BIOCHEMICAL EVALUATION OF RT-TEMPLATE-PRIMER/COMPLEXES

RT 模板引物/复合物的生化评价

• 批准号: 2652106
• 负责人: MARY D BARKLEY
• 金额: $ 27.41万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-09-30 至 2002-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2652106
• 关键词: DNA footprinting; RNA directed DNA polymerase; enzyme activity; enzyme complex; enzyme structure; enzyme substrate; fluorescence spectrometry; human immunodeficiency virus 1; magnesium ion; manganese; metal complex; protein engineering; recombinant proteins; ribonuclease H; site directed mutagenesis

DESCRIPTION: The three dimensional structures of unliganded p66/p51 HIV-1 reverse transcriptase (RT), together with co-crystals containing duplex DNA and non-nucleotide-based inhibitors provide an important framework for examining how the multiple subdomains contribute to the biosynthetic and degradative functions of this key retroviral enzyme. The continuing goal of this project is application of molecular, biochemical and biophysical methodologies to provide both mechanistic and high resolution information on nucleoprotein complexes representative of specific events in the HIV replication cycle. In converting the single-stranded RNA genome of the invading virus into double-stranded pre-integrative DNA, the retroviral replication machinery must accommodate three structurally distinct nucleic acid duplexes, namely B-form duplex DNA, A-form duplex RNA and non-a, non-B RNA DNA hybrids. Recent data also indicates that unusual configurations of certain nucleic acid duplexes provides important control mechanisms for initiation and termination of (+) strand synthesis (the polypurine tract and central termination sequences, respectively). The aim of the proposed studies is to evaluate such replication complexes from the perspective of both the specific nucleic acid duplex and multi-subdomain retroviral reverse transcriptase (RT). In vitro site-directed mutagenesis of subdomains of HIV-1 and related lentiviral RTs interacting with single-stranded template overhand and double-stranded template-primer duplex will be continued, the consequences of which will be evaluated on specific nucleic acid duplexes closely mimicking events in retroviral replication. In parallel, chemical and enzymatic footprinting will be employed to provide high resolution structural data on these nucleoprotein complexes. HIV-1 RT will also be genetically engineered to accommodate nucleic acid cleaving, photoactivatable and fluorescent adducts at rationally designed positions (guided by the three dimensional structure of the HIV-1 enzyme). Such reagents permit a detailed analysis of alterations to subdomain geometry following alteration or removal of structurally important residues. This combination of methodologies will be applied to both the N-terminal DNA polymerase and C-terminal ribonuclease H domains of structurally-related lentiviral enzymes, thereby providing a comprehensive picture of subdomain architecture, offering the possibility of designing a new generation of allosteric inhibitors to impede movement of the translocating enzyme.

描述：未配体的p66/p51 HIV-1的三维结构 逆转录酶（RT），以及含有双链DNA的共晶体 和非核苷酸基抑制剂提供了一个重要的框架， 检查多个子域如何有助于生物合成， 这种关键的逆转录病毒酶的降解功能。 持续的目标 本项目是分子生物化学和生物物理学的应用 提供关于以下方面的机制和高分辨率信息的方法 代表HIV中特定事件的核蛋白复合物 复制周期 在转化的单链RNA基因组的 入侵病毒进入双链前整合DNA，逆转录病毒 复制机器必须容纳三种结构不同的核酸 酸性双链体，即B型双链体DNA、A型双链体RNA和非A、非B RNA DNA杂交体。 最近的数据还表明， 某些核酸双链体提供了重要的控制机制， （+）链合成的起始和终止（多嘌呤段和 分别为中心终止序列）。 建议的目的 研究的目的是从以下角度评估这种复制复合物： 特异性核酸双链体和多亚结构域逆转录病毒逆转录酶 转录酶（RT）。 亚结构域的体外定点诱变 HIV-1和相关慢病毒RT与单链模板相互作用 将继续进行反向和双链模板-引物双链体， 其结果将在特定的核酸双链体上进行评价 密切模仿逆转录病毒复制中的事件。 与此同时，化学 酶足迹法将用于提供高分辨率 这些核蛋白复合物的结构数据。 HIV-1 RT也将 基因工程以适应核酸切割， 在合理设计的位置上的光活化和荧光加合物 （由HIV-1酶的三维结构引导）。 等 试剂允许详细分析亚结构域几何结构的改变 随后改变或去除结构上重要的残基。 这 方法的组合将应用于N-末端DNA 结构相关的聚合酶和C-末端核糖核酸酶H结构域 慢病毒酶，从而提供了一个全面的亚结构域 建筑，提供了设计新一代 别构抑制剂，以阻止易位酶的运动。