FIDELITY OF RETROVIRAL REVERSE TRANSCRIPTASES

因为人类免疫缺陷病毒（HIV-1）的序列变异 很可能是由于逆转录酶的不准确复制， 我们试图了解逆转的机制， 转录酶错误。 我们已经证明这种酶是容易出错的 在DNA和RNA模板复制过程中， RNA比DNA高几倍。 不平等的错误率， RNA和DNA模板表明，在负链和正链 DNA合成可能对HIV-1的突变率没有同等的贡献。 我们专注于模板引物错配介导的RT错误。 均聚物位点的移码错误率受以下因素的影响： 运行侧翼的序列，包括单核苷酸差异 在单链模板链和双链引物中 距离热点多达六个核苷酸的区域。 两 观察到移码保真度的增加和减少，并且大多数 与伴随的概率增加或减少相关， HIV-1 RT在运行中终止了进行性合成。 强 终止位点很少对应于dATP作为"传入"底物 相对模板T，模板中的特定位置引物茎区 比前面模板的顺序更重要 改变引物中的一个核苷酸位置， 模板引物茎在几个核苷酸的位置改变终止 远处 这些结果表明，HIV-1 RT区分两种碱基， 模板中的残基和位置底火杆，并接触 远离活性位点对于保真度是重要的。 我们将 继续侧重于阐明负责 HIV-1 RT的不准确性，希望这将提供深入了解 酶的活性位点与其底物的相互作用， 在设计RT靶向药物方面很有用。

Because sequence variation in the human immunodeficiency virus (HIV-1) most likely results from inaccurate replication by reverse transcriptase, we are attempting to understand the mechanisms responsible for reverse transcriptase errors. We have shown that this enzyme is error-prone during both DNA- and RNA-templated replication, but that fidelity is several-fold higher with RNA than with DNA. The unequal error rates with RNA and DNA templates suggest that mistakes during minus- and plus-strand DNA synthesis may not contribute equally to the mutation rate of HIV-1. We have focused on templateprimer misalignment-mediated RT errors. Frameshift error rates at homopolymeric sites are affected by changes in the sequence 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 are observed, and most correlate with concomitant increases or decreases in the probability that HIV-1 RT terminated processive synthesis within the run. Strong termination sites rarely correspond to dATP as the "incoming" substrate opposite template T, specific positions in the templateprimer stem are more important for termination than the sequence of the template ahead of the primer, and changing one nucleotide position in the template.primer stem alters termination at positions a few nucleotides distant. These results suggest that the HIV-1 RT distinguishes both base residues and positions in the templateprimer stem, and that contacts well-removed from the active site are important for fidelity. We will continue to focus on elucidating the mechanisms responsible for the inaccuracy of HIV-1 RT, in the hope that this will provide insights into the interaction of the enzyme's active site with its substrates that may be useful in designing RT-targeted drugs.