Benarroch Delphine, Shuman Stewart
Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10021, USA.
Virology. 2006 Sep 15;353(1):133-43. doi: 10.1016/j.virol.2006.04.032. Epub 2006 Jul 14.
Mimivirus, a parasite of Acanthamoeba polyphaga, is the largest DNA virus known; it encodes a cornucopia of proteins with imputed functions in DNA replication, modification, and repair. Here we produced, purified, and characterized mimivirus DNA ligase (MimiLIG), an NAD+-dependent nick joining enzyme homologous to bacterial LigA and entomopoxvirus DNA ligase. MimiLIG is a 636-aa polypeptide composed of an N-terminal NAD+ specificity module (domain Ia), linked to nucleotidyltransferase, OB-fold, helix-hairpin-helix, and BRCT domains, but it lacks the tetracysteine Zn-binding module found in all bacterial LigA enzymes. MimiLIG requires conserved domain Ia residues Tyr36, Asp46, Tyr49, and Asp50 for its initial reaction with NAD+ to form the ligase-AMP intermediate, but not for the third step of phosphodiester formation at a preadenylylated nick. MimiLIG differs from bacterial LigA enzymes in that its activity is strongly dependent on the C-terminal BRCT domain, deletion of which reduced its specific activity in nick joining by 75-fold without affecting the ligase adenylylation step. The DeltaBRCT mutant of MimiLIG was impaired in sealing at a preadenylylated nick. We propose that eukaryal DNA viruses acquired the NAD+-dependent ligases by horizontal transfer from a bacterium and that MimiLIG predates entomopoxvirus ligase, which lacks both the tetracysteine and BRCT domains. We speculate that the dissemination of NAD+-dependent ligase from bacterium to eukaryotic virus might have occurred within an amoebal host.
米米病毒是多食棘阿米巴的一种寄生虫，是已知最大的DNA病毒；它编码大量在DNA复制、修饰和修复中具有推测功能的蛋白质。在此，我们制备、纯化并鉴定了米米病毒DNA连接酶（MimiLIG），它是一种与细菌LigA和昆虫痘病毒DNA连接酶同源的依赖NAD⁺的切口连接酶。MimiLIG是一种由636个氨基酸组成的多肽，由一个N端NAD⁺特异性模块（结构域Ia）与核苷酸转移酶、OB折叠、螺旋-发夹-螺旋和BRCT结构域相连，但它缺少所有细菌LigA酶中发现的四半胱氨酸锌结合模块。MimiLIG与NAD⁺的初始反应以形成连接酶-AMP中间体需要保守的结构域Ia残基Tyr36、Asp46、Tyr49和Asp50，但在预腺苷酸化切口处形成磷酸二酯的第三步则不需要。MimiLIG与细菌LigA酶的不同之处在于其活性强烈依赖于C端BRCT结构域，缺失该结构域会使其切口连接的比活性降低75倍，而不影响连接酶腺苷酸化步骤。MimiLIG的DeltaBRCT突变体在预腺苷酸化切口处的封闭功能受损。我们提出真核DNA病毒通过从细菌水平转移获得了依赖NAD⁺的连接酶，并且MimiLIG早于昆虫痘病毒连接酶，后者既缺少四半胱氨酸结构域也缺少BRCT结构域。我们推测依赖NAD⁺的连接酶从细菌传播到真核病毒可能发生在变形虫宿主内。
