Polynucleotide ligase activity of eukaryotic topoisomerase I

Abstract

Introduction of a single ribonucleoside immediately 5' of the scissile phosphate of a duplex DNA substrate converts eukaryotic topoisomerase I into an endoribonuclease. Here, I demonstrate that the RNase reaction is reversible. Vaccinia topoisomerase can ligate 2', 3'-cyclic phosphate and 5'-hydroxyl termini annealed to a bridging template strand. Remarkably, the ligase activity of topoisomerase does not require the active site tyrosine, implying that strand joining can occur via direct attack of the 5' hydroxyl on the cyclic phosphate without a covalent intermediate. Ligation does require other catalytic side chains on the enzyme. These findings underscore how a common ancestral mechanism of phosphoryl and nucleotidyl transfer can be harnessed to perform seemingly diverse tasks through subtle changes at the active site.