ABSTRACT
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.
Subject(s)
Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/metabolism , Adenosine Triphosphatases/metabolism , Acetylation , Chromatids/metabolism , Cell Cycle Proteins/metabolism , CohesinsABSTRACT
Cohesin helps mediate sister chromatid cohesion, chromosome condensation, DNA repair, and transcription regulation. We exploited proximity-dependent labeling to define the in vivo interactions of cohesin domains with DNA or with other cohesin domains that lie within the same or in different cohesin complexes. Our results suggest that both cohesin's head and hinge domains are proximal to DNA, and cohesin structure is dynamic with differential folding of its coiled coil regions to generate butterfly confirmations. This method also reveals that cohesins form ordered clusters on and off DNA. The levels of cohesin clusters and their distribution on chromosomes are cell cycle-regulated. Cohesin clustering is likely necessary for cohesion maintenance because clustering and maintenance uniquely require the same subset of cohesin domains and the auxiliary cohesin factor Pds5p. These conclusions provide important new mechanistic and biological insights into the architecture of the cohesin complex, cohesin-cohesin interactions, and cohesin's tethering and loop-extruding activities.
Subject(s)
Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Cell Cycle Proteins/genetics , Chromatids/physiology , Chromosomal Proteins, Non-Histone/genetics , Chromosomes, Fungal , DNA Repair , Protein Domains , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , CohesinsABSTRACT
The organization of chromatin into higher order structures is essential for chromosome segregation, the repair of DNA-damage, and the regulation of gene expression. Using Micro-C XL to detect chromosomal interactions, we observed the pervasive presence of cohesin-dependent loops with defined positions throughout the genome of budding yeast, as seen in mammalian cells. In early S phase, cohesin stably binds to cohesin associated regions (CARs) genome-wide. Subsequently, positioned loops accumulate with CARs at the bases of the loops. Cohesin regulators Wpl1 and Pds5 alter the levels and distribution of cohesin at CARs, changing the pattern of positioned loops. From these observations, we propose that cohesin with loop extrusion activity is stopped by preexisting CAR-bound cohesins, generating positioned loops. The patterns of loops observed in a population of wild-type and mutant cells can be explained by this mechanism, coupled with a heterogeneous residency of cohesin at CARs in individual cells.
Subject(s)
Cell Cycle Proteins/chemistry , Chromatin/chemistry , Chromosomal Proteins, Non-Histone/chemistry , Animals , Blotting, Western , Cell Cycle Proteins/metabolism , Chromatin/metabolism , Chromatin Immunoprecipitation , Chromosomal Proteins, Non-Histone/metabolism , Chromosome Segregation , DNA/metabolism , Mammals/genetics , Mitosis , S Phase , Saccharomyces cerevisiae/genetics , CohesinsABSTRACT
Condensin mediates chromosome condensation, which is essential for proper chromosome segregation during mitosis. Prior to anaphase of budding yeast, the ribosomal DNA (RDN) condenses to a thin loop that is distinct from the rest of the chromosomes. We provide evidence that the establishment and maintenance of this RDN condensation requires the regulation of condensin by Cdc5p (polo) kinase. We show that Cdc5p is recruited to the site of condensin binding in the RDN by cohesin, a complex related to condensin. Cdc5p and cohesin prevent condensin from misfolding the RDN into an irreversibly decondensed state. From these and other observations, we propose that the spatial regulation of Cdc5p by cohesin modulates condensin activity to ensure proper RDN folding into a thin loop. This mechanism may be evolutionarily conserved, promoting the thinly condensed constrictions that occur at centromeres and RDN of mitotic chromosomes in plants and animals.
