DNA contacts stimulate catalysis by a poxvirus topoisomerase

Eukaryotic type 1B topoisomerases act by forming covalent enzyme-DNA interm ediates that transiently nick DNA and thereby release DNA supercoils. Here we present a study of the topoisomerase encoded by the pathogenic poxvirus molluscum contagiosum, Our studies of DNA sites favored for catalysis revea l a larger recognition site than the 5'-(T/C)CCTT-3' sequence previously id entified for poxvirus topoisomerases. Separate assays of initial DNA bindin g and covalent complex formation revealed that different DNA sequences were important for each reaction step. The location of the protein-DNA contacts was mapped by analyzing mutant sites and inosine-substituted DNAs, Some of the bases flanking the 5'-(T/C)CCTT-3' sequence were selectively important for covalent complex formation but not initial DNA binding. Interactions i mportant for catalysis were probed with 5'-bridging phosphorothiolates at t he site of strand cleavage, which permitted covalent complex formation but prevented subsequent religation. Kinetic studies revealed that the flanking sequences that promoted recovery of covalent complexes increased initial c leavage instead of inhibiting resealing of the nicked intermediate. These d ata 1) indicate that previously unidentified DNA contacts can accelerate a step between initial binding and covalent complex formation and 2) help spe cify models for conformational changes promoting catalysis.