DNA synthesis and recombination by HIV DNA Polymerase

描述（由申请人提供）：已知HIV-1共包装基因组之间的重组可以产生更强效的病毒，从而挫败免疫反应和治疗尝试。重组发生在HIV-1复制的逆转录步骤中，其中一种机制涉及将生长的DNA引物从一个病毒RNA模板转移到另一个病毒RNA模板。我们的工作重点是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，在合成过程中逆转录酶的暂停集中了RNase H对原始模板的定向切割。这为促进转移的第二个模板的交互或入侵清除了一个区域。该流程还通过模板-模板交互得到增强。最近，我们发现转移分两个不同的步骤进行。在第二个模板与DNA的初始相互作用（第一步）之后，DNA- rna杂交区域繁殖到DNA 3'端转移的下游位点，这是第二步。

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.