ABSTRACT
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.
Subject(s)
Cohesins , DNA , Animals , Humans , Adenosine Triphosphate/metabolism , Adenosine Triphosphate/chemistry , Cell Cycle Proteins/metabolism , Cell Cycle Proteins/chemistry , Chromatids/metabolism , Chromatids/chemistry , Chromosomal Proteins, Non-Histone/metabolism , Chromosomal Proteins, Non-Histone/chemistry , Cohesins/chemistry , Cohesins/metabolism , DNA/metabolism , DNA/chemistryABSTRACT
Chromatids of mitotic chromosomes were suggested to coil into a helix in early cytological studies and this assumption was recently supported by chromosome conformation capture (3C) sequencing. Still, direct differential visualization of a condensed chromatin fibre confirming the helical model was lacking. Here, we combined Hi-C analysis of purified metaphase chromosomes, biopolymer modelling and spatial structured illumination microscopy of large fluorescently labeled chromosome segments to reveal the chromonema - a helically-wound, 400 nm thick chromatin thread forming barley mitotic chromatids. Chromatin from adjacent turns of the helix intermingles due to the stochastic positioning of chromatin loops inside the chromonema. Helical turn size varies along chromosome length, correlating with chromatin density. Constraints on the observable dimensions of sister chromatid exchanges further supports the helical chromonema model.
Subject(s)
Chromatids , Hordeum , Metaphase , Chromatids/chemistry , Chromatin/genetics , Chromosomes , Microscopy , Sister Chromatid Exchange , Chromosomes, Plant , Hordeum/cytologyABSTRACT
Eukaryotic genomes are compacted into loops and topologically associating domains (TADs)1-3, which contribute to transcription, recombination and genomic stability4,5. Cohesin extrudes DNA into loops that are thought to lengthen until CTCF boundaries are encountered6-12. Little is known about whether loop extrusion is impeded by DNA-bound machines. Here we show that the minichromosome maintenance (MCM) complex is a barrier that restricts loop extrusion in G1 phase. Single-nucleus Hi-C (high-resolution chromosome conformation capture) of mouse zygotes reveals that MCM loading reduces CTCF-anchored loops and decreases TAD boundary insulation, which suggests that loop extrusion is impeded before reaching CTCF. This effect extends to HCT116 cells, in which MCMs affect the number of CTCF-anchored loops and gene expression. Simulations suggest that MCMs are abundant, randomly positioned and partially permeable barriers. Single-molecule imaging shows that MCMs are physical barriers that frequently constrain cohesin translocation in vitro. Notably, chimeric yeast MCMs that contain a cohesin-interaction motif from human MCM3 induce cohesin pausing, indicating that MCMs are 'active' barriers with binding sites. These findings raise the possibility that cohesin can arrive by loop extrusion at MCMs, which determine the genomic sites at which sister chromatid cohesion is established. On the basis of in vivo, in silico and in vitro data, we conclude that distinct loop extrusion barriers shape the three-dimensional genome.
Subject(s)
Cell Cycle Proteins , Chromosomal Proteins, Non-Histone , DNA , Minichromosome Maintenance Proteins , Animals , CCCTC-Binding Factor/metabolism , Cell Cycle Proteins/metabolism , Chromatids/chemistry , Chromatids/metabolism , Chromosomal Proteins, Non-Histone/metabolism , DNA/chemistry , DNA/metabolism , G1 Phase , HCT116 Cells , Humans , Mice , Minichromosome Maintenance Complex Component 3/chemistry , Minichromosome Maintenance Complex Component 3/metabolism , Minichromosome Maintenance Proteins/chemistry , Minichromosome Maintenance Proteins/metabolism , Multienzyme Complexes/chemistry , Multienzyme Complexes/metabolism , Nucleic Acid Conformation , Saccharomyces cerevisiae , Saccharomyces cerevisiae Proteins/metabolism , CohesinsABSTRACT
Accurate repair of DNA double-strand breaks (DSBs) in developing germ cells is critical to promote proper chromosome segregation and to maintain genome integrity. To directly detect homolog-independent (intersister/intrachromatid) meiotic DSB repair, we exploited the genetics and germline physiology of C. elegans to (1) induce a single DSB in nuclei across discrete stages of meiotic prophase I; (2) detect repair of that DSB as a homolog-independent crossover or noncrossover; and (3) sequence the resultant product to assess mechanisms of recombination. For complete details on the use and execution of this protocol, please refer to Toraason et al. (2021).
