scholarly journals Scc2-mediated loading of cohesin onto chromosomes in G1 yeast cells is insufficient to build cohesion during S phase

2017 ◽  
Author(s):  
Kim A Nasmyth
Keyword(s):  
Replication Fork ◽  
De Novo ◽  
S Phase ◽  
Yeast Cells ◽  
G1 Phase ◽  
Cohesin Complex ◽  
Previous Conclusion ◽  
Sister Chromatids ◽  
Chromatid Cohesion ◽  
Cohesin Subunit

SummarySister chromatids are held together from their replication until mitosis. Sister chromatid cohesion is mediated by the ring-shaped cohesin complex and it is thought that cohesin holds sister chromatids together by entrapping sister DNAs within the cohesin ring (Haering et al., 2008). However, how this occurs is not well understood. Because cohesin binds to DNA prior to replication it is possible that the replication fork passes through the lumen of the ring thereby placing replicated sisters inside cohesin rings. If this is the case, loading of cohesin in the G1 phase may be sufficient to build cohesion.We show here that Scc2, a cohesin subunit required for loading cohesin onto chromosomes de novo, is necessary for establishment of cohesion even after Scc2-mediated loading has already taken place during late G1 or early S phase. Our results challenge a previous conclusion based on related experiments whereby Scc2 was found not to be required for cohesion establishment during S phase (Lengronne et al., 2006).

scholarly journals Quantitative Cytogenetics Reveals Molecular Stoichiometry and Longitudinal Organization of Meiotic Chromosome Axes and Loops

2019 ◽  
Author(s):  
Alexander Woglar ◽  
Kei Yamaya ◽  
Baptiste Roelens ◽  
Alistair Boettiger ◽  
Simone Köhler ◽  
...  
Keyword(s):  
Sister Chromatid ◽  
De Novo ◽  
Meiotic Chromosome ◽  
S Phase ◽  
C Elegans ◽  
Sister Chromatids ◽  
Chromatid Cohesion ◽  
Molecular Bases ◽  
Chromosome Axis ◽  
Specialized Organization

ABSTRACTDuring meiosis, chromosomes adopt a specialized organization involving assembly of a cohesin-based axis along their lengths, with DNA loops emanating from this axis. We applied novel, quantitative and widely applicable cytogenetic strategies to elucidate the molecular bases of this organization using C. elegans. Analyses of WT chromosomes and de novo circular mini-chromosomes revealed that meiosis-specific HORMA-domain proteins assemble into cohorts in defined numbers and co-organize the axis together with two functionally-distinct cohesin complexes (REC-8 and COH-3/4) in defined stoichiometry. We further found that REC-8 cohesins, which load during S phase and mediate sister chromatid cohesion, usually occur as individual complexes, supporting a model wherein sister cohesion is mediated locally by a single cohesin ring. REC-8 complexes are interspersed in an alternating pattern with cohorts of axis-organizing COH-3/4 complexes (averaging three per cohort), which are insufficient to confer cohesion but can bind to individual chromatids, suggesting a mechanism to enable formation of asymmetric sister chromatid loops. Indeed, immuno-FISH assays demonstrate frequent asymmetry in genomic content between the loops formed on sister chromatids. We discuss how features of chromosome axis/loop architecture inferred from our data can help to explain enigmatic, yet essential, aspects of the meiotic program.

scholarly journals A novel mechanism for the establishment of sister chromatid cohesion by the ECO1 acetyltransferase

2015 ◽  
Vol 26 (1) ◽  
pp. 117-133 ◽  
Author(s):  
Vincent Guacci ◽  
Jeremiah Stricklin ◽  
Michelle S. Bloom ◽  
Xuánzōng Guō ◽  
Meghna Bhatter ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Current Model ◽  
S Phase ◽  
High Fidelity ◽  
In Vivo Assays ◽  
Sister Chromatids ◽  
Chromatid Cohesion ◽  
Cohesin Subunit ◽  
Mutant Cells

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.

scholarly journals The budding yeast PP2ACdc55 protein phosphatase prevents the onset of anaphase in response to morphogenetic defects

2007 ◽  
Vol 177 (4) ◽  
pp. 599-611 ◽  
Author(s):  
Elena Chiroli ◽  
Valentina Rossio ◽  
Giovanna Lucchini ◽  
Simonetta Piatti
Keyword(s):  
Protein Phosphatase ◽  
Budding Yeast ◽  
S Phase ◽  
Yeast Cells ◽  
Regulatory Subunit ◽  
Chromosome Transmission ◽  
Anaphase Onset ◽  
Sister Chromatids ◽  
Chromatid Cohesion ◽  
Noncanonical Pathway

