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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

基本信息

批准号：
8499356
负责人：
ANTHONY SCHWACHA
金额：
$ 24.99万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-07-01 至 2015-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8499356
关键词：
ASK Gene ATP phosphohydrolase Active Sites Address Affect Architecture Binding Binding Proteins Binding Sites Biological Assay CDC7 gene 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 reconstitution sensor

项目摘要

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.

本研究的长期目标是阐明真核生物在细胞内的表达调控及其机制。 DNA复制。这一基本过程需要复杂的协调来制作一个精确的副本每个细胞周期的基因组。基本上所有方面的监管和机制的核心 DNA复制的关键是MCM 2 -7复合物，这是一种六聚体ATP酶，被认为是DNA复制的关键。解旋酶-在复制叉处解开双链DNA的分子马达。作为为数不多在DNA复制的起始和延伸阶段都是必不可少的因素，MCM的缺陷活性可以以多种方式损害DNA复制的精确性。特别是， MCM 2 -7复合物从复制叉的位移可能会发生，各种障碍。这种位移将导致复制叉的塌陷，这是一种缺陷，造成了癌症和先天缺陷的基因组不稳定性。以下目的建立在我们以前的工作与MCM 2 -7复合物：1）有缺陷的DNA解旋导致复制分叉崩溃？有证据表明，MCM 2 -7对于复制叉至关重要进展然而，我们的体外分析表明，MCM 2 -7可能本质上在解旋DNA，表明需要额外的因素在体内。我们将量化体外和野生型和突变型MCM 2 -7复合物在体内解开多种DNA的能力底物，评估额外的复制因子对该活性的作用，并使用基因筛选，以确定有助于MCM 2 -7分叉进展的新因子。2)是一种新发现MCM 2 -7复合物的不连续性需要启动和解旋酶激活？我们的体外分析表明，两个MCM亚基在细胞内形成了一个可逆的ATP调节的“门”。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，该门在使用染色质将MCM 2 -7组装到DNA上的体内效用免疫沉淀和通过复制特异性激酶激活MCM 2 -7解旋酶 CDC7/DBF4。(3)ATP结合、水解和DNA解旋之间的关系是什么在MCM 2 -7大楼里ATP酶是丰富的，并执行不同的细胞功能，但关于它们如何将ATP结合和水解偶联到机械作用，存在相当大的争议。工作与大多数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工具。我们建议突变两种特异性的预测参与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-然后使用我们的方法测试对MCM 2 -7活性的影响。建立了体内和体外功能测定。

项目成果

期刊论文数量（5）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

The eukaryotic Mcm2-7 replicative helicase.

DOI：
10.1007/978-94-007-4572-8_7
发表时间：
2012-01-01
期刊：
Sub-cellular biochemistry
影响因子：
0
作者：
Vijayraghavan, Sriram;Schwacha, Anthony
通讯作者：
Schwacha, Anthony

Mcm2-7 Is an Active Player in the DNA Replication Checkpoint Signaling Cascade via Proposed Modulation of Its DNA Gate.

Mcm2-7 通过对其 DNA 门的拟议调制，成为 DNA 复制检查点信号级联中的活跃参与者。

DOI：
10.1128/mcb.01357-14
发表时间：
2015
期刊：
Molecular and cellular biology
影响因子：
5.3
作者：
Tsai,Feng-Ling;Vijayraghavan,Sriram;Prinz,Joseph;MacAlpine,HeatherK;MacAlpine,DavidM;Schwacha,Anthony
通讯作者：
Schwacha,Anthony

The Saccharomyces cerevisiae Mcm6/2 and Mcm5/3 ATPase active sites contribute to the function of the putative Mcm2-7 'gate'.