Cell cycle-specific cleavage of Scc2 regulates its cohesin deposition activity

Abstract

Sister chromatid cohesion (SCC), efficient DNA repair, and the regulation of some metazoan genes require the association of cohesins with chromosomes. Cohesins are deposited by a conserved heterodimeric loading complex composed of the Scc2 and Scc4 proteins in Saccharomyces cerevisiae, but how the Scc2/Scc4 deposition complex regulates the spatiotemporal association of cohesin with chromosomes is not understood. We examined Scc2 chromatin association during the cell division cycle and found that the affinity of Scc2 for chromatin increases biphasically during the cell cycle, increasing first transiently in late G 1 phase and then again later in G 2 /M. Inactivation of Scc2 following DNA replication reduces cellular viability, suggesting that this post S-phase increase in Scc2 chromatin binding affinity is biologically relevant. Interestingly, high and low Scc2 chromatin binding levels correlate strongly with the presence of full-length or amino-terminally cleaved forms of Scc2, respectively, and the appearance of the cleaved Scc2 species is promoted in vitro either by treatment with specific cell cycle-staged cellular extracts or by dephosphorylation. Importantly, Scc2 cleavage eliminates Scc2-Scc4 physical interactions, and an scc2 truncation mutant that mimics in vivo Scc2 cleavage is defective for cohesin deposition. These observations suggest a previously unidentified mechanism for the spatiotemporal regulation of cohesin association with chromosomes through cell cycle regulation of Scc2 cohesin deposition activity by Scc2 dephosphorylation and cleavage.M ultisubunit, ring-shaped cohesin complexes play key roles in chromosome morphogenesis that are required for faithful chromosome transmission to daughter cells. Newly replicated sister chromatids become tethered together by cohesins during S phase, which promotes chromosome biorientation on mitotic spindles (1). Cohesins also mediate efficient DNA double-strand break repair by homologous recombination (2, 3) and the formation or stabilization of chromatin loops that affect various nuclear processes, such as gene expression and Ig gene rearrangements (reviewed in refs. 4 and 5). Altered gene expression resulting from defective cohesinmediated chromatin looping is likely responsible for the pathogenesis of Cornelia de Lange Syndrome (CdLS), a dominantly inherited human developmental disorder (6).Sister chromatid cohesion (Scc) proteins form a heterodimeric cohesin deposition complex, but the complex's activity in deposition is not understood (7). Cohesins copurify with Scc2/Scc4, suggesting that Scc2/Scc4 plays a direct role in deposition (8-11). In the absence of either loader complex subunit, cohesin rings assemble, but fail to be deposited (7,12,13). ATP hydrolysis by cohesin's structural maintenance of chromosome (SMC) subunits is required for cohesin loading, and deposition is inhibited when SMC hinge domains, which mediate Smc1/3 interactions within cohesin, are artificially tethered (8,14,15). Thus, Scc2/ Scc4 may activate cohesin's ATPase activity or f...

Structural Studies Reveal the Functional Modularity of the Scc2-Scc4 Cohesin Loader

“…We also note that the extreme flexibility of the head domain provides an explanation for the proteolytic susceptibility of the Scc2 N terminus previously reported (Woodman et al, 2014) as well as our own limited proteolysis results. Interestingly, we identified several potential Cdk sites [S/T]PX[K/R] around the area susceptible to proteolysis ( Figure S2).…”

“…These two binding sites together form an overall buried interaction area of 6,082 Å 2 . The extended contact between the two proteins promotes high-affinity binding and explains why Scc2 and Scc4 are always observed as a constitutively stable complex (Ciosk et al, 2000;Takahashi et al, 2008;Woodman et al, 2014). The Scc2 N terminus is likely disordered in the absence of Scc4 binding, with the secondary structure elements observed ( Figure 1B) only arising on complex formation.…”

Decision letter: Structural evidence for Scc4-dependent localization of cohesin loading

“…Residues 10-50 of Scc2 snake along the continuous inner cavity of Scc4 and emerge at both ends, with the N-terminus of Scc2 close to the C-terminus of Scc4. Examples of similar TPR-peptide interfaces include the interaction of the kinesin light chain with cargo peptides (Figure 2 (Woodman et al, 2014). (B) Negatively stained Scc2/4 visualized by electron microscopy.…”

Phosphorylation of the Scc2 cohesin deposition complex subunit regulates chromosome condensation through cohesin integrity

“…Although Scc2 and Scc4 protein levels remain constant through the cell division cycle, the chromatin association of Scc2/Scc4 is biphasic, reaching its highest levels in post–S-phase cells ( Kogut et al. , 2009 ; Woodman et al. , 2014 ), suggesting involvement of unknown regulatory mechanisms.…”

“…Furthermore, Scc2 inactivation in postreplicative cells, the period when Scc2/Scc4 achieves its maximal chromatin association levels, results in decreased cell viability ( Furuya et al. , 1998 ; Woodman et al. , 2014 ).…”