Retroviral Integrases Promote Fraying of Viral DNA Ends
Retroviral Integrases Promote Fraying of Viral DNA Ends
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DOI：
10.1074/jbc.m111.229179
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发表时间：
2011-07-22
影响因子：
4.8
通讯作者：
Skalka, Anna Marie
中科院分区：
文献类型：
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作者：
Katz, Richard A.;Merkel, George;Skalka, Anna Marie
在整合的初始阶段，逆转录病毒整合酶（In）在线性病毒DNA末端的退化的短反向重复序列中引入精确的缺口。可剪切的磷酸二酯键位于高度保守的CA/GT二核苷酸的3'处，通常距离末端2 bp。这些缺口产生新的嵌入的3'-OH病毒DNA末端，这是连接宿主细胞DNA所必需的。先前的研究表明，IN对病毒DNA末端的解配对，“磨损”有助于末端识别或催化。在这里，我们报告了利用荧光共振能量转移（FRET）可以独立检测到末端磨损与禽肉瘤病毒（ASV）和人类免疫缺陷病毒1型（HIV-1） IN蛋白的催化作用。结果表明in诱导了病毒DNA末端的分子内构象变化（顺式FRET）。磨损活性与这些酶的DNA结合能力紧密相关，如下所示：一种对两种IN蛋白都有效的抑制剂被证明可以阻断ASV IN DNA结合和终止磨损，其剂量反应相似；减少DNA结合的ASV IN取代也降低了末端磨损活性；在没有金属辅助因子的情况下，HIV-1的DNA结合和末端磨损都无法检测到。与我们之前的结果一致，末端磨损是序列无关的，这表明DNA末端本身是识别的主要结构决定因素。我们得出的结论是，磨损端代表了一个功能性的中间体，在这个中间体中，DNA末端可以被采样以适合于核内溶解处理。
In the initial step of integration, retroviral integrase (IN) introduces precise nicks in the degenerate, short inverted repeats at the ends of linear viral DNA. The scissile phosphodiester bond is located immediately 3' of a highly conserved CA/GT dinucleotide, usually 2 bp from the ends. These nicks create new recessed 3'-OH viral DNA ends that are required for joining to host cell DNA. Previous studies have indicated that unpairing, "fraying," of the viral DNA ends by IN contributes to end recognition or catalysis. Here, we report that end fraying can be detected independently of catalysis with both avian sarcoma virus (ASV) and human immunodeficiency virus type 1 (HIV-1) IN proteins by use of fluorescence resonance energy transfer (FRET). The results were indicative of an IN-induced intramolecular conformational change in the viral DNA ends (cis FRET). Fraying activity is tightly coupled to the DNA binding capabilities of these enzymes, as follows: an inhibitor effective against both IN proteins was shown to block ASV IN DNA binding and end fraying, with similar dose responses; ASV IN substitutions that reduced DNA binding also reduced end fraying activity; and HIV-1 IN DNA binding and end fraying were both undetectable in the absence of a metal cofactor. Consistent with our previous results, end fraying is sequence-independent, suggesting that the DNA terminus per se is a major structural determinant for recognition. We conclude that frayed ends represent a functional intermediate in which DNA termini can be sampled for suitability for endonucleolytic processing.