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′-phosphotyrosyl274)–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....