Publications by authors named "Vincent Guacci"

29 Publications

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Communication between distinct subunit interfaces of the cohesin complex promotes its topological entrapment of DNA.

Elife 2019 06 4;8. Epub 2019 Jun 4.

Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States.

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.
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http://dx.doi.org/10.7554/eLife.46347DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6579514PMC
June 2019

A role for the Smc3 hinge domain in the maintenance of sister chromatid cohesion.

Mol Biol Cell 2018 02 29;29(3):339-355. Epub 2017 Nov 29.

Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720

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.
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http://dx.doi.org/10.1091/mbc.E17-08-0511DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5996953PMC
February 2018

Cohesin Function in Cohesion, Condensation, and DNA Repair Is Regulated by Wpl1p via a Common Mechanism in .

Genetics 2018 01 20;208(1):111-124. Epub 2017 Nov 20.

Department of Molecular and Cell Biology, University of California, Berkeley, California 94720.

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 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.
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http://dx.doi.org/10.1534/genetics.117.300537DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5753852PMC
January 2018

The ATPases of cohesin interface with regulators to modulate cohesin-mediated DNA tethering.

Elife 2015 Nov 19;4. Epub 2015 Nov 19.

Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States.

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.
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http://dx.doi.org/10.7554/eLife.11315DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4709263PMC
November 2015

Interallelic complementation provides functional evidence for cohesin-cohesin interactions on DNA.

Mol Biol Cell 2015 Nov 16;26(23):4224-35. Epub 2015 Sep 16.

Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720

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.
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http://dx.doi.org/10.1091/mbc.E15-06-0331DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4642856PMC
November 2015

A conserved domain in the scc3 subunit of cohesin mediates the interaction with both mcd1 and the cohesin loader complex.

PLoS Genet 2015 Mar 6;11(3):e1005036. Epub 2015 Mar 6.

Faculty of Medicine in The Galilee, Bar-Ilan University, Safed, Israel.

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.
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http://dx.doi.org/10.1371/journal.pgen.1005036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4352044PMC
March 2015

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

Mol Biol Cell 2015 Jan 5;26(1):117-33. Epub 2014 Nov 5.

Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720.

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.
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http://dx.doi.org/10.1091/mbc.E14-08-1268DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4279223PMC
January 2015

ROCC, a conserved region in cohesin's Mcd1 subunit, is essential for the proper regulation of the maintenance of cohesion and establishment of condensation.

Mol Biol Cell 2014 Aug 25;25(16):2351-64. Epub 2014 Jun 25.

Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720

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.
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http://dx.doi.org/10.1091/mbc.E14-04-0929DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4142609PMC
August 2014

Cohesin-independent segregation of sister chromatids in budding yeast.

Mol Biol Cell 2012 Feb 21;23(4):729-39. Epub 2011 Dec 21.

Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.

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.
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http://dx.doi.org/10.1091/mbc.E11-08-0696DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3279399PMC
February 2012

Systematic reduction of cohesin differentially affects chromosome segregation, condensation, and DNA repair.

Curr Biol 2010 May 6;20(10):957-63. Epub 2010 May 6.

Howard Hughes Medical Institute, 3520 San Martin Drive, Baltimore, MD 21218, USA.

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.
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http://dx.doi.org/10.1016/j.cub.2010.04.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2892909PMC
May 2010

The Scc2/Scc4 cohesin loader determines the distribution of cohesin on budding yeast chromosomes.

Genes Dev 2009 Oct;23(19):2345-57

Department of Biochemistry and Molecular Genetics, University of Colorado Denver School of Medicine, Aurora, Colorado 80045, USA.

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.
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http://dx.doi.org/10.1101/gad.1819409DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2758738PMC
October 2009

Pds5 is required for homologue pairing and inhibits synapsis of sister chromatids during yeast meiosis.

J Cell Biol 2009 Sep;186(5):713-25

Department of Biological Science, The Florida State University, Tallahassee, FL 32306, USA.

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.
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http://dx.doi.org/10.1083/jcb.200810107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2742186PMC
September 2009

The zinc finger of Eco1 enhances its acetyltransferase activity during sister chromatid cohesion.

