DNA synthesis and recombination by HIV DNA Polymerase

HIV DNA 聚合酶的 DNA 合成和重组

• 批准号: 6796474
• 负责人: ROBERT A BAMBARA
• 金额: $ 37.8万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-12-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6796474
• 关键词: DNA directed DNA polymerase; DNA replication; RNA directed DNA polymerase; enzyme activity; flow cytometry; genetic recombination; human immunodeficiency virus 1; nucleic acid sequence; polymerase chain reaction; ribonuclease H; tissue /cell culture; virus RNA; virus replication

DESCRIPTION (provided by applicant): Recombination between the co-packaged genomes of HIV-1 is known to produce more potent virus that frustrates immune response and attempts at therapy. Recombination occurs during the reverse transcription steps of HIV-1 replication, with one mechanism involving the transfer of the growing DNA primer from one viral RNA template to the other. Our work is focused on determining the mechanisms that result in recombination during minus strand synthesis. Earlier we advanced evidence that pausing of the reverse transcriptase during synthesis concentrates RNase H directed cutting of the original template. This clears an area for interaction or invasion by the second template that promotes transfer. The process is also enhanced by template-template interactions. More recently we discovered that transfers occur in two distinct steps. After the initial interaction of the second template with the DNA, the first step, the DNA-RNA hybrid region propagates to a downstream site where the DNA 3' terminus transfers, the second step. We propose to determine distinct properties of the reverse transcriptase (RT) and the template structure that promote the second step of transfer. Mutant RTs and altered template structures will also be used to determine the reasons for the previously observed time delay in transfer, and the dynamics with which the propagating hybrid catches the terminus. We have evidence that the transfer of minus strong stop DNA during HIV-1 replication also occurs by a two-step mechanism. It is inefficient when reconstituted with short templates in vitro. However, longer templates show similar efficiency to that in vivo. We will explore how specific long distance interactions within the templates both up- and downstream of the transfer site promote transfers. We have developed an HIV-1 cell culture system designed to determine whether transfer mechanisms observed in vitro occur in the same manner in vivo. Use of RT mutants and viral genomes with specific alterations in template structure will be used for this task. Lastly, recent evidence suggests that cleavage specificity of the RT RNase H is a critical factor in determining the efficiency of transfer reactions. Specifically important is the ability of the RT to make adjacent cuts, by a mechanism of primary-secondary cuts that we previously described. Use of specific RNase H mutants of RT also holds the promise of determining the role of other features of RNase H specificity.

描述（由申请方提供）：已知HIV-1的共包装基因组之间的重叠会产生更强效的病毒，从而阻碍免疫应答和治疗尝试。在HIV-1复制的逆转录步骤期间发生扩增，其中一种机制涉及将生长的DNA引物从一种病毒RNA模板转移到另一种病毒RNA模板。我们的工作重点是确定负链合成过程中导致重组的机制。早先我们提出的证据表明，在合成过程中暂停逆转录酶浓缩了RNA酶H对原始模板的定向切割。这就为促进转移的第二个模板清除了一个相互作用或侵入的区域。该过程还通过模板-模板交互来增强。最近，我们发现转移发生在两个不同的步骤中。在第二模板与DNA的初始相互作用（第一步）之后，DNA-RNA杂交区传播到DNA 3'末端转移的下游位点（第二步）。 我们建议确定逆转录酶（RT）和模板结构，促进转移的第二步的不同属性。突变的RT和改变的模板结构也将用于确定先前观察到的转移时间延迟的原因，以及繁殖杂交体捕获末端的动力学。我们有证据表明，在HIV-1复制过程中，负强终止DNA的转移也是通过两步机制发生的。当在体外用短模板重建时，它是低效的。然而，较长的模板显示出与体内相似的效率。我们将探讨如何在模板内的特定的长距离互动，无论是上游和下游的转移网站促进转移。我们已经开发了一种HIV-1细胞培养系统，旨在确定在体外观察到的转移机制是否以相同的方式在体内发生。将使用RT突变体和模板结构发生特异性改变的病毒基因组来完成这项任务。最后，最近的证据表明，RT RNase H的切割特异性是决定转移反应效率的关键因素。特别重要的是RT通过我们先前描述的初级-次级切割机制进行相邻切割的能力。使用RT的特异性RNase H突变体也有望确定RNase H特异性的其他特征的作用。