Whi5 and Stb1 define redundant pathways through which the G1 cyclin Cln3 promotes cell division

In the budding yeast S. cerevisiae, commitment to cell division, Start, is promoted by a trio of G1 cyclins, Cln1, Cln2, and Cln3, that activate the CDK kinase Cdc28. The active kinases somehow activate two transcription factors, SBF and MBF, leading to induction of about 100 genes for budding, DNA synthesis, and other early cell cycle processes. Activation of the transcription factors is opposed by a repressive protein called Whi5, and also by a second repressive protein called Stb1. Both Whi5 and Stb1 contain many potential sites for phosphorylation by CDK kinase, and is thought that relief of transcriptional repression involves the phosphorylation of Whi5 and Stb1 by CDK. Phosphorylation site mutants have been studied for Whi5, but not for Stb1. Here, we create phosphorylation site mutants of Stb1, and combine them with site mutants of Whi5. We find that the G1 cyclin Cln3 activates cell cycle transcription effectively when at least one of these proteins has its phosphorylation sites. However, when both Whi5 and Stb1 simultaneously lack all consensus phosphorylation sites, Cln3 is unable, or almost unable, to induce any gene expression, or any advancement of Start. Thus the G1 cyclin signaling pathway to Start has a requirement for CDK phosphorylation sites on either Whi5 or Stb1.

A CDK-regulated chromatin segregase promoting chromosome replication

The replication of chromosomes during S phase is critical for cellular and organismal function. Replicative stress can result in genome instability, which is a major driver of cancer. Yet how chromatin is made accessible during eukaryotic DNA synthesis is poorly understood. Here, we report the characterization of a chromatin remodeling enzyme—Yta7—entirely distinct from classical SNF2-ATPase family remodelers. Yta7 is a AAA+ -ATPase that assembles into ~1 MDa hexameric complexes capable of segregating histones from DNA. The Yta7 chromatin segregase promotes chromosome replication both in vivo and in vitro. Biochemical reconstitution experiments using purified proteins revealed that the enzymatic activity of Yta7 is regulated by S phase-forms of Cyclin-Dependent Kinase (S-CDK). S-CDK phosphorylation stimulates ATP hydrolysis by Yta7, promoting nucleosome disassembly and chromatin replication. Our results present a mechanism for how cells orchestrate chromatin dynamics in co-ordination with the cell cycle machinery to promote genome duplication during S phase.

A CDK-regulated chromatin segregase promoting chromosome replication

The replication of chromosomes during S phase is critical for cellular and organismal function. Replicative stress can result in genome instability, which is a major driver of cancer. Yet how chromatin is made accessible during eukaryotic DNA synthesis is poorly understood.Here, we report the identification of a novel class of chromatin remodeling enzyme, entirely distinct from classical SNF2-ATPase family remodelers. Yta7 is a AAA+-ATPase that assembles into ~ 1 MDa hexameric complexes capable of segregating histones from DNA. Yta7 chromatin segregase promotes chromosome replication both in vivo and in vitro. Biochemical reconstitution experiments using purified proteins revealed that Yta7’s enzymatic activity is regulated by S phase-forms of Cyclin-Dependent Kinase (S-CDK). S-CDK phosphorylation stimulates ATP hydrolysis by Yta7, promoting nucleosome disassembly and chromatin replication.Our results present a novel mechanism of how cells orchestrate chromatin dynamics in co-ordination with the cell cycle machinery to promote genome duplication during S phase.

A conserved but plant-specific CDK-mediated regulation of DNA replication protein A2 in the precise control of stomatal terminal division

The R2R3-MYB transcription factor FOUR LIPS (FLP) controls the stomatal terminal division through transcriptional repression of the cell cycle genes CYCLIN-DEPENDENT KINASE (CDK) B1s (CDKB1s), CDKA;1, and CYCLIN A2s (CYCA2s). We mutagenized the weak mutant allele flp-1 seeds with ethylmethane sulfonate and screened out a flp-1 suppressor 1 (fsp1) that suppressed the flp-1 stomatal cluster phenotype. FSP1 encodes RPA2a subunit of Replication Protein A (RPA) complexes that play important roles in DNA replication, recombination, and repair. Here, we show that FSP1/RPA2a functions together with CDKB1s and CYCA2s in restricting stomatal precursor proliferation, ensuring the stomatal terminal division and maintaining a normal guard-cell size and DNA content. Furthermore, we provide direct evidence for the existence of an evolutionarily conserved, but plant-specific, CDK-mediated RPA regulatory pathway. Serine-11 and Serine-21 at the N terminus of RPA2a are CDK phosphorylation target residues. The expression of the phosphorylation-mimic variant RPA2aS11,21/D partially complemented the defective cell division and DNA damage hypersensitivity in cdkb1;1 1;2 mutants. Thus, our study provides a mechanistic understanding of the CDK-mediated phosphorylation of RPA in the precise control of cell cycle and DNA repair in plants.

The Mitotic Exit Network integrates temporal and spatial signals by distributing regulation across multiple components

GTPase signal transduction pathways control cellular decision making by integrating multiple cellular events into a single signal. The Mitotic Exit Network (MEN), a Ras-like GTPase signaling pathway, integrates spatial and temporal cues to ensure that cytokinesis only occurs after the genome has partitioned between mother and daughter cells during anaphase. Here we show that signal integration does not occur at a single step of the pathway. Rather, sequential components of the pathway are controlled in series by different signals. The spatial signal, nuclear position, regulates the MEN GTPase Tem1. The temporal signal, commencement of anaphase, is mediated by mitotic cyclin-dependent kinase (CDK) phosphorylation of the GTPase’s downstream kinases. We propose that integrating multiple signals through sequential steps in the GTPase pathway represents a generalizable principle in GTPase signaling and explains why intracellular signal transmission is a multi-step process. Serial signal integration rather than signal amplification makes multi-step signal transduction necessary.