Abbotts J, Bebenek K, Kunkel T A, Wilson S H
Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
J Biol Chem. 1993 May 15;268(14):10312-23.
During processive DNA synthesis in vitro, the human immunoefficiency virus, type 1 (HIV-1) reverse transcriptase encounters template nucleotide positions at which continued synthesis is difficult. At these positions, the enzyme has a relatively high probability of dissociating from the template, and product molecules of corresponding length accumulate as the incubation proceeds. These positions, which are known as termination sites, could be associated with template secondary structures in some cases, but many termination sites appear to be template sequence-related rather than secondary structure-related. Mechanisms producing these blocks in processive DNA synthesis are not well understood. In this study, to examine further the effects of template sequence on termination, we engineered selected single-base changes in the M13mp2 template, and we found that such changes can influence termination. Several general trends emerged from the study. First, strong termination sites rarely correspond to dATP as the "incoming" substrate opposite template T. Second, the sequence of the template-primer stem is more important for termination than the sequence of the single-stranded template ahead of the primer. Thus, we note the phenomenon of action at a distance: changing sequence at one nucleotide position in the template-primer stem alters termination at other positions, a few nucleotides distant at the primer 3' end. A and C as template bases in the template-primer stem have opposite effects. A is the strongest terminator residue, and C is the weakest terminator residue, followed by G. Since termination sites are produced by reverse transcriptase dissociation from the template-primer, the results suggest that the HIV-1 reverse transcriptase has properties reminiscent of a sequence-specific double-stranded DNA-binding protein in that its binding mechanism can distinguish both base residues and positions in the double-stranded DNA template-primer stem.
在体外进行持续性DNA合成过程中，人类免疫缺陷病毒1型（HIV-1）逆转录酶会遇到模板核苷酸位置，在这些位置上继续合成变得困难。在这些位置，酶从模板上解离的概率相对较高，随着孵育的进行，相应长度的产物分子会积累。这些位置被称为终止位点，在某些情况下可能与模板二级结构有关，但许多终止位点似乎与模板序列相关而非二级结构相关。在持续性DNA合成中产生这些阻断的机制尚未完全了解。在本研究中，为了进一步研究模板序列对终止的影响，我们对M13mp2模板进行了选定的单碱基改变，发现这种改变会影响终止。该研究出现了几个一般趋势。首先，强终止位点很少对应于与模板T相对的“进入”底物dATP。其次，模板引物茎的序列对终止比对引物前单链模板的序列更重要。因此，我们注意到远距离作用现象：改变模板引物茎中一个核苷酸位置的序列会改变引物3'端几个核苷酸远处其他位置的终止。模板引物茎中作为模板碱基的A和C有相反的作用。A是最强的终止残基，C是最弱的终止残基，其次是G。由于终止位点是由逆转录酶从模板引物上解离产生的，结果表明HIV-1逆转录酶具有类似于序列特异性双链DNA结合蛋白的特性，即其结合机制可以区分双链DNA模板引物茎中的碱基残基和位置。
