LIGATION OF RNA-CONTAINING DUPLEXES BY VACCINIA DNA-LIGASE

Vaccinia virus DNA ligase repairs nicked duplex, DNA substrates consis ting of a 5'-phosphate-terminated strand and a 3'-hydroxyl-terminated strand annealed to a bridging template strand. This study addresses th e ability of vaccinia DNA ligase to seal nicked substrates containing one or more]RNA strands. We found that the viral enzyme rapidly and ef ficiently joined a 3'-OH RNA to 5'-phosphate DNA when the reacting pol ynucleotides were annealed to a bridging DNA strand. In contrast, liga tion of 3'-OH DNA to 5'-phosphate RNA was slow (0.2% of the rate of RN A-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 betwe en ligands containing 5'-phosphate DNA versus 5'-phosphate RNA at the nick. The enzyme displayed weak activity in RNA-to-RNA ligation on a b ridging DNA template (0.01% of RNA-to-DNA activity). Vaccinia DNA liga se was incapable of joining two DNAs annealed on an RNA template. Thes e results can be explained by a requirement for B-form helical conform ation on the 5'-phosphate side of the nick. The robust RNA-to-DNA stra nd joining activity underscores the potential for vaccinia DNA ligase to catalyze RNA-based integration of host cell genetic information int o the genome of cytoplasmic poxviruses.