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.

影响体内逆转录屈服的逆转录酶（RT）的结构特征尚未很好地定义。我们进行了体内研究，以鉴定鼠白血病病毒（MLV）RT的结构决定因素，通过分析YXDD基序中突变的影响和DNTP结合位点对DNA合成的准确性，并鉴定出增加逆转突变率的替代方案，从而影响了忠诚度。我们还确定Y586F是MLV RT核糖核酸酶H（RNase H）引物握把子域中的突变，增加了大约5倍至5倍的突变率，这是报道的体内逆转录病毒突变率最大的增加。 Y586F突变增加了在腺嘌呤 - 胸腺氨酸区域18 nt（a-tracts）内的替代频率，从而诱导DNA弯曲。这些结果表明，当野生型RT遇到不规则的模板构象构象时，例如由A诱导的A-586残基和RNase H引物握柄域诱导的构象，这促进了一种模板标准构构，这是维持高忠诚DNA合成所必需的。 为了阐明RT保真度的机制，我们正在尝试识别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 RNase H底漆子域在DNA合成的保真度中的作用。我们还计划确定模板 - 提示结构在RT保真度中的作用。最后，我们通过确定突变器RT和诱变核苷类似物对MLV和HIV-1的复制和进化的影响来测试误差灾难假设。