Subject(s)
Chromatids , Crossing Over, Genetic/genetics , DNA Repair/genetics , Meiotic Prophase I/genetics , Molecular Biology/methods , Animals , Caenorhabditis elegans/genetics , Chromatids/chemistry , Chromatids/genetics , Chromatids/metabolism , Chromosome Segregation/genetics , DNA Breaks, Double-StrandedABSTRACT
During meiosis, homologous chromosomes exchange genetic material. This exchange or meiotic recombination is mediated by a proteinaceous scaffold known as the Synaptonemal complex (SC). Any defects in its formation produce failures in meiotic recombination, chromosome segregation and meiosis completion. It has been proposed that DNA repair events that will be resolved by crossover between homologous chromosomes are predetermined by the SC. Hence, structural analysis of the organization of the DNA in the SC could shed light on the process of crossover interference. In this work, we employed an ultrastructural DNA staining technique on mouse testis and followed nuclei of pachytene cells. We observed structures organized similarly to the SCs stained with conventional techniques. These structures, presumably the DNA in the SCs, are delineating the edges of both lateral elements and no staining was observed between them. DNA in the LEs resembles two parallel tracks. However, a bubble-like staining pattern in certain regions of the SC was observed. Furthermore, this staining pattern is found in SCs formed between non-homologous chromosomes, in SCs formed between sister chromatids and in SCs without lateral elements, suggesting that this particular organization of the DNA is determined by the synapsis of the chromosomes despite their lack of homology or the presence of partially formed SCs.
Subject(s)
DNA-Binding Proteins/metabolism , DNA/metabolism , Meiosis/physiology , Synaptonemal Complex/metabolism , Animals , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/metabolism , Chromatids/chemistry , Chromatids/metabolism , Chromatids/ultrastructure , Chromosome Pairing/physiology , DNA/chemistry , DNA/ultrastructure , Macromolecular Substances/chemistry , Macromolecular Substances/metabolism , Male , Mice , Mice, Knockout , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Nucleic Acid Conformation , Protein Structure, Quaternary , Synaptonemal Complex/physiology , Synaptonemal Complex/ultrastructureABSTRACT
Spatial patterns are ubiquitous in both physical and biological systems. We have recently discovered that mitotic chromosomes sequentially acquire two interesting morphological patterns along their structural axes [L. Chu et al., Mol. Cell, 10.1016/j.molcel.2020.07.002 (2020)]. First, axes of closely conjoined sister chromosomes acquire regular undulations comprising nearly planar arrays of sequential half-helices of similar size and alternating handedness, accompanied by periodic kinks. This pattern, which persists through all later stages, provides a case of the geometric form known as a "perversion." Next, as sister chromosomes become distinct parallel units, their individual axes become linked by bridges, which are themselves miniature axes. These bridges are dramatically evenly spaced. Together, these effects comprise a unique instance of spatial patterning in a subcellular biological system. We present evidence that axis undulations and bridge arrays arise by a single continuous mechanically promoted progression, driven by stress within the chromosome axes. We further suggest that, after sister individualization, this same stress also promotes chromosome compaction by rendering the axes susceptible to the requisite molecular remodeling. Thus, by this scenario, the continuous presence of mechanical stress within the chromosome axes could potentially underlie the entire morphogenetic chromosomal program. Direct analogies with meiotic chromosomes suggest that the same effects could underlie interactions between homologous chromosomes as required for gametogenesis. Possible mechanical bases for generation of axis stress and resultant deformations are discussed. Together, these findings provide a perspective on the macroscopic changes of organized chromosomes.
