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 和非核苷酸抑制剂提供了一个重要的框架 检查多个子域如何促进生物合成和 这种关键逆转录病毒酶的降解功能。 持续的目标是 该项目是分子、生物化学和生物物理的应用 提供机械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。 在转化单链RNA基因组时 逆转录病毒侵入双链预整合DNA 复制机器必须容纳三个结构不同的核酸 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，不寻常的配置 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（多嘌呤束和 分别是中央终止序列）。 拟议的目标 研究的目的是从以下角度评估此类复制复合物 特异性核酸双链体和多亚结构域逆转录病毒逆转 转录酶（RT）。 子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 上手和双链模板引物双链体将继续， 其后果将在特定核酸双链体上进行评估 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 经过基因工程改造以适应核酸切割， 合理设计位置的光活化和荧光加合物 （由 HIV-1 酶的三维结构引导）。 这样的 试剂允许对子域几何结构的变化进行详细分析 改变或去除结构上重要的残留物后。 这 方法组合将应用于 N 端 DNA 结构相关的聚合酶和C端核糖核酸酶H结构域 慢病毒酶，从而提供子域的全面图片 架构，提供了设计新一代的可能性 变构抑制剂阻碍易位酶的运动。