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BIOCHEMICAL EVALUATION OF RT-TEMPLATE-PRIMER/COMPLEXES

RT 模板引物/复合物的生化评价

基本信息

批准号：
6386122
负责人：
MARY D BARKLEY
金额：
$ 25.64万
依托单位：
CASE WESTERN RESERVE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1994
资助国家：
美国
起止时间：
1994-09-30 至 2003-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6386122
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

项目摘要

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：无配体p66/p51 HIV-1的三维结构

项目成果

期刊论文数量（15）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Probing contacts between the ribonuclease H domain of HIV-1 reverse transcriptase and nucleic acid by site-specific photocross-linking.

通过位点特异性光交联探测 HIV-1 逆转录酶的核糖核酸酶 H 结构域与核酸之间的接触。

DOI：
10.1074/jbc.m909808199
发表时间：
2000
期刊：
The Journal of biological chemistry
影响因子：
0
作者：
Rausch,JW;Sathyanarayana,BK;Bona,MK;LeGrice,SF
通讯作者：
LeGrice,SF

Substituting a conserved residue of the ribonuclease H domain alters substrate hydrolysis by retroviral reverse transcriptase.

取代核糖核酸酶 H 结构域的保守残基可改变逆转录病毒逆转录酶对底物的水解作用。

DOI：
10.1074/jbc.272.13.8602
发表时间：
1997
期刊：
The Journal of biological chemistry
影响因子：
0
作者：
Rausch,JW;LeGrice,SF
通讯作者：
LeGrice,SF

Interaction of p55 reverse transcriptase from the Saccharomyces cerevisiae retrotransposon Ty3 with conformationally distinct nucleic acid duplexes.

来自酿酒酵母反转录转座子 Ty3 的 p55 逆转录酶与构象不同的核酸双链体的相互作用。

DOI：
10.1074/jbc.275.18.13879
发表时间：
2000
期刊：
The Journal of biological chemistry
影响因子：
0
作者：
Rausch,JW;Grice,MK;Henrietta,M;Nymark-McMahon;Miller,JT;LeGrice,SF
通讯作者：
LeGrice,SF

RNA polymerase alters the mobility of an A-residue crucial to polymerase-induced melting of promoter DNA.

RNA 聚合酶改变 A 残基的迁移率，这对聚合酶诱导的启动子 DNA 解链至关重要。

DOI：
10.1021/bi026539m
发表时间：
2002
期刊：
Biochemistry
影响因子：
2.9
作者：
Tsujikawa,Laura;Strainic,MichaelG;Watrob,Heather;Barkley,MaryD;DeHaseth,PieterL
通讯作者：
DeHaseth,PieterL

Kinetic analysis of four HIV-1 reverse transcriptase enzymes mutated in the primer grip region of p66. Implications for DNA synthesis and dimerization.

p66 引物夹区域中四种突变的 HIV-1 逆转录酶的动力学分析。