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 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
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
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
The Structural Maintenance of Chromosome (SMC) complex, termed cohesin, is essential for sister chromatid cohesion. Cohesin is also important for chromosome condensation, DNA repair, and gene expression. Cohesin is comprised of Scc3, Mcd1, Smc1, and Smc3. Scc3 also binds Pds5 and Wpl1, cohesin-associated proteins that regulate cohesin function, and to the Scc2/4 cohesin loader. We mutagenized SCC3 to elucidate its role in cohesin function. A 5 amino acid insertion after Scc3 residue I358, or a missense mutation of residue D373 in the adjacent stromalin conservative domain (SCD) induce inviability and defects in both cohesion and cohesin binding to chromosomes. The I358 and D373 mutants abrogate Scc3 binding to Mcd1. These results define an Scc3 region extending from I358 through the SCD required for binding Mcd1, cohesin localization to chromosomes and cohesion. Scc3 binding to the cohesin loader, Pds5 and Wpl1 are unaffected in I358 mutant and the loader still binds the cohesin core trimer (Mcd1, Smc1 and Smc3). Thus, Scc3 plays a critical role in cohesin binding to chromosomes and cohesion at a step distinct from loader binding to the cohesin trimer. We show that residues Y371 and K372 within the SCD are critical for viability and chromosome condensation but dispensable for cohesion. However, scc3 Y371A and scc3 K372A bind normally to Mcd1. These alleles also provide evidence that Scc3 has distinct mechanisms of cohesin loading to different loci. The cohesion-competence, condensation-incompetence of Y371 and K372 mutants suggests that cohesin has at least one activity required specifically for condensation.
Subject(s)
Cell Cycle Proteins/genetics , Chromosomal Proteins, Non-Histone/genetics , Chromosome Segregation/genetics , Saccharomyces cerevisiae Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Nucleus/genetics , Chromatids/genetics , Chromosomal Proteins, Non-Histone/metabolism , Chromosomes, Fungal/genetics , DNA Repair/genetics , Mutation , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Protein Structure, Tertiary , Saccharomyces cerevisiae , Saccharomyces cerevisiae Proteins/metabolism , CohesinsABSTRACT
Cohesin complex mediates cohesion between sister chromatids, which promotes high-fidelity chromosome segregation. Eco1p acetylates the cohesin subunit Smc3p during S phase to establish cohesion. The current model posits that this Eco1p-mediated acetylation promotes establishment by abrogating the ability of Wpl1p to destabilize cohesin binding to chromosomes. Here we present data from budding yeast that is incompatible with this Wpl1p-centric model. Two independent in vivo assays show that a wpl1∆ fails to suppress cohesion defects of eco1∆ cells. Moreover, a wpl1∆ also fails to suppress cohesion defects engendered by blocking just the essential Eco1p acetylation sites on Smc3p (K112, K113). Thus removing WPL1 inhibition is insufficient for generating cohesion without ECO1 activity. To elucidate how ECO1 promotes cohesion, we conducted a genetic screen and identified a cohesion activator mutation in the SMC3 head domain (D1189H). Smc3-D1189H partially restores cohesion in eco1∆ wpl1∆ or eco1 mutant cells but robustly restores cohesion in cells blocked for Smc3p K112 K113 acetylation. These data support two important conclusions. First, acetylation of the K112 K113 region by Eco1p promotes cohesion establishment by altering Smc3p head function independent of its ability to antagonize Wpl1p. Second, Eco1p targets other than Smc3p K112 K113 are necessary for efficient establishment.
Subject(s)
Acetyltransferases/metabolism , Cell Cycle Proteins/genetics , Chromatids/genetics , Chromosomal Proteins, Non-Histone/genetics , Chromosome Segregation , Nuclear Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/genetics , Acetylation , Acetyltransferases/genetics , Amino Acid Sequence , Chromosomes, Fungal , Molecular Sequence Data , Mutation , Nuclear Proteins/genetics , S Phase , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , CohesinsABSTRACT
Cohesin helps orchestrate higher-order chromosome structure, thereby promoting sister chromatid cohesion, chromosome condensation, DNA repair, and transcriptional regulation. To elucidate how cohesin facilitates these diverse processes, we mutagenized Mcd1p, the kleisin regulatory subunit of budding yeast cohesin. In the linker region of Mcd1p, we identified a novel evolutionarily conserved 10-amino acid cluster, termed the regulation of cohesion and condensation (ROCC) box. We show that ROCC promotes cohesion maintenance by protecting a second activity of cohesin that is distinct from its stable binding to chromosomes. The existence of this second activity is incompatible with the simple embrace mechanism of cohesion. In addition, we show that the ROCC box is required for the establishment of condensation. We provide evidence that ROCC controls cohesion maintenance and condensation establishment through differential functional interactions with Pds5p and Wpl1p.
