Structural Basis of Large T Helicase Function in SV40 DNA Replication

描述（申请人提供）：SV40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SV40 DNA复制中发挥重要作用。 SV40复制作为真核DNA复制的模型，因为SV40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），除了解旋酶和由多种启动子因子组成的细胞启动子蛋白（如真核生物中的Orc、Ctd1、Cdc6、MCM或原核生物中的DnaA/DnaC/DnaB）来启动DNA复制，即标记复制起点、招募解旋酶、解链起点和激活解旋酶。对于 SV40 复制，LT 本身基本上实现了所有起始子功能，并且是延伸阶段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，协调其他复制蛋白的 DNA 复制功能，以及 LT 如何转化细胞。具体目标是了解 LT 六聚体和双六聚体解旋酶如何熔化原始 DNA 并解开 dsDNA 以启动 DNA 复制。我们计划主要采用X射线蛋白质晶体学，辅以EM和AFM、单分子分析、计算方法、体外和细胞内的分子生物学和功能生物化学。这项研究的结果预计将对真核细胞 DNA 复制领域和癌症生物学产生潜在影响。公共卫生相关性：SV40 大 T (LT) 具有非常多样化的生物活性。除了能够调节病毒感染和细胞过程的许多方面之外，六聚体和双六聚体形式的 LT 是一种有效的分子机器，可以熔化 dsDNA 并展开复制叉以进行 DNA 复制。 LT 是 SV40 微型染色体 DNA 复制所需的唯一病毒蛋白，所有其余蛋白均来自哺乳动物细胞的细胞复制机制。在这种微型染色体复制中，LT 执行细胞解旋酶 MCM 和其他几种起始蛋白的功能，以进行起点定位和解链。因此，SV40复制系统一直作为研究真核复制的模型系统。 LT 解旋酶机制的研究对于理解其他复制解旋酶，特别是来自真核细胞的复制解旋酶，具有普遍意义。我们的目的是了解LT六聚体和双六聚体解旋酶功能的详细分子机制。这项研究产生的数据将提供有关解旋酶功能和 DNA 复制的有价值的信息。这项研究与癌症生物学具有高度相关性。

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. PUBLIC HEALTH RELEVANCE: SV40 large T (LT) has remarkably diverse biological activities. Besides its ability to regulate many aspects of viral infection and cellular processes, LT in its hexameric and double hexameric forms is an efficient molecular machine that can melt dsDNA and unwind replication forks for DNA replication. LT is the only viral protein required for SV40 minichromosome DNA replication, all the rest proteins are from cellular replication machinery in mammalian cells. In this minichromosome replication, LT performs the functions of the cellular helicase MCM and several other initiator proteins for origin localization and melting. As a result, SV40 replication system has been serving as a model system for studying eukaryotic replication. The study of LT helicase mechanisms will have general implications for understanding other replicative helicases, especially for those from eukaryotic cells. We aim to understand the detailed molecular mechanisms of the helicase function of LT hexameric and double hexameric machine. The data generated from this research will provide valuable information about helicase function and DNA replication. This study bears high relevance to cancer biology.