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.

本提案将重点分析催化机理和DJA