Subject(s)
Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/genetics , Cell Cycle Proteins/genetics , Chromatids/genetics , Chromosomal Proteins, Non-Histone/genetics , Chromosome Segregation , Chromosomes, Fungal , DNA Repair , Nuclear Proteins/metabolism , Saccharomyces cerevisiae Proteins/genetics , CohesinsABSTRACT
Cohesin generates cohesion between sister chromatids, which enables chromosomes to form bipolar attachments to the mitotic spindle and segregate. Cohesin also functions in chromosome condensation, transcriptional regulation, and DNA damage repair. Here we analyze the role of acetylation in modulating cohesin functions and how it affects budding yeast viability. Previous studies show that cohesion establishment requires Eco1p-mediated acetylation of the cohesin subunit Smc3p at residue K113. Smc3p acetylation was proposed to promote establishment by merely relieving Wpl1p inhibition because deletion of WPL1 bypasses the lethality of an ECO1 deletion (eco1Δ wpl1Δ). We find that little, if any, cohesion is established in eco1Δ wpl1Δ cells, indicating that Eco1p performs a function beyond antagonizing Wpl1p. Cohesion also fails to be established when SMC3 acetyl-mimics (K113Q or K112R,K113Q) are the sole functional SMC3s in cells. These results suggest that Smc3p acetylation levels affect establishment. It is remarkable that, despite their severe cohesion defect, eco1Δ wpl1Δ and smc3-K112R,K113Q strains are viable because a cohesin-independent mechanism enables bipolar attachment and segregation. This alternative mechanism is insufficient for smc3-K113Q strain viability. Smc3-K113Q is defective for condensation, whereas eco1Δ wpl1Δ and smc3-K112R,K113Q strains are competent for condensation. We suggest that Smc3p acetylation and Wpl1p antagonistically regulate cohesin's essential role in condensation.
Subject(s)
Cell Cycle Proteins/metabolism , Chromatids/genetics , Chromosomal Proteins, Non-Histone/metabolism , Chromosome Segregation , Chromosomes, Fungal/genetics , Saccharomyces cerevisiae/genetics , Acetylation , Acetyltransferases/genetics , Acetyltransferases/metabolism , Alleles , Cell Cycle Proteins/genetics , Chromosomal Proteins, Non-Histone/genetics , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Spindle Apparatus/genetics , Spindle Apparatus/metabolism , CohesinsABSTRACT
Cohesin's complex distribution on chromosomes and its implication in numerous cellular processes makes it an excellent paradigm for studying the relationship between the in vivo concentration of a protein and its in vivo function. Here, we report a method to generate systematic quantized reductions (QR) in the in vivo concentration of any yeast protein. With QR, we generate strains with 13% and 30% of wild-type levels of the limiting subunit of cohesin, Mcd1p/Scc1p/Rad21p. Reducing cohesin levels reveals a preferential binding of cohesin to pericentric regions over cohesin-associated regions (CAR) on chromosome arms. Chromosome condensation, repetitive DNA stability, and DNA repair are compromised by decreasing cohesin levels to 30% of wild-type levels. In contrast, sister-chromatid cohesion and chromosome segregation are unaffected even when cohesin levels are reduced to 13% of wild-type levels. The requirement for different in vivo cohesin concentrations to achieve distinct cohesin functions provides an explanation for how cohesin mutations can specifically lead to adult disorders such as Cornelia de Lange Syndrome and Roberts Syndrome without compromising the cell divisions needed for development and maturation. Our successful application of QR to cohesin suggests that QR is a powerful tool to study other proteins/pathways with multiple functions.