Subject(s)
Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Chromosomes, Fungal/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Adenosine Triphosphatases/genetics , Chromosomes, Fungal/genetics , DNA-Binding Proteins/genetics , Multiprotein Complexes/genetics , Protein Binding , Protein Folding , Protein Serine-Threonine Kinases/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , CohesinsABSTRACT
Cohesin mediates higher order chromosome structure. Its biological activities require topological entrapment of DNA within a lumen(s) formed by cohesin subunits. The reversible dissociation of cohesin's Smc3p and Mcd1p subunits is postulated to form a regulated gate that allows DNA entry and exit into the lumen. We assessed gate-independent functions of this interface in yeast using a fusion protein that joins Smc3p to Mcd1p. We show that in vivo all the regulators of cohesin promote DNA binding of cohesin by mechanisms independent of opening this gate. Furthermore, we show that this interface has a gate-independent activity essential for cohesin to bind chromosomes. We propose that this interface regulates DNA entrapment by controlling the opening and closing of one or more distal interfaces formed by cohesin subunits, likely by inducing a conformation change in cohesin. Furthermore, cohesin regulators modulate the interface to control both DNA entrapment and cohesin functions after DNA binding.
Subject(s)
Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/metabolism , DNA, Fungal/chemistry , DNA, Fungal/metabolism , Multiprotein Complexes/metabolism , Protein Subunits/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Acetylation , Cell Cycle Proteins/chemistry , Chromatids/metabolism , Chromosomal Proteins, Non-Histone/chemistry , Multiprotein Complexes/chemistry , Mutation/genetics , Protein Domains , Protein Subunits/chemistry , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae Proteins/chemistry , CohesinsABSTRACT
Climate change has accentuated the importance of understanding how organisms respond to stresses imposed by changes to their environment, like water availability. Unusual organisms, called anhydrobiotes, can survive loss of almost all intracellular water. Desiccation tolerance of anhydrobiotes provides an unusual window to study the stresses and stress response imposed by water loss. Because of the myriad of stresses that could be induced by water loss, desiccation tolerance seemed likely to require many established stress effectors. The sugar trehalose and hydrophilins (small intrinsically disordered proteins) had also been proposed as stress effectors against desiccation because they were found in nearly all anhydrobiotes, and could mitigate desiccation-induced damage to model proteins and membranes in vitro. Here, we summarize in vivo studies of desiccation tolerance in worms, yeast, and tardigrades. These studies demonstrate the remarkable potency of trehalose and a subset of hydrophilins as the major stress effectors of desiccation tolerance. They act, at least in part, by limiting in vivo protein aggregation and loss of membrane integrity. The apparent specialization of individual hydrophilins for desiccation tolerance suggests that other hydrophilins may have distinct roles in mitigating additional cellular stresses, thereby defining a potentially new functionally diverse set of stress effectors.
Subject(s)
Stress, Physiological/physiology , Water/metabolism , Adaptation, Physiological/physiology , Animals , Annelida/metabolism , Climate Change , Desiccation , Droughts , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Tardigrada/metabolism , Trehalose/metabolismABSTRACT
Cohesin is a four-subunit ATPase in the family of structural maintenance of chromosomes (SMC). Cohesin promotes sister chromatid cohesion, chromosome condensation, DNA repair, and transcription regulation. Cohesin performs these functions as a DNA tether and potentially a DNA-based motor. At least one of its DNA binding activities involves entrapment of DNA within a lumen formed by its subunits. This activity can be reconstituted in vitro by incubating cohesin with DNA, ATP, and cohesin loader. Previously we showed that a mutant form of cohesin (DE-cohesin) possesses the ability to bind and tether DNA in vivo. Using in vitro reconstitution assays, we show that DE-cohesin can form stable complexes with DNA without ATP hydrolysis. We show that wild-type cohesin with ADP aluminum fluoride (cohesinADP/AlFx) can also form stable cohesin-DNA complexes. These results suggest that an intermediate nucleotide state of cohesin, likely cohesinADP-Pi, is capable of initially dissociating one interface between cohesin subunits to allow DNA entry into a cohesin lumen and subsequently interacting with the bound DNA to stabilize DNA entrapment. We also show that cohesinADP/AlFx binding to DNA is enhanced by cohesin loader, suggesting a function for loader other than stimulating the ATPase. Finally, we show that loader remains stably bound to cohesinADP/AlFx after DNA entrapment, potentially revealing a function for loader in tethering the second DNA substrate. These results provide important clues on how SMC complexes like cohesin can function as both DNA tethers and motors.
