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 (L385, F389, and T390) 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.
References
[1]
Hagstrom KA, Meyer BJ (2003) Condensin and cohesin: more than chromosome compactor and glue. Nat Rev Genet 4: 520–534.
[2]
Losada A, Yokochi T, Kobayashi R, Hirano T (2000) Identification and characterization of SA/Scc3p subunits in the Xenopus and human cohesin complexes. J Cell Biol 150: 405–416.
[3]
Uhlmann F (2001) Chromosome cohesion and segregation in mitosis and meiosis. Curr Opin Cell Biol 13: 754–761.
[4]
Anderson DE, Losada A, Erickson HP, Hirano T (2002) Condensin and cohesin display different arm conformations with characteristic hinge angles. J Cell Biol 156: 419–424.
[5]
Haering CH, Lowe J, Hochwagen A, Nasmyth K (2002) Molecular architecture of SMC proteins and the yeast cohesin complex. Mol Cell 9: 773–788.
[6]
Lowe J, Cordell SC, van den EF (2001) Crystal structure of the SMC head domain: an ABC ATPase with 900 residues antiparallel coiled-coil inserted. J Mol Biol 306: 25–35.
[7]
Schmitz J, Watrin E, Lenart P, Mechtler K, Peters JM (2007) Sororin is required for stable binding of cohesin to chromatin and for sister chromatid cohesion in interphase. Curr Biol 17: 630–636.
[8]
Rankin S, Ayad NG, Kirschner MW (2005) Sororin, a substrate of the anaphase-promoting complex, is required for sister chromatid cohesion in vertebrates. Mol Cell 18: 185–200.
[9]
Panizza S, Tanaka T, Hochwagen A, Eisenhaber F, Nasmyth K (2000) Pds5 cooperates with cohesin in maintaining sister chromatid cohesion. Curr Biol 10: 1557–1564.
[10]
Nishiyama T, Ladurner R, Schmitz J, Kreidl E, Schleiffer A, et al. (2010) Sororin mediates sister chromatid cohesion by antagonizing Wapl. Cell 143: 737–749.
[11]
Losada A, Yokochi T, Hirano T (2005) Functional contribution of Pds5 to cohesin-mediated cohesion in human cells and Xenopus egg extracts. J Cell Sci 118: 2133–2141.
[12]
Zhang N, Panigrahi AK, Mao Q, Pati D (2011) Interaction of Sororin with polo-like kinase 1 mediates the resolution of chromosomal arm cohesion. J Biol Chem 286: 41826–41837.
[13]
Zhang N, Pati D (2012) Sororin is a master regulator of sister chromatid cohesion and separation. Cell Cycle 11: 2073–2083.
[14]
Panigrahi AK, Zhang N, Mao Q, Pati D (2011) Calpain-1 cleaves rad21 to promote sister chromatid separation. Mol Cell Biol 31: 4335–4347.
[15]
Haering CH, Nasmyth K (2003) Building and breaking bridges between sister chromatids. Bioessays 25: 1178–1191.
[16]
Hauf S, Waizenegger IC, Peters JM (2001) Cohesin cleavage by separase required for anaphase and cytokinesis in human cells. Science 293: 1320–1323.
[17]
Zhang N, Kuznetsov SG, Sharan SK, Li K, Rao PH, et al. (2008) A handcuff model for the cohesin complex. J Cell Biol 183: 1019–1031.
[18]
Zhang N, Pati D (2009) Handcuff for sisters: a new model for sister chromatid cohesion. Cell Cycle 8: 399–402.
[19]
Panigrahi AK, Pati D (2009) Road to the crossroads of life and death: Linking sister chromatid cohesion and separation to aneuploidy, apoptosis and cancer. Crit Rev Oncol Hematol.
[20]
Solomon DA, Kim T, az-Martinez LA, Fair J, Elkahloun AG, et al. (2011) Mutational inactivation of STAG2 causes aneuploidy in human cancer. Science 333: 1039–1043.
