Reed Benjamin, Yakovleva Lyudmila, Shuman Stewart, Ghose Ranajeet
Department of Chemistry and Biochemistry, The City College of New York , New York, New York 10031, United States.
Molecular Biology Program, Sloan-Kettering Institute , New York, New York 10021, United States.
Biochemistry. 2017 Jul 5;56(26):3307-3317. doi: 10.1021/acs.biochem.7b00042. Epub 2017 Jun 19.
Vaccinia TopIB (vTopIB), a 314-amino acid eukaryal-type IB topoisomerase, recognizes and transesterifies at the DNA sequence 5'-(T/C)CCTT↓, leading to the formation of a covalent DNA-(3'-phosphotyrosyl)-enzyme intermediate in the supercoil relaxation reaction. The C-terminal segment of vTopIB (amino acids 81-314), which engages the DNA minor groove at the scissile phosphodiester, comprises an autonomous catalytic domain that retains cleavage specificity, albeit with a cleavage site affinity lower than that of the full-length enzyme. The N-terminal domain (amino acids 1-80) engages the major groove on the DNA face opposite the scissile phosphodiester. Whereas DNA contacts of the N-terminal domain have been implicated in the DNA site affinity of vTopIB, it was not known whether the N-terminal domain per se could bind DNA. Here, using isothermal titration calorimetry, we demonstrate the ability of the isolated N-terminal domain to bind a CCCTT-containing 24-mer duplex with an apparent affinity that is ∼2.2-fold higher than that for an otherwise identical duplex in which the pentapyrimidine sequence is changed to ACGTG. Analyses of the interactions of the isolated N-terminal domain with duplex DNA via solution nuclear magnetic resonance methods are consistent with its DNA contacts observed in DNA-bound crystal structures of full-length vTopIB. The chemical shift perturbations and changes in hydrodynamic properties triggered by CCCTT DNA versus non-CCCTT DNA suggest differences in DNA binding dynamics. The importance of key N-terminal domain contacts in the context of full-length vTopIB is underscored by assessing the effects of double-alanine mutations on DNA transesterification and its sensitivity to ionic strength.
痘苗病毒拓扑异构酶IB（vTopIB）是一种含314个氨基酸的真核生物I型拓扑异构酶，它识别5'-(T/C)CCTT↓的DNA序列并进行转酯反应，在超螺旋松弛反应中导致形成共价DNA-(3'-磷酸酪氨酸)-酶中间体。vTopIB的C末端片段（氨基酸81 - 314）在可裂解磷酸二酯处与DNA小沟结合，包含一个自主催化结构域，该结构域保留了切割特异性，尽管其对切割位点的亲和力低于全长酶。N末端结构域（氨基酸1 - 80）与可裂解磷酸二酯相对的DNA面上的大沟结合。虽然N末端结构域与DNA的接触与vTopIB对DNA位点的亲和力有关，但尚不清楚N末端结构域本身是否能结合DNA。在这里，我们使用等温滴定量热法证明了分离的N末端结构域能够结合含有CCCTT的24聚体双链体，其表观亲和力比五嘧啶序列变为ACGTG的相同双链体高约2.2倍。通过溶液核磁共振方法对分离的N末端结构域与双链DNA相互作用的分析与在全长vTopIB的DNA结合晶体结构中观察到的其与DNA的接触一致。CCCTT DNA与非CCCTT DNA引发的化学位移扰动和流体动力学性质变化表明DNA结合动力学存在差异。通过评估双丙氨酸突变对DNA转酯反应及其对离子强度敏感性的影响，强调了全长vTopIB背景下关键N末端结构域接触的重要性。
