Winshell Jamie, Paulson Benjamin A, Buelow Ben D, Champoux James J
Department of Microbiology, School of Medicine, University of Washington, Seattle, WA 98195-7242, USA.
J Biol Chem. 2004 Dec 17;279(51):52924-33. doi: 10.1074/jbc.M409134200. Epub 2004 Sep 30.
DNA displacement synthesis by reverse transcriptase during retroviral replication is required for the production of the linear precursor to integration. The sensitivity of unpaired thymines to KMnO(4) oxidation was used to probe for the extent of DNA melting by human immunodeficiency virus, type 1 (HIV-1) reverse transcriptase in front of the primer terminus in model oligonucleotide-based displacement constructs. Unpairing of the two base pairs downstream of the primer (+1 and +2 positions) requires the presence of the next correct dNTP, indicating that DNA melting only occurs after the formation of the ternary complex with the enzyme tightly clamped around the DNA. The amount or extent of DNA melting is not significantly affected by the length of the already-displaced strand or the base composition of the DNA beyond the +2 position. The F61W mutant form of HIV-1 reverse transcriptase, which is partially impaired for displacement synthesis, exhibits a reduction in the amount of melting at the +1 and +2 positions. These results demonstrate the importance of the observed melting to displacement synthesis and suggest that the unpairing reaction is mediated by an intimate association between the fingers region of the enzyme and the DNA in the closed clamp conformation of the protein.
逆转录病毒复制过程中，逆转录酶进行的DNA置换合成是产生整合所需线性前体的必要条件。利用未配对胸腺嘧啶对KMnO(4)氧化的敏感性，在基于模型寡核苷酸的置换构建体中，探究了人类免疫缺陷病毒1型（HIV-1）逆转录酶在引物末端前方使DNA解链的程度。引物下游两个碱基对（+1和+2位置）的解链需要下一个正确的dNTP存在，这表明DNA解链仅在与紧密夹在DNA周围的酶形成三元复合物后才会发生。DNA解链的量或程度不受已置换链长度或+2位置以外DNA碱基组成的显著影响。HIV-1逆转录酶的F61W突变形式在置换合成方面部分受损，在+1和+2位置的解链量减少。这些结果证明了观察到的解链对置换合成的重要性，并表明解链反应是由酶的指状区域与处于蛋白质闭合钳位构象的DNA之间的紧密结合介导的。
