DNA SYNTHESIS AND RECOMBINATION BY HIV DNA POLYMERASE

本文将重点分析催化机理和DJA 人类免疫缺陷病毒-I（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聚合酶已被 获自Genetics Institute（Boston，MA）。DNA结合特性 可能与DNA合成效率有关的HIV聚合酶将被 使用结构定义的DNA分子进行分析。实验将 评估3 'OH末端或辅因子如dNTPs的结合依赖性。 它们将在合成和RNase H 活性受到不同程度的抑制。聚合酶分布 引物末端和DNA单链区域之间的连接将是 测定了DNA合成过程中的模板链转换 研究RNase Hand模板的作用， 切换发生。持续合成能力是聚合酶的固有特性， 也将研究对潜在治疗性抗病毒药物的反应， 毒品使用特异性引发的噬菌体DNA模板， 作为聚合酶合成屏障模板将被 测定结果将与特定序列相关，或 二级结构。HIV催化DNA合成的保真度 将使用M13 mp21 acZ-α正向突变来研究聚合酶。 分析系统将确定是否产生错误 与DNA合成中停顿的位置相关。潜在的 减弱HIV聚合酶错误掺入宿主细胞将 通过在小牛DNA存在下进行的保真度分析解决 聚合酶delta II，高M.，核聚合酶，具有非- 可分离的3'至5'外切核酸酶。最后，一项关于艾滋病毒作用的研究 将进行重组中的聚合酶。具体实验将 解决HIV聚合酶结合和合成两个 模板同时M13突变试验的新变化 将用于定量HIV聚合酶介导的重组 事件结果将提供基本的见解的性质， HIV聚合酶，复制HIV基因组的酶，和一种主要的 用于艾滋病治疗靶蛋白。

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.