Structural Basis of Large T Helicase Function in SV40 DNA Replication

SV40 DNA 复制中大 T 解旋酶功能的结构基础

• 批准号: 8293210
• 负责人: XIAOJIANG S CHEN
• 金额: $ 50.35万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8293210
• 关键词: Abnormal Cell; Address; Binding; Biochemical; Biochemistry; Biological; Biological Assay; Biological Models; Cancer Biology; Carcinogens; Cell Cycle; Cell physiology; Cells; Complement; Complex; Computational Biology; Computing Methodologies; Coupling; Crystallography; DNA; DNA Binding; DNA Primase; DNA biosynthesis; DNA replication fork; DNA replication origin; Data; Development; Eukaryota; Eukaryotic Cell; Goals; Human; Hydrolysis; In Vitro; Intervention; Iris; Kinetics; Knowledge; Large T Antigen; Lead; Literature; Malignant Neoplasms; Mammalian Cell; Methods; Modeling; Molecular; Molecular Biology; Molecular Machines; Motion; Mutagenesis; Oncogenic Viruses; Pathway interactions; Phase; Play; Polymerase; Power stroke; Process; Prokaryotic Cells; Proteins; Recruitment Activity; Replication Origin; Research; Resolution; Rest; Roentgen Rays; Role; Side; Simian virus 40; Structure; Structure-Activity Relationship; System; Topoisomerase; Ursidae Family; Viral; Viral Proteins; Virus Diseases; base; cell transformation; design; helicase; insight; melting; public health relevance; replication initiator protein; response; single molecule; viral DNA

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.

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。公共卫生相关性：SV 40大T（LT）具有显著多样的生物活性。除了其调节病毒感染和细胞过程的许多方面的能力之外，LT以其六聚体和双六聚体形式是一种有效的分子机器，可以熔化dsDNA并展开用于DNA复制的复制叉。LT是SV 40微型染色体DNA复制所需的唯一病毒蛋白，其余蛋白均来自哺乳动物细胞内的细胞复制机制。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，用于起始定位和解链。因此，SV 40复制系统已成为研究真核生物复制的模型系统。LT解旋酶机制的研究将对理解其他复制解旋酶，特别是真核细胞中的复制解旋酶具有普遍意义。我们的目标是了解LT六聚体和双六聚体机器的解旋酶功能的详细分子机制。从这项研究中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。