Identification of Structural Determinants of Reverse Tra

影响体内逆转录保真度的逆转录酶（RT）的结构特征尚未得到很好的定义。我们发现Y586F是MLV RT核糖核酸酶H （RNase H）引物控制亚域的突变，该突变将突变率提高了约5倍，这是迄今为止报道的体内逆转录病毒突变率中最大的替代突变。Y586F突变使腺嘌呤-胸腺嘧啶束（a -束）在18 nt内的替换频率增加了17倍，从而诱导DNA弯曲。这些结果表明，当野生型RT遇到不规则的模板-引物构象时，例如由a链诱导的构象，Y586残基和RNase H引物控制域促进了模板-引物构象，这是维持DNA合成高保真度所必需的。为了进一步确定MLV RNase H引物对复制保真度和病毒复制的影响，我们进行了额外的突变分析。结果表明，RNase H引物握柄的突变显著增加了引物结合位点（PBS）与PBS下游序列之间的缺失频率。实时荧光定量PCR分析表明，与野生型rt相比，突变体rt在正链DNA转移方面效率低下。这些结果表明，MLV RNase H引物的握力是体内DNA合成保真度的重要决定因素。我们计划确定可能影响逆转录保真度的RT的其他几个结构决定因素的作用。此外，我们正在通过确定突变子RTs和诱变核苷类似物对MLV和HIV-1的复制和进化的影响来验证错误突变假说。

Structural features of reverse transcriptase (RT) that influence fidelity of reverse transcription in vivo are not well defined. We identified Y586F as a mutation in the MLV RT ribonuclease H (RNase H) primer grip subdomain that increased the mutation rate approximately fivefold-to date, the largest reported increase for a substitution mutation in the in vivo retroviral mutation rate. The Y586F mutation increased the frequency of substitutions 17-fold within 18 nt of adenine-thymine tracts (A-tracts), which induce DNA bending. These results suggest that when wild-type RT encounters irregular template-primer conformations such as those induced by A-tracts, the Y586 residue and the RNase H primer grip domain facilitate a template-primer conformation that is necessary for maintaining high fidelity of DNA synthesis. To further determine the effects of MLV RNase H primer grip on replication fidelity and viral replication, we performed additional mutational analysis. The results indicated that mutations in the RNase H primer grip significantly increased the frequency of deletions between the primer-binding site (PBS) and sequences downstream of the PBS. Quantitative real-time PCR analysis indicated that the mutants RTs were substantially inefficient in plus-strand DNA transfer relative to wild-type RT. These results indicate that the MLV RNase H primer grip is an important determinant of the in vivo fidelity of DNA synthesis. We plan to determine the role of several other structural determinants of RT that might influence fidelity of reverse transcription. In addition, we are testing the error catastrophe hypothesis by determining the effects of mutator RTs and mutagenic nucleoside analogs on replication and evolution of MLV and HIV-1.