The capacity to faithfully replicate is one of the hallmarks of living systems. The overall goal of our research program is to understand the control of cell proliferation in eukaryotes. That regulation is imposed during G1 phase of the cell cycle via the regulation of the expression of a large family of G1- specific genes. We have previously described two novel repressors of G1-specific gene expression in yeast. Whi5 binds and represses SBF, one of two G1-specific transcription factors, during early G1 phase whereas Nrm1 acts as a co-repressor with MBF to repress G1 specific transcription as cells exit G1 phase. Both regulators play critical roles in cell cycle checkpoints that impose order on cell cycle events: Whi5 in the G1 cell size checkpoint and Nrm1 in the DNA replication checkpoint. The application is presented in three specific aims. First, we propose to characterize the role of Swi6, a shared component of MBF and SBF, as a platform for regulation of transcription by studying the basis for its interaction with Whi5 and Nrm1 and the regulation of that interaction by protein phosphorylation. Second, we propose to determine the mechanism and role of control of MBF regulated transcription by the DNA replication checkpoint. Nrm1 acts at the nexus between the checkpoint signaling pathway and the cell cycle machinery. The checkpoint regulates the Nrm1/MBF interaction via phosphorylation by checkpoint protein kinases and is important for genomic stability. Third, we propose to establish the role of the RAK motif, an amino acid sequence motif conserved between Nrm1, Whi5 and other proteins that defines a Nrm1/Whi5 superfamily. We expect that this amino acid sequence conservation belies a conserved aspect of cell cycle regulation by those proteins. We anticipate that completion of these specific aims will provide a broader understanding of the regulation of G1-specific transcription both during the cell cycle and in response to activation of the DNA replication checkpoint. Understanding this mechanism in the distantly related budding yeast, Saccharomyces cerevisiae, and fission yeast, Schizosaccharomyces pombe, promises to establish paradigms to be tested in the context of the regulation of cell proliferation and the human DNA replication checkpoint response. Misregulation of both of those processes has been associated with human cancer. We are hopeful that a more general view of the cell cycle-regulated transcriptional machinery and its regulators will offer insight into human biology and medicine.
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