Bebenek K, Abbotts J, 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. 1993 May 15;268(14):10324-34.
We have observed previously that DNA template-directed polymerization by the type 1 human immunodeficiency virus reverse transcriptase is error-prone for single-nucleotide substitution, addition and deletion errors at homopolymeric sequences. We have also noted strong termination of processive synthesis at these positions (Bebenek, K., Abbotts, J., Roberts, J. D., Wilson, S. H., and Kunkel, T. A. (1989) J. Biol. Chem. 264, 16948-16956). Here we have tested three models to explain errors at these hot spots: template-primer misalignment for deletion errors, and dislocation and direct miscoding for substitution errors. The approach involves introducing single-nucleotide changes within or flanking the homopolymeric hot spots and examining the effects that these changes have on human immunodeficiency virus type 1 (HIV-1) reverse transcriptase error rate, error specificity, and termination probability. The results obtained suggest that single-nucleotide deletion errors in homopolymeric runs result from template-primer misalignment and that both direct miscoding and template-primer dislocation contribute to the base substitution hot spots. The data also suggest that base substitution errors at one position can be templated by the preceding nucleotide or either of the next two nucleotides. Frameshift error rates at homopolymeric sites were affected by changes in the sequences flanking the runs, including single-nucleotide differences in the single-stranded template strand and in the double-stranded primer region as many as six nucleotides distant from the hot spot. Both increases and decreases in frameshift fidelity were observed, and most of these correlated with concomitant increases or decreases in the probability that HIV-1 reverse transcriptase terminated processive synthesis within the run. These data provide further support for a relationship between the frameshift fidelity and the processivity of DNA-dependent DNA synthesis by HIV-1 reverse transcriptase.
我们之前观察到，1型人类免疫缺陷病毒逆转录酶进行的DNA模板指导的聚合反应在同聚物序列处易发生单核苷酸取代、添加和缺失错误。我们还注意到在这些位置会强烈终止持续合成（贝贝内克，K.，阿博茨，J.，罗伯茨，J.D.，威尔逊，S.H.，和昆克尔，T.A.（1989年）《生物化学杂志》264卷，16948 - 16956页）。在此，我们测试了三种模型来解释这些热点处的错误：缺失错误的模板 - 引物错配模型，以及取代错误的错位模型和直接错配编码模型。该方法包括在同聚物热点内部或其侧翼引入单核苷酸变化，并检查这些变化对1型人类免疫缺陷病毒（HIV - 1）逆转录酶错误率、错误特异性和终止概率的影响。所得结果表明，同聚物序列中的单核苷酸缺失错误是由模板 - 引物错配导致的，并且直接错配编码和模板 - 引物错位都对碱基取代热点有影响。数据还表明，一个位置的碱基取代错误可以由前一个核苷酸或接下来两个核苷酸中的任何一个作为模板。同聚物位点的移码错误率受到序列侧翼变化的影响，包括单链模板链以及距离热点多达六个核苷酸的双链引物区域中的单核苷酸差异。观察到移码保真度既有增加也有降低，并且其中大多数与HIV - 1逆转录酶在该序列内终止持续合成的概率的相应增加或降低相关。这些数据进一步支持了HIV - 1逆转录酶进行的DNA依赖DNA合成的移码保真度与持续合成能力之间的关系。