Nucleic Acids Res 2009 Oct 19;37(18):6126-34. Epub 2009 Aug 19.

Department of Embryology, Carnegie Institution, Howard Hughes Medical Institute, Baltimore, MD 21218, USA.

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.
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http://dx.doi.org/10.1093/nar/gkp656DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2764436PMC
October 2009

Lampbrush chromosomes enable study of cohesin dynamics.

Chromosome Res 2009 ;17(2):165-84

Cell & Developmental Biology, University of Illinois at Urbana-Champaign, Urbana, IL, USA.

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.
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http://dx.doi.org/10.1007/s10577-008-9015-9DOI Listing
June 2009

Pds1p is required for meiotic recombination and prophase I progression in Saccharomyces cerevisiae.

Genetics 2009 Jan 10;181(1):65-79. Epub 2008 Nov 10.

Department of Biochemistry, Drexel University College of Medicine, Philadelphia, PA 19102, USA.

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.
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http://dx.doi.org/10.1534/genetics.108.095513DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2621190PMC
January 2009

A molecular determinant for the establishment of sister chromatid cohesion.

Science 2008 Jul;321(5888):566-9

Howard Hughes Medical Institute and Department of Embryology, Carnegie Institution, 3520 San Martin Drive, Baltimore, MD 21218, USA.

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.
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http://dx.doi.org/10.1126/science.1157880DOI Listing
July 2008

Sister chromatid cohesion: a simple concept with a complex reality.

Annu Rev Cell Dev Biol 2008 ;24:105-29

Howard Hughes Medical Institute, Carnegie Institution, Baltimore, Maryland 21218, USA.

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.
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http://dx.doi.org/10.1146/annurev.cellbio.24.110707.175350DOI Listing
December 2008

The kleisin subunit of cohesin dictates damage-induced cohesion.

Mol Cell 2008 Jul;31(1):47-56

Department of Embryology, Howard Hughes Medical Institute, Carnegie Institution, 3520 San Martin Drive, Baltimore, MD 21218, USA.

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.
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http://dx.doi.org/10.1016/j.molcel.2008.06.005DOI Listing
July 2008

Sister chromatid cohesion: the cohesin cleavage model does not ring true.

Authors:
Vincent Guacci

Genes Cells 2007 Jun;12(6):693-708

Howard Hughes Medical Institute, Department of Embryology, Carnegie Institution of Washington, Baltimore, MD 21210, USA.

Sister chromatid cohesion is important for high fidelity chromosome segregation during anaphase. Gene products that provide structural components (cohesin complex or cohesin) and regulatory components responsible for cohesion are conserved through eukaryotes. A simple model where cohesion establishment occurs by replication through static cohesin rings and cohesion dissolution occurs by Esp1p/separase mediated cleavage of the cohesin rings (Mcd1p/Rad21p/Scc1p sub-unit cleavage) has become widespread. A growing body of evidence is inconsistent with this ring cleavage model. This review will summarize the evidence showing that cohesin complex is not static but is regulated at multiple cell cycle stages before anaphase in a separase independent manner. Separase is indeed required at anaphase for complete chromosome segregation. However, multiple mechanisms for cohesion dissolution appear to act concurrently during anaphase. Separase is only one such mechanism and its importance varies from organism to organism. The idea that cohesin is a dynamic complex subjected to regulation at various cell cycle stages by multiple mechanisms makes sense in light of the myriad functions in which it has been implicated, such as DNA damage repair, gene silencing and chromosome condensation.
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http://dx.doi.org/10.1111/j.1365-2443.2007.01093.xDOI Listing
June 2007

PIASgamma is required for faithful chromosome segregation in human cells.

PLoS One 2006 Dec 20;1:e53. Epub 2006 Dec 20.

Department of Genetics, Cell Biology and Development, University of Minnesota Medical School, Minneapolis, Minnesota, United States of America.

Background: The precision of the metaphase-anaphase transition ensures stable genetic inheritance. The spindle checkpoint blocks anaphase onset until the last chromosome biorients at metaphase plate, then the bonds between sister chromatids are removed and disjoined chromatids segregate to the spindle poles. But, how sister separation is triggered is not fully understood.

