Structural Basis of Large T Helicase Function in SV40 DNA Replication

描述（由申请方提供）：SV 40大T抗原（LT）是一种强效致癌物，作为复制解旋酶和复制起始蛋白，在SV 40 DNA复制中发挥重要作用。SV 40复制用作真核DNA复制的模型，因为SV 40使用所有必需的细胞复制蛋白（引物酶、聚合酶、PCNA、拓扑异构酶等），除了解旋酶和由多个起始因子（如真核生物中的Orc、Ctd 1、Cdc 6、MCM，或原核生物中的DnaA/DnaC/DnaB）组成的细胞起始蛋白以起始DNA复制，即标记复制起点、募集解旋酶、解链起点和激活解旋酶。对于SV 40复制，LT单独实现基本上所有的起始功能，并且是在延伸阶段期间用于复制叉解旋的解旋酶。这项研究的长期目标是了解LT如何作为解旋酶发挥作用，以协调其他复制蛋白的DNA复制功能，以及LT如何转化细胞。具体的目的是为了了解LT六聚体和双六聚体解旋酶如何融化的起源DNA和解旋dsDNA启动DNA复制。我们计划主要使用X射线蛋白质晶体学，辅以EM和AFM，单分子测定，计算方法，分子生物学和功能生物化学在体外和细胞中。这项研究的结果有望对真核细胞中的DNA复制和癌症生物学领域产生潜在影响。

DESCRIPTION (provided by applicant): SV40 large T antigen (LT) is a potent carcinogen, and plays an essential role for SV40 DNA replication as the replicative helicase and replication initiator protein. SV40 replication serves as a model for eukaryotic DNA replication, as SV40 uses all the essential cellular replication proteins (primase, polymerase, PCNA, Topoisomerases, etc.), except for the helicase and cellular initiator proteins that consist of multiple initiator factors (such as Orc, Ctd1, Cdc6, MCM in eukaryotes, or DnaA/DnaC/DnaB in prokaryotes) to initiate DNA replication, i.e. marking the replication origin, recruiting helicase, melting origin, and activating helicase. For SV40 replication, LT alone fulfills essentially all the initiator functions and is the helicase for replication fork unwinding during elongation phase. The long-term goal of this research is to understand how LT functions as a helicase to coordinate the functions of the other replication proteins for DNA replication, as well as how LT transforms cells. Specific aims are designed to understand how LT hexameric and double hexameric helicase melt the origin DNA and unwinds dsDNA to initiate DNA replication. We plan to use mainly X-ray protein crystallography, assisted with EM and AFM, single molecule assay, computational method, molecular biology and functional biochemistry in vitro and in cells. The results from this research are expected to have potential impact on the field of DNA replication in eukaryotic cells and on cancer biology.