INITIATION OF HSV DNA REPLICATION--UL9 INTERACTIONS

HSV DNA 复制的启动--UL9 相互作用

• 批准号: 6386389
• 负责人: Deborah S. Parris
• 金额: $ 27.62万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2003-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6386389
• 关键词: DNA binding protein; DNA directed DNA polymerase; DNA replication; DNA replication origin; adenosinetriphosphatase; binding proteins; chemical association; chemical kinetics; enzyme activity; enzyme complex; enzyme mechanism; helicase; herpes simplex virus 1; molecular assembly /self assembly; protein binding; protein protein interaction; surface plasmon resonance; virus DNA; virus protein; virus replication

Herpes simplex virus (HSV) provides a powerful genetic model for understanding eukaryotic DNA replication because of its relative simplicity compared to eukaryotic cells and because its haploid genome renders it more genetically tractable. HSV encodes 7 proteins directly involved in and required for viral DNA synthesis: a ss DNA binding protein (ICP8), a processive DNA polymerase (pol/UL42 heterodimer), a heterotrimeric helicase/primase complex (UL5, UL52, and UL8), and a protein mechanism of initiation of DNA replication of native viral DNA genomes in infected cells. Initiation of viral DNA synthesis is likely to be dependent upon the ability of UL9 to form an open complex at ori's present in the input viral DNA. A multiplicity of protein-protein interactions among the HSV DNA replication proteins has been described, through the functions of very few have been elucidated. UL9 most likely plays a central role in assembling the other proteins at ori's since UL9 interacts with a member of each of the complexes described above (ICP8, UL8, and UL42). We hypothesize that UL42 acts as an adapter protein which facilitates the entry of the processive pol (perhaps together with ICP8 and helicase/primase) into an activated ori occupied by UL9. We will combine biochemical and immunologic approaches to better understand the function of the UL9-UL42 interaction on the known UL9 activities. Three specific aims are proposed: 1) To measure the affinity of UL9 for UL42, pol, and pol/UL42 complex using the BIAcore 2000 and solution competition experiments with fusion proteins. The ability of UL9 to displace pol when it is complexed to UL42 will be determined as well as the stoichiometry of proteins in stable complexes. 2) To determine the ability and affinity to bind to different DNA substrates which vary in their structure and ori content, and the effect of UL42 on these properties. 3) To determine the functional significance of UL9-UL42 interaction by examining enzymatic mechanisms using pre-steady state and steady-state kinetic analysis. The role of UL42 in enhancing ATPase and helicase activities will be studied to differentiate specific from non-specific mechanisms of action. Effects of UL42 on load, initial rate constants, and processivity of helicase activities will be distinguished. The knowledge we gain in understanding the importance of interactions among DNA replciation proteins will help in defining an in vitro ori-dependent DNA replication system and in defining targets for the development of novel anti-viral compounds designed to disrupt the viral DNA replication complex.

单纯疱疹病毒 (HSV) 为理解真核 DNA 复制提供了强大的遗传模型，因为它与真核细胞相比相对简单，而且其单倍体基因组使其在遗传上更容易处理。 HSV 编码直接参与病毒 DNA 合成并为病毒 DNA 合成所需的 7 种蛋白质：单链 DNA 结合蛋白 (ICP8)、持续性 DNA 聚合酶 (pol/UL42 异二聚体)、异三聚解旋酶/引物酶复合物 (UL5、UL52 和 UL8)，以及在受感染细胞中启动天然病毒 DNA 基因组 DNA 复制的蛋白质机制。病毒 DNA 合成的启动可能取决于 UL9 在输入病毒 DNA 中的 ori 处形成开放复合物的能力。 HSV DNA 复制蛋白之间的多种蛋白质-蛋白质相互作用已被描述，但只有极少数的功能已被阐明。 UL9 很可能在 ori 处组装其他蛋白质中发挥核心作用，因为 UL9 与上述每个复合体（ICP8、UL8 和 UL42）的成员相互作用。我们假设 UL42 作为衔接蛋白，促进持续性 pol（可能与 ICP8 和解旋酶/引物酶一起）进入由 UL9 占据的激活 ori。我们将结合生化和免疫学方法来更好地了解 UL9-UL42 相互作用对已知 UL9 活性的功能。提出了三个具体目标： 1) 使用 BIAcore 2000 和融合蛋白的溶液竞争实验来测量 UL9 对 UL42、pol 和 pol/UL42 复合物的亲和力。将确定 UL9 与 UL42 复合时取代 pol 的能力以及稳定复合物中蛋白质的化学计量。 2) 确定与结构和ori含量不同的不同DNA底物结合的能力和亲和力，以及UL42对这些特性的影响。 3) 通过使用前稳态和稳态动力学分析检查酶机制来确定 UL9-UL42 相互作用的功能意义。将研究 UL42 在增强 ATP 酶和解旋酶活性中的作用，以区分特异性和非特异性作用机制。将区分 UL42 对解旋酶活性的负载、初始速率常数和持续合成能力的影响。我们在了解 DNA 复制蛋白之间相互作用的重要性方面获得的知识将有助于定义体外 ori 依赖性 DNA 复制系统，并确定开发旨在破坏病毒 DNA 复制复合物的新型抗病毒化合物的目标。