Subject(s)
Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Chromosome Segregation , Chromosomes/metabolism , DNA Repair , Adult , Cell Cycle Proteins/genetics , Cell Division/physiology , Centromere/metabolism , Chromosomal Proteins, Non-Histone/genetics , De Lange Syndrome/genetics , Humans , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , CohesinsSubject(s)
Cell Cycle Proteins/metabolism , Centromere/physiology , Endopeptidases/metabolism , Mitosis , Protein Kinases/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Small Ubiquitin-Related Modifier Proteins/metabolism , Catalytic Domain , Chromosome Segregation , Phosphorylation , Protein Serine-Threonine Kinases , Saccharomyces cerevisiae Proteins/antagonists & inhibitorsABSTRACT
Cohesins mediate sister chromatid cohesion and DNA repair and also function in gene regulation. Chromosomal cohesins are distributed nonrandomly, and their deposition requires the heterodimeric Scc2/Scc4 loader. Whether Scc2/Scc4 establishes nonrandom cohesin distributions on chromosomes is poorly characterized, however. To better understand the spatial regulation of cohesin association, we mapped budding yeast Scc2 and Scc4 chromosomal distributions. We find that Scc2/Scc4 resides at previously mapped cohesin-associated regions (CARs) in pericentromeric and arm regions, and that Scc2/Scc4-cohesin colocalization persists after the initial deposition of cohesins in G1/S phase. Pericentromeric Scc2/Scc4 enrichment is kinetochore-dependent, and both Scc2/Scc4 and cohesin associations are coordinately reduced in these regions following chromosome biorientation. Thus, these characteristics of Scc2/Scc4 binding closely recapitulate those of cohesin. Although present in G1, Scc2/Scc4 initially has a poor affinity for CARs, but its affinity increases as cells traverse the cell cycle. Scc2/Scc4 association with CARs is independent of cohesin, however. Taken together, these observations are inconsistent with a previous suggestion that cohesins are relocated by translocating RNA polymerases from separate loading sites to intergenic regions between convergently transcribed genes. Rather, our findings suggest that budding yeast cohesins are targeted to CARs largely by Scc2/Scc4 loader association at these locations.
Subject(s)
Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Chromosomes, Fungal/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Cell Cycle/physiology , Chromatin/metabolism , Kinetochores/metabolism , CohesinsABSTRACT
During meiosis, homologues become juxtaposed and synapsed along their entire length. Mutations in the cohesin complex disrupt not only sister chromatid cohesion but also homologue pairing and synaptonemal complex formation. In this study, we report that Pds5, a cohesin-associated protein known to regulate sister chromatid cohesion, is required for homologue pairing and synapsis in budding yeast. Pds5 colocalizes with cohesin along the length of meiotic chromosomes. In the absence of Pds5, the meiotic cohesin subunit Rec8 remains bound to chromosomes with only minor defects in sister chromatid cohesion, but sister chromatids synapse instead of homologues. Double-strand breaks (DSBs) are formed but are not repaired efficiently. In addition, meiotic chromosomes undergo hypercondensation. When the mitotic cohesin subunit Mcd1 is substituted for Rec8 in Pds5-depleted cells, chromosomes still hypercondense, but synapsis of sister chromatids is abolished. These data suggest that Pds5 modulates the Rec8 activity to facilitate chromosome morphological changes required for homologue synapsis, DSB repair, and meiotic chromosome segregation.
Subject(s)
Cell Cycle Proteins/metabolism , Chromatids/metabolism , Chromosome Pairing , Meiosis/physiology , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae , Cell Cycle Proteins/genetics , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , Chromosome Segregation , Chromosomes, Fungal/metabolism , Chromosomes, Fungal/ultrastructure , DNA Breaks, Double-Stranded , DNA Repair , DNA Topoisomerases, Type II/genetics , DNA Topoisomerases, Type II/metabolism , Endodeoxyribonucleases , Rad51 Recombinase/genetics , Rad51 Recombinase/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Recombination, Genetic , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolismABSTRACT
Eco1p/Ctf7p is an essential acetyltransferase required for the establishment of sister chromatid cohesion. Eco1p acetylates Smc3p and Mcd1p (Scc1p or Rad21p) to establish cohesion during S phase and in response to DNA damage, respectively. In addition to its acetyltransferase domain, Eco1p harbors a conserved zinc finger domain. The zinc finger has been implicated in the establishment of sister chromatid cohesion in S phase, yet its function on the molecular level and its contribution to damage-induced cohesion are unknown. Here, we show that the zinc finger is essential for the establishment of cohesion in both S phase and in response to DNA damage. Our results suggest that the zinc finger augments the acetylation of Eco1p itself, Smc3p and likely Mcd1p. We propose that the zinc finger is a general enhancer of substrate recognition, thereby enhances the ability of Eco1p to acetylate its substrates above a threshold needed to generate cohesion during DNA replication and repair. Finally our studies of the zinc finger led to the discovery that Eco1 is a multimer, a property that could be exploited to coordinate acetylation of substrates either spatially or temporally for establishment of sister chromatid cohesion.
