CELLULAR PROLIFERATION AND THE CDC7 PROTEIN KINASE

细胞生物学中的一个中心问题是，细胞之间的过渡是如何 控制细胞分裂周期的各个阶段，确保 关键细胞组件的有序复制和分离。 利用酵母遗传学和动物细胞生物学获得的结果 生物化学最近在我们的 对规范和促进进入的机制的理解 有丝分裂。相比之下，人们对G1/S的控制知之甚少 过渡和DNA复制的启动。在酵母菌 Cerevisiae，似乎相同的蛋白激酶参与了 同时调节G1/S和G2/M转换，CDC2/CDC28/MPF 激活剂。我们的研究旨在定义 在酵母中通过激活G1中的CDC28蛋白进行运动。这个 我们研究的总体目标是了解CDC7的作用 蛋白质，也是一种蛋白激酶，进入S阶段。CDC7基因 功能在G1中较晚。我们的初步结果表明，cdc7p是 参与了G1期潜在的磷酸化级联反应，从而导致 DNA复制的启动。我们发现CDC28免疫 络合物能在体外磷酸化CDC7P和CDC7免疫 在体外，复合体可以使复制蛋白RP-A磷酸化。 如果得到证实，这些研究表明，cdc7作为一种分子 活跃在DNA承诺中的监管机构之间的联系 DNA合成及其引发DNA的催化装置 综合。我们将进一步描述体外相互作用的特征 这些蛋白质并试图表明它们反映了体内的细胞周期。 首先，我们发现从细菌中纯化的CDC7蛋白是 作为一种激活剂不活跃。我们将调查 翻译后修饰，包括CDC28的磷酸化， 以及与其他酵母蛋白在激活CDC7中的相互作用 激活剂。CDC28蛋白的激酶活性在整个过程中都受到调节。 由共价修饰和结合引起的细胞周期 其他蛋白质，包括细胞周期蛋白和cdc7也可能经历类似的过程。 修改。第二，我们将测定cdc7中的氨基酸。 它们在体内和体外都是磷酸化的。我们将测试他们的 位点定向改变对CDC7功能的影响 突变及其互补cdc7突变体能力的分析。 第三，我们将试图验证RP-A是Cdc7P在 并研究其他潜在的底物。

A central problem in cell biology is how the transitions between the various phases of the cell division cycle are controlled, ensuring orderly duplication and segregation of crucial cellular components. Results obtained using yeast genetics and animal cell biology and biochemistry have recently led to significant breakthroughs in our understanding of the mechanisms that regulate and promote entry into mitosis. By comparison, less is known about control of the G1/S transition and the initiation of DNA replication. In Saccharomyces cerevisiae, it appears that the same protein kinase is involved in regulating both the G1/S and the G2/M transitions, the cdc2/CDC28/MPF kinase. Our studies have been aimed at defining the events set in motion by activation of the Cdc28 protein in G1 in yeast. The overall gaol of our studies is to understand the role of the Cdc7 protein, also a protein kinase, in entry into S phase. The CDC7 gene functions late in G1. Our preliminary results suggest that Cdc7p is involved in a potential phosphorylation cascade during G1 that leads to initiation of DNA replication. We have found the Cdc28 immune complexes can phosphorylate Cdc7p in vitro and that Cdc7 immune complexes can phosphorylate the replication protein, RP-A, in vitro. If verified, these studies suggest that Cdc7 serves as a molecular link between the regulatory apparatus active in commitment to DNA synthesis and the catalytic apparatus involved in initiating DNA synthesis. We will further characterize the in vitro interactions of these proteins and try to show they reflect the cell cycle in vivo. First, we have found that Cdc7 protein purified from bacteria is inactive as a kinase. We will investigate the role of posttranslational modifications, including phosphorylation by Cdc28, and interactions with other yeast proteins in activating the Cdc7 kinase. The kinase activity of Cdc28 protein is regulated throughout the cell cycle by both covalent modifications and association with other proteins, including the cyclins, and Cdc7 may undergo similar modifications. Second, we will determine the amino acids in Cdc7 that are phosphorylated in vivo and in vitro. We will test their effect on the functions of Cdc7 by altering them by site directed mutagenesis and analysis of their ability to complement cdc7 mutants. Third, we will try to verify that RP-A is a substrate of Cdc7p in vivo and investigate other potential substrates.