DNA SYNTHESIS AND RECOMBINATION BY HIV DNA POLYMERASE

HIV DNA 聚合酶的 DNA 合成和重组

• 批准号: 2187114
• 负责人: PHILIP J. FAY
• 金额: $ 20.24万
• 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 1996-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2187114
• 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.

这项建议将重点分析催化机理和DJA 人类免疫缺陷病毒I型(HIV)DNA的模板相互作用 聚合酶。这种病毒是获得性肺炎的病原体 免疫缺陷综合征(艾滋病)。重组HIV-I聚合酶已经被 从遗传学研究所(马萨诸塞州波士顿)获得。DNA结合特性的研究 可能与DNA合成效率相关的艾滋病毒聚合酶将是 使用结构定义的DNA分子进行分析。实验将会 评估3‘羟基末端结合或dNTPs等辅因子的依赖性。 它们将在合成和核糖核酸酶H 活动受到不同程度的抑制。聚合酶的分布 在DNA的引物末端和单链区域之间 量过了。DNA合成过程中模板链的切换将 关于核糖核酸酶手部模板的作用进行研究 切换到发生。加工性，聚合酶的固有属性， 也将研究对潜在的治疗性抗病毒的反应 毒品。使用专门标记的噬菌体DNA模板， 作为聚合酶合成障碍的模板将是 下定决心。结果将与特定序列相关，或者 二次结构。人类免疫缺陷病毒催化DNA合成的保真度 聚合酶将使用M13mp21acZ-α正向突变进行研究 化验系统。将确定是否生成错误 与DNA合成中停顿的位置有关。潜在的 减少HIV聚合酶误掺入的宿主细胞将是 通过在存在小牛DNA的情况下执行的保真度分析来解决 聚合酶增量II，一种高M，核聚合酶，具有非 可解离的3‘至5’核酸外切酶。最后，对艾滋病毒的作用进行了研究 将进行聚合酶的重组。具体的实验将 解决HIV聚合酶结合和合成两个 同时创建模板。M13基因突变检测的新变种 将被用来定量HIV聚合酶介导的重组 事件。这些结果将为我们提供对 HIV聚合酶，复制HIV基因组的酶，也是主要的 艾滋病治疗的靶蛋白。