Examining the ribonuclease H primer grip of HIV-1 reverse transcriptase by charge neutralization of RNA/DNA hybrids.
Examining the ribonuclease H primer grip of HIV-1 reverse transcriptase by charge neutralization of RNA/DNA hybrids.
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DOI：
10.1093/nar/gkn678
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发表时间：
2008-11
影响因子：
14.9
通讯作者：
Le Grice, Stuart F. J.
中科院分区：
文献类型：
--
作者：
Dash, Chandravanu;Scarth, Brian J.;Badorrek, Christopher;Goette, Matthias;Le Grice, Stuart F. J.
与RNA/DNA杂交体结合的人类免疫缺陷病毒1型（HIV-1）逆转录酶（RT）的晶体结构揭示了与核糖核酸酶H（RNase H）催化中心下游约4-9 bp的DNA链磷酸骨架的广泛接触网络。该基序被统称为“RNA酶H引物夹”，含有类似于人和耐盐芽孢杆菌RNA酶H的磷酸盐结合口袋。RNA酶H引物夹介导RNA/DNA杂交体进入RNA酶H活性位点的轨迹的概念表明，可以利用局部中和磷酸骨架来操纵核酸柔性。为了检验这一点，我们通过与RNase H引物夹接触的DNA引物区域引入了单个和串联的甲基膦酸酯取代，并进入RNase H催化中心。此后检查了突变杂交体支持RNA酶H和DNA聚合酶活性的能力。此外，位点特异性化学足迹法用于评估DNA聚合酶和RNA酶H结构域的移动。我们在这里表明，微小的改变，RNA酶H引物可以有一个显着的影响酶的定位，并讨论了这些发现，在最近的结晶学的人RNA酶H含有RNA/DNA杂交。
The crystal structure of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) bound to an RNA/DNA hybrid reveals an extensive network of contacts with the phosphate backbone of the DNA strand ∼4–9 bp downstream from the ribonuclease H (RNase H) catalytic center. Collectively designated as ‘the RNase H primer grip’, this motif contains a phosphate binding pocket analogous to the human and Bacillus halodurans RNases H. The notion that the RNase H primer grip mediates the trajectory of RNA/DNA hybrids accessing the RNase H active site suggests that locally neutralizing the phosphate backbone may be exploited to manipulate nucleic acid flexibility. To examine this, we introduced single and tandem methylphosphonate substitutions through the region of the DNA primer contacted by the RNase H primer grip and into the RNase H catalytic center. The ability of mutant hybrids to support RNase H and DNA polymerase activity was thereafter examined. In addition, site-specific chemical footprinting was used to evaluate movement of the DNA polymerase and RNase H domains. We show here that minor alteration to the RNase H primer can have a dramatic effect on enzyme positioning, and discuss these findings in light of recent crystallography of human RNase H containing an RNA/DNA hybrid.
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