BIOCHEMICAL EVALUATION OF RT-TEMPLATE-PRIMER/COMPLEXES

RT 模板引物/复合物的生化评价

• 批准号: 6180611
• 负责人: MARY D BARKLEY
• 金额: $ 24.91万
• 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/6180611
• 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：未连接的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 和非核苷酸类抑制剂提供了一个重要的框架 研究多个亚区如何对生物合成和 这种关键的逆转录病毒酶的降解功能。持续的目标是 本项目是分子、生物化学和生物物理学的应用。 提供机械性和高分辨率信息的方法 代表HIV中特定事件的核蛋白复合体 复制周期。在将单链RNA基因组转化为 病毒侵入双链整合前DNA，逆转录病毒 复制机制必须容纳三个结构不同的核 酸性双链，即B型双链DNA、A型双链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 某些核酸双链提供了重要的调控机制 (+)链合成的开始和终止(多聚尿路和 中央终止序列)。建议的目的是 研究是从以下角度评价这种复制复合体 特异性核酸双链和多亚域逆转录病毒均逆转 转录酶(RT)。亚区基因的体外定点突变 HIV-1及其相关慢病毒逆转录酶与单链模板相互作用 上手和双链模板-引物双链将继续存在， 其结果将在特定的核酸双链上进行评估 与逆转录病毒复制中的事件非常相似。与之平行的是，化学 并将使用酶足迹来提供高分辨率 这些核蛋白复合体的结构数据。HIV-1 RT也将是 通过基因工程来适应核酸的裂解， 在合理设计的位置上的可光激活和荧光加合物 (由HIV-1酶的三维结构指导)。是这样的 试剂允许对子区域几何结构的变化进行详细分析 在改变或移除结构上重要的残基之后。这 两种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区 慢病毒酶，从而提供了亚区的全面图像 架构，提供了设计新一代 变构抑制剂以阻止转运酶的移动。