SPECIFICITY AND FIDELITY OF STRAND JOINING BY CHLORELLA VIRUS-DNA LIGASE

Chlorella virus PBCV-1 DNA ligase seals nicked duplex DNA substrates c onsisting of a 5'-phosphate-terminated strand and a 3'-hydroxyl-termin ated strand annealed to a bridging template strand, but cannot ligate a nicked duplex composed of two DNAs annealed on an RNA template, Wher eas PBCV-1 ligase efficiently joins a 3'-OH RNA to a 5'-phosphate DNA, it is unable to join a 3'-OH DNA to a 5'-phosphate RNA. The ligase di scriminates at the substrate binding step between nicked duplexes cont aining 5'-phosphate DNA versus 5'-phosphate RNA strands. PBCV-1 ligase readily seals a nicked duplex DNA containing a single ribonucleotide substitution at the reactive 5'-phosphate end. These results suggest a requirement for a B-form helical conformation of the polynucleotide o n the 9'-phosphate side of the nick, Single base mismatches at the nic k exert disparate effects on DNA ligation efficiency, PBCV-1 ligase to lerates mismatches involving the 5'-phosphate nucleotide, with the exc eption of 5'-A:G and 5'-G:A mispairs, which reduce ligase activity by two orders of magnitude. Inhibitory configurations at the 3'-OH nucleo tide include 3'-G:A, 3'-G:T, 3'-T:T, 3'-A:G, 3'-G:G, 3'-A:C and 3'-C:C , Our findings indicate that Chlorella virus DNA ligase has the potent ial to affect genome integrity by embedding ribonucleotides in viral D NA and by sealing nicked molecules with mispaired ends, thereby genera ting missense mutations.