ENHANCED AND COORDINATED PROCESSING OF SYNAPSED VIRAL-DNA ENDS BY RETROVIRAL INTEGRASES IN-VITRO
ENHANCED AND COORDINATED PROCESSING OF SYNAPSED VIRAL-DNA ENDS BY RETROVIRAL INTEGRASES IN-VITRO
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DOI：
10.1101/gad.9.20.2556
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发表时间：
1995-10-15
影响因子：
10.5
通讯作者：
SKALKA, AM
中科院分区：
文献类型：
--
作者：
KUKOLJ, G;SKALKA, AM
我们设计了新的底物来研究逆转录病毒整合的第一步：来自病毒DNA 3'端的两个核苷酸的位点特异性加工。底物由短的双工寡脱氧核苷酸组成，其序列与病毒DNA的U3和U5末端相匹配，但通过短的单链核苷酸连接物在末端共价连接。我们在这里表明，禽肉瘤/白血病病毒（ASV） in的突触端底物的末端之间的最佳间隔为2个核苷酸。这使得两个保守的5‘-CA-3’加工位点相距6个核苷酸，这一距离等于该酶在随后的连接反应中产生的靶DNA的交错切割。根据初始反应速率的估计，这种突触端底物被IN处理的效率比U3和U5单端（不偶联）底物的等效混合物高10倍。增强的加工在低IN浓度下保持，这表明突触端底物可能促进酶的多聚。HIV-1 IN在整合过程中产生5 bp的错开，从而增强了其加工能力，在突触端底物中，加工位点之间的距离为5个核苷酸。这些观察结果提供了ASV和HIV-1多聚体in - dna复合物活性位点之间距离的估计。我们的研究结果还表明，配对的U3和U5端加工不需要在时间上耦合。最后，我们观察到，将野生型与突变末端配对的底物在两端都被切割得很差。因此，突触末端底物的体外加工需要对两端的序列进行特异性识别。这些发现为逆转录病毒整合酶以及其他与病毒酶相关的原核和真核转座酶的整合重组机制提供了新的见解。
We have designed novel substrates to investigate the first step in retroviral integration: the site-specific processing of two nucleotides from the 3' ends of viral DNA. The substrates consist of short duplex oligodeoxynucleotides whose sequences match those of the U3 and U5 ends of viral DNA but are covalently synapsed across the termini by short, single-strand nucleotide linkers. We show here that the optimal separation between termini in a synapsed-end substrate for avian sarcoma/leukosis virus (ASV) IN is 2 nucleotides. This places the two conserved 5'-CA-3' processing sites 6 nucleotides apart, a separation equal to the staggered cut in target DNA produced by this enzyme during the subsequent joining reaction. Based on estimates of initial reaction rates, this synapsed-end substrate is processed by IN at >10-fold higher efficiency than observed with an equivalent mixture of U3 and U5 single-end (uncoupled) substrates. Enhanced processing is maintained at low IN concentrations, suggesting that the synapsed-end substrate may facilitate enzyme multimerization. Enhanced processing by HIV-1 IN, which produces a 5-bp stagger during integration, was observed with a synapsed-end substrate in which the separation between processing sites was 5 nucleotides. These observations provide estimates of the distances between active sites in the multimeric IN-DNA complexes of ASV and HIV-1. Our results also show that processing of paired U3 and U5 ends need not be coupled temporally. Finally, we observed that substrates that paired a wild-type with a mutated terminus were cleaved poorly at both ends. Thus, in vitro processing of the synapsed-end substrates requires specific recognition of the sequences at both ends. These findings provide new insights into the mechanism of integrative recombination by retroviral integrases and, by extension, other prokaryotic and eukaryotic transposases that are related to the viral enzymes.