NICK SENSING BY VACCINIA VIRUS-DNA LIGASE REQUIRES A 5'-PHOSPHATE AT THE NICK AND OCCUPANCY OF THE ADENYLATE BINDING-SITE ON THE ENZYME

Vaccinia virus DNA ligase has an intrinsic nick-sensing function. The enzyme discriminates at the substrate binding step between a DNA conta ining a 5' phosphate and a DNA containing a 5' hydroxyl at the nick. F urther insights into nick recognition and catalysis emerge from studie s of the active-site mutant K231A, which is unable to form the covalen t ligase-adenylate intermediate and hence cannot activate a nicked DNA substrate via formation of the DNA-adenylate intermediate. Nonetheles s, K231A does catalyze phosphodiester bond formation at a preadenylate d nick. Hence, the active-site lysine of DNA ligase is not required fo r the strand closure step of the ligation reaction. The K231A mutant b inds tightly to nicked DNA-adenylate but has low affinity for a standa rd DNA nick. The wild-type vaccinia virus ligase, which is predominant ly ligase-adenylate, binds tightly to a DNA nick. This result suggests that occupancy of the AMP binding pocket of DNA ligase is essential f or stable binding to DNA. Sequestration of an extrahelical nucleotide by DNA-bound ligase is reminiscent of the base-flipping mechanism of t arget-site recognition and catalysis used by other DNA modification an d repair enzymes.