Sororin is an evolutionary conserved antagonist of WAPL.

Cohesin mediates sister chromatid cohesion to enable chromosome segregation and DNA damage repair. To perform these functions, cohesin needs to be protected from WAPL, which otherwise releases cohesin from DNA. It has been proposed that cohesin is protected from WAPL by SORORIN. However, in vivo evidence for this antagonism is missing and SORORIN is only known to exist in vertebrates and insects. It is therefore unknown how important and widespread SORORIN's functions are. Here we report the identification of SORORIN orthologs in Schizosaccharomyces pombe (Sor1) and Arabidopsis thaliana (AtSORORIN). sor1Δ mutants display cohesion defects, which are partially alleviated by wpl1Δ. Atsororin mutant plants display dwarfism, tissue specific cohesion defects and chromosome mis-segregation. Furthermore, Atsororin mutant plants are sterile and separate sister chromatids prematurely at anaphase I. The somatic, but not the meiotic deficiencies can be alleviated by loss of WAPL. These results provide in vivo evidence for SORORIN antagonizing WAPL, reveal that SORORIN is present in organisms beyond the animal kingdom and indicate that it has acquired tissue specific functions in plants.

Cohesin Complex: Structure and Principles of Interaction with DNA.

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.

Cohesin-Dependent Loop Extrusion: Molecular Mechanics and Role in Cell Physiology.

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.

Human AAA+ ATPase FIGNL1 suppresses RAD51-mediated ultra-fine bridge formation.

RAD51 filament is crucial for the homology-dependent repair of DNA double-strand breaks and stalled DNA replication fork protection. Positive and negative regulators control RAD51 filament assembly and disassembly. RAD51 is vital for genome integrity but excessive accumulation of RAD51 on chromatin causes genome instability and growth defects. However, the detailed mechanism underlying RAD51 disassembly by negative regulators and the physiological consequence of abnormal RAD51 persistence remain largely unknown. Here, we report the role of the human AAA+ ATPase FIGNL1 in suppressing a novel type of RAD51-mediated genome instability. FIGNL1 knockout human cells were defective in RAD51 dissociation after replication fork restart and accumulated ultra-fine chromosome bridges (UFBs), whose formation depends on RAD51 rather than replication fork stalling. FIGNL1 suppresses homologous recombination intermediate-like UFBs generated between sister chromatids at genomic loci with repeated sequences such as telomeres and centromeres. These data suggest that RAD51 persistence per se induces the formation of unresolved linkage between sister chromatids resulting in catastrophic genome instability. FIGNL1 facilitates post-replicative disassembly of RAD51 filament to suppress abnormal recombination intermediates and UFBs. These findings implicate FIGNL1 as a key factor required for active RAD51 removal after processing of stalled replication forks, which is essential to maintain genome stability.

DNA repair is efficient in irradiated M phase zygotes.

In somatic cells, DNA repair is attenuated during mitosis to prevent the formation of anaphase bridges and facilitate the proper segregation of sister chromatids. Irradiation-induced γH2AX foci persist for hours in M phase somatic cells. However, we observed that anaphase bridges formed in a significant fraction of mouse zygotes irradiated during mitosis. Additionally, γH2AX signals in M phase zygotes peaked 30 min after irradiation and subsequently reduced with a half-life within 1-2 h. These results suggest that the DNA repair system may operate efficiently in M phase zygotes following irradiation, leading to the frequent formation of anaphase bridges. The absence of H2AX promoted the successful segregation of sister chromatids and enhanced the development of embryos to the blastocyst stage. The DNA repair system may be differentially regulated during the M phase of the first cell cycle to ensure the immediate elimination of damaged zygotes, thereby efficiently preventing transmission of mutations to subsequent generations.

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.

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.

Mapping of DDX11 genetic interactions defines sister chromatid cohesion as the major dependency.

DDX11/Chl1R is a conserved DNA helicase with roles in genome maintenance, DNA replication, and chromatid cohesion. Loss of DDX11 in humans leads to the rare cohesinopathy Warsaw breakage syndrome. DDX11 has also been implicated in human cancer where it has been proposed to have an oncogenic role and possibly to constitute a therapeutic target. Given the multiple roles of DDX11 in genome stability and its potential as an anticancer target, we set out to define a complete genetic interaction profile of DDX11 loss in human cell lines. Screening the human genome with clustered regularly interspaced short palindromic repeats (CRISPR) guide RNA drop out screens in DDX11-wildtype (WT) or DDX11-deficient cells revealed a strong enrichment of genes with functions related to sister chromatid cohesion. We confirm synthetic lethal relationships between DDX11 and the tumor suppressor cohesin subunit STAG2, which is frequently mutated in several cancer types and the kinase HASPIN. This screen highlights the importance of cohesion in cells lacking DDX11 and suggests DDX11 may be a therapeutic target for tumors with mutations in STAG2.

