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

MCM2-7 复合体在复制叉过程中的作用

• 批准号: 7858284
• 负责人: ANTHONY SCHWACHA
• 金额: $ 26.33万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7858284
• 关键词: ATP phosphohydrolase; Active Sites; Address; Affect; Architecture; Binding; Binding Proteins; Biological Assay; Cell Cycle; Cell physiology; Characteristics; Chromosomes; Complex; Congenital Abnormality; DNA; DNA Binding; DNA Double Strand Break; DNA Sequence; DNA biosynthesis; Daughter; Defect; Electron Microscopy; Genetic Screening; Genome; Genomic Instability; Genomics; Goals; Health; Human; Hydrolysis; In Vitro; Lead; Left; MCM2 gene; MCM5 gene; Macromolecular Complexes; Malignant Neoplasms; Measures; Mechanics; Molecular; Molecular Motors; Motion; Motor; Mutate; Phase; Phosphotransferases; Play; Pre-Replication Complex; Predisposition; Process; Property; Proteins; Regulation; Research; Role; Shapes; Structure; System; Testing; Work; chromatin immunoprecipitation; helicase; in vitro activity; in vitro testing; in vivo; interest; mutant; novel; prevent; public health relevance; reconstitution; sensor

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.

描述（由申请人提供）：我们研究的长期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，以在每个细胞周期产生基因组的精确拷贝。对DNA复制的基本上所有方面的调节和机制至关重要的是MCM 2 -7复合物，一种六聚体ATP酶，被认为是复制解旋酶-在复制叉处解旋双链DNA的分子马达。作为在DNA复制的起始和延伸阶段中必不可少的少数因素之一，MCM活性的缺陷可以以多种方式损害DNA复制的精度。特别是，MCM 2 -7复合物从复制叉的位移可能会发生，因为它遇到了各种障碍。这种置换将导致复制叉的崩溃，这种缺陷会产生癌症和出生缺陷所特有的基因组不稳定性。以下目的建立在我们先前对MCM 2 -7复合物的工作基础上：1）缺陷DNA解旋是否导致复制叉崩溃？有证据表明，MCM 2 -7对复制叉进展至关重要。然而，我们的体外分析表明，MCM 2 -7在解旋DNA时可能本质上是低效的，这表明需要在体内使用额外的因子。我们将量化野生型和突变型MCM 2 -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，并使用遗传筛选来鉴定有助于MCM 2 -7分叉进展的新因子。2)新发现的MCM 2 -7复合物中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亚基形成一个可逆的ATP调节的“门”的环形结构。使用我们的MCM突变体缺陷的这种活性在体外，我们建议测试这个门的效用在体内组装的MCM 2 -7到DNA上使用染色质免疫沉淀，并激活的MCM 2 -7解旋酶的复制特异性激酶CDC 7/DBF 4。(3)MCM 2 -7复合物中ATP结合、水解和DNA解旋之间的关系是什么？ATP酶是丰富的，并执行不同的细胞功能，但相当大的争议存在，因为他们如何耦合ATP结合和水解机械功。与大多数ATP酶不同，MCM 2 -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功能的理想选择。我们建议突变两个特定的结构基序，预测参与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测定来测试对MCM 2 -7活性的影响。DNA复制缺陷会导致基因组不稳定，这种情况会导致癌症、出生缺陷和其他各种人类健康问题。MCM 2 -7复合物对于基因组的调控和精确复制都是至关重要的。我们建议利用我们最近在了解这种复合物的机制方面取得的进展来研究MCM 2 -7的调节及其在基因组不稳定性中的参与。