Studies on Eukaryotic Replication

描述(由申请人提供)：我们研究的目标是确定和描述涉及真核DNA复制的机制。我们专注于分离DNA合成所需的单个蛋白质和蛋白质复合体，并确定它们在这一关键的大分子过程中的作用。所有正在生长的细胞必须准确复制它们的DNA，并在每个细胞周期内进行一次且仅一次这一壮举。干扰这些要求的事件可能会导致细胞死亡或改变增殖，从而导致癌症。目前，支持复制及其控制的许多步骤要么未知，要么特征不佳。 我们计划扩展我们之前在各种关键复制蛋白及其复合体上的工作，这些复制蛋白及其复合体支持DNA链延长所需的起始复合体的激活。为此，我们将研究：a)包含CDC45、MCM2-7复合体和GINS(CMG)的复合体的作用，CMG含有DNA解旋酶活性，可能对复制叉处的解离反应起重要作用；b)GINS与显著刺激其DNA聚合酶活性的Pol1-Primase复合体之间的相互作用；c)克隆的Pol5的性质及其与增殖细胞核抗原的相互作用，这是其对启动的DNA模板进行延伸所必需的。我们还观察到GINS与Pol5和Pol4的功能相互作用。我们计划进一步检查这些影响，并评估CMG联合体是否具有同样的作用。将进行耦合的复制和解卷反应，这可能有助于链的选择；d)我们计划探索CDC7激酶在CMG复合体的活性和形成中的作用。在体内，该激酶被证明影响了CDC45与MCM2-7复合体的相互作用。我们将进一步描述最近在CDC7激酶复合体和粘附素加载复合体Scc2/Scc4之间发现的一种新的相互作用。我们将调查这种相互作用的要求及其后果。所有细胞都必须复制相同的DNA才能生存。我们的研究针对的是这些副本是如何制作和监管的。我们计划分离与复制DNA有关的蛋白质，并确定它们的功能。这些研究的结果将对遗传病和癌症产生直接影响。

DESCRIPTION (provided by applicant): The goal of our research is to identify and characterize the machinery involved in eukaryotic DNA replication. We focus on the isolation of the individual proteins and protein complexes required for DNA synthesis and define their role in this critical macromolecular process. All growing cells must duplicate their DNA accurately and perform this feat once and only once per cell cycle. Events that interfere with these requirements can lead to cell death or altered proliferation that result in cancers. At present, many steps involved in supporting replication and its control are either unknown or poorly characterized. We plan to extend our previous work on various key replication proteins and their complexes that support the activation of initiation complexes required for the elongation of DNA chains. For this purpose, we will study: a) the role of a complex, containing Cdc45, the MCM2-7 complex and GINS (CMG) which contains DNA helicase activity and may contribute important to the unwinding reaction at the replication fork; b) the interaction between GINS and the Pol 1-primase complex which markedly stimulates its DNA polymerase activity; c) the properties of cloned Pol5 and its interaction with PCNA required for its processive elongation of primed DNA templates. We have also observed functional interactions of GINS with Pol5 as well as Pol4. We plan to examine these effects further and evaluate whether the CMG complex does the same. Coupled replication and unwinding reactions will be carried out which may contribute to strand selection; d) We plan to explore the role of the Cdc7 kinase on the activity and formation of the CMG complex. In vivo, this kinase was shown to influence the interaction of Cdc45 with the MCM2-7 complex. We will further characterize a novel interaction recently detected between the Cdc7 kinase complex and the cohesin loader complex Scc2/Scc4. The requirements for this interaction and its consequences will be investigated. All cells must make identical copies of their DNA to survive. Our studies are directed at how these copies are made and regulated. We plan to isolate the proteins involved in copying DNA and determine how they function. Results from these studies will have a direct impact on genetic diseases and cancer.