Identification and Characterization of Sa/Scc3p Subunits in the Xenopus and Human Cohesin Complexes

Abstract

A multisubunit protein complex, termed cohesin, plays an essential role in sister chromatid cohesion in yeast and in Xenopus laevis cell-free extracts. We report here that two distinct cohesin complexes exist in Xenopus egg extracts. A 14S complex (x-cohesinSA1) contains XSMC1, XSMC3, XRAD21, and a newly identified subunit, XSA1. In a second 12.5S complex (x-cohesinSA2), XSMC1, XSMC3, and XRAD21 associate with a different subunit, XSA2. Both XSA1 and XSA2 belong to the SA family of mammalian proteins and exhibit similarity to Scc3p, a recently identified component of yeast cohesin. In Xenopus egg extracts, x-cohesinSA1 is predominant, whereas x-cohesinSA2 constitutes only a very minor population. Human cells have a similar pair of cohesin complexes, but the SA2-type is the dominant form in somatic tissue culture cells. Immunolocalization experiments suggest that chromatin association of cohesinSA1 and cohesinSA2 may be differentially regulated. Dissociation of x-cohesinSA1 from chromatin correlates with phosphorylation of XSA1 in the cell-free extracts. Purified cdc2-cyclin B can phosphorylate XSA1 in vitro and reduce the ability of x-cohesinSA1 to bind to DNA or chromatin. These results shed light on the mechanism by which sister chromatid cohesion is partially dissolved in early mitosis, far before the onset of anaphase, in vertebrate cells.

Shugoshin regulates cohesion by driving relocalization of PP2A in Xenopus extracts

“…Rabbit polyclonal sera against Xenopus CENP-A were obtained using a synthetic peptide as immunogen (MRPGSTPPSRRKSRPPRRVC). Other antibodies used in this study were as follows: anti-SMC1, anti-SMC3, and anti-Scc1 (Losada et al 1998); anti-SA1 (Losada et al 2000); anti-XCAP-G (Hirano et al 1997); anti-XINCENP and anti-XAurora B (MacCallum et al 2002); and anti-Plx1 . A mouse monoclonal antibody against the catalytic subunit of PP2A (clone 1D6) was purchased from Upstate.…”

“…Soluble cohesin was purified from HeLa cell nuclear extracts with anti-SMC3 as described (Losada et al 2000) and was first incubated with a Polo kinase fraction (or an Aurora B kinase fraction as control) immunoprecipitated from a CSF extract on Dynabeads protein A. After 20 min, the Polo-incubated cohesin was incubated with a Sgo fraction immunoprecipitated from a CSF extract in the presence or absence of 1 μM okadaic acid.…”

Cohesin Associates with Spindle Poles in a Mitosis-specific Manner and Functions in Spindle Assembly in Vertebrate Cells

“…Cohesin is composed of two structural maintenance of chromosomes family proteins, SMC1 and SMC3, which form a stable heterodimer that associates with the two non-SMC components Rad21 (Scc1/Mcd1) and SA protein (Scc3) (Toth et al, 1999;Losada et al, 2000;Sumara et al, 2000;Tomonaga et al, 2000). In vertebrates, two mitotic SA protein homologues, SA1 and SA2, were found to form distinct cohesin complexes (Losada et al, 2000).In metazoans, cohesin is loaded onto chromosomes in interphase to establish sister chromatid cohesion. During G2/prophase, the majority of cohesin dissociates from chromosomes as an intact complex in a Polo-like kinase-reguThis article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E08 -04 -0419) on January 7, 2009.…”

“…During G2/prophase, the majority of cohesin dissociates from chromosomes as an intact complex in a Polo-like kinase-reguThis article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E08 -04 -0419) on January 7, 2009. Address correspondence to: Kyoko Yokomori (kyokomor@uci.edu).Abbreviations used: histone H3 lysine 9 methylation (H3K9me), histone methyltransferase (HMTase), microtubules (MTs), nuclear mitotic apparatus protein (NuMA), polo-like kinase 1 (plk1), structural maintenance of chromosomes (SMC).© 2009 by The American Society for Cell Biology 1289 lated manner (Losada et al, 2000;Sumara et al, 2000;Gregson et al, 2001). Only a small amount of cohesin (ϳ5%) remains at the chromosome-pairing domain to mediate sister chromatid cohesion, which is eventually terminated by the cleavage of Rad21 at the onset of anaphase leading to chromosome segregation (Waizenegger et al, 2000;Hoque and Ishikawa, 2001;Gimenez-Abian et al, 2004).…”

Familial skewed X-chromosome inactivation linked to a component of the cohesin complex, SA2

“…SA2 is a core component of the ringlike cohesin complex. [22][23][24] Cohesin is best known for its role in sister chromatid cohesion before and during mitosis, 23,24 however, other functions have recently been attributed to cohesin, 25 including nuclear re-organisation, 26 S-phase check point activation, DNA repair 27,28 and gene regulation. [29][30][31] SA2 itself has been shown to have transcriptional co-activator function.…”