Control of Simian Virus 40 and Cellular DNA Replication

描述（由申请人提供）：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DNA的蛋白质机制是维持所有细胞基因组稳定性的高度整合的蛋白质网络的一部分。基因组维护中的错误和故障可导致细胞活力丧失，并在很大程度上导致癌症等疾病，包括病毒相关癌症。然而，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了解仍然存在重大差距。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。染色体DNA复制始于RNA引物的合成，RNA引物可以通过DNA聚合酶进行延伸。在原核生物中，一种被称为“原核体”的动态纳米机器将双链DNA解绕与RNA引物合成结合在一起，但真核生物的原核体仍然难以捉摸。对猿猴病毒40 (SV40) DNA复制的分析提供了对真核原体的第一次深入了解，该原体可以在体外用纯化的重组蛋白重建。SV40引物体由病毒解旋酶大T抗原（Tag）、宿主DNA聚合酶α -引物酶（pol-prim）和宿主单链DNA结合蛋白RPA组成。我们之前的工作表明，病毒解旋酶标签与人类pol-prim和RPA之间至少有六对物理相互作用的网络产生了原体活性。同样，保守的脊椎动物解旋酶B （HELB/HDHB）与pol-prim和RPA相互作用，并在体外显示出原体活性。HDHB与人类细胞DNA损伤后的染色体复制和复制重启有关。提出的研究计划旨在(1)在原子水平上对SV40启动体进行全面的机制理解，(2)将这种机制分析扩展到HDHB启动体的活性，并确定该活性是否与DNA损伤后HDHB介导的复制重启有关。我们预计，这些“简单”的原体机制将作为识别和理解复杂的原体的有用模型，这些原体可以启动真核生物的染色体复制，也可以重新启动被DNA损伤阻止的复制叉。

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.