The Role of the MCM2-7 Complex in the Replication Fork Processivity

我们研究的长期目标是阐明真核生物的调控和机制。 DNA复制。这一基本过程需要复杂的协调才能产生一个完全相同的副本 基因组的每一个细胞周期。基本上是所有方面的监管和机制的核心 DNA复制的关键是MCM2-7复合体，一种被认为是复制物的六聚体ATPase 解旋酶--在复制叉处解开双链DNA的分子马达。作为为数不多的 DNA复制的起始和延伸阶段的关键因素，MCM中的缺陷 活动会以多种方式影响DNA复制的精确度。特别是， 当MCM2-7复合体遇到复制叉时，可能会发生移位 各种障碍。这种移位将导致复制叉子的坍塌，这是 造成癌症和出生缺陷所特有的基因组不稳定类型。以下是 我的目标是在我们之前对MCM2-7复合体的工作的基础上：1)解开有缺陷的DNA 导致复制分叉崩溃？有证据表明，MCM2-7对复制分叉是必不可少的 进步。然而，我们的体外分析表明，MCM2-7可能在 解开DNA，这表明体内需要额外的因素。我们将对体外培养的 野生型和突变型MCM2-7复合体对多种DNA的体内解离能力 底物，评估其他复制因素对此活动的作用，并使用 进行基因筛查，以确定有助于MCM2-7分叉进展的新因素。2)是一种新的 发现启动和解旋酶激活所需的MCM2-7复合体不连续？ 我们的体外分析表明，两个MCM亚基形成了一个可逆的受ATP调节的门。 环形结构。使用我们的MCM突变体在体外具有这种活性缺陷，我们建议测试 该门在利用染色质将MCM2-7组装到DNA上的体内应用 MCM2-7解旋酶的免疫沉淀和复制特异性激酶的激活 CDC7/DBF4。(3)三磷酸腺苷结合、水解和DNA解离之间的关系是什么 在MCM2-7复合体中？ATPase含量丰富，具有多种细胞功能，但 关于它们如何将三磷酸腺苷结合和水解与机械作用相结合，存在很大的争议 工作。与大多数ATPase不同，MCM2-7有六个不同的亚基，可以单独修饰， 使其成为研究这些机器的功能的理想之选。我们建议突变两个特定的 预测参与DNA结合和活性位点协调的结构基序-前 传感器I插入和传感器II基序-然后使用我们的 建立了体内和体外功能检测方法。

The long-term goal of our research is to elucidate the regulation and mechanism of eukaryotic DNA replication. This essential process requires complex coordination to produce an exact copy of the genome every cell cycle. Central to the regulation and mechanism of essentially all aspects of DNA replication is the MCM2-7 complex, a hexameric ATPase believed to be the replicative helicase - the molecular motor that unwinds duplex DNA at the replication fork. As one of the few factors essential in both the initiation and elongation phases of DNA replication, defects in MCM activity can compromise the precision of DNA replication in multiple ways. In particular, displacement of the MCM2-7 complex from replication forks can potentially occur as it encounters various obstacles. Such displacement will lead to collapse of the replication fork, a defect that creates the types of genomic instability characteristic of cancer and birth defects. The following Aims build upon our previous work with the MCM2-7 complex: 1) Does defective DNA unwinding lead to replication fork collapse? Evidence suggests that MCM2-7 is essential for replication fork progression. However, our in vitro analysis indicates that MCM2-7 may be intrinsically inefficient at unwinding DNA, suggesting the need for additional factors in vivo. We will quantify the in vitro and in vivo ability of both wild type and mutant MCM2-7 complexes to unwind a variety of DNA substrates, assess the role that additional replication factors have on this activity, and use a genetic screen to identify novel factors that assist MCM2-7 fork progression. 2) Is a newly discovered discontinuity in the MCM2-7 complex required for initiation and helicase activation? Our in vitro analysis indicates that two MCM subunits form a reversible ATP-regulated "gate" in the toroidal structure. Using our MCM mutants defective for this activity in vitro, we propose to test the utility of this gate in vivo on the assembly of MCM2-7 onto DNA using chromatin immunoprecipitation, and activation of the MCM2-7 helicase by the replication specific kinase CDC7/DBF4. (3) What is the relationship between ATP binding, hydrolysis and DNA unwinding within the MCM2-7 complex? ATPases are abundant and perform diverse cellular functions, but considerable controversy exists as to how they couple ATP binding and hydrolysis to mechanical work. Unlike most ATPases, MCM2-7 has six distinct subunits that can be individually modified, making it ideal for studying the function these machines. We propose to mutate two specific structural motifs with predicted involvement in DNA binding and active site coordination - the pre- Sensor I insert and the Sensor II motif - then test the consequences on MCM2-7 activity using our established in vivo and in vitro functional assays.