Subject(s)
Adenosine Triphosphatases/metabolism , Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/metabolism , DNA/metabolism , ATP-Binding Cassette Transporters/metabolism , DNA Repair , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Schizosaccharomyces/genetics , Schizosaccharomyces/metabolism , Schizosaccharomyces pombe Proteins/metabolism , Translocation, Genetic , CohesinsABSTRACT
DNA-RNA hybrids associated with R-loops promote DNA damage and genomic instability. The capacity of hybrids at different genomic sites to cause DNA damage was not known, and the mechanisms leading from hybrid to damage were poorly understood. Here, we adopt a new strategy to map and characterize the sites of hybrid-induced damage genome-wide in budding yeast. We show that hybrid removal is essential for life because persistent hybrids cause irreparable DNA damage and cell death. We identify that a subset of hybrids is prone to cause damage, and the chromosomal context of hybrids dramatically impacts their ability to induce damage. Furthermore, persistent hybrids affect the repair pathway, generating large regions of single-stranded DNA (ssDNA) by two distinct mechanisms, likely resection and re-replication. These damaged regions may act as potential precursors to gross chromosomal rearrangements like deletions and duplications that are associated with R-loops and cancers.
Subject(s)
DNA, Single-Stranded/genetics , Gene Expression Regulation, Fungal , Genome, Fungal , Genomic Instability , RNA/genetics , Saccharomyces cerevisiae/genetics , DNA Cleavage , DNA Damage , DNA Helicases/genetics , DNA Helicases/metabolism , DNA Replication , DNA, Single-Stranded/chemistry , DNA, Single-Stranded/metabolism , G2 Phase Cell Cycle Checkpoints/drug effects , G2 Phase Cell Cycle Checkpoints/genetics , Hydroxyurea/pharmacology , Indoleacetic Acids/pharmacology , Nucleic Acid Conformation , Nucleic Acid Hybridization , RNA/chemistry , RNA/metabolism , RNA Helicases/genetics , RNA Helicases/metabolism , Rad52 DNA Repair and Recombination Protein/genetics , Rad52 DNA Repair and Recombination Protein/metabolism , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
Anhydrobiotes are rare microbes, plants and animals that tolerate severe water loss. Understanding the molecular basis for their desiccation tolerance may provide novel insights into stress biology and critical tools for engineering drought-tolerant crops. Using the anhydrobiote, budding yeast, we show that trehalose and Hsp12, a small intrinsically disordered protein (sIDP) of the hydrophilin family, synergize to mitigate completely the inviability caused by the lethal stresses of desiccation. We show that these two molecules help to stabilize the activity and prevent aggregation of model proteins both in vivo and in vitro. We also identify a novel in vitro role for Hsp12 as a membrane remodeler, a protective feature not shared by another yeast hydrophilin, suggesting that sIDPs have distinct biological functions.
Subject(s)
Dehydration , Heat-Shock Proteins/metabolism , Intrinsically Disordered Proteins/metabolism , Microbial Viability , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/physiology , Stress, Physiological , Trehalose/metabolism , Cell Membrane/metabolism , Protein Aggregation, Pathological/prevention & controlABSTRACT
Cohesin tethers DNA to mediate sister chromatid cohesion, chromosome condensation, and DNA repair. How the cell regulates cohesin to perform these distinct functions remains to be elucidated. One cohesin regulator, Wpl1p, was characterized in Saccharomyces cerevisiae as a promoter of efficient cohesion and an inhibitor of condensation. Wpl1p is also required for resistance to DNA-damaging agents. Here, we provide evidence that Wpl1p promotes the timely repair of DNA damage induced during S-phase. Previous studies have indicated that Wpl1p destabilizes cohesin's binding to DNA by modulating the interface between the cohesin subunits Mcd1p and Smc3p Our results suggest that Wpl1p likely modulates this interface to regulate all of cohesin's biological functions. Furthermore, we show that Wpl1p regulates cohesion and condensation through the formation of a functional complex with another cohesin-associated factor, Pds5p In contrast, Wpl1p regulates DNA repair independently of its interaction with Pds5p Together, these results suggest that Wpl1p regulates distinct biological functions of cohesin by Pds5p-dependent and -independent modulation of the Smc3p/Mcd1p interface.
