Identification of Structural Determinants of Reverse Tra

反向传输的结构决定因素的识别

• 批准号: 7291894
• 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/7291894
• 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 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.

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。我们确定Y 586 F是MLV RT核糖核酸酶H（RNase H）引物夹亚结构域中的突变，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中最大的置换突变增加。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。为了进一步确定MLV RNase H引物夹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引物夹中的突变显著增加了引物结合位点（PBS）和PBS下游序列之间的缺失频率。定量实时PCR分析表明，突变体RT是基本上无效的正链DNA转移相对于野生型RT。这些结果表明，MLV RNA酶H引物抓地力是一个重要的决定因素，在体内的DNA合成的保真度。我们计划确定RT的其他几个结构决定因素的作用，可能会影响逆转录的保真度。此外，我们正在通过确定突变体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。