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.

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。我们将Y586F确定为MLV RT核糖核酸酶H（RNase H）引物握把子域中的突变，该突变率增加了大约5倍至5倍，这是报道的替代逆转录病毒突变替代突变的最大增加。 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。为了进一步确定MLV RNase H引物握把对复制忠诚度和病毒复制的影响，我们进行了其他突变分析。结果表明，RNase H引物握把中的突变显着增加了引物结合位点（PBS）和PBS下游序列之间缺失的频率。定量实时PCR分析表明，相对于野生型RT，突变体RT在链DNA转移方面效率低下。这些结果表明，MLV RNase H底漆握把是DNA合成体内保真度的重要决定因素。我们计划确定RT的其他几种结构决定因素的作用，这些决定因素可能影响逆转录的保真度。此外，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。