Alterations to the primer grip of p66 HIV-1 reverse transcriptase and their consequences for template-primer utilization
Alterations to the primer grip of p66 HIV-1 reverse transcriptase and their consequences for template-primer utilization
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DOI：
10.1021/bi952773j
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发表时间：
1996-07-02
期刊：
影响因子：
2.9
通讯作者：
leGrice, SFJ
中科院分区：
文献类型：
--
作者：
Ghosh, M;Jacques, PS;leGrice, SFJ
用丙氨酸扫描突变法研究了人类免疫缺陷病毒逆转录酶（HIV-1 RT）p66/p51亚基的Met（230）-His（235）结构意义。与Glu（224）-Trp（229）一起，这些残基提供p66“引物夹”的框架，其建议的作用是将引物末端保持在适合于对进入的dNTP进行亲核攻击的方向上。在这些残基中，改变Leu（234）导致p66亚基不能缔合成异二聚体。剩余的选择性突变的酶成功地重建和纯化的同质性的RT相关的活动进行评估。我们在此表明，p66-Trp（229）-Met（230）-Gly（231）-Tyr（232）-四联体内任何残基的改变都会改变与DNA聚合酶和核糖核酸酶H（RNase H）结构域相关的功能。对具有完整或模型“预切割”RNA-DNA杂合体的突变型p66（Y232 A）/p51的详细分析表明，改变的RNase H表型可能是由于模板-引物在核酸结合裂缝中的重新定位。因此，模板核苷酸-8位于紧邻RNase H催化中心的位置，而不是核苷酸-17。
Alanine scanning mutagenesis was undertaken to evaluate the structural significance of Met(230)-His(235) Of the 66 kDa subunit of p66/p51 human immunodeficiency virus reverse transcriptase (HIV-1 RT). Together with Glu(224)-Trp(229), these residues provide the framework of the p66 ''primer grip'', whose proposed role is maintaining the primer terminus in an orientation appropriate for nucleophilic attack on an incoming dNTP. Of these residues, altering Leu(234) results in a p66 subunit incapable of associating into heterodimer. The remaining selectively mutated enzymes were successfully reconstituted and purified to homogeneity for evaluation of RT-associated activities. We show here that alterations to any residue within the p66-Trp(229)-Met(230)-Gly(231)-Tyr(232)-quartet alter functions associated with both the DNA polymerase and ribonuclease H (RNase H) domains. Detailed analysis of mutant p66(Y232A)/p51 with an intact or a model ''precleaved'' RNA-DNA hybrid suggests an altered RNase H phenotype could result from relocation of template-primer in the nucleic acid binding cleft. As a consequence, template nucleotide -8 is positioned in the immediate vicinity of the RNase H catalytic center rather than nucleotide -17.