Scottoline B P, Chow S, Ellison V, Brown P O
Department of Biochemistry, Stanford University Medical School, California 94305-5428, USA.
Genes Dev. 1997 Feb 1;11(3):371-82. doi: 10.1101/gad.11.3.371.
Integrase catalyzes two essential steps in the integration of the retroviral genome--end processing and strand transfer--both of which require the interaction of integrase with viral att sites located at the ends of viral genomic DNA. These two different polynucleotidyl transfer reactions are apparently carried out by a single active site. The end product of these reactions, the integrated provirus, does not undergo transposition and remains a stable part of the host cell genome. A central question in understanding the mechanism of integration is how a single active site accomplishes two distinct polynucleotidyl transfer reactions. We propose that integrase distorts DNA substrates to accommodate both reactions within the active site. Evidence is provided for disruption of base-pairing at the terminus of viral DNA during end processing. Furthermore, we show that this end fraying is a required step in end processing and that it appears to occur after initial binding of the viral DNA end. This requirement for base-pair disruption may account for the inability of integrase to use internal sites on DNA molecules as viral att sites. The specificity of integrase for DNA ends solves a problem posed by the long terminal repeat structure of the viral genome, and may help to prevent transposition of integrated proviruses.
整合酶催化逆转录病毒基因组整合过程中的两个关键步骤——末端加工和链转移，这两个步骤都需要整合酶与位于病毒基因组DNA末端的病毒附着位点相互作用。这两种不同的多聚核苷酸转移反应显然是由单一活性位点进行的。这些反应的最终产物，即整合后的前病毒，不会发生转座，而是宿主细胞基因组的一个稳定组成部分。理解整合机制的一个核心问题是，单一活性位点如何完成两种不同的多聚核苷酸转移反应。我们提出，整合酶会使DNA底物发生扭曲，以便在活性位点内适应这两种反应。有证据表明，在末端加工过程中，病毒DNA末端的碱基配对会被破坏。此外，我们表明这种末端解链是末端加工中的一个必要步骤，并且它似乎发生在病毒DNA末端最初结合之后。这种对碱基对破坏的需求可能解释了整合酶无法将DNA分子上的内部位点用作病毒附着位点的原因。整合酶对DNA末端的特异性解决了由病毒基因组的长末端重复结构带来的问题，并且可能有助于防止整合后的前病毒发生转座。
