Yu H, Goodman M F
Department of Biological Sciences, University of Southern California, Los Angeles 90089-1340.
J Biol Chem. 1992 May 25;267(15):10888-96.
A comparison of the fidelity of reverse transcriptases (RT) from human immunodeficiency virus (HIV-1) and avian myeloblastosis virus (AMV) is made using RNA and DNA primer-template molecules in vitro. Selected template target sites containing either uracil or thymine are used to measure nucleotide insertion fidelities and to compare the efficiency of extending mismatched nucleotides at primer 3'-termini. HIV-1 reverse transcriptase is observed to incorporate as many as three consecutive mismatches and to continue efficient elongation from mismatched primer 3'-termini without discernible pausing. Nucleotide misinsertion and mispair extension efficiencies are similar for both enzymes on RNA and DNA templates having identical surrounding sequence. HIV-1 and AMV reverse transcriptases form G.T and G.U mismatches most efficiently, between 1.6 x 10(-4) and 7 x 10(-4), and both enzymes extend G.U with exceptionally high efficiencies, 2.7 x 10(-2) for HIV-1 RT and 4.5 x 10(-2) for AMV RT. Extension of the G.T mismatch is similar for AMV RT (5.8 x 10(-2) but 20-fold less efficient for HIV-1 RT. C.U and C.T mismatches are formed by both enzymes in a frequency range of 4.4 x 10(-5)-2.4 x 10(-4). HIV-1 RT extends these mismatches with slightly higher efficiencies (5.5 x 10(-3)-5.9 x 10(-3)) than AMV RT (5.6 x 10(-4)-2.1 x 10(-3)). Insertion of dTMP opposite U and T occur at about 1 x 10(-4)-2 x 10(-4) for HIV-1 RT. For AMV RT, formation of T.U mispairs occurs with an 8-fold lower efficiency, whereas insertion of dTMP opposite T is not detected. This particular DNA template sequence generates a pause site for AMV RT but not HIV-1 RT. HIV-1 RT dissociation rate constants are about 8-fold larger from a DNA primer bound to a DNA template (0.5 s-1), as compared with an RNA template (0.06 s-1) at one site, and are at most 2-fold larger at another site. The equilibrium binding constant for HIV-1 RT bound to DNA primed RNA and DNA templates appears to be similar, KD approximately 2.5 nM. Values of kpol from 0.3 to 1.5 nucleotides/s are obtained for HIV-1 RT at the RNA and DNA template sites used to measure insertion and extension fidelity. The relatively high efficiency of mispair extension catalyzed by reverse transcriptases with both RNA and DNA templates suggests that a significant component of retroviral genetic variability may be related to the ability of reverse transcriptases to continue efficient synthesis of DNA containing mismatches on both RNA and DNA templates.
利用RNA和DNA引物-模板分子在体外对来自人类免疫缺陷病毒（HIV-1）和禽成髓细胞瘤病毒（AMV）的逆转录酶（RT）保真度进行了比较。使用含有尿嘧啶或胸腺嘧啶的选定模板靶位点来测量核苷酸插入保真度，并比较在引物3'末端延伸错配核苷酸的效率。观察到HIV-1逆转录酶可掺入多达三个连续错配，并从错配的引物3'末端继续高效延伸而无明显停顿。对于具有相同周围序列的RNA和DNA模板，两种酶的核苷酸错插入和错配延伸效率相似。HIV-1和AMV逆转录酶最有效地形成G.T和G.U错配，效率在1.6×10⁻⁴至7×10⁻⁴之间，并且两种酶都以极高的效率延伸G.U，HIV-1 RT为2.7×10⁻²，AMV RT为4.5×10⁻²。AMV RT延伸G.T错配的情况类似（5.8×10⁻²），但HIV-1 RT的效率低20倍。两种酶形成C.U和C.T错配的频率范围为4.4×10⁻⁵至2.4×10⁻⁴。HIV-1 RT延伸这些错配的效率略高于AMV RT（5.5×10⁻³至5.9×10⁻³）（AMV RT为5.6×10⁻⁴至2.1×10⁻³）。HIV-1 RT在U和T相对处插入dTMP的发生率约为1×10⁻⁴至2×10⁻⁴。对于AMV RT，T.U错配的形成效率低8倍，而未检测到T相对处插入dTMP。这种特定的DNA模板序列为AMV RT产生了一个停顿位点，但对HIV-1 RT没有。与RNA模板（0.06 s⁻¹）在一个位点相比，HIV-1 RT从与DNA模板结合的DNA引物上的解离速率常数大约大8倍，在另一个位点最多大2倍。HIV-1 RT与DNA引发的RNA和DNA模板结合的平衡结合常数似乎相似，KD约为2.5 nM。在用于测量插入和延伸保真度的RNA和DNA模板位点，HIV-1 RT的kpol值为0.3至1.5个核苷酸/秒。逆转录酶对RNA和DNA模板催化错配延伸的相对高效率表明，逆转录病毒遗传变异性的一个重要组成部分可能与逆转录酶在RNA和DNA模板上继续高效合成含错配DNA的能力有关。