Subject(s)
Chromatin/chemistry , Chromosomes/chemistry , Mitosis/genetics , Morphogenesis/genetics , Cell Line , Chromatids/chemistry , Chromatids/genetics , Chromatids/metabolism , Chromatin/genetics , Chromatin/metabolism , Chromosomes/genetics , Chromosomes/metabolism , HumansABSTRACT
The three-dimensional organization of the genome supports regulated gene expression, recombination, DNA repair, and chromosome segregation during mitosis. Chromosome conformation capture (Hi-C)1,2 analysis has revealed a complex genomic landscape of internal chromosomal structures in vertebrate cells3-7, but the identical sequence of sister chromatids has made it difficult to determine how they topologically interact in replicated chromosomes. Here we describe sister-chromatid-sensitive Hi-C (scsHi-C), which is based on labelling of nascent DNA with 4-thio-thymidine and nucleoside conversion chemistry. Genome-wide conformation maps of human chromosomes reveal that sister-chromatid pairs interact most frequently at the boundaries of topologically associating domains (TADs). Continuous loading of a dynamic cohesin pool separates sister-chromatid pairs inside TADs and is required to focus sister-chromatid contacts at TAD boundaries. We identified a subset of TADs that are overall highly paired and are characterized by facultative heterochromatin and insulated topological domains that form separately within individual sister chromatids. The rich pattern of sister-chromatid topologies and our scsHi-C technology will make it possible to investigate how physical interactions between identical DNA molecules contribute to DNA repair, gene expression, chromosome segregation, and potentially other biological processes.
Subject(s)
Chromatids/chemistry , Chromosome Pairing , DNA Replication , Genome, Human/genetics , Nucleic Acid Conformation , Cell Cycle Proteins/metabolism , Chromatids/genetics , Chromatids/metabolism , Chromosomal Proteins, Non-Histone/metabolism , DNA/analysis , DNA/biosynthesis , Heterochromatin/chemistry , Heterochromatin/genetics , Heterochromatin/metabolism , Humans , CohesinsABSTRACT
At each round of cell division, the DNA must be correctly duplicated and distributed between the two daughter cells to maintain genome identity. In order to achieve proper chromosome replication and segregation, sister chromatids must be recognized as such and kept together until their separation. This process of cohesion is mainly achieved through proteinaceous linkages of cohesin complexes, which are loaded on the sister chromatids as they are generated during S phase. Cohesion between sister chromatids must be fully removed at anaphase to allow chromosome segregation. Other (non-proteinaceous) sources of cohesion between sister chromatids consist of DNA linkages or sister chromatid intertwines. DNA linkages are a natural consequence of DNA replication, but must be timely resolved before chromosome segregation to avoid the arising of DNA lesions and genome instability, a hallmark of cancer development. As complete resolution of sister chromatid intertwines only occurs during chromosome segregation, it is not clear whether DNA linkages that persist in mitosis are simply an unwanted leftover or whether they have a functional role. In this review, we provide an overview of DNA linkages between sister chromatids, from their origin to their resolution, and we discuss the consequences of a failure in their detection and processing and speculate on their potential role.
Subject(s)
Anaphase , DNA, Catenated/genetics , Genomic Instability , Animals , Chromatids/chemistry , Chromatids/genetics , Chromosome Segregation , DNA, Catenated/chemistry , HumansABSTRACT
Sister chromatid cohesion essential for mitotic chromosome segregation is thought to involve the co-entrapment of sister DNAs within cohesin rings. Although cohesin can load onto chromosomes throughout the cell cycle, it only builds cohesion during S phase. A key question is whether cohesion is generated by conversion of cohesin complexes associated with un-replicated DNAs ahead of replication forks into cohesive structures behind them, or from nucleoplasmic cohesin that is loaded de novo onto nascent DNAs associated with forks, a process that would be dependent on cohesin's Scc2 subunit. We show here that in S. cerevisiae, both mechanisms exist and that each requires a different set of replisome-associated proteins. Cohesion produced by cohesin conversion requires Tof1/Csm3, Ctf4 and Chl1 but not Scc2 while that created by Scc2-dependent de novo loading at replication forks requires the Ctf18-RFC complex. The association of specific replisome proteins with different types of cohesion establishment opens the way to a mechanistic understanding of an aspect of DNA replication unique to eukaryotic cells.
