BIOCHEMICAL EVALUATION OF RT-TEMPLATE-PRIMER/COMPLEXES

RT 模板引物/复合物的生化评价

• 批准号: 6019052
• 负责人: MARY D BARKLEY
• 金额: $ 24.19万
• 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/6019052
• 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：无配合的P66/p51 HIV-1的三维结构 逆转录酶（RT），以及含有双工DNA的共晶 基于非核苷酸的抑制剂为 研究多个子域如何促进生物合成和 该关键逆转录病毒酶的降解功能。 持续的目标 该项目是分子，生化和生物物理的应用 提供有关机械和高分辨率信息的方法论 核蛋白复合物代表了HIV中特定事件的代表 复制周期。 在转换单链的RNA基因组时 入侵病毒成双链的前综合DNA，逆转录病毒 复制机制必须容纳三种结构上不同的核心 酸双链体，即B形式双链DNA，A形式双链RNA和非A，非B RNA DNA杂种。 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 （+）链合成的启动和终止 中央终止序列分别）。 提议的目的 研究是为了评估此类复制复合物的角度 特定的核酸双链体和多屈服逆转录病毒反向 转录酶（RT）。 体外位置定向的亚域的诱变 HIV-1和相关的慢病毒RTS与单链模板相互作用 将继续进行双链模板 - 纸条复合体， 其后果将在特定的核酸双链体上进行评估 在逆转录病毒复制中紧密模仿事件。 并行，化学 并将采用酶足迹来提供高分辨率 这些核蛋白复合物的结构数据。 HIV-1 RT也将 经过基因设计以适应核酸切割， 在理性设计的位置处的光活化和荧光加合物 （以HIV-1酶的三维结构为指导）。 这样的 试剂允许对亚域几何变化的详细分析 改变或去除结构上重要的残基。 这 方法的组合将应用于两个N末端DNA 与结构相关的聚合酶和C末端核糖核酸酶H结构 慢病毒酶，从而提供了子域的全面图片 建筑，提供设计新一代的可能性 变构抑制剂阻碍易位酶的运动。