Subject(s)
Acetyltransferases/chemistry , Chromatids/enzymology , Nuclear Proteins/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Zinc Fingers , Acetyltransferases/genetics , Acetyltransferases/metabolism , Amino Acid Sequence , DNA Damage , Molecular Sequence Data , Mutagenesis, Site-Directed , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Protein Multimerization , S Phase , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
The lampbrush chromosomes present in the nuclei of amphibian oocytes offer unique biological approaches for study of the mechanisms that regulate chromatin structure with high spatial resolution. We discuss fundamental aspects of the remarkable organization and plasticity exhibited by lampbrush chromosomes. We then utilize lampbrush chromosomes to characterize the chromosomal distribution and dynamics of cohesin, the four-protein complex (RAD21/MCD1/SCC1, SMC1, SMC3, SCC3/SA2) responsible for sister chromatid cohesion. We find that endogenous SMC3 and newly expressed hRAD21 co-localize on chromosomal axes, sites where sister chromatids are tightly paired. We present evidence suggesting that hRAD21 recruitment to lampbrush chromosomes is modulated by chromosomal SMC1 and SMC3. Notably, using a technique for de novo chromosome assembly, we demonstrate that both SMC3 and hRAD21 are recruited to single, unreplicated lampbrush chromatids. Finally, we used our novel method of analyzing the oocyte nucleus under oil combined with fluorescence recovery after photobleaching, to provide direct evidence that cohesin is highly dynamic at discrete, condensed chromosomal regions. Collectively, these data demonstrate that lampbrush chromosomes provide a unique and powerful tool for combining biochemical and cytological analyses for dissection of complex chromosomal processes.
Subject(s)
Cell Cycle Proteins/metabolism , Cell Cycle Proteins/physiology , Chromosomal Proteins, Non-Histone/metabolism , Chromosomal Proteins, Non-Histone/physiology , Chromosomes/metabolism , Nuclear Proteins/physiology , Phosphoproteins/physiology , Xenopus Proteins/physiology , Animals , Apoptosis Regulatory Proteins , Cell Cycle Proteins/analysis , Cell Nucleus/ultrastructure , Chromatids/metabolism , Chromatids/ultrastructure , Chromosomal Proteins, Non-Histone/analysis , Chromosomes/ultrastructure , DNA-Binding Proteins , Female , Fluorescence Recovery After Photobleaching , Humans , Microscopy, Phase-Contrast , Nuclear Proteins/analysis , Nuclear Proteins/genetics , Oocytes/ultrastructure , Phosphoproteins/analysis , Phosphoproteins/genetics , Protein Interaction Mapping , Recombinant Fusion Proteins/analysis , Recombinant Fusion Proteins/physiology , Xenopus Proteins/analysis , Xenopus laevis , CohesinsABSTRACT
Sister-chromatid separation at the metaphase-anaphase transition is regulated by a proteolytic cascade. Destruction of the securin Pds1p liberates the Esp1p separase, which ultimately targets the mitotic cohesin Mcd1p/Scc1p for destruction. Pds1p stabilization by the spindle or DNA damage checkpoints prevents sister-chromatid separation while mutants lacking PDS1 (pds1Delta) are temperature sensitive for growth due to elevated chromosome loss. This report examined the role of the budding yeast Pds1p in meiotic progression using genetic, cytological, and biochemical assays. Similar to its mitotic function, Pds1p destruction is required for metaphase I-anaphase I transition. However, even at the permissive temperature for growth, pds1Delta mutants arrest with prophase I spindle and nuclear characteristics. This arrest was partially suppressed by preventing recombination initiation or by inactivating a subset of recombination checkpoint components. Further studies revealed that Pds1p is required for recombination in both double-strand-break formation and synaptonemal complex assembly. Although deleting PDS1 did not affect the degradation of the meiotic cohesin Rec8p, Mcd1p was precociously destroyed as cells entered the meiotic program. This role is meiosis specific as Mcd1p destruction is not altered in vegetative pds1Delta cultures. These results define a previously undescribed role for Pds1p in cohesin maintenance, recombination, and meiotic progression.
