Separase and Roads to Disengage Sister Chromatids during Anaphase

Abstract

Receiving complete and undamaged genetic information is vital for the survival of daughter cells after chromosome segregation. The most critical steps in this process are accurate DNA replication during S phase and a faithful chromosome segregation during anaphase. Any errors in DNA replication or chromosome segregation have dire consequences, since cells arising after division might have either changed or incomplete genetic information. Accurate chromosome segregation during anaphase requires a protein complex called cohesin, which holds together sister chromatids. This complex unifies sister chromatids from their synthesis during S phase, until separation in anaphase. Upon entry into mitosis, the spindle apparatus is assembled, which eventually engages kinetochores of all chromosomes. Additionally, when kinetochores of sister chromatids assume amphitelic attachment to the spindle microtubules, cells are finally ready for the separation of sister chromatids. This is achieved by the enzymatic cleavage of cohesin subunits Scc1 or Rec8 by an enzyme called Separase. After cohesin cleavage, sister chromatids remain attached to the spindle apparatus and their poleward movement on the spindle is initiated. The removal of cohesion between sister chromatids is an irreversible step and therefore it must be synchronized with assembly of the spindle apparatus, since precocious separation of sister chromatids might lead into aneuploidy and tumorigenesis. In this review, we focus on recent discoveries concerning the regulation of Separase activity during the cell cycle.

Keywords: CDK1; Cyclin B1; Mad2; Sgo2; aneuploidy; chromosome division; cohesin; securin; segregation errors; separase.

Figure 1

Two-step cohesin removal in mitosis. The prophase pathway removes the cohesin complex from chromosome arms [29]. Inactivation and destruction of Sororin by APC/C Cdh1 activates Wapl protein and leads to loss of cohesion at chromosome arms [21,31]. The PP2A and Shugoshin protect the centromeric cohesin [32,33,34] by counteracting phosphorylation caused by Plk1, CDK1 and Aurora B [35,36,37]). The separation of sister chromatids during anaphase requires the cleavage of remaining cohesion by Separase.

Figure 2

Structural motifs of Separase. (A) Human separase consists of super-helical N-terminal and C-terminal domains, which is also called proteolytic domain [50]. The N-terminus consists of a HEAT-repeat domain [50] and a TPR-like domain [49]. The C-terminus contains pseudo protease domain (PPD) and active protease domain (APD) [50,58]. (B) The N-terminus contains securin binding site [50] and Pin1 binding domain [56], whose binding depends on Ser1153 residue. The mutation of Ser1126 prevents inhibition of Separase by Cdk1/cyclin B [59]. The unstructured region of separase also contains a cell division cycle 6 (Cdc6)-like domain [60], with two important phosphorylation sites (Thr1346 and Ser1399) [61]. PP2A binds to a 55 amino acids motif (residues 1419–1473). The unstructured domain also contains three Separase auto-cleavage sites (residues Arg1486, Arg1506 and Arg1535 in human Separase) [62,63,64]. The APD contains two conserved residues, a histidine (His2003) and a cysteine (Cys2029) [41]. The function of individual domains is discussed further in the text.

Figure 3

Schematic illustration of mechanisms of Separase inhibition. (A) Separase activation is controlled by APC/C activity [57], whose activation is controlled by SAC [92]. After ubiquitination and destruction of Securin and Cyclin B, and disassembly of Sgo2/Mad 2 complex, the Separase is activated and subsequently the sister chromatids separate in anaphase. (B,C) After activation of APC/C, Securin and Cyclin B ubiquitination is commenced and simultaneously Pin1 isomerase changes the Separase conformation [56]. (D) The recently discovered third pathway involves the Sgo2/Mad2 complex, and upon its removal from Separase, after prometaphase and metaphase, the Separase is activated [81]. During the metaphase to anaphase transition, Separase shows abrupt activation and cleaves cohesion. Consequently, the separase undergoes autocleavage [63,64,72].

Figure 4

Separase key roles during cell cycle. The proteolytic activity of Separase is required for cohesion and Meikin cleavage and also for autocleavage and centrosome disengagement. For some other roles of Separase, the proteolytic activity is not required, partially for FEAR network in yeasts, although the cleavage of the kinetochore-associated protein Slk19 during anaphase requires Separase proteolytic activity. In vertebrates, Separase is involved in CDK1 inactivation during mitotic exit by formation of a complex with Cyclin B/CDK1 (MPF).