Faithful chromosome transmission requires establishment of sister chromatid cohesion during S phase, followed by its removal at anaphase onset. Sister chromatids are tethered together by cohesin, which is displaced from chromosomes through cleavage of its Mcd1 subunit by the separase protease. Separase is in turn inhibited, up to this moment, by securin. Budding yeast cells respond to morphogenetic defects by a transient arrest in G2 with high securin levels and unseparated chromatids. We show that neither securin elimination nor forced cohesin cleavage is sufficient for anaphase in these conditions, suggesting that other factors contribute to cohesion maintainance in G2. We find that the protein phosphatase PP2A bound to its regulatory subunit Cdc55 plays a key role in this process, uncovering a new function for PP2ACdc55 in controlling a noncanonical pathway of chromatid cohesion removal.

scholarly journals Evidence for cohesin sliding along budding yeast chromosomes

Open Biology ◽  
2016 ◽  
Vol 6 (6) ◽  
pp. 150178 ◽  
Author(s):  
Maria Ocampo-Hafalla ◽  
Sofía Muñoz ◽  
Catarina P. Samora ◽  
Frank Uhlmann
Keyword(s):  
Chromosome Segregation ◽  
Sister Chromatid ◽  
Budding Yeast ◽  
Transcription Termination ◽  
Gene Activation ◽  
S Phase ◽  
Cohesin Complex ◽  
Sister Chromatids ◽  
Chromatid Cohesion ◽  
Active Genes

The ring-shaped cohesin complex is thought to topologically hold sister chromatids together from their synthesis in S phase until chromosome segregation in mitosis. How cohesin stably binds to chromosomes for extended periods, without impeding other chromosomal processes that also require access to the DNA, is poorly understood. Budding yeast cohesin is loaded onto DNA by the Scc2–Scc4 cohesin loader at centromeres and promoters of active genes, from where cohesin translocates to more permanent places of residence at transcription termination sites. Here we show that, at the GAL2 and MET17 loci, pre-existing cohesin is pushed downstream along the DNA in response to transcriptional gene activation, apparently without need for intermittent dissociation or reloading. We observe translocation intermediates and find that the distribution of most chromosomal cohesin is shaped by transcription. Our observations support a model in which cohesin is able to slide laterally along chromosomes while maintaining topological contact with DNA. In this way, stable cohesin binding to DNA and enduring sister chromatid cohesion become compatible with simultaneous underlying chromosomal activities, including but maybe not limited to transcription.

scholarly journals MCM2–7-dependent cohesin loading during S phase promotes sister-chromatid cohesion

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Ge Zheng ◽  
Mohammed Kanchwala ◽  
Chao Xing ◽  
Hongtao Yu
Keyword(s):  
Sister Chromatid ◽  
Replication Fork ◽  
Sister Chromatid Cohesion ◽  
S Phase ◽  
Nucleosome Assembly ◽  
Okazaki Fragment ◽  
Replicative Helicase ◽  
Sister Chromatids ◽  
Chromatid Cohesion ◽  
Okazaki Fragment Processing

DNA replication transforms cohesin rings dynamically associated with chromatin into the cohesive form to establish sister-chromatid cohesion. Here, we show that, in human cells, cohesin loading onto chromosomes during early S phase requires the replicative helicase MCM2–7 and the kinase DDK. Cohesin and its loader SCC2/4 (NIPBL/MAU2 in humans) associate with DDK and phosphorylated MCM2–7. This binding does not require MCM2–7 activation by CDC45 and GINS, but its persistence on activated MCM2–7 requires fork-stabilizing replisome components. Inactivation of these replisome components impairs cohesin loading and causes interphase cohesion defects. Interfering with Okazaki fragment processing or nucleosome assembly does not impact cohesion. Therefore, MCM2–7-coupled cohesin loading promotes cohesion establishment, which occurs without Okazaki fragment maturation. We propose that the cohesin–loader complex bound to MCM2–7 is mobilized upon helicase activation, transiently held by the replisome, and deposited behind the replication fork to encircle sister chromatids and establish cohesion.

scholarly journals The yeast S phase checkpoint enables replicating chromosomes to bi-orient and restrain spindle extension during S phase distress