Subject(s)
Cell Cycle Proteins , Chromatids , Chromosomal Proteins, Non-Histone , DNA Replication/genetics , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Chromatids/chemistry , Chromatids/genetics , Chromatids/metabolism , Chromosomal Proteins, Non-Histone/chemistry , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , DNA, Fungal/chemistry , DNA, Fungal/genetics , DNA, Fungal/metabolism , S Phase/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , CohesinsABSTRACT
Sister chromatid cohesion requires cohesin to act as a protein linker to hold chromatids together. How cohesin tethers chromatids remains poorly understood. We have used optical tweezers to visualize cohesin as it holds DNA molecules. We show that cohesin complexes tether DNAs in the presence of Scc2/Scc4 and ATP demonstrating a conserved activity from yeast to humans. Cohesin forms two classes of tethers: a "permanent bridge" resisting forces over 80 pN and a force-sensitive "reversible bridge." The establishment of bridges requires physical proximity of dsDNA segments and occurs in a single step. "Permanent" cohesin bridges slide when they occur in trans, but cannot be removed when in cis. Therefore, DNAs occupy separate physical compartments in cohesin molecules. We finally demonstrate that cohesin tetramers can compact linear DNA molecules stretched by very low force (below 1 pN), consistent with the possibility that, like condensin, cohesin is also capable of loop extrusion.
Subject(s)
Adenosine Triphosphate/chemistry , Cell Cycle Proteins/chemistry , Chromosomal Proteins, Non-Histone/chemistry , DNA, Fungal/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae/chemistry , Adenosine Triphosphate/metabolism , Cell Cycle Proteins/metabolism , Chromatids/chemistry , Chromatids/metabolism , Chromosomal Proteins, Non-Histone/metabolism , DNA, Fungal/metabolism , Humans , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , CohesinsABSTRACT
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.
Subject(s)
Chromatids , Chromosomes/metabolism , DNA/chemistry , DNA/metabolism , Multiprotein Complexes/metabolism , Adenosine Triphosphatases/metabolism , Animals , Cell Cycle Proteins/metabolism , Chromatids/chemistry , Chromatids/genetics , Chromosomal Proteins, Non-Histone/metabolism , Chromosome Segregation , Chromosomes/chemistry , Chromosomes/genetics , DNA-Binding Proteins/metabolism , Enhancer Elements, Genetic , Mitosis , Multiprotein Complexes/chemistry , Promoter Regions, Genetic , V(D)J Recombination , CohesinsABSTRACT
Linear chromosome ends are capped by telomeres that have been previously reported to adopt a t-loop structure. The lack of simple methods for detecting t-loops has hindered progress in understanding the dynamics of t-loop formation and its function in protecting chromosome ends. Here, we employed a classical two-dimensional agarose gel method (2D gel method) to innovatively apply to t-loop detection. Briefly, restriction fragments of genomic DNA were separated in a 2D gel, and the telomere sequence was detected by in-gel hybridization with telomeric probe. Using this method, we found that t-loops are present throughout the cell cycle, and t-loop formation tightly couples to telomere replication. We also observed that t-loop abundance positively correlates with chromatin condensation, i.e. cells with less compact telomeric chromatin (ALT cells and trichostatin A (TSA)-treated HeLa cells) exhibited fewer t-loops. Moreover, we observed that telomere dysfunction-induced foci, ALT-associated promyelocytic leukemia bodies, and telomere sister chromatid exchanges are activated upon TSA-induced loss of t-loops. These findings confirm the importance of the t-loop in protecting linear chromosomes from damage or illegitimate recombination.
