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首页> 外文学位 >Interactions between HIV-1 reverse transcriptase and chain-terminated primer/template: Discovery of a novel primer unblocking activity involved in resistance to AZT.
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Interactions between HIV-1 reverse transcriptase and chain-terminated primer/template: Discovery of a novel primer unblocking activity involved in resistance to AZT.

机译：HIV-1逆转录酶和链终止引物/模板之间的相互作用：发现了一种新型的引物解封闭活性，涉及对AZT的抗性。

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摘要

HIV-1 reverse transcriptase is the key enzyme responsible for replication of the HIV-1 genome. It acts as an RNA-dependent DNA polymerase, an RNaseH, and a DNA-dependent DNA polymerase to convert the HIV-1 positive single stranded RNA into double stranded DNA that is incorporated by HIV-1 integrase into the host call genome. DNA polymerization by HIV-1 RT is a sequential process in which the rate limiting step is a conformational change that occurs after binding of the dNTP complementary to the next nucleotide position on the template, but before phosphodiester bond formation.;Due to its vital role in the HIV-1 life cycle, HIV-1 RT has been one of the main targets of pharmaceutical intervention. Inhibitors of HIV-1 RT include analogs of deoxynucleosides which lack a functional 3' -OH and are phosphorylated by host-cell kinases to their active triphosphorylated form. Once the nucleotide analog is incorporated, the kick of a functional 3'-OH on the primer terminus blocks any further elongation of the primer (chain-terminators). Resistance to 3'-azido,3 '-deoxythymidine (AZT) does not seem to involve discrimination by mutant RT against AZTTP. This suggests that perhaps resistance instead is due to increased removal of AZTMP from blocked primers.;The main goal of my research was to better understand the interactions that occur between HIV-1 RT and chain-terminated primer/template in the absence and presence of the dNTP complementary to the nod nucleotide position (+1) on the template. I used DNAse-1 protection assays to probe the binding of HIV-1 RT to primer/template. The pattern of protection suggests that RT slides forward by one base relative to the primer/template in the presence of the dNTP complementary to the nucleotide at template position +1, and by two bases in the presence of dNTP complementary to the nucleotide at template position +2. The forward movement by RT suggests that the translocation stop of polymerization occurs before chemical bond formation, not after, which is the commonly held belief.;While doing DNase-1 protection assays I uncovered a novel, nucleotide-dependent activity of HIV-1 RT that can unblock chain-terminated primers. The enzyme is capable of transferring the 3'-terminal nucleotide from the primer terminus to a free acceptor nucleotide, such as ATP. The mechanism of the reaction is related to pyrophosphorolysis, but instead of using PPi as a substrate, HIV-1 RT uses the distal two phosphate groups of the acceptor nucleotide as a PPi analog. Importantly, the presence of the dNTP complementary to the nucleotide at the +1 position on the template strongly inhibits the novel unblocking reaction. This indicates that chain-terminated primer/template is not a substrate for the unblocking reaction while present in the stable DEC.;Experiments will be presented that show that the mechanism of AZT-resistance involves both increased unblocking of AZTMP-terminated primers through the novel nucleotide-dependent mechanism, but not through pyrophosphorolysis, as well as decreased sensitivity to inhibition by the complementary dNTP.;Although mutant RT can remove either 2',3 '-dideoxyadenosine monophosphate (ddAMP) or AZTMP through nucleotide-dependent primer unblocking more efficiently than WT RT, removal of ddAMP is strongly inhibited by the presence of complementary dNTP, while removal of AZTMP is insensitive to the presence of dNTPs. (Abstract shortened by UMI.)
机译：HIV-1逆转录酶是负责HIV-1基因组复制的关键酶。它充当RNA依赖的DNA聚合酶，RNaseH和DNA依赖的DNA聚合酶，将HIV-1阳性单链RNA转换成双链DNA，并通过HIV-1整合到宿主调用基因组中。通过HIV-1 RT进行的DNA聚合是一个顺序过程，其中限速步骤是构象变化，发生在dNTP与模板上下一个核苷酸位置互补后结合，但在磷酸二酯键形成之前发生。在HIV-1生命周期中，HIV-1 RT已成为药物干预的主要目标之一。 HIV-1 RT的抑制剂包括缺乏功能性3'-OH的脱氧核苷类似物，并被宿主细胞激酶磷酸化为活性三磷酸化形式。一旦掺入了核苷酸类似物，引物末端上的功能性3'-OH的反冲作用会阻止引物（链终止剂）的进一步延伸。对3'-叠氮基，3'-脱氧胸苷（AZT）的抗性似乎不涉及突变RT对AZTTP的区分。这表明耐药性可能是由于增加了从封闭引物上去除AZTMP所致。我的主要研究目的是更好地了解在不存在和存在以下条件的情况下，HIV-1 RT与链终止引物/模板之间发生的相互作用。 dNTP与模板上的nod核苷酸位置（+1）互补。我使用DNAse-1保护试验来检测HIV-1 RT与引物/模板的结合。保护模式表明，在与模板位置+1的核苷酸互补的dNTP存在下，RT相对于引物/模板向前滑动一个碱基，在与模板位置的核苷酸互补的dNTP存在下RT向前滑动两个碱基。 +2。 RT的向前运动表明，聚合的易位停止发生在化学键形成之前，而不是之后，这是普遍认为的观点。；在进行DNase-1保护试验时，我发现了一种新的，核苷酸依赖性的HIV-1 RT活性。可以解开链终止的引物。该酶能够将3'末端核苷酸从引物末端转移至游离受体核苷酸，例如ATP。该反应的机理与焦磷酸解有关，但是HIV-1 RT不用受体PPi作为底物，而是使用受体核苷酸的远端两个磷酸基团作为PPi类似物。重要的是，与模板+1位核苷酸互补的dNTP的存在强烈抑制了新的解封反应。这表明链终止的引物/模板虽然存在于稳定的DEC中，但不是解封闭反应的底物。;实验将显示AZT抗性的机制涉及通过新颖的方式增加AZTMP终止的引物的解封闭核苷酸依赖性机制，但不是通过焦磷酸解，也不是通过互补dNTP抑制作用的敏感性降低；尽管突变体RT可以通过核苷酸依赖性引物更有效地解封闭来去除2'，3'-二脱氧腺苷单磷酸（ddAMP）或AZTMP。与WT RT相比，互补dNTP的存在强烈抑制ddAMP的去除，而AZTMP的去除对dNTP的存在不敏感。 （摘要由UMI缩短。）

著录项

  • 作者

    Meyer, Peter Robert.;

  • 作者单位

    University of Miami.;

  • 授予单位 University of Miami.;
  • 学科 Biology Molecular.;Chemistry Biochemistry.;Health Sciences Immunology.
  • 学位 Ph.D.
  • 年度 1999
  • 页码 112 p.
  • 总页数 112
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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