Mechanism and fidelity of HIV reverse transcriptase.
Mechanism and fidelity of HIV reverse transcriptase.
复制标题
DOI：
10.1016/s0021-9258(18)35706-5
复制
发表时间：
1992-12
期刊：
影响因子：
--
通讯作者：
W. Kati;K. Johnson;L. F. Jerva;K. Anderson
中科院分区：
文献类型：
--
作者：
W. Kati;K. Johnson;L. F. Jerva;K. Anderson
我们用确定的合成25/45聚合体DNA/RNA和DNA/DNA引物/模板，采用快速瞬变动力学方法检测了人类免疫缺陷病毒逆转录酶的RNA依赖性和DNA依赖性聚合酶和核糖核酸酶H催化活性。酶与双工DNA相互作用的Kd值为4.7 nM，与RNA/DNA异双工的Kd值相似。在DNA和RNA模板中观察到三磷酸核苷结合的预稳态爆发。dATP浓度对爆发率的依赖性分析显示，DNA和RNA模板的dATP的Kd值分别为4和14微米，单核苷酸结合的最大速率kpol分别为33和74秒-1。对于双工DNA和RNA/DNA异双工，引物/模板从酶解离的速率分别为0.18和0.06 s-1，限制了随后的翻转。利用RNA模板对DNA聚合速率和RNA裂解速率进行分析，发现这两种活性是相互独立的。聚合速率（4-70 s-1）依赖于dATP浓度，而在100倍dATP浓度范围内（2-200微米），RNA的切割速率为10 s-1。对由单次转换产生的RNA切割产物的检测表明，该酶的聚合酶和核糖核酸酶结构域之间的距离相当于RNA/DNA异双工的19个碱基，与最近发表的晶体结构一致（Kohlstaedt， L. a ., Wang， J., Friedman， J., Rice， P. a .和Steitz， T. a . (1992) Science 256, 1783-1790）。过程合成动力学分析表明，dNTP的初始结合导致DNA从酶解离的速度更快。进一步的研究支持两步dNTP结合机制，即初始E' dna .dNTP复合物的形成，然后是更稳定的E'. dna。核苷酸复杂。与DNA模板胸苷相反的不正确三磷酸核苷结合的Kd值为dGTP为1010微米，dCTP为1240微米，dtp为840微米。dGTP的最大kpol速率为4.8 s-1， dCTP为0.52 s-1， dtp为0.41 s-1。这些值提供了该站点对dGTP歧视的保真度估计为1740，对dCTP的保真度估计为19,700，对dtp错误合并的保真度估计为16,900。
We have examined the RNA-dependent and DNA-dependent polymerase and ribonuclease H catalytic activities of human immunodeficiency virus reverse transcriptase using rapid transient kinetic methods with defined synthetic 25/45-mer DNA/RNA and DNA/DNA primer/templates. The Kd value for interaction of the enzyme with duplex DNA was 4.7 nM, and the value for RNA/DNA heteroduplex was of similar magnitude. A pre-steady state burst of nucleoside triphosphate incorporation was observed for both DNA and RNA templates. Analysis of the dATP concentration dependence of the burst rate provided Kd values for dATP of 4 and 14 microM and maximum rates of single nucleotide incorporation, kpol, of 33 and 74 s-1, for DNA and RNA templates, respectively. Subsequent turnovers were limited by the rate of dissociation of the primer/template from the enzyme at rates of 0.18 and 0.06 s-1 for duplex DNA and RNA/DNA heteroduplex, respectively. Analysis of rates of DNA polymerization and RNA cleavage using the RNA template revealed that the two activities are independent of one another. The polymerization rate (4-70 s-1) was dependent on dATP concentration, whereas the RNA cleavage occurred at a constant rate of 10 s-1 over the 100-fold dATP concentration range (2-200 microM). Examination of the RNA cleavage products resulting from a single turnover indicates that the polymerase and ribonuclease domains of the enzyme are separated by a distance corresponding to 19 bases of RNA/DNA heteroduplex, consistent with the recently published crystal structure (Kohlstaedt, L. A., Wang, J., Friedman, J., Rice, P. A., and Steitz, T. A. (1992) Science 256, 1783-1790). Analysis of the kinetics of processive synthesis suggested that the initial binding of dNTP leads to a faster rate of dissociation of DNA from the enzyme. Further investigation supported a two-step dNTP binding mechanism with the formation of an initial E.DNA.dNTP complex followed by a more stable E'.DNA.dNTP complex. The Kd values for incorporation of incorrect nucleoside triphosphates opposite a DNA template thymidine were 1010 microM for dGTP, 1240 microM for dCTP, and 840 microM for dTTP. The corresponding maximum kpol rates were 4.8 s-1 for dGTP, 0.52 s-1 for dCTP, and 0.41 s-1 for dTTP. These values provide fidelity estimates of 1740 for discrimination against dGTP, 19,700 for dCTP, and 16,900 for dTTP misincorporations at this site.