Properties of the eukaryotic replicative DNA helicase

真核复制 DNA 解旋酶的特性

• 批准号: 7646455
• 负责人: Johannes Walter
• 金额: $ 33.74万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7646455
• 关键词: ATP phosphohydrolase; Address; Aphidicolin; Binding; Biochemical; Biological Assay; Cell Cycle; Cell-Free System; Chromatin; Chromosomes; Complex; DNA; DNA Polymerase Inhibitor; DNA biosynthesis; DNA lesion; Environment; Enzymatic Biochemistry; Enzymes; Eukaryota; Eukaryotic Cell; Event; Exhibits; G1 Phase; Genomic Instability; Grant; MCM10 gene; MCM4 gene; MCM6 gene; MCM7 gene; Malignant Neoplasms; Measures; Metabolism; Molecular; Mutation; Plasmids; Polymerase; Pre-Replication Complex; Preparation; Property; Protein Kinase; Proteins; Recruitment Activity; Research Personnel; Site; Structure; System; Xenopus; Xenopus laevis; biotin-streptavidin complex; chemotherapy; chromatin immunoprecipitation; ds-DNA; egg; enzyme mechanism; helicase; human disease; in vitro activity; inhibitor/antagonist; insight; interest; novel; programs; reconstitution

DESCRIPTION (provided by applicant): Replicative DNA helicases provide a critical function in DNA metabolism as they separate the 2 strands of the parental DNA in preparation for chromosome duplication. In eukaryotic cells, a hexameric ATPase called the MCM2-7 complex is an excellent candidate for the replicative DNA helicase, but little is known about how it unwinds DNA in the context of DNA replication. To study the biochemical mechanism of the replicative DNA helicase, a cell-free system derived from Xenopus laevis eggs is being used that recapitulates efficient, cell-cycle regulated DNA replication in a soluble protein environment. In this system, when DNA replication is initiated in the presence of DNA a polymerase inhibitor, many kilobasepairs of DNA are unwound by a highly processive DNA helicase in the absence of detectable DNA synthesis. This "hyper-unwinding" is dependent on MCM2-7 and the replication factor Cdc45, indicating it reflects functional uncoupling of the replicative DNA helicase from the polymerase. The hyperunwinding assay will be used to study the biochemical properties of the helicase in its native chromosomal context. In Specific Aim 1, chromatin immunoprecipitation will be used to determine which of the known DNA replication proteins are physically associated with the uncoupled helicase. In Specific Aim 2, proteins other than MCM2-7 and Cdc45 that are required for DNA unwinding by the helicase will be identified. In Specific Aim 3, the mechanism by which Cdc45 stimulates helicase activity will be studied. In Specific Aim 4, we address whether MCM2-7 unwinds DNA by translocating along single-stranded or double-stranded DNA. These studies are expected to yield important insights into the molecular mechanisms underlying DNA replication. While mutations that severely inhibit the function of the replicative DNA helicase will cause lethality, subtle mutations might cause genomic instability, and ultimately, cancer. In addition, this helicase is an attractive target for chemotherapy. Therefore, understanding the enzymology of this important enzyme is potentially of great interest for the treatment of human disease.

描述（由申请人提供）：复制性 DNA 解旋酶在 DNA 代谢中提供关键功能，因为它们分离亲本 DNA 的 2 条链，为染色体复制做准备。在真核细胞中，一种称为 MCM2-7 复合体的六聚体 ATP 酶是复制 DNA 解旋酶的绝佳候选者，但人们对其如何在 DNA 复制过程中解旋 DNA 知之甚少。为了研究复制 DNA 解旋酶的生化机制，我们使用了一种源自非洲爪蟾卵的无细胞系统，该系统在可溶性蛋白质环境中重现了有效的、细胞周期调节的 DNA 复制。在该系统中，当DNA复制在DNA聚合酶抑制剂存在的情况下启动时，在没有可检测到的DNA合成的情况下，许多数千碱基对的DNA被高度持续的DNA解旋酶解开。这种“超解旋”依赖于 MCM2-7 和复制因子 Cdc45，表明它反映了复制 DNA 解旋酶与聚合酶​​的功能性解偶联。超解旋测定将用于研究解旋酶在其天然染色体背景下的生化特性。在具体目标 1 中，染色质免疫沉淀将用于确定哪些已知的 DNA 复制蛋白与未偶联的解旋酶物理相关。在特定目标 2 中，将鉴定解旋酶解旋 DNA 所需的除 MCM2-7 和 Cdc45 之外的蛋白质。在具体目标3中，将研究Cdc45刺激解旋酶活性的机制。在具体目标 4 中，我们探讨了 MCM2-7 是否通过沿单链或双链 DNA 易位来解旋 DNA。这些研究有望对 DNA 复制的分子机制产生重要的见解。虽然严重抑制复制 DNA 解旋酶功能的突变会导致死亡，但细微的突变可能会导致基因组不稳定，并最终导致癌症。此外，这种解旋酶是化疗的一个有吸引力的靶点。因此，了解这种重要酶的酶学对于人类疾病的治疗可能具有重大意义。