Properties of the eukaryotic replicative DNA helicase

描述（由申请人提供）：复制性DNA解旋酶在DNA代谢中起关键作用，因为它们分离亲本DNA的两条链，为染色体复制做准备。在真核细胞中，一种名为MCM2-7复合体的六聚三磷酸腺苷酶是复制DNA解旋酶的绝佳候选者，但人们对它在DNA复制过程中如何解旋DNA知之甚少。为了研究复制DNA解旋酶的生化机制，研究人员使用了一种来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再现了有效的、细胞周期调节的DNA复制。在这个系统中，当DNA复制在DNA聚合酶抑制剂的存在下开始时，在没有可检测到的DNA合成的情况下，许多DNA的千碱基对被高度进展的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旋。超解旋试验将用于研究解旋酶在其天然染色体背景下的生化特性。在特异性目的1中，染色质免疫沉淀将用于确定哪些已知的DNA复制蛋白与解偶联解旋酶物理相关。在Specific Aim 2中，将鉴定除MCM2-7和Cdc45以外的DNA解旋酶解绕所需的蛋白质。在Specific Aim 3中，我们将研究Cdc45刺激解旋酶活性的机制。在Specific Aim 4中，我们讨论了MCM2-7是否通过沿单链或双链DNA易位来解绕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人类疾病的治疗具有潜在的巨大兴趣。

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.