Principal Findings: We identify PIASgamma as a human E3 sumo ligase required for timely and efficient sister chromatid separation. In cells lacking PIASgamma, normal metaphase plates form, but the spindle checkpoint is activated, leading to a prolonged metaphase block. Sister chromatids remain cohered even if cohesin is removed by depletion of hSgo1, because DNA catenations persist at centromeres. PIASgamma-depleted cells cannot properly localize Topoisomerase II at centromeres or in the cores of mitotic chromosomes, providing a functional link between PIASgamma and Topoisomerase II.

Conclusions: PIASgamma directs Topoisomerase II to specific chromosome regions that require efficient removal of DNA catenations prior to anaphase. The lack of this activity activates the spindle checkpoint, protecting cells from non-disjunction. Because DNA catenations persist without PIASgamma in the absence of cohesin, removal of catenations and cohesin rings must be regulated in parallel.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0000053PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1762334PMC
December 2006

Intersection between the regulators of sister chromatid cohesion establishment and maintenance in budding yeast indicates a multi-step mechanism.

Cell Cycle 2006 Nov 13;5(21):2528-36. Epub 2006 Sep 13.

Fox Chase Cancer Center, Philadelphia, Pennsylvania, USA.

Sister chromatid cohesion is established during S phase and maintained until anaphase. The cohesin complex (Mcd1p/Scc1p, Smc1p, Smc3p Irr1p/Scc3p in budding yeast) serves a structural role as it is required at all times when cohesion exists. Pds5p colocalizes temporally and spatially with cohesin on chromosomes but is thought to serve as a regulator of cohesion maintenance during mitosis. In contrast, Ctf7p/Eco1p is required during S phase for establishment but is not required during mitosis. Here we provide genetic and biochemical evidence that the pathways of cohesion establishment and maintenance are intimately linked. Our results show that mutants in ctf7 and pds5 are synthetically lethal. Moreover, over-expression of either CTF7 or PDS5 exhibits reciprocal suppression of the other mutant's temperature sensitivity. The suppression by CTF7 is specific for pds5 mutants as CTF7 over-expression increases the temperature sensitivity of an mcd1 mutant but has no effect on smc1 or smc3 mutants. Three additional findings provide new insights into the process of cohesion establishment. First, over-expression of ctf7 alleles deficient in acetylase activity exhibit significantly reduced suppression of the pds5 mutant but exacerbated toxicity to the mcd1 mutant. Second, using chromosome spreads and chromatin immuno-precipitation, we find either cohesin complex or Pds5p chromosomal localization is altered in ctf7 mutants. Finally, biochemical analysis reveals that Ctf7p and Pds5p coimmunoprecipitate, which physically links these regulators of cohesion establishment and maintenance. We propose a model whereby Ctf7p and Pds5p cooperate to facilitate efficient establishment by mediating changes in cohesin complex on chromosomes after its deposition.
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http://dx.doi.org/10.4161/cc.5.21.3405DOI Listing
November 2006

Phosphorylation of histone H4 Ser1 regulates sporulation in yeast and is conserved in fly and mouse spermatogenesis.

Genes Dev 2006 Sep;20(18):2580-92

Gene Expression and Regulation Program, The Wistar Institute, Philadelphia, Pennsylvania 19104, USA.

Sporulation in Saccharomyces cerevisiae is a highly regulated process wherein a diploid cell gives rise to four haploid gametes. In this study we show that histone H4 Ser1 is phosphorylated (H4 S1ph) during sporulation, starting from mid-sporulation and persisting to germination, and is temporally distinct from earlier meiosis-linked H3 S10ph involved in chromosome condensation. A histone H4 S1A substitution mutant forms aberrant spores and has reduced sporulation efficiency. Deletion of sporulation-specific yeast Sps1, a member of the Ste20 family of kinases, nearly abolishes the sporulation-associated H4 S1ph modification. H4 S1ph may promote chromatin compaction, since deletion of SPS1 increases accessibility to antibody immunoprecipitation; furthermore, either deletion of Sps1 or an H4 S1A substitution results in increased DNA volume in nuclei within spores. We find H4 S1ph present during Drosophila melanogaster and mouse spermatogenesis, and similar to yeast, this modification extends late into sperm differentiation relative to H3 S10ph. Thus, H4 S1ph may be an evolutionarily ancient histone modification to mark the genome for gamete-associated packaging.
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http://dx.doi.org/10.1101/gad.1457006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1578680PMC
September 2006

