DNA SYNTHESIS AND RECOMBINATION BY HIV DNA POLYMERASE

HIV DNA 聚合酶的 DNA 合成和重组

• 批准号: 2187113
• 负责人: PHILIP J. FAY
• 金额: $ 21.78万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-12-01 至 1995-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2187113
• 关键词: AIDS; DNA directed DNA polymerase; DNA replication; Escherichia coli; HIV infections; RNA directed DNA polymerase; complementary DNA; human immunodeficiency virus; microorganism genetics; molecular cloning; nucleic acid sequence; transfection; transposon /insertion element; virus DNA

This proposal will focus on analysis of the catalytic mechanism and DJA template interactions of the human immunodeficiency virus-I (HIV) DNA poluymerase. This virus is the etiologic agent of acquired immunodeficiency syndrome (AIDS). Recombinant HIV-I polymerase has been obtained from Genetics Institute (Boston, MA). DNA binding properties of HIV polymerase that could relate to DNA synthesis efficiency will be analyzed using structurally defined DNA molecules. Experiments will assess dependence of binding of 3'OH termini or cofactors such as dNTPs. They will be performed under conditions where synthetic and RNase H activities are differentially inhibited. Distribution of polymerase between primer termini and single-stranded regions of DNA will be measured. Template strand switching during processive DNA synthesis will be studied with regard to the role of RNase Hand template requiremtns for switching to occur. Processivity, an inherent property of a polymerase, also will be studied in response to potentially therapeutic anti-viral drugs. Using specifically primed phage DNA templates, positions on the template that act as barriers to synthesis by the polymerase will be determined. Results will be correlated to particular sequences or secondary structures. The fidelity of DNA synthesis catalyzed by HIV polymerase will be studied using an M13mp21acZ-alpha forward mutational assay system. It will be determined whether generation of errors correlates with positions of pauses in DNA synthesisl. Tjhe potential for the host cell to attenuate misincorporation by HIV polymerase will be addressed by fidelity analyses performed in the presence of calf DNA polymerase delta II, a high M., nuclear polymerase, having a non- dissociable 3' to 5' exonuclease. Finally, a study of the role of HIV polymerase in recombination will be undertaken. Specific experiments will address the ability of HIV polymerase to bind and synthesize on two templates simultaneously. Novel variations of the M13 mutational assay will be used to quantitate HIV poluymerase-mediated recombinational events. Results will provide fundamental insights into the properties of HIV polymerase, the enzyme that replicates the HIV genome, and a primary target protein for AIDS therapy.

该建议将重点放在分析催化机制和DJA 人类免疫缺陷病毒-I（HIV）DNA的模板相互作用 致态酶。该病毒是获得的病因学药物 免疫缺陷综合征（AIDS）。重组HIV-I聚合酶已经 从遗传学研究所（马萨诸塞州波士顿）获得。 DNA结合特性 可能与DNA合成效率有关的HIV聚合酶将是 使用结构定义的DNA分子分析。实验会 评估3'OH末端或辅助因子（例如DNTP）的结合依赖性。 它们将在合成和RNase H的条件下进行 活动被差异抑制。聚合酶的分布 DNA的引物末端和单链区域之间 测量。过程中DNA合成过程中的模板链切换将 研究RNase手工模板的作用 切换到发生。加工性，聚合酶的固有特性， 还将研究潜在的治疗性抗病毒 毒品。使用明确的噬菌体DNA模板，在 由聚合酶作为合成障碍的模板将是 决定。结果将与特定序列或 二级结构。由HIV催化的DNA合成的保真度 聚合酶将使用M13MP21ACZ-ALPHA正向突变进行研究 测定系统。将确定是否产生错误 与DNA合成中的停顿位置相关。潜力 通过HIV聚合酶减弱失物结构的宿主细胞将是 通过在小牛DNA存在下进行的富达分析来解决 聚合酶Delta II，A高M，核聚合酶，具有非 - 可分离的3'至5'外切酶。最后，研究艾滋病毒的作用 将进行重组中的聚合酶。特定的实验将 解决HIV聚合酶结合和合成两个的能力 模板同时。 M13突变测定的新型变化 将用于定量HIV杂种酶介导的重组 事件。结果将提供对特性的基本见解 HIV聚合酶，复制HIV基因组的酶，主要 靶蛋白进行艾滋病治疗。