👤 K Nasmyth

When yeast cells reach a critical size in late G1 they simultaneously start budding, initiate DNA synthesis, and activate transcription of a set of genes that includes G1 cyclins CLN1, CLN2, and many DNA synthesis genes. Cell cycle-regulated expression of CLN1, CLN2 genes is attributable to the heteromeric transcription factor complex SBF. SBF is composed of Swi4 and Swi6 and binds to the promoters of CLN1 and CLN2. Different cyclin-Cdc28 complexes have different effects on late G1-specific transcription. Activation of transcription at the G1/S boundary requires Cdc28 and one of the G1 cyclins Cln1-Cln3, whereas repression of SBF-regulated genes in G2 requires the association of Cdc28 with G2-specific cyclins Clb1-Clb4. Using in vivo genomic footprinting, we show that SBF (Swi4/Swi6) binding to SCB elements (Swi4/Swi6 cell cycle box) in the CLN2 promoter is cell cycle regulated. SBF binds to the promoter prior to the activation of transcription in late G1, suggesting that Cln/Cdc28 kinase regulates the ability of previously bound SBF to activate transcription. In contrast, SBF dissociates from the CLN2 promoter when transcription is repressed during G2 and M phases, suggesting that Clb1-Clb4 repress SBF activity by inhibiting its DNA-binding activity. Switching transcription on and off by different mechanisms could be important to ensure that Clns are activated only once per cell cycle and could be a conserved feature of cell cycle-regulated transcription. Show less

In budding yeast G1 cells increase in cell mass until they reach a critical cell size, at which point (called Start) they enter S phase, bud and duplicate their spindle pole bodies. Activation of the Cdc28 protein kinase by G1-specific cyclins Cln1, Cln2 or Cln3 is necessary for all three Start events. Transcriptional activation of CLN1 and CLN2 by SBF and MBF transcription factors also requires an active Cln-Cdc28 kinase and it has therefore been proposed that the sudden accumulation of CLN1 and CLN2 transcripts during late G1 occurs via a positive feedback loop. We report that whereas Cln1 and Cln2 are required for the punctual execution of most, if not all, other Start-related events, they are not required for the punctual activation of SBF- or MBF-driven transcription. Cln3, on the other hand, is essential. By turning off cyclin B proteolysis and turning on proteolysis of the cyclin B-Cdc28 inhibitor p40SIC1, Cln1 and Cln2 kinases activate cyclin B-Cdc28 kinases and thereby trigger S phase. Thus the accumulation of Cln1 and Cln2 kinases which starts the yeast cell cycle is set in motion by prior activation of SBF- and MBF-mediated transcription by Cln3-Cdc28 kinase. This dissection of regulatory events during late G1 demands a rethinking of Start as a single process that causes cells to be committed to the mitotic cell cycle. Show less

Cyclin-dependent protein kinases have a central role in cell cycle regulation. In Saccharomyces cerevisiae, Cdc28 kinase and the G1 cyclins Cln1, 2 and 3 are required for DNA replication, duplication of the spindle pole body and bud emergence. These three independent processes occur simultaneously in late G1 when the cells reach a critical size, an event known as Start. At least one of the three Clns is necessary for Start. Cln3 is believed to activate Cln1 and Cln2, which can then stimulate their own accumulation by means of a positive feedback loop. They (or Cln3) also activate another pair of cyclins, Clb5 and 6, involved in initiating S phase. Little is known about the role of Clns in spindle pole body duplication and budding. We report here the isolation of a gene (CLA2/BUD2/ERC25) that codes for a homologue of mammalian Ras-associated GTPase-activating proteins (GAPs) and is necessary for budding only in cln1 cln2 cells. This suggests that Cln1 and Cln2 may have a direct role in bud formation. Show less

The functions of the Cdc28 protein kinase in DNA replication and mitosis in Saccharomyces cerevisiae are thought to be determined by the type of cyclin subunit with which it is associated. G1-specific cyclins encoded by CLN1, CLN2, and CLN3 are required for entry into the cell cycle (Start) and thereby for S phase, whereas G2-specific B-type cyclins encoded by CLB1, CLB2, CLB3, and CLB4 are required for mitosis. We describe a new family of B-type cyclin genes, CLB5 and CLB6, whose transcripts appear in late G1 along with those of CLN1, CLN2, and many genes required for DNA replication. Deletion of CLB6 has little or no effect, but deletion of CLB5 greatly extends S phase, and deleting both genes prevents the timely initiation of DNA replication. Transcription of CLB5 and CLB6 is normally dependent on Cln activity, but ectopic CLB5 expression allows cells to proliferate in the absence of Cln cyclins. Thus, the kinase activity associated with Clb5/6 and not with Cln cyclins may be responsible for S-phase entry. Clb5 also has a function, along with Clb3 and Clb4, in the formation of mitotic spindles. Our observation that CLB5 is involved in the initiation of both S phase and mitosis suggests that a single primordial B-type cyclin might have been sufficient for regulating the cell cycle of the common ancestor of many, if not all, eukaryotes. Show less

Entry into the mitotic cycle (START) requires a protein kinase encoded by the CDC28 gene and one of three redundant G1-specific cyclins encoded by CLN1, -2, and -3. SWI4 and SWI6 are transcription factors required for the START-dependent activation of the HO endonuclease gene. They also fulfill an overlapping but essential role for cell division since cells deleted for both genes are inviable. We show that the essential role of SWI4 and SWI6 is to ensure the activity of G1-specific cyclin genes. SWI4 and SWI6 appear necessary for the transcription of CLN1 and CLN2, but not for that of CLN3. CLN3 function is, however, also dependent on SWI4 and SWI6. Show less

Yeast cells become committed to the mitotic cell cycle at a stage during G1 called Start. To enter Start, cells must grow to a critical size. They also require the CDC28 protein kinase and at least one of three G1-specific cyclins encoded by CLN1, 2, and 3. It is thought that Start is triggered by the accumulation of G1 cyclins that bind to the CDC28 kinase and activate it. So what determines the accumulation of G1 cyclins? For CLN1 and CLN2, transcriptional activation could be involved because their RNAs appear transiently during the cell cycle as cells undergo Start. Here we report that the appearance of CLN1 and CLN2 RNAs depends on an active CDC28 kinase and is stimulated by CLN3 activity. We propose that CDC28 kinase activity due to CLN1 and CLN2 proteins arises through a positive feedback loop which allows CLN proteins to promote their own synthesis. Show less