Mechanism of Herpes DNA Replication

疱疹 DNA 复制机制

• 批准号: 8296513
• 负责人: ROBERT D KUCHTA
• 金额: $ 30.04万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-05 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8296513
• 关键词: Acyclovir; Address; Area; Binding; Binding Proteins; Biology; Budgets; Chemicals; Chickenpox; Complex; Coupled; Coupling; Cytomegalovirus Retinitis; DNA; DNA Primase; DNA biosynthesis; DNA-Binding Proteins; Development; Disease; Double Stranded DNA Virus; Exonuclease; Future; Genome; Goals; Grant; Herpesviridae; Human; Human Resources; Immunocompromised Host; In Vitro; Individual; Infection; Kinetics; Knowledge; Lead; Mouth Diseases; Neuraxis; Pharmaceutical Preparations; Polymerase; Process; Proteins; Recruitment Activity; Research; Research Priority; Role; Series; Simplexvirus; Small Interfering RNA; System; Testing; Time; UL5 protein; Vertebrates; Viral; Viral Encephalitis; Viral Genome; Virus; Work; genital herpes; helicase; inhibitor/antagonist; insight; mortality; neonate; novel; pathogen; protein function; small molecule

DESCRIPTION (provided by applicant): The long term goal of these studies is to develop a detailed understanding of the mechanism by which herpes viruses replicate their DNA. This process requires a series of herpes encoded proteins and may also involve cellular proteins. The proposed studies will focus on two complexes containing 5 essential proteins - the herpes polymerase complex (UL30/UL42) and the primase-helicase complex (UL5/UL52/UL8). The specific aims of the proposal are: (1) Determine if the cellular pol 1 is required for herpes replication using both siRNA knockdown of pol 1 and a specific small molecule inhibitor of pol 1. (2) Develop a mechanistic understanding of how the interplay between polymerase activity, exonuclease activity, and UL42 enhances the fidelity of replication using a variety of kinetic and mechanistic probes (3) Elucidate the mechanistic consequences of coupled activity between the polymerase and helicase and if the helicase can displace tightly bound proteins from DNA. Both steady- state and pre-steady state kinetic approaches will be used. (4) Using purified proteins and a minicircle template that gives efficient leading and lagging strand, examine the mechanistic coupling of the leading and lagging strand replication apparatus and the effects of bound proteins on the progression of the replication fork. These studies will provide novel insights into how the different proteins functionally interact with each other and the mechanism of herpes DNA replication.

描述（由申请人提供）：这些研究的长期目标是详细了解疱疹病毒复制 DNA 的机制。这个过程需要一系列疱疹编码的蛋白质，也可能涉及细胞蛋白质。拟议的研究将集中于含有 5 种必需蛋白质的两种复合物 - 疱疹聚合酶复合物 (UL30/UL42) 和引物酶-解旋酶复合物 (UL5/UL52/UL8)。该提案的具体目标是：(1) 使用 pol 1 的 siRNA 敲低和 pol 1 的特定小分子抑制剂确定细胞 pol 1 是否是疱疹复制所必需的。(2) 深入了解细胞 pol 1 之间如何相互作用聚合酶活性、核酸外切酶活性和 UL42 之间的分析可使用各种动力学和机械探针增强复制的保真度 (3) 阐明耦合活性的机械后果聚合酶和解旋酶之间的关系，以及解旋酶是否可以从 DNA 中取代紧密结合的蛋白质。将使用稳态和预稳态动力学方法。 (4) 使用纯化的蛋白质和提供有效前导链和滞后链的小环模板，检查前导链和滞后链复制装置的机械耦合以及结合蛋白对复制叉进展的影响。这些研究将为不同蛋白质如何在功能上相互作用以及疱疹 DNA 复制机制提供新的见解。