Nowak Matthew G, Sudol Malgorzata, Lee Noelle E, Konsavage Wesley M, Katzman Michael
Cell and Molecular Biology Graduate Program, Penn State College of Medicine, The Milton S. Hershey Medical Center, P.O. Box 850, Hershey, PA 17033, USA.
Virology. 2009 Jun 20;389(1-2):141-8. doi: 10.1016/j.virol.2009.04.014. Epub 2009 May 17.
Although retroviral integrase specifically trims the ends of viral DNA and inserts these ends into any sequence in cellular DNA, little information is available to explain how integrase distinguishes between its two DNA substrates. We recently described novel integrase mutants that were improved for specific nicking of viral DNA but impaired at joining these ends into nonviral DNA. An acidic or bulky substitution at one particular residue was critical for this activity profile, and the prototypic protein--Rous sarcoma virus integrase with an S124D substitution--was defective at nonspecifically binding DNA. We have now characterized 19 (including 16 new) mutants that contain one or more aspartic acid substitutions at residues that extend over the surface of the protein and might participate with residue 124 in binding cellular DNA. In particular, every mutant with an aspartate substitution at residue 98 or 128, similar to the original S124D protein, showed improved specific nicking of viral DNA but disturbed nonspecific nicking of nonviral DNA. These data describe a probable cellular-DNA binding platform that involves at least 5 amino acids, in the following order of importance: 124>128>(98, 125)>123. These experimental data are vital for new models of integrase and will contribute to identifying targets for the next generation of integrase inhibitors.
尽管逆转录病毒整合酶能特异性地修剪病毒DNA的末端，并将这些末端插入细胞DNA的任何序列中，但目前几乎没有信息能解释整合酶如何区分其两种DNA底物。我们最近描述了新型整合酶突变体，这些突变体在病毒DNA的特异性切口方面有所改进，但在将这些末端连接到非病毒DNA上存在缺陷。在一个特定残基处的酸性或大体积取代对于这种活性特征至关重要，具有S124D取代的原型蛋白——劳斯肉瘤病毒整合酶在非特异性结合DNA方面存在缺陷。我们现在对19个（包括16个新的）突变体进行了表征，这些突变体在延伸于蛋白质表面且可能与124位残基一起参与结合细胞DNA的残基处含有一个或多个天冬氨酸取代。特别是，每个在98位或128位残基处有天冬氨酸取代的突变体，类似于原始的S124D蛋白，都显示出病毒DNA特异性切口的改善，但非病毒DNA非特异性切口受到干扰。这些数据描述了一个可能的细胞DNA结合平台，该平台至少涉及5个氨基酸，按重要性顺序排列为：124>128>(98, 125)>123。这些实验数据对于整合酶的新模型至关重要，并将有助于确定下一代整合酶抑制剂的靶点。