Subject(s)
Cell Cycle/physiology , Chromatids/metabolism , Heterochromatin/metabolism , Telomere/metabolism , Chromatids/chemistry , Electrophoresis, Gel, Two-Dimensional , HeLa Cells , Heterochromatin/chemistry , Humans , Hydroxamic Acids/pharmacology , Telomere/chemistryABSTRACT
As predicted by the notion that sister chromatid cohesion is mediated by entrapment of sister DNAs inside cohesin rings, there is perfect correlation between co-entrapment of circular minichromosomes and sister chromatid cohesion. In most cells where cohesin loads without conferring cohesion, it does so by entrapment of individual DNAs. However, cohesin with a hinge domain whose positively charged lumen is neutralized loads and moves along chromatin despite failing to entrap DNAs. Thus, cohesin engages chromatin in non-topological, as well as topological, manners. Since hinge mutations, but not Smc-kleisin fusions, abolish entrapment, DNAs may enter cohesin rings through hinge opening. Mutation of three highly conserved lysine residues inside the Smc1 moiety of Smc1/3 hinges abolishes all loading without affecting cohesin's recruitment to CEN loading sites or its ability to hydrolyze ATP. We suggest that loading and translocation are mediated by conformational changes in cohesin's hinge driven by cycles of ATP hydrolysis.
Subject(s)
Cell Cycle Proteins/chemistry , Chromatids/chemistry , Chromosomal Proteins, Non-Histone/chemistry , DNA/chemistry , Adenosine Triphosphate/chemistry , Animals , Binding Sites , Chromatin/chemistry , Humans , Hydrolysis , Lysine/chemistry , Mice , Mutation , Nuclear Proteins/genetics , Protein Conformation , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , CohesinsABSTRACT
The FANCJ DNA helicase is linked to hereditary breast and ovarian cancers as well as bone marrow failure disorder Fanconi anemia (FA). Although FANCJ has been implicated in the repair of DNA double-strand breaks (DSBs) by homologous recombination (HR), the molecular mechanism underlying the tumor suppressor functions of FANCJ remains obscure. Here, we demonstrate that FANCJ deficient human and hamster cells exhibit reduction in the overall gene conversions in response to a site-specific chromosomal DSB induced by I-SceI endonuclease. Strikingly, the gene conversion events were biased in favour of long-tract gene conversions in FANCJ depleted cells. The fine regulation of short- (STGC) and long-tract gene conversions (LTGC) by FANCJ was dependent on its interaction with BRCA1 tumor suppressor. Notably, helicase activity of FANCJ was essential for controlling the overall HR and in terminating the extended repair synthesis during sister chromatid recombination (SCR). Moreover, cells expressing FANCJ pathological mutants exhibited defective SCR with an increased frequency of LTGC. These data unravel the novel function of FANCJ helicase in regulating SCR and SCR associated gene amplification/duplications and imply that these functions of FANCJ are crucial for the genome maintenance and tumor suppression.
Subject(s)
BRCA1 Protein/genetics , Basic-Leucine Zipper Transcription Factors/genetics , Chromatids/chemistry , DNA/genetics , Fanconi Anemia Complementation Group Proteins/genetics , Recombinational DNA Repair , Animals , BRCA1 Protein/metabolism , Basic-Leucine Zipper Transcription Factors/metabolism , CHO Cells , Cell Line, Tumor , Chromatids/metabolism , Cricetulus , DNA/metabolism , DNA Breaks, Double-Stranded , Deoxyribonucleases, Type II Site-Specific/pharmacology , Fanconi Anemia Complementation Group Proteins/metabolism , G2 Phase Cell Cycle Checkpoints , Gene Expression Regulation , Homologous Recombination/drug effects , Humans , Mutation , Osteoblasts/cytology , Osteoblasts/drug effects , Osteoblasts/metabolism , Protein Binding , Saccharomyces cerevisiae Proteins/pharmacologyABSTRACT
Condensins I and II are multisubunit complexes that play a central role in mitotic chromosome assembly. Although both complexes become concentrated along the axial region of each chromatid by metaphase, it remains unclear exactly how such axes might assemble and contribute to chromosome shaping. To address these questions from a physico-chemical point of view, we have established a set of two-step protocols for inducing reversible assembly of chromosome structure in situ, namely within a whole cell. In this assay, mitotic chromosomes are first expanded in a hypotonic buffer containing a Mg2+-chelating agent and then converted into different shapes in a NaCl concentration-dependent manner. Both chromatin and condensin-positive chromosome axes are converted into near-original shapes at 100 mM NaCl. This assay combined with small interfering RNA depletion demonstrates that the recovery of chromatin shapes and the reorganization of axes are highly sensitive to depletion of condensin II but less sensitive to depletion of condensin I or topoisomerase IIα. Furthermore, quantitative morphological analyses using the machine-learning algorithm wndchrm support the notion that chromosome shaping is tightly coupled to the reorganization of condensin II-based axes. We propose that condensin II makes a primary contribution to mitotic chromosome architecture and maintenance in human cells.
