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

描述（申请人提供）：我们的长期研究目标是阐明真核生物DNA复制的调控和机制。这个重要的过程需要复杂的协调才能在每个细胞周期产生一个精确的基因组拷贝。MCM2-7复合体是DNA复制所有方面的调控和机制的核心，它是一种六聚三磷酸腺苷酶，被认为是复制解旋酶，是在复制叉上解开双链DNA的分子马达。MCM作为DNA复制起始和延伸阶段的重要因素之一，其活性缺陷会以多种方式影响DNA复制的精度。特别是，MCM2-7复合体在遇到各种障碍时可能会从复制分叉中移位。这种位移将导致复制叉的崩溃，这种缺陷会造成癌症和出生缺陷的基因组不稳定类型。以下目标建立在我们之前对MCM2-7复合体的研究基础上：1)有缺陷的DNA解绕是否会导致复制叉崩溃？有证据表明MCM2-7对复制分叉的进展至关重要。然而，我们的体外分析表明，MCM2-7在解绕DNA时可能本质上效率低下，这表明体内需要其他因素。我们将量化野生型和突变型MCM2-7复合物在体外和体内解开各种DNA底物的能力，评估额外的复制因子对这种活性的作用，并使用遗传筛选来确定协助MCM2-7分叉进展的新因子。2)新发现的MCM2-7复合体的不连续性是起始和解旋酶激活所必需的吗？我们的体外分析表明，两个MCM亚基在环形结构中形成一个可逆的atp调节“门”。利用体外缺乏这种活性的MCM突变体，我们建议使用染色质免疫沉淀和复制特异性激酶CDC7/DBF4激活MCM2-7解旋酶，在体内测试该门在MCM2-7组装到DNA上的效用。(3) MCM2-7复合物内ATP结合、水解和DNA解绕之间的关系是什么？ATP酶种类丰富，具有多种细胞功能，但它们是如何将ATP结合和水解与机械作用结合起来的，存在很大的争议。与大多数atp酶不同，MCM2-7有六个不同的亚基，可以单独修改，使其成为研究这些机器功能的理想选择。我们建议突变两个特定的结构基序，它们预计会参与DNA结合和活性位点协调-前传感器I插入和传感器II基序-然后使用我们建立的体内和体外功能分析来测试对MCM2-7活性的影响。DNA复制中的缺陷导致基因组不稳定，这种情况会导致癌症、出生缺陷和各种其他人类健康问题的易感性。MCM2-7复合体是调控和精确复制基因组的核心。我们建议利用我们最近在了解该复合物机制方面的进展来研究MCM2-7调控及其在基因组不稳定性中的参与。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE Defects in DNA replication lead to genomic instability, a condition that causes a predisposition to cancer, birth defects, and a variety of other human health issues. The MCM2-7 complex is central to both the regulation and precise replication of the genome. We propose to capitalize on our recent progress on understanding the mechanism of this complex to examine MCM2-7 regulation and its involvement in genomic instability.