Evidence that the yeast spindle assembly checkpoint has a target other than the anaphase promoting complex.

Cell Cycle 2005 Nov 5;4(11):1555-7. Epub 2005 Nov 5.

Department of Genetics, Cell Biology & Development, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.

The spindle assembly checkpoint monitors biorientation of chromosomes on the metaphase spindle and inhibits the Anaphase Promoting Complex (APC) specificity factor Cdc20. If APC-Cdc20 is the sole target of the spindle checkpoint, then cells lacking APC and its targets, B-type cyclin and securin, would lack spindle checkpoint function. We tested this hypothesis in yeast cells that are APC-null. Surprisingly, we find that such yeast cells are able to activate the spindle assembly checkpoint, delaying cell cycle progression in G2/M phase. These data suggest that the spindle checkpoint has a non-APC target that can restrain anaphase onset.
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http://dx.doi.org/10.4161/cc.4.11.2144DOI Listing
November 2005

Recombination protein Tid1p controls resolution of cohesin-dependent linkages in meiosis in Saccharomyces cerevisiae.

J Cell Biol 2005 Oct 17;171(2):241-53. Epub 2005 Oct 17.

Program in Molecular, Cell, and Developmental Biology, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104.

Sister chromatid cohesion and interhomologue recombination are coordinated to promote the segregation of homologous chromosomes instead of sister chromatids at the first meiotic division. During meiotic prophase in Saccharomyces cerevisiae, the meiosis-specific cohesin Rec8p localizes along chromosome axes and mediates most of the cohesion. The mitotic cohesin Mcd1p/Scc1p localizes to discrete spots along chromosome arms, and its function is not clear. In cells lacking Tid1p, which is a member of the SWI2/SNF2 family of helicase-like proteins that are involved in chromatin remodeling, Mcd1p and Rec8p persist abnormally through both meiotic divisions, and chromosome segregation fails in the majority of cells. Genetic results indicate that the primary defect in these cells is a failure to resolve Mcd1p-mediated connections. Tid1p interacts with recombination enzymes Dmc1p and Rad51p and has an established role in recombination repair. We propose that Tid1p remodels Mcd1p-mediated cohesion early in meiotic prophase to facilitate interhomologue recombination and the subsequent segregation of homologous chromosomes.
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http://dx.doi.org/10.1083/jcb.200505020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2171180PMC
October 2005

Topoisomerase II suppresses the temperature sensitivity of Saccharomyces cerevisiae pds5 mutants, but not the defect in sister chromatid cohesion.

Cell Cycle 2005 Sep 7;4(9):1294-304. Epub 2005 Sep 7.

Basic Science Division, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA.

Sister chromatid cohesion enables chromosomes to achieve bipolar attachment to the mitotic spindle and its dissolution is required for chromosome segregation. The cohesin complex serves as the primary molecular glue responsible for cohesion. Pds5p binds to the same chromosomal loci as the cohesin complex but plays a distinct role as a regulator of cohesion maintenance. Catenation between sister chromatids must also be removed by Topoisomerase II (Top2p) enzymatic activity to enable chromosome segregation. We identified TOP2 as a high-copy suppressor of the temperature sensitivity of pds5 mutants. TOP2 suppression is specific for pds5 mutants as it does not suppress mutants in the cohesin complex. TOP2 suppresses mini-chromosome loss in pds5 mutants indicating that it rescues a chromosome segregation defect. Surprisingly, TOP2 overexpression fails to suppress the cohesion defect of pds5 mutants, suggesting that it suppresses an additional and as yet uncharacterized defect in pds5 mutants that is essential for viability. A catalytically dead TOP2 allele suppresses pds5 temperature sensitivity, suggesting that suppression is unrelated to Top2p enzymatic function. Consistent with this idea, when the pds5 mutant is combined with the top2-4 mutant, which accumulates DNA catenanes due to defective enzymatic activity, the double mutants exhibit synthetic sickness indicating that increased catenation is toxic to pds5 cells. Our results suggest that Pds5p and Top2p cooperate to promote proper chromosome segregation by a mechanism unrelated to either cohesion or catenation/decatenation.
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http://dx.doi.org/10.4161/cc.4.9.1997DOI Listing
September 2005

