Control of Simian Virus 40 and Cellular DNA Replication

描述(申请人提供)：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。SV40DNA在感染细胞和无细胞反应中的复制已成为真核细胞DNA复制的模式系统，有助于鉴定和鉴定与病毒T抗原一起在体外重建SV40DNA复制所必需的10种人类蛋白。宿主细胞DNA复制需要相同的细胞蛋白，这表明用于宿主DNA复制的某些机制类似于病毒系统中阐明的机制。然而，SV40DNA复制的早期步骤：识别DNA复制的起始点，向起始点招募复制酶，以及在复制叉处解开双亲DNA时的DNA解旋酶活性都是由T抗原完成的。在哺乳动物DNA复制中，需要多种细胞蛋白来执行这些早期步骤，并在复制压力下对它们进行调节。拟议的研究计划旨在增加我们对病毒和宿主细胞DNA复制之间的差异和相似之处的理解。具体目标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相互作用的机制，以介导DNA聚合酶a-Primase的引物合成。在特定的目标2中，我们将继续探索DNA聚合酶a-Primase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。在具体目标3中，我们的目标是确定HDHb的功能(S)，这是一种新的人类DNA解旋酶，具有与T抗原相同的某些特性。在存在和不存在T抗原的情况下，将使用生化、遗传学和细胞生物学方法研究HDHB与Mre11/Nbs1/Rad50和其他未知蛋白的关联。这些研究将提供对SV40T抗原如何选择宿主复制蛋白的更深层次的理解，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.