Control of Simian Virus 40 and Cellular DNA Replication

描述(申请人提供)：完整和忠实的基因组复制是发育过程中细胞分裂和组织更新的基本前提。复制DNA的蛋白质机制是高度整合的蛋白质网络的一部分，该网络在所有细胞中维持基因组的稳定性。基因组维护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疾病，包括病毒相关性癌症。然而，关于哺乳动物基因组是如何复制的，这一过程是如何调控的，以及故障是如何纠正的，我们的知识仍然存在重大差距。这项拟议研究的长期目标是从分子细节上阐明控制哺乳动物细胞中DNA复制的机制。染色体DNA复制始于合成可由DNA聚合酶延伸的RNA引物。在原核生物中，一种被称为“原始体”的动态纳米机器与RNA引物合成结合在一起，使DNA双链解离，但真核生物的原始体仍然难以捉摸。对猴病毒40(SV40)DNA复制的分析提供了第一次洞察到可以在体外用纯化的重组蛋白重组的真核原粒。SV40原始体由病毒解旋酶大T抗原(Tag)、宿主DNA聚合酶α-Primase(PolPrim)和宿主单链DNA结合蛋白RPA组成。我们以前的工作表明，病毒解旋酶标签与人polPrim和RPA至少由六对物理相互作用组成的网络能够产生原粒活性。类似地，一个保守的脊椎动物解旋酶B(Helb/HDHB)与polPrim和RPA相互作用，并在体外显示出原粒体活性。HDHB与人类细胞的染色体复制和DNA损伤后的复制重新启动有关。该研究计划的目的是(1)在原子水平上对SV40原粒进行全面的机制理解，(2)将这种机制分析扩展到HDHB的原粒活性，并确定这一活性是否与HDHB介导的DNA损伤后的复制重新启动有关。我们预计，这些“简单”的原始体机制将作为有用的模型来识别和理解复杂的原始体(S)，它启动了真核生物中的染色体复制，并可能重新启动因DNA损伤而停止的复制分叉。 与公共卫生相关：准确和及时地复制基因组DNA是每个细胞在所有生命王国中繁殖的基本先决条件。面对环境和内源性DNA损伤，细胞进化出了一个复杂的蛋白质网络，以维持基因组的稳定性。由于病毒已经进化到利用这些途径进行自身传播，通常会给宿主带来致病后果，例如癌症，这些途径的发现将为抗病毒药物的设计和更有效的癌症治疗发现新的靶点。

DESCRIPTION (provided by applicant): Complete and faithful duplication of the genome is a fundamental prerequisite for cell division in development and tissue renewal. The protein machinery that replicates DNA is part of a highly integrated protein network that maintains genomic stability in all cells. Errors and malfunctions in genome maintenance can result in loss of cell viability and are responsible in large part for diseases such as cancer, including virus-associated cancers. However, major gaps remain in our knowledge of how the mammalian genome is duplicated, how this process is regulated, and how malfunctions are corrected. The long-term goal of the proposed research is to elucidate in molecular detail the mechanisms that control DNA replication in mammalian cells. Chromosomal DNA replication begins with the synthesis of RNA primers that can be extended by a DNA polymerase. In prokaryotes, a dynamic nanomachine known as a 'primosome' couples duplex DNA unwinding with RNA primer synthesis, but eukaryotic primosomes remain elusive. Analysis of simian virus 40 (SV40) DNA replication provides the first insight into a eukaryotic primosome that can be reconstituted with purified recombinant proteins in vitro. The SV40 primosome consists of the viral helicase large T antigen (Tag), the host DNA polymerase alpha-primase (pol-prim), and the host single-strand DNA-binding protein RPA. Our previous work suggests that a network of at least six pairs of physical interactions of the viral helicase Tag with human pol-prim and RPA gives rise to primosome activity. Similarly, a conserved vertebrate helicase B (HELB/HDHB) interacts with pol-prim and RPA, and displays primosome activity in vitro. HDHB has been implicated in chromosomal replication and in replication restart after DNA damage in human cells. The proposed research program is designed (1) to develop a comprehensive mechanistic understanding of the SV40 primosome at the atomic level, and (2) to extend this mechanistic analysis to the HDHB primosome activity and determine whether this activity is linked to HDHB-mediated replication restart after DNA damage. We anticipate that these "simple" primosome mechanisms will serve as useful models for identifying and understanding the complex primosome(s) that initiates chromosomal replication in eukaryotes and others that may restart replication forks arrested by DNA damage. PUBLIC HEALTH RELEVANCE: Accurate and timely replication of genomic DNA is a fundamental prerequisite for the propagation of each cell in all kingdoms of life. Cells have evolved a complex network of proteins to maintain genome stability in the face of environmental and endogenous DNA damage. Since viruses have evolved to exploit these pathways for their own propagation, often with pathogenic consequences for the host, e.g. cancer, discovery of these pathways will uncover new targets for the design of anti-viral drugs and more effective cancer therapy.