POLYNUCLEOTIDE LIGASE ACTIVITY OF EUKARYOTIC TOPOISOMERASE-I

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 reacti on is reversible. Vaccinia topoisomerase can ligate 2',3'cyclic phosph ate and 5'-hydroxyl termini annealed to a bridging template strand. Re markably, the ligase activity of topoisomerase does not require the ac tive site tyrosine, implying that strand joining can occur via direct attack of the 5' hydroxyl on the cyclic phosphate without a covalent i ntermediate. 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 seemin gly diverse tasks through subtle changes at the active site.