Subject(s)
Adenosine Triphosphatases/physiology , Chromatin/physiology , Chromosomes, Human/physiology , DNA-Binding Proteins/physiology , Multiprotein Complexes/physiology , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Cell Cycle Proteins/genetics , Chromatids/chemistry , Chromatids/physiology , Chromatin/chemistry , Chromosomal Proteins, Non-Histone/genetics , Chromosomes, Human/genetics , Chromosomes, Human/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , HeLa Cells , Humans , Mitosis/physiology , Multiprotein Complexes/genetics , Multiprotein Complexes/metabolism , Nuclear Proteins/genetics , RNA, Small InterferingABSTRACT
Tudor containing protein 6 (TDRD6) is a male germ line-specific protein essential for chromatoid body (ChB) structure, elongated spermatid development and male fertility. Here we show that in meiotic prophase I spermatocytes TDRD6 interacts with the key protein arginine methyl transferase PRMT5, which supports splicing. TDRD6 also associates with spliceosomal core protein SmB in the absence of RNA and in an arginine methylation dependent manner. In Tdrd6-/- diplotene spermatocytes PRMT5 association with SmB and arginine dimethylation of SmB are much reduced. TDRD6 deficiency impairs the assembly of spliceosomes, which feature 3.5-fold increased levels of U5 snRNPs. In the nucleus, these deficiencies in spliceosome maturation correlate with decreased numbers of SMN-positive bodies and Cajal bodies involved in nuclear snRNP maturation. Transcriptome analysis of TDRD6-deficient diplotene spermatocytes revealed high numbers of splicing defects such as aberrant usage of intron and exons as well as aberrant representation of splice junctions. Together, this study demonstrates a novel function of TDRD6 in spliceosome maturation and mRNA splicing in prophase I spermatocytes.
Subject(s)
Protein-Arginine N-Methyltransferases/metabolism , Ribonucleoprotein, U5 Small Nuclear/metabolism , Ribonucleoproteins/genetics , Ribonucleoproteins/physiology , Spermatocytes/metabolism , Spliceosomes/metabolism , Animals , Arginine/chemistry , Chromatids/chemistry , Coiled Bodies/metabolism , DNA Methylation , Deoxyadenosines/chemistry , Exons , Fatty Acids, Unsaturated/chemistry , Introns , Male , Methylation , Mice , Mice, Transgenic , Microscopy, Fluorescence , Protein Domains , RNA Splicing , RNA, Messenger/metabolism , Spermatocytes/cytology , Thionucleosides/chemistry , TranscriptomeABSTRACT
The assembly of rod-shaped chromosomes during mitosis is an essential prerequisite for faithful segregation of genetic information into daughter cells. Despite the long history of chromosome research, it is only recently that we have acquired powerful approaches and crucial tools that help to unlock the secret of this seemingly complex process. In particular, in vitro assays, mammalian genetics, Hi-C analyses and computer simulations have provided valuable information during the past two years. These studies are now beginning to elucidate how the core components of mitotic chromosomes, namely, histones, topoisomerase IIα and condensins, cooperate with each other to convert very long stretches of DNA into rod-shaped chromosomes.
