Molecular mechanisms of DNA replication

DNA复制的分子机制

• 批准号: 8160834
• 负责人: Eric Enemark
• 金额: $ 32.38万
• 依托单位: ST. JUDE CHILDREN'S RESEARCH HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-01 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8160834
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; ATPase Domain; Address; Baculoviruses; Base Pairing; Binding; Biochemical; Biological; Biological Models; Cell division; Cells; Clinical Trials; Complex; DNA Binding; Data; Defect; Disease; Drug Design; Enzymes; Eukaryota; Eukaryotic Cell; Event; Face; Genetic; Genetic Materials; Goals; Growth; Human; Human Biology; Image; In Vitro; Insecta; Investigation; Knowledge; Lead; Length; Life; MCM2 gene; Maintenance; Malignant Neoplasms; Mediating; Methods; Modeling; Molecular; Movement; Mutagenesis; N-terminal; Nucleotides; Oligonucleotides; Oligosaccharides; Organism; Phosphotransferases; Polymerase; Preparation; Process; Protein Binding; Proteins; Reagent; Recombinants; Reporting; Research; Single-Stranded DNA; Structure; Structure-Activity Relationship; Techniques; Work; X ray diffraction analysis; X-Ray Crystallography; X-Ray Diffraction; base; cancer therapy; cofactor; helicase; human MCM2 protein; inhibitor/antagonist; insight; interest; pathogen; reconstitution; tumorigenesis

DESCRIPTION (provided by applicant): ABSTRACT DNA replication is a fundamental process for all organisms to precisely duplicate genetic material prior to cell division. Central to the process is a helicase enzyme that utilizes ATP-hydrolysis to separate base- paired DNA to allow polymerases to gain access to synthesize complementary strands as well as to drive the replication machinery along the DNA. In human and other eukaryotic cells, the helicase engine is the hexameric MCM complex. The mechanisms that MCMs use to operate upon DNA are poorly understood at the molecular level. The proposed research will fill a knowledge gap by providing detailed pictures of MCM proteins interacting with DNA and ATP compounds. These will be studied at the molecular level by a coordinated approach involving structural studies by X-ray crystallography and in vitro methods to study their functions and interactions. A considerable body of preliminary data has been obtained for this project that includes MCM:DNA co-crystal structures, important preliminary diffracting crystals of MCM:ADP, purified proteins, and functional information from SPR studies. PUBLIC HEALTH RELEVANCE: RELEVANCE STATEMENT This project focuses on DNA replication, a fundamental event that is required in all life forms. Defects in the replication machinery lead to genetic instability and potentiates tumorigenesis. The central engine of the replication machinery is the hexameric MCM complex that is loaded onto DNA in a regulated process and ultimately unwinds DNA at the replication fork once replication begins. MCMs and the kinases that activate them are logical targets for anti-cancer therapies, and inhibitors of the Cdc7 kinase that phosphorylates MCMs in order to activate its helicase function are currently in clinical trials. The actual mechanisms used by the MCM complex during assembly, activation, and progression upon DNA are not well understood at the structural level. The goals of the project are to study the interactions of MCMs with DNA and how the MCM complex is transformed by molecules of ATP and the ATPase cycle. The project encompasses structural and biochemical techniques working in tandem to study these important biological questions and will provide fundamental insights into mechanisms of replication in human cells.

描述（由申请人提供）： 摘要DNA复制是所有生物体在细胞分裂之前精确复制遗传物质的基本过程。该过程的核心是一种解旋酶，它利用ATP-Hydrolsys分离碱基对DNA，以使聚合酶可以访问合成互补链，并驱动沿DNA的复制机械。在人和其他真核细胞中，解旋酶发动机是六聚体MCM复合物。在分子水平上，MCM用于在DNA上使用的机制对DNA的作用很少。拟议的研究将通过提供与DNA和ATP化合物相互作用的MCM蛋白的详细图片来填补知识空白。这些将通过涉及X射线晶体学和体外方法的结构研究的协调方法在分子水平上进行研究，以研究其功能和相互作用。该项目已经获得了包括MCM：DNA共结构结构，MCM的重要初步衍射晶体：ADP，ADP，纯化的蛋白质以及SPR研究的功能信息的大量初步数据。 公共卫生相关性： 相关声明该项目的重点是DNA复制，这是所有生命形式中需要的基本事件。复制机制中的缺陷导致遗传不稳定性并增强肿瘤发生。复制机械的中央发动机是六聚体MCM复合物，在调节过程中加载到DNA上，并最终在复制开始后在复制叉处放松DNA。 MCMS和激活它们的激酶是抗癌疗法的逻辑靶标，以及磷酸化MCMs以激活其解旋酶功能的CDC7激酶的抑制剂，目前正在临床试验中。在结构水平上，MCM复合物在组装，激活和进展过程中使用的实际机制在结构水平上尚未得到充分了解。该项目的目标是研究MCM与DNA的相互作用，以及如何通过ATP分子和ATPase循环转化MCM复合物。该项目涵盖了在同步研究这些重要生物学问题的结构和生化技术，并将提供对人类细胞复制机制的基本见解。