K65R and K65A substitutions in HIV-1 reverse transcriptase enhance polymerase fidelity by decreasing both dNTP misinsertion and mispaired primer extension efficiencies.
K65R and K65A substitutions in HIV-1 reverse transcriptase enhance polymerase fidelity by decreasing both dNTP misinsertion and mispaired primer extension efficiencies.
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DOI：
10.1016/j.jmb.2010.06.001
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发表时间：
2010-08-06
影响因子：
5.6
通讯作者：
Prasad VR
中科院分区：
文献类型：
--
作者：
Garforth SJ;Domaoal RA;Lwatula C;Landau MJ;Meyer AJ;Anderson KS;Prasad VR
Lys65残基位于HIV逆转录酶（RT）的手指结构域，与传入的dNTP以序列独立的方式相互作用。之前，我们发现跨越Lys65的5个氨基酸的缺失和K65A的替换都提高了dNTP插入的保真度。我们假设Lys65残基通过与传入dNTP的γ-磷酸相互作用增强了dNTP误插入。我们现在用野生型HIV-1 RT和两个替代突变体K65A和K65R在稳态前动力学研究中检验了这一假设。K65R突变没有显著增加误插入保真度，但K65A突变导致更高的插入保真度。误插入要成为永久性错误，就必须伴随着由此形成的错配末端的延伸。两种突变体和野生型酶在催化步骤（kpol）上都对不匹配的引物进行了区分。此外，K65A和K65R突变体的错配扩展效率进一步降低，主要是在dNTP结合水平上。我们使用羟基自由基足迹来确定RT在引物模板上的位置。野生型酶和Lys65取代酶在引物末端占据相同的位置；不匹配的引物末端的存在导致所有三种酶相对于引物3 '端移位到−2位置。在有效延长错配末端的情况下，下一个互补核苷酸的存在克服了位移，导致类似于匹配末端的复合体。结果与在错配引物延伸中观察到的kpol减少是一致的，这是由于酶在错配末端的位置。我们的工作显示了稳定Lys65与传入的dNTP相互作用对聚合酶保真度的两个不同方面的影响。
The Lys65 residue, in the fingers domain of HIV reverse transcriptase (RT), interacts in a sequence independent fashion with the incoming dNTP. Previously, we showed that a 5 amino acid deletion spanning Lys65 and a K65A substitution both enhanced the fidelity of dNTP insertion. We hypothesized that the Lys65 residue enhances dNTP misinsertion via interactions with the γ-phosphate of the incoming dNTP. We now examine this hypothesis in pre-steady state kinetic studies using wild type HIV-1 RT and two substitution mutants: K65A and K65R. The K65R mutation did not greatly increase misinsertion fidelity, but the K65A mutation led to higher incorporation fidelity. For a misinsertion to become a permanent error, it needs to be accompanied by the extension of the mispaired terminus thus formed. Both mutants, and the wild-type enzyme, discriminated against the mismatched primer at the catalytic step (kpol). Additionally, K65A and K65R mutants displayed a further decrease in mismatch extension efficiency, primarily at the level of dNTP binding. We employed hydroxyl radical footprinting to determine the position of the RT on the primer-template. The wild-type and Lys65 substituted enzymes occupied the same position at the primer terminus; the presence of a mismatched primer terminus caused all three enzymes to be displaced to a −2 position relative to the primer 3’ end. In the context of an efficiently extended mismatched terminus, the presence of the next complementary nucleotide overcame the displacement, resulting in a complex resembling the matched terminus. The results are consistent with the observed reduction in kpol in mispaired primer extension being due to the position of the enzyme at a mismatched terminus. Our work shows the influence of stabilizing interactions of Lys65 with the incoming dNTP in affecting two different aspects of polymerase fidelity.
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影响因子：
14.9
作者：
Gutiérrez-Rivas, M;Menéndez-Arias, L
通讯作者：
Menéndez-Arias, L
影响因子：
4.8
作者：
Götte, M;Maier, G;Heumann, H
通讯作者：
Heumann, H
影响因子：
4.8
作者：
Fisher, TS;Prasad, VR
通讯作者：
Prasad, VR
DOI：
10.1016/j.biocel.2004.02.029
发表时间：
2004-09-01
影响因子：
4
作者：
Marchand, B;Götte, M
通讯作者：
Götte, M
DOI：
10.1073/pnas.86.21.8343
发表时间：
1989-11-01
影响因子：
11.1
作者：
PERRINO, FW;PRESTON, BD;LOEB, LA
通讯作者：
LOEB, LA