YEAST CDC6 GENE CELL CYCLE PROGRESSION

这项拨款的总体目标是研究CDC6基因的功能和它的 细胞周期调节。早期的遗传学研究表明，酵母 温敏突变体cdc6在g1和S后期有缺陷 相边界，其功能可以是直接或 间接参与DNA复制。我们之前已经隔离了 描述了CDC6基因的特征。我们还证明了CDC6的mRNA CDC6蛋白的核进入依赖于细胞周期。在这 提交期间，我们发现GTS：：CDC6融合蛋白具有 依赖于DNA的ATPase活性。在本提案中，我们将使用 分子遗传学和生物化学相结合的方法研究 CDC6的生物学功能(S)及其细胞周期调控。 首先，我们将确定与之相互作用的细胞组件 CDC6基因产物。为了实现这一点，我们将隔离和 刻画抑制子(S)以建立CDC6之间的连锁通路 以及细胞周期进程中的其他相关基因。我们还将使用 一种分子遗传学方法，即双杂交系统，用于鉴定 体内与CDC6相互作用的蛋白质组分(S)。结果将会是 补充我们的基因研究，提供更多信息 建立一条细胞周期进程的途径。 其次，我们将通过CDC6的基因功能(S)来探索 生化方法。我们将对纯化的CDC6蛋白进行鉴定 研究其核苷酸结合、ATPase和/或GTPase活性。 根据序列基序，我们还将检测可能的解旋酶 活动。点突变的cdc6蛋白也将被提纯到 补充上述生化研究。据预计， 这些结果将有助于揭示CDC6的生物学功能(S)。 我们期待着对酵母CDC6基因及其基因的研究 抑制者将导致对 其他真核生物中的相应基因，并可能为我们提供有用的 揭示细胞深刻控制机制的信息 组织周期。

The overall goal of this grant is to study CDC6 gene function and its cell cycle regulation. Early genetic studies revealed that yeast temperature-sensitive mutant, cdc6, is defective in the late G1 and S phase boundary, and that its function may be, either directly or indirectly, involved in DNA replication. We have previously isolated and characterized the CDC6 gene. We have also demonstrated that CDC6 mRNA and nuclear entry of CDC6 protein are cell cycle dependent. In this submission period, we have found that the GTS::CDC6 fusion protein has a DNA-dependent ATPase activity. In this proposal, we will use a combined molecular genetic and biochemical approach to investigate the biological function(s) of CDC6 and its cell cycle regulation. Initially, we will identify the cellular components which interact with CDC6 gene products. In order to accomplish this, we will isolate and characterize the suppressor(s) to establish a linked pathway between CDC6 and other related genes in the cell cycle progression. We will also use a molecular genetic approach, the 2-hybrid system, to identify the protein component(s) which interact with CDC6 in vivo. The results will complement our genetic studies, providing additional information to establish a pathway of cell cycle progression. Secondly, we will explore the gene function(s) of CDC6 via the biochemical approach. We will characterize the purified CDC6 protein by investigating its nucleotide-binding, ATPase, and/or GTPase activities. Based on the sequence motif, we will also detect possible helicase activity. Point-mutated cdc6 proteins will also be purified to complement the above biochemical studies. It is anticipated that the results will shed light on the biological function(s) of CDC6. We expect that the investigation of the yeast CDC6 gene and its suppressors will lead to an understanding of the functions of corresponding genes in other eukaryotes, and may provide us with useful information to disclose the profound control mechanism of the cell division cycle.