REVERSE TRANSCRIPTASE TEMPLATE SWITCHING AND FIDELITY

逆转录酶模板切换和保真度

• 批准号: 2462203
• 负责人: VINAY K. PATHAK
• 金额: $ 18.02万
• 依托单位: WEST VIRGINIA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-01-01 至 2002-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2462203
• 关键词: DNA replication; RNA directed DNA polymerase; Retroviridae; adenine deaminase; antiAIDS agent; enzyme structure; gene mutation; nucleic acid structure; ribonuclease H; virus genetics; virus replication

DESCRIPTION (Adapted from the applicant's abstract): Genetic variation in the retroviral populations depend on the mutation and recombination rates/replication cycle, the rate of replication/time and the selective forces that exist on the population. The high mutational rate of retroviruses is due to the virally encoded reverse transcriptase, which lacks proofreading function and has low processivity. Mutations occur through misincorporation, dislocation mutagenesis, and intramolecular or intermolecular strand switching. The template switching, which is a process required for viral replication, can lead to deletions, duplications, and recombination. Previous studies has defined the mutation rates and the spectrum of mutations possible through viral replication. The current application aims at defining the mechanism that RT switches templates and the structural determinants of RT and the reverse transcription complex that are important for fidelity. Four specific aims are proposed: 1) Utilize retroviral vectors encoding directly repeated sequences to determine in vivo the relative rates of template switching in RNA and DNA- dependent DNA synthesis, and the effects of distance between direct repeats, RNA dimer linkage signal, and template-primer hydrogen bonding on template switching; 2) Perform targeted and random mutational analysis to define structural features of reverse transcriptase and RNase H that are important for accuracy of DNA synthesis. 3) Determine whether environmental factors such as nucleotide pool imbalances and anti-viral drugs affect retroviral mutation rates. 4) Probe the structure of the reverse transcription complex by determining whether DNA synthesis initiates on both co-packaged RNAs and whether minus-strand transfer is promoted by a putative circular structure of the viral RNAs.

描述（改编自申请人的摘要）：遗传变异 逆转录病毒群体取决于突变和重组 速率/复制周期、复制速率/时间和选择性 存在于人口中的力量。 突变率高 逆转录病毒是由病毒编码的逆转录酶引起的， 缺乏校对功能，处理能力低。 发生突变 通过错误掺入、位错诱变和分子内或 分子间链转换。 模板切换，这是一个过程 病毒复制所需的，可能导致删除、重复和 重组。 先前的研究已经定义了突变率和 通过病毒复制可能发生的突变谱。 目前的 应用程序旨在定义 RT 切换模板和 RT 的结构决定因素和逆转录复合物 对于保真度很重要。 提出了四个具体目标： 1) 利用 逆转录病毒载体编码直接重复序列以确定体内 RNA 和 DNA 依赖性 DNA 中模板转换的相对速率 合成，以及直接重复之间距离的影响，RNA二聚体 模板切换时的连锁信号和模板-引物氢键； 2) 进行靶向和随机突变分析以定义结构 逆转录酶和 RNase H 的重要特征 DNA 合成的准确性。 3) 确定是否有环境因素 由于核苷酸库失衡和抗病毒药物影响逆转录病毒 突变率。 4) 探究逆转录复合物的结构 通过确定 DNA 合成是否在共同包装的 RNA 上启动， 负链转移是否是由假定的圆形结构促进的 病毒RNA。