The Transcription Factor Swi5 Regulates Expression of the Cyclin Kinase Inhibitor p40^SIC1

Abstract

DNA replication in budding yeast cells depends on the activation of the Cdc28 kinase (Cdk1 of Saccharomyces cerevisiae) associated with B-type cyclins Clb1 to Clb6. Activation of the kinase depends on proteolysis of the Cdk inhibitor p40SIC1 in late G 1 , which is mediated by the ubiquitin-conjugating enzyme Cdc34 and two other proteins, Cdc4 and Cdc53. Inactivation of any one of these three proteins prevents p40 SIC1 degradation and causes cells to arrest in G 1 with active Cln kinases but no Clb-associated Cdc28 kinase activity. Deletion of SIC1 allows these mutants to replicate. p40 SIC1 disappears at the G 1 /S transition and reappears only after nuclear division. Cell cycle-regulated proteolysis seems largely responsible for this pattern, but transcriptional control could also contribute; SIC1 RNA accumulates to high levels as cells exit M phase. To identify additional factors necessary for the inhibition of the Cdk1/Cdc28 kinase in G 1 , we isolated mutants that can replicate DNA in the absence of Cdc4 function. Mutations in three loci (SIC1, SWI5, and RIC3) were identified. We have shown that high SIC1 transcript levels at late M phase depend on Swi5. Swi5 accumulates in the cytoplasm during S, G 2 , and M phases of the cell cycle but enters the nuclei at late anaphase. Our data suggest that cell cycle-regulated nuclear accumulation of Swi5 is responsible for the burst of SIC1 transcription at the end of anaphase. This transcriptional control may be important for inactivation of the Clb/Cdk1 kinase in G 2 /M transition and during the subsequent G 1 period.DNA replication in budding yeast cells depends on the activation of Cdk1/Cdc28 kinase associated with six different B-type cyclins, Clb1 to Clb6. Clb-associated Cdk1 kinase is regulated by transcription of cyclin genes, by proteolysis of cyclin proteins, and by accumulation of inhibitory proteins. Due largely to the onset of CLB5 and CLB6 transcription in late G 1 (CLB1 to CLB4 RNAs do not appear until later), DNA replication is normally triggered by the appearance of active Clb5 or Clb6/Cdc28 kinases (10, 26). In the absence of Clb5 and Clb6, Clb1 to Clb4 trigger replication. Normally, DNA replication is simultaneous with bud formation, but in the clb5 clb6 mutant, DNA replication is delayed relative to bud emergence (26). Clb/Cdc28 kinase activity is also regulated by changes in cyclin stability (1) and by binding of the inhibitory protein p40 SIC1 . p40 SIC1 does not inhibit Cdc28 kinase associated with Cln cyclins which also appear in late G 1 and are necessary for activating CLB gene transcription, turning off Clb proteolysis, and triggering p40 SIC1 proteolysis (19,22,25). p40 SIC1 protein accumulates to high levels in G 1 , disappears at the G 1 /S transition, and does not reaccumulate until cells complete nuclear division and reenter G 1 . To understand how p40 SIC1 regulates the activity of cyclin B/Cdc28 kinases, we need to know how p40 SIC1 accumulation is regulated. It has been demonstrated that the appearance of Cln1 and Cln2/ Cdc28 kinases ...

Swi5 Controls a Novel Wave of Cyclin Synthesis in Late Mitosis

“…Two transcription factors, Swi5 and Ace2, are active in cell cycle regulation of gene expression in late M/early G1. We and others have recently shown that Swi5 does indeed regulate SIC1 (Knapp et al, 1996;Toyn et al, 1997).…”

“…To identify further genes regulated by Swi5, which might have a role in late M phase, we searched the EMBL database for genes containing putative Swi5 binding sites in their promoter. The motif used in our search was ACCAGC (Knapp et al, 1996). Interestingly, two putative binding sites were found in the promoter of PCL9 (Figure 1), which encodes a protein showing significant homology to Pcl2, a cyclin-like protein interacting with Pho85 (Measday et al, 1997).…”

“…The identification of further genes regulated by Swi5 might therefore reveal novel genes with roles in late M phase. Accordingly, we carried out a computer search for genes with Swi5-binding sites (Knapp et al, 1996) in their promoters. Surprisingly, we identified PCL9, a gene with high homology to Pcl2, a cyclin-like protein that associates with the cyclin-dependent protein kinase (Cdk) Pho85.…”

A role for the Pcl9‐Pho85 cyclin–cdk complex at the M/G1 boundary in Saccharomyces cerevisiae

“…The ASH1 and HO genes are both involved in regulating the initiation of mating type switching; the timing of the mating type switching event is precisely controlled and restricted to late G 1 phase before the initiation of DNA replication (Amon, 1996;Babola et al, 1996). The CDC6 and SIC1 genes are Swi5 targets involved in the proper timing and initiation of DNA replication (Piatti et al, 1995;Knapp et al, 1996). SIC1 encodes an inhibitor of Cdc28 kinase complexes required for S phase (Schwob et al, 1994), while CDC6 is required for the establishment of the so-called prereplicative complex during G 1 phase that is a prerequisite for the initiation of DNA replication later in the cell cycle (reviewed in Stillman, 1996).…”

“…1A). Furthermore, examination of the sequences upstream of PCL9 identified two putative binding sites for Swi5 located at ¹278 and ¹320 relative to the presumptive initiator codon (Stillman et al, 1988;Knapp et al, 1996).…”