Title of project : Studies on mechanisms of regulation of initiation and progression of S phase

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We previously identified Cdc7-related kinase complexes, composed of Cdc7-like catalytic and Dbf4-like regulatory subunits, from various eukaryotes and presented evidence that initiation of eukaryotic DNA replication may be regulated by conserved mechanisms which involve this class of kinases. In order to clarify molecular mechanisms underlying how Cdc7-mediated phosphorylation of MCM leads to origin firing, we are precisely mapping Cdc7-specific phosphorylation sites on MCM2 and are characterizing the phosphorylation site mutants. We propose that MCM2 phosphorylation by Cdc7 may lead to alteration of subunit structures and modification of DNA helicase and ATP hydrolysis activities. We are also using genetic approaches to dissect the regulatory pathways in which Gl regulation leads to activation of Cdc7 at the Gl/S boundary in mammalian cells. Toward this goal, we have generated mutant ES cells and genetically manipulated mice in which inactivation of muCdc7 genes can be artificially in … More duced. The results indicate that loss of Cdc7 functions in ES cells lead to cessation of DNA synthesis and subsequently to cell death. Genetic analyses of fission yeast hskl+ (Cdc7 homologue) demonstrated its genetic interactions with Cdc19 (Mcm2), Rad3 (ATM) and Rad21 (Cohesion component). Hsk1 phosphorylates Cdc19 in vivo and in vitro, and this phosphorylation plays crucial roles in initiation of DNA replication. A hsk1 (ts) mutant undergoes aberrant mitosis in the absence of DNA replication, and this phenotype is enhanced in combination with rad3 mutation. HU-induced activation of Cds1 kinase is significantly reduced in the hsk1 (Us) mutant, and this may be partly responsible for the impaired DNA replication checkpoint control in the mutant. Introduction of the hsk1 (ts) into a rad21 (ts) resulted in enhanced aberrant chromatin structures and abnormal chromosomal DNA content characteristic to the rad21 mutant. hsk1 (ts) alone exhibited very similar phenotypes at 37C.We propose that Cdc7 kinase may regulate directly or indirectly S phase-specific chromatin structures through modulating Rad21 Cohesin functions. Less