DNA synthesis and recombination by HIV DNA Polymerase

描述(由申请人提供)：HIV-1共同包装的基因组之间的重组已知会产生更强大的病毒，使免疫反应和治疗尝试受挫。重组发生在HIV-1复制的逆转录步骤中，一种机制涉及将不断增长的DNA引物从一个病毒RNA模板转移到另一个模板。我们的工作集中在确定在负链合成过程中导致重组的机制。早些时候，我们提出了证据，证明在合成过程中暂停逆转录酶会浓缩RNaseH定向切割原始模板。这清除了促进转移的第二模板相互作用或侵入的区域。模板与模板之间的相互作用也增强了这一过程。最近，我们发现，转移发生在两个截然不同的步骤。在第二模板与DNA的初始相互作用之后，第一步，DNA-RNA杂交区传播到DNA 3‘末端转移的下游位置，第二步。 我们建议确定逆转录酶(RT)的不同性质和促进第二步转移的模板结构。突变的RTS和改变的模板结构也将被用来确定先前观察到的转移时间延迟的原因，以及传播的杂种抓住末端的动力学。我们有证据表明，在HIV-1复制过程中负的强终止DNA的转移也是通过两步机制发生的。当在体外用短模板重组时，效率不高。然而，较长的模板显示出与体内相似的效率。我们将探索转移站点上下游模板内的特定远程交互如何促进转移。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在体内以同样的方式发生。这项任务将使用具有特定模板结构变化的RT突变体和病毒基因组。最后，最近的证据表明，RT RNaseH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通过我们前面描述的一次-二次切割机制进行相邻切割的能力。使用RT的特定RNase H突变体也有望确定RNase H特异性的其他特征的作用。

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.