Induction of somatic cell haploidy by premature cell division.

Canonical mitotic and meiotic cell divisions commence with replicated chromosomes consisting of two sister chromatids. Here, we developed and explored a model of premature cell division, where nonreplicated, G0/G1-stage somatic cell nuclei are transplanted to the metaphase cytoplasm of mouse oocytes. Subsequent cell division generates daughter cells with reduced ploidy. Unexpectedly, genome sequencing analysis revealed proper segregation of homologous chromosomes, resulting in complete haploid genomes. We observed a high occurrence of somatic genome haploidization in nuclei from inbred genetic backgrounds but not in hybrids, emphasizing the importance of sequence homology between homologs. These findings suggest that premature cell division relies on mechanisms similar to meiosis I, where genome haploidization is facilitated by homologous chromosome interactions, recognition, and pairing. Unlike meiosis, no evidence of recombination between somatic cell homologs was detected. Our study offers an alternative in vitro gametogenesis approach by directly reprogramming diploid somatic cells into haploid oocytes.

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.

DNA combing versus DNA spreading and the separation of sister chromatids.

DNA combing and DNA spreading are two central approaches for studying DNA replication fork dynamics genome-wide at single-molecule resolution by distributing labeled genomic DNA on coverslips or slides for immunodetection. Perturbations in DNA replication fork dynamics can differentially affect either leading or lagging strand synthesis, for example, in instances where replication is blocked by a lesion or obstacle on only one of the two strands. Thus, we sought to investigate whether the DNA combing and/or spreading approaches are suitable for resolving adjacent sister chromatids during DNA replication, thereby enabling the detection of DNA replication dynamics within individual nascent strands. To this end, we developed a thymidine labeling scheme that discriminates between these two possibilities. Our data suggests that DNA combing resolves sister chromatids, allowing the detection of strand-specific alterations, whereas DNA spreading typically does not. These findings have important implications when interpreting DNA replication dynamics from data obtained by these two commonly used techniques.

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.

Spindle assembly checkpoint insensitivity allows meiosis-II despite chromosomal defects in aged eggs.

Chromosome segregation errors in mammalian oocyte meiosis lead to developmentally compromised aneuploid embryos and become more common with advancing maternal age. Known contributors include age-related chromosome cohesion loss and spindle assembly checkpoint (SAC) fallibility in meiosis-I. But how effective the SAC is in meiosis-II and how this might contribute to age-related aneuploidy is unknown. Here, we developed genetic and pharmacological approaches to directly address the function of the SAC in meiosis-II. We show that the SAC is insensitive in meiosis-II oocytes and that as a result misaligned chromosomes are randomly segregated. Whilst SAC ineffectiveness in meiosis-II is not age-related, it becomes most prejudicial in oocytes from older females because chromosomes that prematurely separate by age-related cohesion loss become misaligned in meiosis-II. We show that in the absence of a robust SAC in meiosis-II these age-related misaligned chromatids are missegregated and lead to aneuploidy. Our data demonstrate that the SAC fails to prevent cell division in the presence of misaligned chromosomes in oocyte meiosis-II, which explains how age-related cohesion loss can give rise to aneuploid embryos.

Cosegregation of recombinant chromatids maintains genome-wide heterozygosity in an asexual nematode.

In asexual animals, female meiosis is modified to produce diploid oocytes. If meiosis still involves recombination, this is expected to lead to a rapid loss of heterozygosity, with adverse effects on fitness. Many asexuals, however, have a heterozygous genome, the underlying mechanisms being most often unknown. Cytological and population genomic analyses in the nematode Mesorhabditis belari revealed another case of recombining asexual being highly heterozygous genome-wide. We demonstrated that heterozygosity is maintained despite recombination because the recombinant chromatids of each chromosome pair cosegregate during the unique meiotic division. A theoretical model confirmed that this segregation bias is necessary to account for the observed pattern and likely to evolve under a wide range of conditions. Our study uncovers an unexpected type of non-Mendelian genetic inheritance involving cosegregation of recombinant chromatids.