Subject(s)
Cell Cycle Proteins/metabolism , Chromatids/genetics , Chromosomal Proteins, Non-Histone/metabolism , DNA Repair , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Binding Sites , Cell Cycle/drug effects , Cell Cycle Proteins/chemistry , Chromatids/metabolism , Chromosomal Proteins, Non-Histone/chemistry , Chromosomes, Fungal , DNA Damage/drug effects , Models, Biological , Models, Molecular , Protein Binding , Protein Conformation , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Topoisomerase I Inhibitors/pharmacology , CohesinsABSTRACT
Cohesin is a conserved protein complex required for sister chromatid cohesion, chromosome condensation, DNA damage repair, and regulation of transcription. Although cohesin functions to tether DNA duplexes, the contribution of its individual domains to this activity remains poorly understood. We interrogated the Smc3p subunit of cohesin by random insertion mutagenesis. Analysis of a mutant in the Smc3p hinge revealed an unexpected role for this domain in cohesion maintenance and condensation. Further investigation revealed that the Smc3p hinge functions at a step following cohesin's stable binding to chromosomes and independently of Smc3p's regulation by the Eco1p acetyltransferase. Hinge mutant phenotypes resemble loss of Pds5p, which binds opposite the hinge near Smc3p's head domain. We propose that a specific conformation of the Smc3p hinge and Pds5p cooperate to promote cohesion maintenance and condensation.
Subject(s)
Cell Cycle Proteins/genetics , Cell Cycle Proteins/physiology , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/physiology , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/physiology , Cell Cycle Proteins/metabolism , Chromatids/genetics , Chromatids/physiology , Chromosomal Proteins, Non-Histone/metabolism , Chromosome Segregation/genetics , Chromosomes, Fungal/metabolism , DNA Repair , Mutation , Nuclear Proteins/metabolism , Protein Domains/genetics , Protein Domains/physiology , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , CohesinsABSTRACT
R-loops, three-stranded structures that form when transcripts hybridize to chromosomal DNA, are potent agents of genome instability. This instability has been explained by the ability of R-loops to induce DNA damage. Here, we show that persistent R-loops also compromise DNA repair. Depleting endogenous RNase H activity impairs R-loop removal in Saccharomyces cerevisiae, causing DNA damage that occurs preferentially in the repetitive ribosomal DNA locus (rDNA). We analyzed the repair kinetics of this damage and identified mutants that modulate repair. We present a model that the persistence of R-loops at sites of DNA damage induces repair by break-induced replication (BIR). This R-loop induced BIR is particularly susceptible to the formation of lethal repair intermediates at the rDNA because of a barrier imposed by RNA polymerase I.
Subject(s)
DNA Damage , DNA Repair , Ribonuclease H/metabolism , Saccharomyces cerevisiae/enzymologyABSTRACT
DNA:RNA hybrids can lead to DNA damage and genome instability. This damage can be prevented by degradation of the RNA in the hybrid by two evolutionarily conserved enzymes, RNase H1 and H2. Indeed, RNase H-deficient cells have increased chromosomal rearrangements. However, the quantitative and spatial contributions of the individual enzymes to hybrid removal have been unclear. Additionally, RNase H2 can remove single ribonucleotides misincorporated into DNA during replication. The relative contribution of DNA:RNA hybrids and misincorporated ribonucleotides to chromosome instability also was uncertain. To address these issues, we studied the frequency and location of loss-of-heterozygosity (LOH) events on chromosome III in Saccharomyces cerevisiae strains that were defective for RNase H1, H2, or both. We showed that RNase H2 plays the major role in preventing chromosome III instability through its hybrid-removal activity. Furthermore, RNase H2 acts pervasively at many hybrids along the chromosome. In contrast, RNase H1 acts to prevent LOH within a small region of chromosome III where the instability is dependent upon two hybrid-prone sequences. This restriction of RNase H1 activity to a subset of hybrids is not the result of its constrained localization, because we found it at hybrids genome-wide. This result suggests that the genome-protection activity of RNase H1 is regulated at a step after hybrid recognition. The global function of RNase H2 and the region-specific function of RNase H1 provide insight into why these enzymes with overlapping hybrid-removal activities have been conserved throughout evolution.
