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

描述(申请人提供)：我们研究的长期目标是阐明真核DNA复制的调节和机制。这一必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副本。在DNA复制的几乎所有方面的调控和机制的中心是MCM2-7复合体，一个被认为是复制解旋酶的六聚体ATPase-在复制分叉处解开双链DNA的分子马达。作为DNA复制起始和延伸阶段为数不多的关键因素之一，MCM活性缺陷会以多种方式影响DNA复制的精确度。特别是，当MCM2-7复合体遇到各种障碍时，它可能会从复制叉子上移位。这种移位将导致复制叉的崩溃，这种缺陷会造成癌症和先天缺陷所特有的基因组不稳定类型。以下目标建立在我们先前对MCM2-7复合体的工作基础上：1)有缺陷的DNA解离是否会导致复制叉崩溃？有证据表明，MCM2-7对于复制分叉的进展是必不可少的。然而，我们的体外分析表明，MCM2-7在解开DNA方面可能本质上是低效的，这表明在体内需要额外的因素。我们将量化野生型和突变型MCM2-7复合体在体外和体内解离各种DNA底物的能力，评估额外的复制因子对这一活性的作用，并使用遗传筛选来识别帮助MCM2-7分叉进展的新因素。2)新发现的MCM2-7复合体的不连续是启动和解旋酶激活所必需的吗？我们的体外分析表明，两个MCM亚基在环状结构中形成了一个可逆的受ATP调节的“门”。使用我们的MCM突变体在体外具有这一活性缺陷，我们建议在体内测试该门在染色质免疫沉淀法将MCM2-7组装到DNA上以及通过复制特异性激酶CDC7/DBF4激活MCM2-7解旋酶方面的实用性。(3)在MCM2-7复合体中，ATP结合、水解和DNA解离之间有什么关系？ATPase含量丰富，具有多种细胞功能，但关于它们如何将ATP结合和水解与机械功结合在一起仍存在相当大的争议。与大多数ATPase不同，MCM2-7有六个可以单独修饰的不同亚基，这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，预计参与DNA结合和活性部位协调-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复制中与公共卫生相关的缺陷会导致基因组不稳定，这种情况会导致癌症、出生缺陷和各种其他人类健康问题。MCM2-7复合体是基因组调控和精确复制的核心。我们建议利用我们在了解这个复合体的机制方面的最新进展来研究MCM2-7的调节及其参与基因组不稳定的过程。

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.