Processing of viral DNA ends channels the HIV-1 integration reaction to concerted integration.
Processing of viral DNA ends channels the HIV-1 integration reaction to concerted integration.
复制标题
DOI：
10.1074/jbc.m505367200
复制
发表时间：
2005-08-12
期刊：
影响因子：
--
通讯作者：
Craigie R
中科院分区：
文献类型：
--
作者：
Li M;Craigie R
逆转录产生的逆转录病毒DNA是钝端的，病毒整合酶蛋白在整合到染色体DNA之前必须从每一个3‘端去除两个核苷酸。在大多数体外整合的反应条件下，大多数反应产物是由于只将一个病毒DNA末端整合到靶DNA的一条链中而产生的“半部位”产物。前处理的DNA底物是比钝端底物更有效的半位反应底物，后者需要在整合之前去除两个核苷酸。相反，我们发现钝端的DNA是一对病毒DNA末端协同整合的生物学相关反应的更好底物。反应途径被引导到协同整合，半位整合产物被钝端的DNA底物还原，这必须首先由整合酶处理。此外，协同整合的末端核苷酸要求比半位点反应更严格。在协同反应中，较长的DNA比能够有效整合半个位点的较短的DNA更有效。这表明整合酶与远离末端的病毒DNA的非特异性相互作用有助于组装具有协同整合能力的复合体。最后，整合酶C末端残基突变对协同整合和半部位整合的不同影响意味着涉及该区域的蛋白质-蛋白质相互作用对协同整合非常重要。
Retroviral DNA made by reverse transcription is blunt-ended, and the viral integrase protein must remove two nucleotides from each 3′ end prior to integration into chromosomal DNA. Under most reaction conditions for integration in vitro, the majority of the reaction products are “half-site” products that result from integration of only one viral DNA end into one strand of the target DNA. Preprocessed DNA substrates are more efficient substrates for half-site reactions than are blunt-ended substrates, which require the removal of two nucleotides prior to integration. In contrast, we find that blunt-ended DNA is a better substrate for the biologically relevant reaction of concerted integration of pairs of viral DNA ends. The reaction pathway is channeled to concerted integration, and half-site integration products are reduced with blunt-ended DNA substrate that must first be processed by integrase. In addition, the terminal nucleotide requirements for concerted integration are more stringent than for the half-site reaction. Longer DNA is more efficient for the concerted reaction than is shorter DNA that is capable of efficient half-site integration. This suggests that nonspecific interactions of integrase with viral DNA distant from the termini contribute to the assembly of a complex that is competent for concerted integration. Finally, differential effects of mutation of a residue in the C-terminal domain of integrase on concerted versus half-site integration implicate protein-protein interactions involving this domain as important for concerted integration.