Brin E, Yi J, Skalka A M, Leis J
Department of Microbiology and Immunology, Northwestern University School of Medicine, Chicago, Illinois 60611, USA.
J Biol Chem. 2000 Dec 15;275(50):39287-95. doi: 10.1074/jbc.M006929200.
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)-catalyzed 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.
使用代表具有缺口和两个核苷酸5'突出端的病毒DNA整合中间体的模型寡脱氧核糖核苷酸底物，来研究1型人类免疫缺陷病毒（HIV-1）逆转录病毒整合酶（IN）催化的体外DNA整合的后期步骤。HIV-1或禽成髓细胞瘤病毒逆转录酶（RT）能够定量填补缺口以产生带切口的底物，但不会去除5'突出端。HIV-1 IN也无法去除带缺口底物的5'突出端。然而，对于由RT形成的带切口底物，HIV-1 IN去除了突出端并在类似解离的反应中使切口共价闭合。这种闭合反应的效率非常低。添加HMG-(I/Y)并不会刺激这种闭合反应，这表明该蛋白仅在早期加工和连接反应中起作用。添加已知可去除5'突出端的核酸酶Flap内切核酸酶-1，可消除IN催化的闭合反应。在与保守的CA二核苷酸相邻和/或包含该二核苷酸的HIV-1 U5长末端重复序列中引入一系列碱基对倒位，对HIV-1 IN依赖性链闭合反应没有影响或仅有小幅降低。这些相同的突变导致体外修饰供体DNA协同DNA整合效率显著降低，这表明长末端重复序列末端的识别仅在DNA整合的早期步骤中是必需的。最后，HIV-1 RT、Flap内切核酸酶-1和DNA连接酶的组合能够定量地与这些模型底物形成共价闭合的DNA。这些结果支持细胞酶可能催化逆转录病毒DNA整合后期步骤的假设。
