetic lethal with PICH and mediates RAD51 dynamics.

Joint DNA molecules are natural byproducts of DNA replication and repair. Persistent joint molecules give rise to ultrafine DNA bridges (UFBs) in mitosis, compromising sister chromatid separation. The DNA translocase PICH (ERCC6L) has a central role in UFB resolution. A genome-wide loss-of-function screen is performed to identify the genetic context of PICH dependency. In addition to genes involved in DNA condensation, centromere stability, and DNA-damage repair, we identify FIGNL1-interacting regulator of recombination and mitosis (FIRRM), formerly known as C1orf112. We find that FIRRM interacts with and stabilizes the AAA+ ATPase FIGNL1. Inactivation of either FIRRM or FIGNL1 results in UFB formation, prolonged accumulation of RAD51 at nuclear foci, and impaired replication fork dynamics and consequently impairs genome maintenance. Combined, our data suggest that inactivation of FIRRM and FIGNL1 dysregulates RAD51 dynamics at replication forks, resulting in persistent DNA lesions and a dependency on PICH to preserve cell viability.

Replisome-cohesin interactions provided by the Tof1-Csm3 and Mrc1 cohesion establishment factors.

The chromosomal cohesin complex establishes sister chromatid cohesion during S phase, which forms the basis for faithful segregation of DNA replication products during cell divisions. Cohesion establishment is defective in the absence of either of three non-essential Saccharomyces cerevisiae replication fork components Tof1-Csm3 and Mrc1. Here, we investigate how these conserved factors contribute to cohesion establishment. Tof1-Csm3 and Mrc1 serve known roles during DNA replication, including replication checkpoint signaling, securing replication fork speed, as well as recruiting topoisomerase I and the histone chaperone FACT. By modulating each of these functions independently, we rule out that one of these known replication roles explains the contribution of Tof1-Csm3 and Mrc1 to cohesion establishment. Instead, using purified components, we reveal direct and multipronged protein interactions of Tof1-Csm3 and Mrc1 with the cohesin complex. Our findings open the possibility that a series of physical interactions between replication fork components and cohesin facilitate successful establishment of sister chromatid cohesion during DNA replication.

Structural basis of centromeric cohesion protection.

In the early stages of mitosis, cohesin is released from chromosome arms but not from centromeres. The protection of centromeric cohesin by SGO1 maintains the sister chromatid cohesion that resists the pulling forces of microtubules until all chromosomes are attached in a bipolar manner to the mitotic spindle. Here we present the X-ray crystal structure of a segment of human SGO1 bound to a conserved surface of the cohesin complex. SGO1 binds to a composite interface formed by the SA2 and SCC1RAD21 subunits of cohesin. SGO1 shares this binding interface with CTCF, indicating that these distinct chromosomal regulators control cohesin through a universal principle. This interaction is essential for the localization of SGO1 to centromeres and protects centromeric cohesin against WAPL-mediated cohesin release. SGO1-cohesin binding is maintained until the formation of microtubule-kinetochore attachments and is required for faithful chromosome segregation and the maintenance of a stable karyotype.

A model for Scc2p stimulation of cohesin's ATPase and its inhibition by acetylation of Smc3p.

The evolutionarily conserved cohesin complex mediates sister chromatid cohesion and facilitates mitotic chromosome condensation, DNA repair, and transcription regulation. These biological functions require cohesin's two ATPases, formed by the Smc1p and Smc3p subunits. Cohesin's ATPase activity is stimulated by the Scc2p auxiliary factor. This stimulation is inhibited by Eco1p acetylation of Smc3p at an interface with Scc2p. It was unclear how cohesin's ATPase activity is stimulated by Scc2p or how acetylation inhibits Scc2p, given that the acetylation site is distal to cohesin's ATPase active sites. Here, we identify mutations in budding yeast that suppressed the in vivo defects caused by Smc3p acetyl-mimic and acetyl-defective mutations. We provide compelling evidence that Scc2p activation of cohesin ATPase depends on an interface between Scc2p and a region of Smc1p proximal to cohesin's Smc3p ATPase active site. Furthermore, substitutions at this interface increase or decrease ATPase activity to overcome ATPase modulation by acetyl-mimic and acetyl-null mutations. Using these observations and an existing cryo-EM structure, we propose a model for regulating cohesin ATPase activity. We suggest that Scc2p binding to Smc1p causes the adjacent Smc1p residues and ATP to shift, stimulating Smc3p's ATPase. This stimulatory shift is inhibited through acetylation of the distal Scc2p-Smc3p interface.

Chromosome organization by fine-tuning an ATPase.

Cohesin is an ATPase that drives chromosome organization through the generation of intramolecular loops and sister chromatid cohesion. Cohesin's ATPase is stimulated by Scc2 binding but attenuated by acetylation of its Smc3 subunit. In this issue of Genes & Development, Boardman and colleagues (pp. 277-290) take a genetic approach to generate a mechanistic model for the opposing regulation of cohesin's ATPase by Scc2 and Smc3 acetylation. Their findings provide in vivo insight into how this important genome organizer functions in vivo.

Separase and Roads to Disengage Sister Chromatids during Anaphase.

Receiving complete and undamaged genetic information is vital for the survival of daughter cells after chromosome segregation. The most critical steps in this process are accurate DNA replication during S phase and a faithful chromosome segregation during anaphase. Any errors in DNA replication or chromosome segregation have dire consequences, since cells arising after division might have either changed or incomplete genetic information. Accurate chromosome segregation during anaphase requires a protein complex called cohesin, which holds together sister chromatids. This complex unifies sister chromatids from their synthesis during S phase, until separation in anaphase. Upon entry into mitosis, the spindle apparatus is assembled, which eventually engages kinetochores of all chromosomes. Additionally, when kinetochores of sister chromatids assume amphitelic attachment to the spindle microtubules, cells are finally ready for the separation of sister chromatids. This is achieved by the enzymatic cleavage of cohesin subunits Scc1 or Rec8 by an enzyme called Separase. After cohesin cleavage, sister chromatids remain attached to the spindle apparatus and their poleward movement on the spindle is initiated. The removal of cohesion between sister chromatids is an irreversible step and therefore it must be synchronized with assembly of the spindle apparatus, since precocious separation of sister chromatids might lead into aneuploidy and tumorigenesis. In this review, we focus on recent discoveries concerning the regulation of Separase activity during the cell cycle.