FIDELITY OF RETROVIRAL REVERSE TRANSCRIPTASE

人类免疫缺陷病毒（HIV-1）在体内有很高的突变率。 基因组的某些部分，允许新形式的快速进化 对药物治疗有抵抗力或能逃避 宿主的免疫反应。 这个项目调查了最有可能的原因 HIV-1逆转录酶的DNA合成不准确 （RT）。 RT的错误率足以解释病毒 可变性 在体外所犯的大多数错误都是由 模板和引物链，也许当聚合酶解离时， 然后与DNA重新结合 为了深入了解 HIV-1 RT对于这些类型的错误是如此容易出错， 检查RT的突变衍生物，重点是氨基酸 被认为对于模板-引物相互作用是重要的。 突变体 丙氨酸取代的α螺旋H中的两个特定氨基酸 在位置3、4和5处接触引物链的拇指亚结构域 从3’OH末端返回的核苷酸具有降低的DNA结合 亲和力、对AZTTP的抗性、持续合成能力降低和移码减少 忠诚 这些氨基酸可能在次要的 模板引物的凹槽，这是一个假设，正在检查 确定额外的氨基酸取代在这些位点的作用， 关键残留物 基于X射线测定的RT结构 晶体学，研究也正在进行中，以分析突变体在我 螺旋和“引物抓地力”。 的比较测定 HIV-2和SIV RT的保真度和持续合成能力已经完成。 对这些野生型和突变型逆转录酶的分析将 增强我们对RT如何与其底物相互作用的理解， 或许能让我们更深入地了解艾滋病病毒的超变异性。 以来 这些研究集中在由链滑移引起的突变上， 还提供了与几种人类疾病相关的见解， 由DNA复制过程中的链滑动引起的突变引起。 这包括几种类型的癌症和遗传性疾病。

The human immunodeficiency virus (HIV-1) has high mutation rates within certain portions of its genome, permitting rapid evolution of new forms of the the virus that are resistant to drug treatments or can evade the host's immune response. This project investigates the most likely cause for this, inaccurate DNA synthesis by the HIV-1 reverse transcriptase (RT). The RT makes mistakes at a rate sufficient to explain viral variability. Most mistakes made in vitro are initiated by slippage of the template and primer strands, perhaps when the polymerase dissociates and then reassociates with the DNA. 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 two specific amino acids in alpha helix H of the thumb subdomain that contact the primer strand at positions 3, 4 and 5 nucleotides back from the 3'OH terminus have reduced DNA binding affinity, resistance to AZTTP, reduced processivity and reduced framshift fidelity. These amino acids may make important contacts in the minor groove of the template-primer, a hypothesis that is being examined by determining the effects of additional amino acid substitutions at these key residues. Based on RT structure as determined by X-ray crystallography, studies are also underway to analyze mutants in the I helix and in the "primer grip". Comparative determinations of the fidelity and processivity of HIV-2 and SIV RTs have been completed. 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. This includes several types of cancer and hereditary diseases.