Reed, Benjamin Yakovleva, Lyudmila Shuman, Stewart Ghose, Ranajeet Characterization of DNA Binding by the Isolated N‑Terminal Domain of Vaccinia Virus DNA Topoisomerase IB 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<sup>274</sup>)–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. supercoil relaxation reaction;CCCTT-containing 24- mer duplex;DNA site affinity;N-terminal domain;N-terminal domain contacts;DNA binding dynamics;DNA contacts;acid eukaryal-type IB topoisomerase;DNA-bound crystal structures;scissile phosphodiester;Vaccinia Virus DNA Topoisomerase IB Vaccinia TopIB;chemical shift perturbations;ACGTG;cleavage site affinity 2017-06-01
    https://acs.figshare.com/articles/journal_contribution/Characterization_of_DNA_Binding_by_the_Isolated_N_Terminal_Domain_of_Vaccinia_Virus_DNA_Topoisomerase_IB/5117650
10.1021/acs.biochem.7b00042.s001