Budding yeast PDS5 plays an important role in meiosis and is required for sister chromatid cohesion.

Mol Microbiol 2005 May;56(3):670-80

Department of Zoology and Physiology, University of Wyoming; Laramie, WY 82071, USA.

Budding yeast PDS5 is an essential gene in mitosis and is required for chromosome condensation and sister chromatid cohesion. Here we report that PDS also is required in meiosis. Pds5p localizes on chromosomes at all stages during meiotic cycle, except anaphase I. PDS5 plays an important role at first meiotic prophase. Failure in function of PDS5 causes premature separation of chromosomes. The loading of Pds5p onto chromosome requires the function of REC8, but the association of Rec8p with chromosome is independent of PDS5. Mutant analysis and live cell imaging indicate that PDS5 play a role in meiosis II as well.
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http://dx.doi.org/10.1111/j.1365-2958.2005.04582.xDOI Listing
May 2005

Mutation of the cohesin related gene PDS5 causes cell death with predominant apoptotic features in Saccharomyces cerevisiae during early meiosis.

Mutat Res 2005 Mar;570(2):163-73

Department of Zoology and Physiology, University of Wyoming, Laramie, WY 82071, USA.

Pds5p is a cohesin related protein. It is required for maintenance of sister chromatid cohesion in mitosis and meiosis. Here we report that pds5-1 causes cell death in yeast Saccharomyces cerevisiae during early meiosis. The pds5-1 caused cell death possesses characteristics of apoptosis and necrosis, including externalization of phosphatidylserine at cytoplasmic membrane, accumulation of DNA breaks, chromatin condensation and fragmentation, nuclei fragmentation, membrane degeneration and cell size enlargement. Our results also suggest that (1) The defect of DNA repair; (2) The production of reactive oxygen species, in pds5-1 mutant are involved in pds5-1 induced cell death.
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http://dx.doi.org/10.1016/j.mrfmmm.2004.11.014DOI Listing
March 2005

Pds5p regulates the maintenance of sister chromatid cohesion and is sumoylated to promote the dissolution of cohesion.

J Cell Biol 2003 Nov 17;163(4):729-41. Epub 2003 Nov 17.

Basic Science Division, Fox Chase Cancer Center, Philadelphia, PA 19111, USA.

Pds5p and the cohesin complex are required for sister chromatid cohesion and localize to the same chromosomal loci over the same cell cycle window. However, Pds5p and the cohesin complex likely have distinct roles in cohesion. We report that pds5 mutants establish cohesion, but during mitosis exhibit precocious sister dissociation. Thus, unlike the cohesin complex, which is required for cohesion establishment and maintenance, Pds5p is required only for maintenance. We identified SMT4, which encodes a SUMO isopeptidase, as a high copy suppressor of both the temperature sensitivity and precocious sister dissociation of pds5 mutants. In contrast, SMT4 does not suppress temperature sensitivity of cohesin complex mutants. Pds5p is SUMO conjugated, with sumoylation peaking during mitosis. SMT4 overexpression reduces Pds5p sumoylation, whereas smt4 mutants have increased Pds5p sumoylation. smt4 mutants were previously shown to be defective in cohesion maintenance during mitosis. These data provide the first link between a protein required for cohesion, Pds5p, and sumoylation, and suggest that Pds5p sumoylation promotes the dissolution of cohesion.
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http://dx.doi.org/10.1083/jcb.200305080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2173684PMC
November 2003