Subject(s)
Adenosine Triphosphatases/metabolism , Chromatids/chemistry , Chromosomes/chemistry , DNA-Binding Proteins/metabolism , Mitosis , Multiprotein Complexes/metabolism , Adenosine Triphosphatases/chemistry , Animals , Chromatids/metabolism , Chromosomes/metabolism , DNA/chemistry , DNA/metabolism , DNA Topoisomerases, Type II/chemistry , DNA Topoisomerases, Type II/metabolism , DNA-Binding Proteins/chemistry , Histones/chemistry , Histones/metabolism , Humans , Multiprotein Complexes/chemistry , Nucleosomes/chemistry , Nucleosomes/metabolismABSTRACT
Replication across damaged DNA templates is accompanied by transient formation of sister chromatid junctions (SCJs). Cells lacking Esc2, an adaptor protein containing no known enzymatic domains, are defective in the metabolism of these SCJs. However, how Esc2 is involved in the metabolism of SCJs remains elusive. Here we show interaction between Esc2 and a structure-specific endonuclease Mus81-Mms4 (the Mus81 complex), their involvement in the metabolism of SCJs, and the effects Esc2 has on the enzymatic activity of the Mus81 complex. We found that Esc2 specifically interacts with the Mus81 complex via its SUMO-like domains, stimulates enzymatic activity of the Mus81 complex in vitro, and is involved in the Mus81 complex-dependent resolution of SCJs in vivo Collectively, our data point to the possibility that the involvement of Esc2 in the metabolism of SCJs is, in part, via modulation of the activity of the Mus81 complex.
Subject(s)
Chromatids/metabolism , DNA, Cruciform/metabolism , DNA-Binding Proteins/genetics , Endonucleases/genetics , Gene Expression Regulation, Fungal , Nuclear Proteins/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/genetics , Cell Cycle Proteins , Chromatids/chemistry , Cloning, Molecular , DNA Damage , DNA Replication , DNA, Cruciform/chemistry , DNA, Fungal/genetics , DNA, Fungal/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Endonucleases/chemistry , Endonucleases/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Genomic Instability , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Protein Binding , Protein Domains , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Small Ubiquitin-Related Modifier Proteins/chemistry , Small Ubiquitin-Related Modifier Proteins/genetics , Small Ubiquitin-Related Modifier Proteins/metabolismABSTRACT
The clinical relevance of cohesin in DNA repair, tumorigenesis, and severe birth defects continues to fuel efforts in understanding cohesin structure, regulation, and enzymology. Early models depicting huge cohesin rings that entrap two DNA segments within a single lumen are fading into obscurity based on contradictory findings, but elucidating cohesin structure amid a myriad of functions remains challenging. Due in large part to integrated uses of a wide range of methodologies, recent advances are beginning to cast light into the depths that previously cloaked cohesin structure. Additional efforts similarly provide new insights into cohesin enzymology: specifically, the discoveries of ATP-dependent transitions that promote cohesin binding and release from DNA. In combination, these efforts posit a new model that cohesin exists primarily as a relatively flattened structure that entraps only a single DNA molecule and that subsequent ATP hydrolysis, acetylation, and oligomeric assembly tether together individual DNA segments.
Subject(s)
Cell Cycle Proteins/chemistry , Chromatids/chemistry , Chromosomal Proteins, Non-Histone/chemistry , DNA/chemistry , Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/genetics , Adenosine Triphosphate/chemistry , Adenosine Triphosphate/metabolism , Carcinogenesis/genetics , Cell Cycle Proteins/genetics , Chromatids/genetics , Chromosomal Proteins, Non-Histone/genetics , DNA/genetics , DNA Repair/genetics , Humans , Nucleic Acid Conformation , Protein Conformation , CohesinsABSTRACT
Cohesin is a ring-shaped protein complex that is capable of embracing DNA. Most of the ring circumference is comprised of the anti-parallel intramolecular coiled coils of the Smc1 and Smc3 proteins, which connect globular head and hinge domains. Smc coiled coil arms contain multiple acetylated and ubiquitylated lysines. To investigate the role of these modifications, we substituted lysines for arginines to mimic the unmodified state and uncovered genetic interaction between the Smc arms. Using scanning force microscopy, we show that wild-type Smc arms associate with each other when the complex is not on DNA. Deacetylation of the Smc1/Smc3 dimers promotes arms' dissociation. Smc arginine mutants display loose packing of the Smc arms and, although they dimerize at the hinges, fail to connect the heads and associate with the DNA. Our findings highlight the importance of a "collapsed ring," or "rod," conformation of cohesin for its loading on the chromosomes.