Resolution of a Holliday junction by vaccinia topoisomerase requires a spacer DNA segment 3 ' of the CCCTT down arrow cleavage sites

Vaccinia virus DNA topoisomerase catalyzes resolution of synthetic Holliday junctions in vitro. The mechanism entails concerted transesterifications a t two recognition sites, 5'-CCCTT down arrow, that are opposed within a par tially mobile four-way junction. Efficient resolution occurs on a junction with a 10 bp segment of branch mobility (5'-GCCCTTATCG) that extends 4 bp 3 ' of the scissile phosphate. Here we report that resolution is decreased wh en branch mobility is limited to an 8 bp segment extending 2 bp 3' of the c leavage site and then eliminated when branch mobility is confined to the 6 bp GCCCTT sequence 5' of the scissile phosphate. We surmise that a spacer r egion 3' of CCCTT is needed for simultaneous cleavage at two opposing sites at the junction. Branch mobility is not required for reaction chemistry at a junction, because topoisomerase cleaves a single CCCTT site in a non-mob ile four-way junction where the scissile phosphate is at the crossover poin t. The junction resolvase activity of topoisomerase may be involved in form ing the hairpin telomeres of the vaccinia genome.