Impact of the translocational equilibrium of HIV-1 reverse transcriptase on the efficiency of mismatch extensions and the excision of mispaired nucleotides
Impact of the translocational equilibrium of HIV-1 reverse transcriptase on the efficiency of mismatch extensions and the excision of mispaired nucleotides
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DOI：
10.1016/j.biocel.2004.02.029
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发表时间：
2004-09-01
影响因子：
4
通讯作者：
Götte, M
中科院分区：
文献类型：
--
作者：
Marchand, B;Götte, M
人类免疫缺陷病毒I型（HIV-1 RT）的逆转录酶不具有核酸外切校正活性;然而，以前的研究表明，这种酶可以在焦磷酸盐或ATP存在下切除掺入的链终止子。这种反应为HIV-1对几种核苷类似物抑制剂的耐药性提供了一种合理的机制。在这里，我们研究了在错配核苷酸的情况下焦磷酸解的效率，发现与T相反的dCMP和dTMP的去除被字面上阻断。因此，焦磷酸解可能不能提供一种替代的、通用的校对机制，尽管切除dGMP和正确的dAMP对T可以相当有效地发生。位点特异性足迹法实验显示，C：T-和T：T-错配引物链的3'末端主要存在于易位后构型中，这阻止了末端核苷酸的去除。相反，含有G：T和A：T碱基对的复合物可以存在于易位后和易位前阶段。切除只能发生在后者，这有助于解释所观察到的反应的选择性。错配扩展的效率似乎并不依赖于预先存在的变化的易位平衡。然而，含有3'倒数第二个错配的复合物的足迹表明，错配后第一个核苷酸的掺入可以迫使酶向后滑动，这可以抑制随后的聚合事件。错误掺入的核苷酸会影响RT的精确定位，这一事实为开发在碱基部分含有修饰的新型核苷类似物抑制剂提供了理论依据。(C)2004 Elsevier Ltd.保留所有权利。
The reverse transcriptase of the human immunodeficiency virus type I (HIV-1 RT) does not possess an exonucleolytic proofreading activity; however, previous studies have shown that this enzyme can excise incorporated chain-terminators in the presence of pyrophosphate or ATP. This type of reaction provides a plausible mechanism for HIV-1 resistance to several nucleoside analogue inhibitors. Here we studied the efficiency of pyrophosphorolysis in the context of mismatched nucleotides, and found that the removal of dCMP and dTMP opposite T is literally blocked. Thus, pyrophosphorolysis may not provide an alternative, universal proofreading mechanism, although excision of dGMP and the correct dAMP opposite T can occur with considerable efficiency. Site-specific footprinting experiments revealed that the 3' end of C:T- and T:T-mispaired primer strands is predominantly found in a post-translocational configuration, which prevents the removal of terminal nucleotides. In contrast, complexes containing G:T and A:T base pairs can exist in both post- and pre-translocational stages. Excision can only occur in the latter, which helps to explain the observed selectivity of the reaction. The efficiency of mismatch extensions does not appear to depend on pre-existing changes of the translocational equilibrium. However, footprints of complexes containing 3' penultimate mismatches suggest that the incorporation of the first nucleotide following the mispair can force the enzyme to slide backwards, which can inhibit ensuing polymerization events. The fact that misincorporated nucleotides can affect the precise positioning of RT provides a rational for the development of novel nucleoside analogue inhibitors that contain modifications in the base moiety. (C) 2004 Elsevier Ltd. All rights reserved.