FIDELITY OF RETROVIRAL REVERSE TRANSCRIPTASE

人类免疫缺陷病毒(HIV-1)在 其基因组的某些部分，允许新形式的快速进化 对药物治疗有抵抗力或可以逃避 宿主的免疫反应。这个项目调查了最有可能的原因 为此，HIV-1逆转录酶合成的DNA不准确 (RT)。RT犯错误的比率足以解释病毒 可变性。大多数在试管中犯的错误都是由细胞的滑脱引起的 模板和引物链，可能是当聚合酶解离时 然后与DNA重新关联。为了深入了解为什么 HIV-1 RT很容易犯这些类型的错误，我们 检查RT的突变衍生物，重点是氨基酸 被认为对模板-引物相互作用很重要。突变体具有 丙氨酸取代α螺旋H上的两个特定氨基酸 在位置3、4和5处与引物链接触的拇指亚区 从3‘OH端返回的核苷酸减少了DNA结合 亲和力、对AZTTP的抵抗力、处理能力降低和帧偏移减少 富达。这些氨基酸可能在辅助性疾病中起着重要的作用 模板-底漆的凹槽，这是一个正在进行检验的假设 确定这些额外氨基酸取代的影响 关键残留物。基于X射线确定的RT结构 结晶学方面，分析i中突变的研究也在进行中 螺旋和“底漆握把”中。比较确定的方法 HIV-2和SIV RT的保真度和处理已经完成。 对这些野生型和突变型逆转录酶的分析将 加深我们对RTS如何与其底物相互作用的理解 可能为了解艾滋病病毒的高度变异性提供洞察力。自.以来 这些研究集中在链滑动导致的突变上，他们可能 还提供了与几种人类疾病相关的见解 可能由DNA复制过程中的链滑移引起的突变。 这包括几种癌症和遗传性疾病。

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.