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首页> 外文期刊>Gene Therapy and Molecular Biology >Identification of a negative regulatory mechanism for the repair of U5 long terminal repeat DNA by the human immunodeficiency virus type 1 integrase DNA polymerase
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Identification of a negative regulatory mechanism for the repair of U5 long terminal repeat DNA by the human immunodeficiency virus type 1 integrase DNA polymerase

机译：鉴定人类免疫缺陷病毒1型整合酶DNA聚合酶修复U5长末端重复DNA的负调控机制

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The quasi-random integration of retroviral DNA elements into the chromosomes of infected cells is believed to proceed by a four-step mechanism. The 3'- ends of the long terminal repeats (LTRs) are processed by the endonucleolytic cleavage and removal, usually, of a GT dinucleotide (step 1); the 3'-processed DNA ends are inserted at staggered nicks in the host DNA via a DNA strand transfer reaction, simultaneously generating short gaps at the sites of insertion (step 2); the gaps are repaired by a DNA polymerase (step 3); and the 5'-ends of the viral DNA are joined to the host DNA (step 4). Human immunodeficiency virus type 1 (HIV-1) integrase was previously reported to possess enzymatic activities capable of performing at least the first 3 steps of the integration process including an intrinsic DNA - dependent polymerase activity capable of short gap repair (Acel et. al., 1998 J. Virol. 72: 2062-2071). In the present study, the behavior of the integrase DNA polymerase was examined in a DNA end-repair assay in which the frequency of polymerization on 3'-processed HIV-1 U5 LTRs was examined. The frequency of polymerization was negatively regulated by the 5'-AC sequence comprising the 2-nucleotide template and by the sequence of the adjacent conserved 5'TG/CA dinucleotide. Mutations within these DNA elements of the LTR enhanced the polymerization frequency on 2-nucleotide templates between 3- and 100-fold. In most cases, the integrase DNA polymerase added only one nucleotide to 3'-processed LTRs even though the DNA was comprised of 2-nucleotide template-primers. This level of regulation was controlled by a DNA binding and/or zinc finger domain in the integrase protein. By contrast, the integrase DNA polymerase behaved in a processive manner with homopolymeric pyrimidine templates, extending nascent DNA chains up to at least 20 nucleotides, whereas DNA polymerization with an oligo dA template exhibited a lower processivity of 1-7 nucleotides. The results suggest a model whereby an interaction between integrase and specific DNA elements in the HIV-1 LTRs prevents the repair of 3'-processed LTRs by the integrase DNA polymerase. Drugs with the ability to alter this regulatory aspect of integrase DNA polymerase function and thus induce repair of processed LTRs are predicted to block integration of HIV-1 DNA, and thus have potentially lethal consequences for HIV-1 replication.
机译：逆转录病毒DNA元件向感染细胞染色体的准随机整合被认为是通过四步机制进行的。长末端重复序列（LTR）的3'-末端通常通过内切核酸酶切和GT二核苷酸的去除来处理（步骤1）;通过DNA链转移反应，将3'处理的DNA末端通过DNA链转移反应插入到宿主DNA的交错切口处，同时在插入位点产生短缺口（步骤2）;缺口由DNA聚合酶修复（步骤3）;病毒DNA的5'末端与宿主DNA相连（步骤4）。先前已报道人类1型免疫缺陷病毒（HIV-1）整合酶具有酶活性，能够执行整合过程的至少前3个步骤，包括能够短间隙修复的固有的DNA依赖性聚合酶活性（Acel等。 ，1998 J.Virol.72：2062-2071）。在本研究中，在DNA末端修复测定中检查了整合酶DNA聚合酶的行为，在该测定中检查了在3'处理的HIV-1 U5 LTR上的聚合频率。聚合的频率由包含2-核苷酸模板的5'-AC序列和相邻的保守的5'TG / CA二核苷酸的序列负调控。 LTR的这些DNA元素内的突变将2-核苷酸模板上的聚合频率提高了3到100倍。在大多数情况下，即使DNA由2个核苷酸的模板引物组成，整合酶DNA聚合酶也仅向3'处理的LTR中添加了一个核苷酸。该调节水平由整合酶蛋白中的DNA结合和/或锌指结构域控制。相比之下，整合酶DNA聚合酶与均聚嘧啶模板的杂交过程，将新生的DNA链延伸至至少20个核苷酸，而具有oligo dA模板的DNA聚合酶则具有较低的1-7个核苷酸的合成能力。该结果提出了一种模型，其中整合酶与HIV-1 LTR中特定DNA元素之间的相互作用阻止了整合酶DNA聚合酶修复3'处理的LTR。能够改变整合酶DNA聚合酶功能这一调控方面并因此诱导已加工LTR修复的药物预计会阻断HIV-1 DNA的整合，因此对HIV-1复制具有潜在的致命后果。

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