[21]
Canudas S, Smith S (2009) Differential regulation of telomere and centromere cohesion by the Scc3 homologues SA1 and SA2, respectively, in human cells. J Cell Biol 187: 165–173.
[22]
Shintomi K, Hirano T (2009) Releasing cohesin from chromosome arms in early mitosis: opposing actions of Wapl-Pds5 and Sgo1. Genes Dev 23: 2224–2236.
[23]
Rost B, Yachdav G, Liu J (2004) The PredictProtein server. Nucleic Acids Res 32: W321–W326.
[24]
Linding R, Russell RB, Neduva V, Gibson TJ (2003) GlobPlot: Exploring protein sequences for globularity and disorder. Nucleic Acids Res 31: 3701–3708.
[25]
Arumugam P, Gruber S, Tanaka K, Haering CH, Mechtler K, et al. (2003) ATP hydrolysis is required for cohesin's association with chromosomes. Curr Biol 13: 1941–1953.
[26]
Gimenez-Abian JF, Sumara I, Hirota T, Hauf S, Gerlich D, et al. (2004) Regulation of sister chromatid cohesion between chromosome arms. Curr Biol 14: 1187–1193.
[27]
Sumara I, Vorlaufer E, Gieffers C, Peters BH, Peters JM (2000) Characterization of vertebrate cohesin complexes and their regulation in prophase. J Cell Biol 151: 749–762.
[28]
Sumara I, Vorlaufer E, Stukenberg PT, Kelm O, Redemann N, et al. (2002) The dissociation of cohesin from chromosomes in prophase is regulated by Polo-like kinase. Mol Cell 9: 515–525.
[29]
Hauf S, Roitinger E, Koch B, Dittrich CM, Mechtler K, et al. (2005) Dissociation of Cohesin from Chromosome Arms and Loss of Arm Cohesion during Early Mitosis Depends on Phosphorylation of SA2. PLoS Biol 3: e69.
[30]
Pezzi N, Prieto I, Kremer L, Perez Jurado LA, Valero C, et al. (2000) STAG3, a novel gene encoding a protein involved in meiotic chromosome pairing and location of STAG3-related genes flanking the Williams-Beuren syndrome deletion. FASEB J 14: 581–592.
[31]
Uhlmann F, Lottspeich F, Nasmyth K (1999) Sister-chromatid separation at anaphase onset is promoted by cleavage of the cohesin subunit Scc1. Nature 400: 37–42.
[32]
Uhlmann F, Wernic D, Poupart MA, Koonin EV, Nasmyth K (2000) Cleavage of cohesin by the CD clan protease separin triggers anaphase in yeast. Cell 103: 375–386.
[33]
Kueng S, Hegemann B, Peters BH, Lipp JJ, Schleiffer A, et al. (2006) Wapl controls the dynamic association of cohesin with chromatin. Cell 127: 955–967.
[34]
Rowland BD, Roig MB, Nishino T, Kurze A, Uluocak P, et al. (2009) Building sister chromatid cohesion: smc3 acetylation counteracts an antiestablishment activity. Mol Cell 33: 763–774.
[35]
Pati D, Zhang N, Plon SE (2002) Linking sister chromatid cohesion and apoptosis: role of Rad21. Mol Cell Biol 22: 8267–8277.
[36]
Freshney RI (2000) Culture of animal cells: a manual of basic technique. New York:Wiley-Liss, Inc.
[37]
Demeler B (2005) Ultrascan: a comprehensive data analysis software package for analytical ultracentrifugation experiments. In: Scott DJ, Harding SE, Rowe Aj, editors. In Modern Ananlytical Ultracentrifugation: Techniques and Methods. London: Royal Society of Chemistry, pp. 210–229.
[38]
Brookes E, Cao W, Demeler B (2010) A two-dimensional spectrum analysis for sedimentation velocity experiments of mixtures with heterogeneity in molecular weight and shape. Eur Biophys J 39: 405–414.
[39]
Demeler B, van Holde KE (2004) Sedimentation velocity analysis of highly heterogeneous systems. Anal Biochem 335: 279–288.