Subject(s)
Chromosomal Instability/genetics , Loss of Heterozygosity/genetics , Ribonuclease H/genetics , Chromosomes, Fungal/genetics , DNA Damage/genetics , DNA Replication/genetics , DNA, Fungal/genetics , RNA, Fungal/genetics , Ribonucleotides/genetics , Saccharomyces cerevisiae/geneticsABSTRACT
Innumerable breakthroughs in many fundamental areas of biology have come from unbiased screens and selections for mutations, either across the genome or within a gene. However, long-standing hurdles to key elements of mutant hunts (mutagenesis, phenotypic characterization, and linkage of phenotype to genotype) have limited the organisms in which mutant hunts could be used. These hurdles are now being eliminated by an explosion of new technologies. We believe that a renewed emphasis on unbiased mutant hunts, in both existing model systems and in those where genetics is just now becoming feasible, will lead to new seminal discoveries and surprises.
Subject(s)
DNA Mutational Analysis , Genome , Mutation/genetics , Genotype , Mutagenesis/geneticsABSTRACT
R loops form when transcripts hybridize to homologous DNA on chromosomes, yielding a DNA:RNA hybrid and a displaced DNA single strand. R loops impact the genome of many organisms, regulating chromosome stability, gene expression, and DNA repair. Understanding the parameters dictating R-loop formation in vivo has been hampered by the limited quantitative and spatial resolution of current genomic strategies for mapping R loops. We report a novel whole-genome method, S1-DRIP-seq (S1 nuclease DNA:RNA immunoprecipitation with deep sequencing), for mapping hybrid-prone regions in budding yeast Saccharomyces cerevisiae Using this methodology, we identified â¼800 hybrid-prone regions covering 8% of the genome. Given the pervasive transcription of the yeast genome, this result suggests that R-loop formation is dictated by characteristics of the DNA, RNA, and/or chromatin. We successfully identified two features highly predictive of hybrid formation: high transcription and long homopolymeric dA:dT tracts. These accounted for >60% of the hybrid regions found in the genome. We demonstrated that these two factors play a causal role in hybrid formation by genetic manipulation. Thus, the hybrid map generated by S1-DRIP-seq led to the identification of the first global genomic features causal for R-loop formation in yeast.
Subject(s)
Gene Expression , Genome, Fungal/genetics , Poly A/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Chromosome Mapping , DNA, Fungal/metabolism , Genomics , Histones/metabolism , Poly A/chemistry , Poly A/metabolism , Protein Conformation , RNA, Fungal/metabolism , Single-Strand Specific DNA and RNA Endonucleases/metabolismABSTRACT
Cohesin is essential for the hierarchical organization of the eukaryotic genome and plays key roles in many aspects of chromosome biology. The conformation of cohesin bound to DNA remains poorly defined, leaving crucial gaps in our understanding of how cohesin fulfills its biological functions. Here, we use single-molecule microscopy to directly observe the dynamic and functional characteristics of cohesin bound to DNA. We show that cohesin can undergo rapid one-dimensional (1D) diffusion along DNA, but individual nucleosomes, nucleosome arrays, and other protein obstacles significantly restrict its mobility. Furthermore, we demonstrate that DNA motor proteins can readily push cohesin along DNA, but they cannot pass through the interior of the cohesin ring. Together, our results reveal that DNA-bound cohesin has a central pore that is substantially smaller than anticipated. These findings have direct implications for understanding how cohesin and other SMC proteins interact with and distribute along chromatin.
