Ligation of RNA-Containing Duplexes by Vaccinia DNA Ligase

Abstract

Vaccinia virus DNA ligase repairs nicked duplex DNA substrates consisting of a 5‘-phosphate-terminated strand and a 3‘-hydroxyl-terminated strand annealed to a bridging template strand. This study addresses the ability of vaccinia DNA ligase to seal nicked substrates containing one or more RNA strands. We found that the viral enzyme rapidly and efficiently joined a 3‘-OH RNA to 5‘-phosphate DNA when the reacting polynucleotides were annealed to a bridging DNA strand. In contrast, ligation of 3‘-OH DNA to 5‘-phosphate RNA was slow (0.2% of the rate of RNA-to-DNA ligation) and entailed the accumulation of high levels of RNA−adenylate intermediate. A native gel mobility shift assay showed that vaccinia DNA ligase discriminates at the substrate binding step between ligands containing 5‘-phosphate DNA versus 5‘-phosphate RNA at the nick. The enzyme displayed weak activity in RNA-to-RNA ligation on a bridging DNA template (0.01% of RNA-to-DNA activity). Vaccinia DNA ligase was incapable of joining two DNAs annealed on an RNA template. These results can be explained by a requirement for B-form helical conformation on the 5‘-phosphate side of the nick. The robust RNA-to-DNA strand joining activity underscores the potential for vaccinia DNA ligase to catalyze RNA-based integration of host cell genetic information into the genome of cytoplasmic poxviruses.