Control of Simian Virus 40 and Cellular DNA Replication

猿猴病毒 40 和细胞 DNA 复制的控制

• 批准号: 7999919
• 负责人: ELLEN Hope FANNING
• 金额: $ 17.54万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-08 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7999919
• 关键词: Base Pairing; Binding; Biochemistry; Biological Models; Cell Cycle; Cell Extracts; Cell Survival; Cell division; Cell-Free System; Cells; Chromatin; Chromosomal Duplication; Chromosomes; Complex; Copying Processes; DNA; DNA Damage; DNA Maintenance; DNA Primase; DNA Repair; DNA biosynthesis; DNA polymerase alpha-primase; Development; Eukaryota; Genome; Genome Stability; Genomics; Goals; Heart; Hereditary Disease; Human; In Vitro; Knowledge; Large T Antigen; Maintenance; Malignant Neoplasms; Mammalian Cell; Maps; Mediating; Molecular; Molecular Genetics; Mutagens; Pathway interactions; Polymerase; Polyomavirus; Primates; Primer Extension; Process; Proteins; Reaction; Regulation; Replication-Associated Process; Research; Role; SS DNA BP; Signal Pathway; Signal Transduction; Simian virus 40; Single-Stranded DNA; Site; Stress; Tissues; Type I DNA Topoisomerases; Viral; Viral Proteins; Viral Tumor Antigens; Virus; Work; base; design; helicase; homologous recombination; human DNA; mammalian genome; novel; programs; recombinational repair; reconstitution; repaired; replication factor A; sialosyl-T antigen; structural biology; viral DNA

DESCRIPTION: Complete and faithful duplication of the genome is a fundamental prerequisite for cell division in development and tissue renewal. Errors and malfunctions in the replication process can result in loss of cell viability and are responsible in large part for genetic diseases such as cancer, but our understanding of how the mammalian genome is duplicated, how this process is regulated, and how malfunctions are corrected remains incomplete. The long-term goal of the proposed research is to elucidate in molecular detail the mechanisms that control DNA replication in mammalian cells. Replication of the simian virus 40 mini-chromosome in infected cells and in cell-free reactions has served as a simple model system to identify and characterize ten human proteins that, together with the viral protein T antigen, are necessary and sufficient to reconstitute SV40 DNA replication in vitro. These ten human proteins are conserved among eukaryotes and essential for cell DNA replication, suggesting that the virus and its host use similar mechanisms to replicate their genomes. However, the mechanism of viral DNA replication differs from that of the host in several key features. These differences suggest that viral DNA replication mechanisms may resemble those of host pathways that are activated by DNA damage signaling to rescue or restart stalled replication forks. The proposed research program is designed to explore this possibility by first determining the detailed molecular mechanisms of the early steps in viral DNA replication and then applying this knowledge to elucidate the role of a novel human DNA helicase (HDHB) in DNA damage repair. Specific Aim 1 combines molecular genetics, biochemistry, and structural biology to determine the detailed interactions of T antigen with human DNA polymerase alpha-primase in initiation of SV40 replication in a cell-free system and to characterize host proteins that interact similarly with the polymerase-primase. Specific Aim 2 uses the same approaches to investigate the interaction of the single-strand DNA-binding protein replication protein A with the polymerase-primase in primer synthesis and elongation. Specific Aim 3 uses molecular genetics and biochemistry to determine the functional domains of HDHB, characterize in detail its interactions with DNA and other proteins, and elucidate its roles in genotoxin-regulated chromatin-binding, primosome activity, and DNA repair.

描述：基因组的完整和忠实复制是发育和组织更新中细胞分裂的基本先决条件。复制过程中的错误和故障可能导致细胞活力丧失，并在很大程度上导致癌症等遗传疾病，但我们对哺乳动物基因组如何复制、该过程如何调节以及如何纠正故障的理解仍然不完整。该研究的长期目标是从分子细节上阐明哺乳动物细胞中控制 DNA 复制的机制。猿猴病毒 40 微型染色体在受感染细胞和无细胞反应中的复制已成为识别和表征 10 种人类蛋白的简单模型系统，这些蛋白与病毒蛋白 T 抗原一起对于体外重建 SV40 DNA 复制是必要且充分的。这十种人类蛋白质在真核生物中是保守的，对于细胞 DNA 复制至关重要，表明病毒及其宿主使用相似的机制来复制其基因组。然而，病毒 DNA 复制机制在几个关键特征上与宿主不同。这些差异表明，病毒 DNA 复制机制可能类似于宿主途径，通过 DNA 损伤信号激活来挽救或重新启动停滞的复制叉。拟议的研究计划旨在探索这种可能性，首先确定病毒 DNA 复制早期步骤的详细分子机制，然后应用这些知识来阐明新型人类 DNA 解旋酶 (HDHB) 在 DNA 损伤修复中的作用。具体目标 1 结合了分子遗传学、生物化学和结构生物学，以确定在无细胞系统中启动 SV40 复制过程中 T 抗原与人类 DNA 聚合酶 α-引物酶的详细相互作用，并表征与聚合酶-引物酶类似相互作用的宿主蛋白。具体目标 2 使用相同的方法来研究单链 DNA 结合蛋白复制蛋白 A 与聚合酶引物酶在引物合成和延伸中的相互作用。具体目标 3 使用分子遗传学和生物化学来确定 HDHB 的功能域，详细描述其与 DNA 和其他蛋白质的相互作用，并阐明其在基因毒素调节的染色质结合、引发体活性和 DNA 修复中的作用。