FIDELITY OF RETROVIRAL REVERSE TRANSCRIPTASE

这个项目调查了高突变率的最可能的原因。 在HIV-1基因组的某些部分，不准确的DNA合成 HIV-1逆转录酶(RT)。大多数在试管中犯下的错误是 由链条滑移引发。为了深入了解为什么 HIV-1 RT对于这些类型的错误是如此容易出错，我们正在检查 RT的突变衍生物，重点是据信 对模板和底物的相互作用很重要。含丙氨酸的突变体 拇指α螺旋H上四种特定氨基酸的替代 亚区和一个手掌残基都降低了DNA结合亲和力， 对AZTTP的抵抗、处理能力降低和/或帧移位减少 富达。这些表型与分子模型一致 研究表明，这五种氨基酸构成了一种“疏水性” 与模板底漆接触的“小槽跟踪模块” 从活动站点。我们还分析了I螺旋中的突变和 表明该元素中的单个变化在动力学上是静默的，在 与“螺旋钳”概念的预测形成对比的是 文学。这些野生型和突变型反转基因的分析 转录酶将增强我们对RTS如何与 它们的底物，并可能提供对超可变的洞察 艾滋病病毒。由于这些研究侧重于链导致的突变 它们还可能提供与几个人类相关的见解 以可能由链滑动导致的突变为特征的疾病 在DNA复制过程中。

This project investigates the most likely cause for high mutation rates within certain portions of the HIV-1 genome, inaccurate DNA synthesis by the HIV-1 reverse transcriptase (RT). Most mistakes made in vitro are initiated by strand slippage. In order to gain insight into why the HIV-1 RT is so error-prone for these types of mistakes, we are examining mutant derivatives of the RT, with emphasis on amino acids believed to be important for template-primer interactions. Mutants with alanine substituted for four specific amino acids in alpha helix H of the thumb subdomain and one palm residue all have reduced DNA binding affinity, resistance to AZTTP, reduced processivity and/or reduced framshift fidelity. These phenotypes are consistent with molecular modeling studies suggesting that these five amino acids comprise a "hydrophobic minor groove tracking module" that contacts the template-primer just back from the active site. We have also analyzed mutants in the I helix and shown that single changes in this element are kinetically silent, in contrast to the predictions of the "helix-clamp" concept in the literature. Analysis of these wild-type and mutant reverse transcriptases will enhance our understanding of how RTs interact with their substrates and may provide insights into the hypermutability of the AIDS virus. Since these studies focus on mutations resulting from strand slippage, they may also provide insights relevant to several human diseases characterized by mutations that may result from strand slippage during DNA replication.