Subject(s)
Cell Cycle Proteins/metabolism , Meiotic Prophase I , Nuclear Proteins/metabolism , Recombination, Genetic/genetics , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/genetics , Cell Cycle Proteins/genetics , Chromatin/metabolism , Chromosomal Proteins, Non-Histone/metabolism , DNA Breaks, Double-Stranded , Gene Deletion , Gene Expression Regulation, Fungal , Genes, Fungal , Mutation/genetics , Nuclear Proteins/genetics , Phosphoproteins/metabolism , Saccharomyces cerevisiae Proteins/genetics , Securin , Signal Transduction , Spindle Apparatus/metabolism , Synaptonemal Complex/metabolism , Transcription, GeneticABSTRACT
Cohesin, the protein complex that mediates sister chromatid cohesion, is required for faithful chromosome segregation and efficient repair of double-strand breaks (DSBs). Cohesion generation is normally restricted to S phase. However, in G2/M, a DSB activates cohesion generation near the DSB and genome-wide. Here, using budding yeast, we show that DSB-induced cohesion occurs when cohesin contains the kleisin subunit, Mcd1 (Scc1), but not when Mcd1 is replaced by its meiotic isoform, Rec8. We exploit this divergence to demonstrate that serine 83 of Mcd1 and the Chk1 kinase are critical determinants for DSB-induced cohesion. We propose that a DSB in G2/M activates Mec1 (ATR), which in turn stimulates Chk1-dependent phosphorylation of Mcd1 at serine 83. Serine 83 phosphorylation promotes chromatin-bound cohesin to become cohesive.
Subject(s)
Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/metabolism , DNA Damage , Nuclear Proteins/metabolism , Phosphoproteins/metabolism , Protein Subunits/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Sister Chromatid Exchange , Amino Acid Sequence , Amino Acid Substitution , Cell Cycle Proteins/chemistry , Checkpoint Kinase 1 , DNA Breaks, Double-Stranded , DNA Repair , DNA Replication , Models, Biological , Molecular Sequence Data , Nuclear Proteins/chemistry , Phosphoproteins/chemistry , Phosphorylation , Protein Isoforms/metabolism , Protein Kinases/metabolism , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae Proteins/chemistry , Serine/metabolism , Structure-Activity Relationship , CohesinsABSTRACT
In eukaryotes, the process of sister chromatid cohesion holds the two sister chromatids (the replicated chromosomes) together from DNA replication to the onset of chromosome segregation. Cohesion is mediated by cohesin, a four-subunit SMC (structural maintenance of chromosome) complex. Cohesin and cohesion are required for proper chromosome segregation, DNA repair, and gene expression. To carry out these functions, cohesion is regulated by elaborate mechanisms involving a growing list of cohesin auxiliary factors. These factors control the timing and position of cohesin binding to chromatin, activate chromatin-bound cohesin to become cohesive, and orchestrate the orderly dissolution of cohesion. The 45-nm ringlike architecture of soluble cohesin is compatible with dramatically different mechanisms for both chromatin binding and cohesion generation. Solving the mechanism of cohesion and its complex regulation presents significant challenges but offers the potential to provide important insights into higher-order chromosome organization and chromosome biology.
Subject(s)
Cell Cycle Proteins , Chromatids/metabolism , Chromosomal Proteins, Non-Histone , Chromosome Segregation , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/metabolism , Cell Division/physiology , Chromosomal Proteins, Non-Histone/chemistry , Chromosomal Proteins, Non-Histone/metabolism , DNA Repair , Macromolecular Substances/chemistry , Macromolecular Substances/metabolism , Models, Molecular , Protein Binding , Protein Isoforms/chemistry , Protein Isoforms/metabolism , CohesinsABSTRACT
Chromosome segregation, transcriptional regulation, and repair of DNA double-strand breaks require the cohesin protein complex. Cohesin holds the replicated chromosomes (sister chromatids) together to mediate sister chromatid cohesion. The mechanism of how cohesion is established is unknown. We found that in budding yeast, the head domain of the Smc3p subunit of cohesin is acetylated by the Eco1p acetyltransferase at two evolutionarily conserved residues, promoting the chromatin-bound cohesin to tether sister chromatids. Smc3p acetylation is induced in S phase after the chromatin loading of cohesin and is suppressed in G(1) and G(2)/M. Smc3 head acetylation and its cell cycle regulation provide important insights into the biology and mechanism of cohesion establishment.