Bebenek K, Abbotts J, Roberts J D, Wilson S H, Kunkel T A
Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709.
J Biol Chem. 1989 Oct 5;264(28):16948-56.
DNA-dependent DNA synthesis in vitro by human immunodeficiency virus-1 reverse transcriptase is relatively error-prone. The enzyme, whether recombinant or from virus particles, produces errors while replicating M13mp2 DNA at a rate that, if operative in vivo, would produce about five mutations per genome per round of replication. Sequence analysis of mutants resulting from in vitro synthesis demonstrates that errors are nonrandomly distributed. Base substitution and one-base frameshift mutational hot spots are observed. The specificity and position of errors suggest that most of the frameshifts and many of the base substitutions are initiated by template-primer slippage. Processivity analysis for the enzyme on the M13mp2 DNA template reveals strong termination at specific sites. Termination sites within homopolymer sequences correlate with frameshift mutational hot spots. The results suggest that the formation and/or utilization of misaligned template-primers is increased during the dissociation-reinitiation phase of the reaction.
人免疫缺陷病毒1型逆转录酶在体外进行的依赖DNA的DNA合成相对容易出错。该酶无论是重组的还是来自病毒颗粒，在复制M13mp2 DNA时都会产生错误，其速率如果在体内起作用，每轮复制每个基因组会产生约五个突变。对体外合成产生的突变体进行序列分析表明，错误是非随机分布的。观察到碱基替换和单碱基移码突变热点。错误的特异性和位置表明，大多数移码和许多碱基替换是由模板-引物滑动引发的。对该酶在M13mp2 DNA模板上的持续合成能力分析揭示了在特定位点的强烈终止。同聚物序列内的终止位点与移码突变热点相关。结果表明，在反应的解离-重新起始阶段，错配模板-引物的形成和/或利用增加。
