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.

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。我们进行了体内研究，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，确定了影响MLV RT保真度的结构决定因素，并发现了增加逆转录病毒突变率的替换。我们还发现Y586F是MLV RT核糖核酸酶H （RNase H）引物控制亚域的一个突变，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的最大增幅。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。