Identification of Structural Determinants of Reverse Tra

反向传输的结构决定因素的识别

• 批准号: 6952145
• 负责人: VINAY K. PATHAK
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6952145
• 关键词: Identification; Structural; Determinants; Reverse; Tra

Structural features of reverse transcriptase (RT) that influence fidelity of reverse transcription in vivo are not well defined. We performed in vivo studies to identify structural determinants of murine leukemia virus (MLV) RT that influence fidelity by analyzing the effects of mutations in the YXDD motif and the dNTP binding site on the accuracy of DNA synthesis, and identified substitutions that increased the retroviral mutation rate. We also 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 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 elucidate mechanisms of RT fidelity, we are attempting to identify and characterize structural determinants of MLV and HIV-1 RTs that affect the retroviral mutation rate. We are examining the role of the MLV and HIV-1 RNase H primer grip subdomains in fidelity of DNA synthesis through mutational analysis. We also plan to determine the role of template-primer structure in RT fidelity. Finally, 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.

逆转录酶（RT）的结构特征，影响逆转录在体内的保真度没有得到很好的定义。我们进行了体内研究，以确定影响保真度的鼠白血病病毒（MLV）RT的结构决定因素，通过分析YXDD基序和dNTP结合位点突变对DNA合成准确性的影响，并确定了增加逆转录病毒突变率的取代。我们还确定Y 586 F是MLV RT核糖核酸酶H（RNase H）引物夹亚结构域的突变，该突变使突变率增加约5倍，这是迄今为止报告的体内逆转录病毒突变率的最大增加。Y 586 F突变使腺嘌呤-胸腺嘧啶束（A-束）18 nt内的取代频率增加了17倍，这会诱导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，如那些诱导的A-tracts，Y 586残基和RNase H引物抓持域促进模板引物构象，这是必要的保持高保真的DNA合成。 为了阐明RT保真度的机制，我们正试图确定和表征影响逆转录病毒突变率的MLV和HIV-1 RT的结构决定因素。我们正在通过突变分析研究MLV和HIV-1 RNA酶H引物夹亚结构域在DNA合成保真度中的作用。我们还计划确定模板-引物结构在RT保真度中的作用。最后，我们通过确定突变体RT和诱变核苷类似物对MLV和HIV-1复制和进化的影响来检验错误突变假说。