Control of Simian Virus 40 and Cellular DNA Replication

描述（由申请人提供）：拟议研究的长期目标是在分子细节上阐明控制哺乳动物细胞DNA复制的机制。SV40 DNA在感染细胞和无细胞反应中的复制已成为真核生物DNA复制的模型系统，促进了10种人类蛋白质的鉴定和表征，这些蛋白质与病毒T抗原一起，是体外重建SV40 DNA复制所必需和充分的。宿主细胞DNA复制需要相同的细胞蛋白，这表明宿主DNA复制的一些机制与病毒系统中阐明的机制相似。然而，SV40 DNA复制的早期步骤：识别DNA复制的起点，将复制酶募集到起点，以及在复制叉处解绕亲本DNA时的DNA解旋酶活性都是由T抗原完成的。哺乳动物DNA复制的这些早期步骤需要多种细胞蛋白来完成，同时也需要多种细胞蛋白来调节它们以应对复制压力。提出的研究计划旨在增加我们对病毒和宿主细胞DNA复制之间差异和相似之处的理解。Specific Aim 1将结合遗传学、生物化学和结构生物学来研究sv40t抗原与复制蛋白A相互作用介导DNA聚合酶A -引物酶合成引物的机制。在特异性目标2中，我们将继续探索DNA聚合酶a-引物酶的结构和功能，它与T抗原的相互作用，以及它对复制应激的反应。在特异性目标3中，我们的目标是确定HDHB的功能，HDHB是一种新型的人类DNA解旋酶，与T抗原具有某些特性。HDHB与Mre11/Nbs1/Rad50和其他未知蛋白的关联将在T抗原存在和不存在的情况下通过生化、遗传学和细胞生物学方法进行研究。这些研究将为SV40 T抗原如何选择宿主复制蛋白提供更深入的了解，并可能揭示这些解旋酶影响宿主基因组完整性的新机制。

DESCRIPTION (provided by applicant): The long-term goal of the proposed research is to elucidate in molecular detail the mechanisms that control DNA replication in mammalian cells. SV40 DNA replication in infected cells and in cell-free reactions has served as a model system for eukaryotic DNA replication, facilitating the identification and characterization of 10 human proteins that, together with the viral T antigen, are necessary and sufficient to reconstitute SV40 DNA replication in vitro. The same cellular proteins are required for host cell DNA replication, suggesting that some mechanisms used for host DNA replication resemble those elucidated in the viral system. However, the early steps of SV40 DNA replication: recognition of the origin of DNA replication, recruitment of replication enzymes to the origin, and DNA helicase activity during unwinding of parental DNA at the replication forks are all performed by T antigen. Multiple cellular proteins are required to perform these early steps in mammalian DNA replication, as well as to regulate them in response to replication stress. The proposed research program is designed to increase our understanding of the differences and similarities between viral and host cell DNA replication. Specific Aim 1 will combine genetics, biochemistry, and structural biology to investigate the mechanism by which SV40 T antigen interacts with Replication Protein A to mediate primer synthesis by DNA polymerase a-primase. In Specific Aim 2, we will continue to explore the structure and function of DNA polymerase a-primase, its interaction with T antigen, and its response to replication stress. In Specific Aim 3, our goal is to determine the function(s) of HDHB, a novel human DNA helicase that shares some properties with T antigen. The association of HDHB with Mre11/Nbs1/Rad50 and other unknown proteins will be studied using biochemical, genetic, and cell biological approaches, in the presence and absence of T antigen. These studies will provide a deeper understanding of how SV40 T antigen co-opts host replication proteins and may uncover novel mechanisms by which these helicases affect host genomic integrity.