INTERACTIONS OF THE MCM PROTEINS AT REPLICATION ORIGINS

DNA复制的启动标志着一个细胞承诺进入S 并完成下一个细胞周期。在会议上作出的这一承诺 Gi/S边界是细胞生长和细胞生长的控制点 组织是协调一致的。癌细胞的特征是它们的 细胞分裂不协调。我们目前对这个问题的理解 真核生物中DNA复制的启动主要来自病毒 提供引发事件的分子细节的模型系统 缺乏对这些事件的监管信息。我们的实验室有 发现了一个名为MCM的基因家族，其产品参与了 在酵母中复制起始处DNA合成的启动。MCM1是 一种转录因子，也起复制启动的作用 在酵母复制起始点与多个位点结合时的因子。MCM2， MCM3和MCM5是一个结构和功能相关的家族 具有依赖于细胞周期的核定位的蛋白质。最多的 这一蛋白质家族的保守区包含一个潜在的DNA 解旋酶基序。这些蛋白质中每一种的哺乳动物同源物都是 鉴定表明这些MCM蛋白的功能是 可能在所有真核生物中都是保守的。在这项提案中， MCM蛋白的功能关系及其在细胞周期调控中的作用 将研究复制起始处DNA合成的启动 再远一点。MCM1的结构和功能将通过 生化和突变分析。两者之间的函数关系 MCM1和MCM2-3-5蛋白家族将通过DNA结合进行检测 研究，双杂交和抑制物分析。分子的结合部位 复制起点上的MCM蛋白将通过DNase I和 化学足迹分析。成员之间的交互 MCM2-3-5蛋白家族将从遗传学和生化角度进行研究 分析。纯化的MCM蛋白将单独进行分析，并作为 ATPase和DNA解旋酶活性的重组复合体。其他基因 与MCM蛋白相互作用的产品将从 两个杂交体文库和通过抑制分析。我们的长期目标 是重建一个复制启动复合体，它引导起源- 体外DNA合成的特异性启动。

Initiation of DNA replication signals commitment of a cell to enter S phase and to complete the next cell cycle. This commitment made at the GI/S boundary is the control point at which cell growth and cell division are coordinated. Cancer cells are characterized by their uncoordinated cell divisions. Our current understanding of the initiation of DNA replication in eukaryotes is derived mostly from viral model systems which provide molecular detail of the initiation event but lack information on the regulation of these events. Our laboratory has identified a family of genes, called MCM, whose products participate in the initiation of DNA synthesis at replication origins in yeast. MCM1 is a transcription factor which also acts as a replication initiation factor when bound to multiple sites at yeast replication origins. MCM2, MCM3 and MCM5 are a family of structurally and functionally related proteins which has a cell cycle dependent nuclear localization. The most conserved region of this family of proteins contains a potential DNA helicase motif. Mammalian homologs for each of these proteins have been identified suggesting that the functions of these MCM proteins are likely to be conserved in all eukaryotes. In this proposal, the functional relationship between the MCM proteins and their roles in the initiation of DNA synthesis at replication origins will be investigated further. The structure and function of MCM1 will be examined by biochemical and mutational analyses. The functional relationship between the MCM1 and MCM2-3-5 protein family will be examined by DNA binding studies, two-hybrids and suppressor analyses. The binding sites of the MCM proteins on replication origins will be analyzed by DNase I and chemical footprinting analyses. The interactions between members of the MCM2-3-5 protein family will be investigated by genetic and biochemical analyses. Purified MCM proteins will be analyzed individually and as a reconstituted complex for ATPase and DNA helicase activities. Other gene products that interact with the MCM proteins will be identified from the two hybrids library and by suppressor analyses. Our long term objective is to reconstitute a replication initiation complex that directs origin- specific initiation of DNA synthesis in vitro.