Subject(s)
Cell Cycle Proteins/chemistry , Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/chemistry , Chromosomal Proteins, Non-Histone/metabolism , DNA/metabolism , Schizosaccharomyces pombe Proteins/chemistry , Schizosaccharomyces pombe Proteins/metabolism , Single Molecule Imaging/methods , AT Rich Sequence/genetics , Base Sequence , Diffusion , Models, Biological , Molecular Motor Proteins/metabolism , Nucleosomes/metabolism , Porosity , Protein Binding , Protein Conformation , Protein Transport , Schizosaccharomyces/metabolism , CohesinsABSTRACT
Cohesin tethers together regions of DNA, thereby mediating higher order chromatin organization that is critical for sister chromatid cohesion, DNA repair and transcriptional regulation. Cohesin contains a heterodimeric ATP-binding Cassette (ABC) ATPase comprised of Smc1 and Smc3 ATPase active sites. These ATPases are required for cohesin to bind DNA. Cohesin's DNA binding activity is also promoted by the Eco1 acetyltransferase and inhibited by Wpl1. Recently we showed that after cohesin stably binds DNA, a second step is required for DNA tethering. This second step is also controlled by Eco1 acetylation. Here, we use genetic and biochemical analyses to show that this second DNA tethering step is regulated by cohesin ATPase. Furthermore, our results also suggest that Eco1 promotes cohesion by modulating the ATPase cycle of DNA-bound cohesin in a state that is permissive for DNA tethering and refractory to Wpl1 inhibition.
Subject(s)
Acetyltransferases/metabolism , Adenosine Triphosphatases/metabolism , Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/metabolism , DNA/metabolism , Nuclear Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Cell Cycle Proteins/genetics , Chromatin/metabolism , Chromosomal Proteins, Non-Histone/genetics , Saccharomyces cerevisiae Proteins/genetics , CohesinsABSTRACT
The cohesin complex (Mcd1p, Smc1p, Smc3p, and Scc3p) has multiple roles in chromosome architecture, such as promoting sister chromatid cohesion, chromosome condensation, DNA repair, and transcriptional regulation. The prevailing embrace model for sister chromatid cohesion posits that a single cohesin complex entraps both sister chromatids. We report interallelic complementation between pairs of nonfunctional mcd1 alleles (mcd1-1 and mcd1-Q266) or smc3 alleles (smc3-42 and smc3-K113R). Cells bearing individual mcd1 or smc3 mutant alleles are inviable and defective for both sister chromatid cohesion and condensation. However, cells coexpressing two defective mcd1 or two defective smc3 alleles are viable and have cohesion and condensation. Because cohesin contains only a single copy of Smc3p or Mcd1p, these examples of interallelic complementation must result from interplay or communication between the two defective cohesin complexes, each harboring one of the mutant allele products. Neither mcd1-1p nor smc3-42p is bound to chromosomes when expressed individually at its restrictive temperature. However, their chromosome binding is restored when they are coexpressed with their chromosome-bound interallelic complementing partner. Our results support a mechanism by which multiple cohesin complexes interact on DNA to mediate cohesion and condensation.
Subject(s)
Alleles , Cell Cycle Proteins/genetics , Chromosomal Proteins, Non-Histone/genetics , DNA, Fungal/genetics , Cell Cycle , Cell Cycle Proteins/metabolism , Chromatids/genetics , Chromatids/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Chromosome Segregation , Chromosomes/genetics , Chromosomes/metabolism , DNA, Fungal/metabolism , DNA-Binding Proteins , Genetic Complementation Test/methods , Mutation , Structure-Activity Relationship , Yeasts/genetics , Yeasts/metabolism , CohesinsABSTRACT
RNA performs diverse functions in cells, directing translation, modulating transcription and catalyzing enzymatic reactions. Remarkably RNA can also anneal to its genomic template co- or post-transcriptionally to generate an RNA-DNA hybrid and a displaced single-stranded DNA. These unusual nucleic acid structures are called R-loops. Studies in the last decades concentrated on the detrimental effects of R-loop formation, particularly on genome stability. In fact, R-loops are thought to play a role in several human diseases like cancer and neurodegenerative syndromes. But recent data has revealed that R-loops can also have a positive impact on cell processes, like regulating gene expression, chromosome structure and DNA repair. Here we summarize our current understanding of the formation and dissolution of R-loops, and discuss their negative and positive impact on genome structure and function.