Control of Simian Virus 40 and Cellular DNA Replication

完整和忠实的基因组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、如何调节这一过程以及如何纠正故障的理解仍然不完整。这项研究的长期目标是阐明哺乳动物细胞中控制DNA复制的分子细节机制。猿猴病毒40迷你染色体在感染细胞和无细胞反应中的复制作为一种简单的模型系统，用于鉴定和表征十种人类蛋白质，这些蛋白质与病毒蛋白T抗原一起，是体外重建SV40 DNA复制所必需和充分的。这10种人类蛋白在真核生物中是保守的，对细胞DNA复制至关重要，这表明病毒及其宿主使用类似的机制来复制其基因组。然而，病毒DNA复制的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，这些途径被DNA损伤信号激活，以挽救或重新启动停滞的复制分叉。提出的研究计划旨在通过首先确定病毒DNA复制早期步骤的详细分子机制，然后应用这些知识阐明一种新型人类DNA解旋酶（HDHB）在DNA损伤修复中的作用来探索这种可能性。特异性Aim 1结合分子遗传学、生物化学和结构生物学来确定T抗原与人DNA聚合酶α引物酶在无细胞系统中SV40复制起始的详细相互作用，并表征与聚合酶引物酶类似相互作用的宿主蛋白。特异性目标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。特异性目的3利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、原体活性和DNA修复中的作用。

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.