INTERACTIONS OF THE MCM PROTEINS AT REPLICATION ORIGINS

DNA复制的起始是细胞进入S 完成下一个细胞周期。这一承诺在 GI/S边界是细胞生长和细胞增殖的控制点。 各部门协调一致。癌细胞的特征在于它们 不协调的细胞分裂我们目前对 真核生物中DNA复制的起始主要来自病毒 提供起始事件的分子细节的模型系统， 缺乏关于这些事件的管理的信息。本实验室 发现了一个基因家族，称为MCM，其产物参与了 酵母中DNA合成在复制起点的起始。MCM 1是 一种也作为复制起始的转录因子 当结合到酵母复制起点的多个位点时，MCM2， MCM 3和MCM 5是一个结构和功能相关的家族。 具有细胞周期依赖性核定位的蛋白质。最 这个蛋白质家族的保守区域含有潜在的DNA 解旋酶基序这些蛋白质中的每一种的哺乳动物同源物已经被发现。 这表明这些MCM蛋白的功能是 可能在所有真核生物中都是保守的。在本提案中， MCM蛋白之间的功能关系及其在 将研究DNA合成在复制起点的起始 进一步. MCM 1的结构和功能将通过 生化和突变分析。之间的函数关系 将通过DNA结合检测MCM 1和MCM 2 -3-5蛋白家族 研究、双杂交和抑制基因分析。的结合位点 复制起点上的MCM蛋白将通过DNA酶I和DNA酶D分析。 化学足迹分析成员之间的互动 MCM 2 -3-5蛋白家族将通过遗传学和生物化学方法进行研究 分析。纯化的MCM蛋白将单独分析，并作为 重组复合物的ATP酶和DNA解旋酶活性。其他基因 与MCM蛋白相互作用的产物将从 两个杂交体文库和抑制子分析。长期不变的宗旨和追求 是重建一个复制起始复合物， 特异性启动体外DNA合成。

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.