2005 ◽  
Vol 168 (7) ◽  
pp. 999-1012 ◽  
Author(s):  
Jeff Bachant ◽  
Shannon R. Jessen ◽  
Sarah E. Kavanaugh ◽  
Candida S. Fielding
Keyword(s):  
Dna Replication ◽  
Chromosome Segregation ◽  
Replication Fork ◽  
S Phase ◽  
Origin Of Replication ◽  
Independent Manner ◽  
Checkpoint Signaling ◽  
Hydroxyurea Treatment ◽  
Chromatid Cohesion ◽  
S Phase Checkpoint

The budding yeast S phase checkpoint responds to hydroxyurea-induced nucleotide depletion by preventing replication fork collapse and the segregation of unreplicated chromosomes. Although the block to chromosome segregation has been thought to occur by inhibiting anaphase, we show checkpoint-defective rad53 mutants undergo cycles of spindle extension and collapse after hydroxyurea treatment that are distinct from anaphase cells. Furthermore, chromatid cohesion, whose dissolution triggers anaphase, is dispensable for S phase checkpoint arrest. Kinetochore–spindle attachments are required to prevent spindle extension during replication blocks, and chromosomes with two centromeres or an origin of replication juxtaposed to a centromere rescue the rad53 checkpoint defect. These observations suggest that checkpoint signaling is required to generate an inward force involved in maintaining preanaphase spindle integrity during DNA replication distress. We propose that by promoting replication fork integrity under these conditions Rad53 ensures centromere duplication. Replicating chromosomes can then bi-orient in a cohesin-independent manner to restrain untimely spindle extension.

scholarly journals CLN3 expression is sufficient to restore G1-to-S-phase progression in Saccharomyces cerevisiae mutants defective in translation initiation factor eIF4E

1999 ◽  
Vol 340 (1) ◽  
pp. 135-141 ◽  
Author(s):  
Parisa DANAIE ◽  
Michael ALTMANN ◽  
Michael N. HALL ◽  
Hans TRACHSEL ◽  
Stephen B. HELLIWELL
Keyword(s):  
Cell Cycle ◽  
S Phase ◽  
Translation Initiation Factor ◽  
Yeast Cells ◽  
Initiation Factor ◽  
G1 Phase ◽  
Mrna Levels ◽  
Wild Type ◽  
Phase Progression ◽  
Type Gene

The essential cap-binding protein (eIF4E) of Saccharomycescerevisiae is encoded by the CDC33 (wild-type) gene, originally isolated as a mutant, cdc33-1, which arrests growth in the G1 phase of the cell cycle at 37 °C. We show that other cdc33 mutants also arrest in G1. One of the first events required for G1-to-S-phase progression is the increased expression of cyclin 3. Constructs carrying the 5ʹ-untranslated region of CLN3 fused to lacZ exhibit weak reporter activity, which is significantly decreased in a cdc33-1 mutant, implying that CLN3 mRNA is an inefficiently translated mRNA that is sensitive to perturbations in the translation machinery. A cdc33-1 strain expressing either stable Cln3p (Cln3-1p) or a hybrid UBI4 5ʹ-CLN3 mRNA, whose translation displays decreased dependence on eIF4E, arrested randomly in the cell cycle. In these cells CLN2 mRNA levels remained high, indicating that Cln3p activity is maintained. Induction of a hybrid UBI4 5ʹ-CLN3 message in a cdc33-1 mutant previously arrested in G1 also caused entry into a new cell cycle. We conclude that eIF4E activity in the G1-phase is critical in allowing sufficient Cln3p activity to enable yeast cells to enter a new cell cycle.

scholarly journals Checkpoint-Dependent Regulation of Origin Firing and Replication Fork Movement in Response to DNA Damage in Fission Yeast

2008 ◽  
Vol 29 (2) ◽  
pp. 602-611 ◽  
Author(s):  
Sanjay Kumar ◽  
Joel A. Huberman
Keyword(s):  
Fission Yeast ◽  
Replication Fork ◽  
S Phase ◽  
Yeast Cells ◽  
Methyl Methanesulfonate ◽  
Cyclin Dependent Kinase ◽  
Cdc25 Phosphatase ◽  
Origin Firing ◽  
Replication Intermediates ◽  
In Cells

ABSTRACT To elucidate the checkpoint mechanism responsible for slowing passage through S phase when fission yeast cells are treated with the DNA-damaging agent methyl methanesulfonate (MMS), we carried out two-dimensional gel analyses of replication intermediates in cells synchronized by cdc10 block (in G1) followed by release into synchronous S phase. The results indicated that under these conditions early-firing centromeric origins were partially delayed but late-firing telomeric origins were not delayed. Replication intermediates persisted in MMS-treated cells, suggesting that replication fork movement was inhibited. These effects were dependent on the Cds1 checkpoint kinase and were abolished in cells overexpressing the Cdc25 phosphatase, suggesting a role for the Cdc2 cyclin-dependent kinase. We conclude that both partial inhibition of the firing of a subset of origins and inhibition of replication fork movement contribute to the slowing of S phase in MMS-treated fission yeast cells.

