Katzman M, Sudol M, Pufnock J S, Zeto S, Skinner L M
Department of Medicine, Section of Infectious Diseases, Pennsylvania State University College of Medicine, The Milton S. Hershey Medical Center, 500 University Drive, Mail Services H036, Hershey, PA 17033-2390, USA.
Virus Res. 2000 Jan;66(1):87-100. doi: 10.1016/s0168-1702(99)00126-4.
To identify the parts of retroviral integrase that interact with its DNA substrates, we compared the patterns of target site usage by chimeric enzymes and protein fragments in assays that reveal integrase's non-specific nuclease activities. The central region of 12 chimeric proteins between the human immunodeficiency virus type 1 and visna virus integrases was found to be responsible for selecting non-viral target DNA sites when small alcohols provide the attacking nucleophilic OH group during non-specific alcoholysis assays. Testing deletion derivatives of the integrase protein in this assay, which has similarities to the DNA joining reaction that occurs during retroviral integration, defined a smaller central domain that is sufficient for activity. Thus, this core domain likely contains both the host DNA site and the nucleophile site. Surprisingly, the region of integrase responsible for selecting non-viral target DNA sites when the viral DNA end is the attacking nucleophile could not similarly be mapped with the standard oligonucleotide joining assay. We therefore tested the proteins in a more sensitive assay that displays preferred sites of viral DNA insertion in a plasmid DNA target. All 12 chimeras yielded novel patterns compared with the wild-type enzymes in this assay, although local insertion patterns indicated that the central domain plays an important role in target site selection. Together, these data suggest that other protein regions must be involved when the attacking nucleophilic group is provided by viral DNA. Because specific recognition of viral DNA ends was previously mapped to the central domain, two different regions of integrase must interact with retroviral DNA.
为了鉴定逆转录病毒整合酶中与DNA底物相互作用的部分，我们在揭示整合酶非特异性核酸酶活性的实验中，比较了嵌合酶和蛋白质片段的靶位点使用模式。在非特异性醇解实验中，当小分子醇提供亲核攻击的OH基团时，发现人免疫缺陷病毒1型和维斯纳病毒整合酶之间的12种嵌合蛋白的中央区域负责选择非病毒靶DNA位点。在该与逆转录病毒整合过程中发生的DNA连接反应相似的实验中，测试整合酶蛋白的缺失衍生物，确定了一个足以发挥活性的较小中央结构域。因此，该核心结构域可能同时包含宿主DNA位点和亲核位点。令人惊讶的是，当病毒DNA末端作为亲核攻击基团时，负责选择非病毒靶DNA位点的整合酶区域无法通过标准的寡核苷酸连接实验类似地定位。因此，我们在一个更灵敏的实验中测试了这些蛋白，该实验显示了病毒DNA插入质粒DNA靶标的偏好位点。在该实验中，与野生型酶相比，所有12种嵌合体都产生了新的模式，尽管局部插入模式表明中央结构域在靶位点选择中起重要作用。这些数据共同表明，当亲核攻击基团由病毒DNA提供时，其他蛋白质区域肯定也参与其中。由于病毒DNA末端的特异性识别先前已定位到中央结构域，整合酶的两个不同区域必定与逆转录病毒DNA相互作用。
