Modeling the late steps in HIV-1 retroviral integrase-catalyzed DNA integration
Modeling the late steps in HIV-1 retroviral integrase-catalyzed DNA integration
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DOI：
10.1074/jbc.m006929200
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发表时间：
2000-12-15
影响因子：
4.8
通讯作者：
Leis, J
中科院分区：
文献类型：
--
作者：
Brin, E;Yi, JZ;Leis, J
用代表病毒DNA整合中间体的模型寡脱氧核糖核苷酸底物（具有缺口和两个核苷酸5 '突出端）检测人免疫缺陷病毒1型（HIV-1）逆转录病毒整合酶（IN）体外催化的DNA整合的后期步骤。HIV-1或禽成髓细胞瘤病毒逆转录酶（RT）能够定量地填充差距以产生带切口的底物，但不去除5 '突出端。HIV-1 IN也不能用有缺口的底物去除5 '突出端。然而，通过RT形成的切口底物，HIV-1 IN去除了突出端，并在类似解离的反应中共价闭合切口。这种闭合反应的效率非常低，这种闭合不受HMG-（I/Y）的加入的刺激，这表明该蛋白质仅在早期加工和连接反应中起作用。添加Flap内切核酸酶-1（一种已知可去除5 '突出端的核酸酶），废除了IN催化的闭合反应，IN是一系列碱基对倒位，引入到邻近和/或包括保守CA二核苷酸的HIV-1 U 5长末端重复序列中，不产生或仅产生少量。HIV-1 IN依赖性链闭合反应减少。这些相同的突变导致体外经修饰的供体DNA的协同DNA整合效率的显著降低，表明仅在DNA整合的早期步骤中需要识别长末端重复序列的末端。最后，HIV-1 RT、Flap内切核酸酶-1和DNA连接酶的组合能够与这些模型底物定量地形成共价闭合的DNA，这些结果支持细胞酶可能催化逆转录病毒DNA整合的后期步骤的假设。
Model oligodeoxyribonucleotide substrates representing viral DNA integration intermediates with a gap and a two-nucleotide 5 ' overhang were used to examine late steps in human immunodeficiency virus, type 1 (HIV-1) retroviral integrase (IN)-cataIyzed DNA integration in vitro. HIV-1 or avian myeloblastosis virus reverse transcriptase (RT) were capable of quantitatively filling in the gap to create a nicked substrate but did not remove the 5 ' overhang. HIV-1 IN also failed to remove the 5 ' overhang with the gapped substrate. However, with a nicked substrate formed by RT, HIV-1 IN removed the overhang and covalently closed the nick in a disintegration-like reaction. The efficiency of this closure reaction was very low, Such closure was not stimulated by the addition of HMG-(I/Y), suggesting that this protein only acts during the early processing and joining reactions. Addition of Flap endonuclease-1, a nuclease known to remove 5 ' overhangs, abolished the closure reaction catalyzed by IN, A series of base pair inversions, introduced into the HIV-1 U5 long terminal repeat sequence adjacent to and/or including the conserved CA dinucleotide, produced no or only a small. decrease in the HIV-1 IN-dependent strand closure reaction. These same mutations caused a significant decrease in the efficiency of concerted DNA integration by a modified donor DNA in vitro, suggesting that recognition of the ends of the long terminal repeat sequence is required only in the early steps of DNA integration, Finally, a combination of HIV-1 RT,Flap endonuclease-1, and DNA ligase is capable of quantitatively forming covalently closed DNA with these model substrates, These results support the hypothesis that cellular enzyme(s) may catalyze the late steps of retroviral DNA integration.