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      Characterization of the Interaction between the Cohesin Subunits Rad21 and SA1/2

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          Abstract

          The cohesin complex is responsible for the fidelity of chromosomal segregation during mitosis. It consists of four core subunits, namely Rad21/Mcd1/Scc1, Smc1, Smc3, and one of the yeast Scc3 orthologs SA1 or SA2. Sister chromatid cohesion is generated during DNA replication and maintained until the onset of anaphase. Among the many proposed models of the cohesin complex, the 'core' cohesin subunits Smc1, Smc3, and Rad21 are almost universally displayed as tripartite ring. However, other than its supportive role in the cohesin ring, little is known about the fourth core subunit SA1/SA2. To gain deeper insight into the function of SA1/SA2 in the cohesin complex, we have mapped the interactive regions of SA2 and Rad21 in vitro and ex vivo. Whereas SA2 interacts with Rad21 through a broad region (301–750 aa), Rad21 binds to SA proteins through two SA-binding motifs on Rad21, namely N-terminal (NT) and middle part (MP) SA-binding motif, located at 60–81 aa of the N-terminus and 383–392 aa of the MP of Rad21, respectively. The MP SA-binding motif is a 10 amino acid, α-helical motif. Deletion of these 10 amino acids or mutation of three conserved amino acids (L 385, F 389, and T 390) in this α-helical motif significantly hinders Rad21 from physically interacting with SA1/2. Besides the MP SA-binding motif, the NT SA-binding motif is also important for SA1/2 interaction. Although mutations on both SA-binding motifs disrupt Rad21-SA1/2 interaction, they had no apparent effect on the Smc1-Smc3-Rad21 interaction. However, the Rad21-Rad21 dimerization was reduced by the mutations, indicating potential involvement of the two SA-binding motifs in the formation of the two-ring handcuff for chromosomal cohesion. Furthermore, mutant Rad21 proteins failed to significantly rescue precocious chromosome separation caused by depletion of endogenous Rad21 in mitotic cells, further indicating the physiological significance of the two SA-binding motifs of Rad21.

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          PredictProtein (http://www.predictprotein.org) is an Internet service for sequence analysis and the prediction of protein structure and function. Users submit protein sequences or alignments; PredictProtein returns multiple sequence alignments, PROSITE sequence motifs, low-complexity regions (SEG), nuclear localization signals, regions lacking regular structure (NORS) and predictions of secondary structure, solvent accessibility, globular regions, transmembrane helices, coiled-coil regions, structural switch regions, disulfide-bonds, sub-cellular localization and functional annotations. Upon request fold recognition by prediction-based threading, CHOP domain assignments, predictions of transmembrane strands and inter-residue contacts are also available. For all services, users can submit their query either by electronic mail or interactively via the World Wide Web.
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            Sister-chromatid separation at anaphase onset is promoted by cleavage of the cohesin subunit Scc1.

            Cohesion between sister chromatids is established during DNA replication and depends on a multiprotein complex called cohesin. Attachment of sister kinetochores to the mitotic spindle during mitosis generates forces that would immediately split sister chromatids were it not opposed by cohesion. Cohesion is essential for the alignment of chromosomes in metaphase but must be abolished for sister separation to start during anaphase. In the budding yeast Saccharomyces cerevisiae, loss of sister-chromatid cohesion depends on a separating protein (separin) called Esp1 and is accompanied by dissociation from the chromosomes of the cohesion subunit Scc1. Here we show that Esp1 causes the dissociation of Scc1 from chromosomes by stimulating its cleavage by proteolysis. A mutant Scc1 is described that is resistant to Esp1-dependent cleavage and which blocks both sister-chromatid separation and the dissociation of Scc1 from chromosomes. The evolutionary conservation of separins indicates that the proteolytic cleavage of cohesion proteins might be a general mechanism for triggering anaphase.
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              Cleavage of cohesin by the CD clan protease separin triggers anaphase in yeast.

              In eukaryotic cells, replicated DNA strands remain physically connected until their segregation to opposite poles of the cell during anaphase. This "sister chromatid cohesion" is essential for the alignment of chromosomes on the mitotic spindle during metaphase. Cohesion depends on the multisubunit cohesin complex, which possibly forms the physical bridges connecting sisters. Proteolytic cleavage of cohesin's Sccl subunit at the metaphase to anaphase transition is essential for sister chromatid separation and depends on a conserved protein called separin. We show here that separin is a cysteine protease related to caspases that alone can cleave Sccl in vitro. Cleavage of Sccl in metaphase arrested cells is sufficient to trigger the separation of sister chromatids and their segregation to opposite cell poles.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2013
                12 July 2013
                : 8
                : 7
                : e69458
                Affiliations
                [1 ]Texas Children' Cancer Center, Department of Pediatric Hematology/Oncology, Baylor College of Medicine, Houston, Texas, United States of America
                [2 ]Department of Biochemistry and Cell Biology, Rice University, Houston, Texas, United States of America
                [3 ]Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas, United States of America
                Virginia Tech, United States of America
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Performed the experiments: YJ NZ QM. Analyzed the data: NZ YJ BD YJT DP. Contributed reagents/materials/analysis tools: BD. Wrote the paper: NZ YJ. Conceived the project: DP. Designed the experiments: YJ NZ YJT DP. Contributed to the manuscript: BD YJT DP.

                Article
                PONE-D-11-08013
                10.1371/journal.pone.0069458
                3709894
                23874961
                9dc7cdf0-c2bc-4ee1-95c5-8b6e4c37e97c
                Copyright @ 2013

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 6 May 2011
                : 11 June 2013
                Page count
                Pages: 13
                Funding
                This study was supported by award 1RO1 CA109330 from the National Cancer Institute to D. Pati, the 2008 Virginia and L.E. Simmons Family Foundation Collaborative research award ( https://www.collaborativeresearchfund.org/) to D. Pati and Y. J. Tao, and the Welch Foundation award R4079 to Y. J. Tao. The Ultrascan development is supported by National Institutes of Health grant RO1 RR022200 to B. Demeler. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology
                Biochemistry
                Molecular Cell Biology
                Chromosome Biology
                Mitosis
                Cell Division
                Proteomics
                Protein Interactions

                Uncategorized
                Uncategorized

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