scholarly journals Meiotic cohesin REC8 marks the axial elements of rat synaptonemal complexes before cohesins SMC1β and SMC3

2003 ◽  
Vol 160 (5) ◽  
pp. 657-670 ◽  
Author(s):  
Maureen Eijpe ◽  
Hildo Offenberg ◽  
Rolf Jessberger ◽  
Ekaterina Revenkova ◽  
Christa Heyting
Keyword(s):  
Synaptonemal Complex ◽  
Sister Chromatid ◽  
Meiotic Prophase ◽  
S Phase ◽  
Synaptonemal Complexes ◽  
Sister Chromatids ◽  
Chromatid Cohesion ◽  
Axial Element ◽  
Striking Contrast ◽  
Metaphase I

In meiotic prophase, the sister chromatids of each chromosome develop a common axial element (AE) that is integrated into the synaptonemal complex (SC). We analyzed the incorporation of sister chromatid cohesion proteins (cohesins) and other AE components into AEs. Meiotic cohesin REC8 appeared shortly before premeiotic S phase in the nucleus and formed AE-like structures (REC8-AEs) from premeiotic S phase on. Subsequently, meiotic cohesin SMC1β, cohesin SMC3, and AE proteins SCP2 and SCP3 formed dots along REC8-AEs, which extended and fused until they lined REC8-AEs along their length. In metaphase I, SMC1β, SMC3, SCP2, and SCP3 disappeared from the chromosome arms and accumulated around the centromeres, where they stayed until anaphase II. In striking contrast, REC8 persisted along the chromosome arms until anaphase I and near the centromeres until anaphase II. We propose that REC8 provides a basis for AE formation and that the first steps in AE assembly do not require SMC1β, SMC3, SCP2, and SCP3. Furthermore, SMC1β, SMC3, SCP2, and SCP3 cannot provide arm cohesion during metaphase I. We propose that REC8 then provides cohesion. RAD51 and/or DMC1 coimmunoprecipitates with REC8, suggesting that REC8 may also provide a basis for assembly of recombination complexes.

scholarly journals Physical Association of Saccharomyces cerevisiae Polo-like Kinase Cdc5 with Chromosomal Cohesin Facilitates DNA Damage Response

2016 ◽  
Vol 291 (33) ◽  
pp. 17228-17246 ◽  
Author(s):  
Sujiraporn Pakchuen ◽  
Mai Ishibashi ◽  
Emi Takakusagi ◽  
Katsuhiko Shirahige ◽  
Takashi Sutani
Keyword(s):  
Direct Interaction ◽  
Mitotic Cell ◽  
Yeast Cells ◽  
Interaction Domain ◽  
Additional Layer ◽  
Sister Chromatids ◽  
Physiological Relevance ◽  
Damage Response ◽  
M Phase ◽  
Cohesin Subunit

At the onset of anaphase, a protease called separase breaks the link between sister chromatids by cleaving the cohesin subunit Scc1. This irreversible step in the cell cycle is promoted by degradation of the separase inhibitor, securin, and polo-like kinase (Plk) 1-dependent phosphorylation of the Scc1 subunit. Plk could recognize substrates through interaction between its phosphopeptide interaction domain, the polo-box domain, and a phosphorylated priming site in the substrate, which has been generated by a priming kinase beforehand. However, the physiological relevance of this targeting mechanism remains to be addressed for many of the Plk1 substrates. Here, we show that budding yeast Plk1, Cdc5, is pre-deposited onto cohesin engaged in cohesion on chromosome arms in G2/M phase cells. The Cdc5-cohesin association is mediated by direct interaction between the polo-box domain of Cdc5 and Scc1 phosphorylated at multiple sites in its middle region. Alanine substitutions of the possible priming phosphorylation sites (scc1-15A) impair Cdc5 association with chromosomal cohesin, but they make only a moderate impact on mitotic cell growth even in securin-deleted cells (pds1Δ), where Scc1 phosphorylation by Cdc5 is indispensable. The same scc1-15A pds1Δ double mutant, however, exhibits marked sensitivity to the DNA-damaging agent phleomycin, suggesting that the priming phosphorylation of Scc1 poses an additional layer of regulation that enables yeast cells to adapt to genotoxic environments.

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