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

我们以前确定了Cdc 7相关的激酶复合物，Cdc 7样催化和Dbf 4样调节亚基组成，从各种真核生物，并提出证据表明，启动真核DNA复制可能是由保守的机制，涉及这类激酶。为了阐明Cdc 7介导的MCM磷酸化如何导致起源放电的分子机制，我们正在精确地绘制MCM 2上Cdc 7特异性磷酸化位点，并表征磷酸化位点突变体。我们建议，MCM 2磷酸化Cdc 7可能会导致改变亚基结构和修改的DNA解旋酶和ATP水解活性。我们还使用遗传方法来剖析其中Gl调节导致哺乳动物细胞中Gl/S边界处Cdc 7活化的调节途径。为了实现这一目标，我们已经产生了突变的ES细胞和遗传操作的小鼠，其中muCdc 7基因的失活可以被人为地抑制。 ...更多信息 被引诱结果表明，在ES细胞中Cdc 7功能的丧失导致DNA合成的停止，随后导致细胞死亡。对裂殖酵母hskl+（Cdc 7同源物）的遗传分析表明其与Cdc 19（Mcm 2）、Rad 3（ATM）和Rad 21（内聚组分）的遗传相互作用。Hsk 1在体内和体外磷酸化Cdc 19，这种磷酸化在DNA复制的启动中起着至关重要的作用。hsk 1（ts）突变体在DNA复制不存在的情况下经历异常有丝分裂，并且这种表型在与rad 3突变组合时增强。HU诱导的Cds 1激酶激活在hsk 1（Us）突变体中显著降低，这可能是突变体中DNA复制检查点控制受损的部分原因。将hsk 1（ts）导入rad 21（ts）中导致rad 21突变体特有的异常染色质结构和异常染色体DNA含量的增强。结论Cdc 7激酶可能通过调节Rad 21 Cohesin的功能直接或间接调控S期特异性染色质结构。少

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