Resolution of a Holliday junction by vaccinia topoisomerase requires a spacer DNA segment 3' of the CCCTT/ cleavage sites.
Vaccinia virus DNA topoisomerase catalyzes resolution of synthetic Holliday junctions in vitro. The mechanism entails concerted transesterifications at two recognition sites, 5'-CCCTT/, that are opposed within a partially 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 when branch mobility is limited to an 8 bp segment extending 2 bp 3' of the cleavage 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 region 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-mobile four-way junction where the scissile phosphate is at the crossover point. The junction resolvase activity of topo-isomerase may be involved in forming the hairpin telomeres of the vaccinia genome.
Sekiguchi
J
J
Molecular Biology Program, Sloan-Kettering Institute, 1275 York Avenue, New York, NY 10021, USA.
Cheng
C
C
Shuman
S
S
D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D004264:DNA Topoisomerases, Type I / Q000378:metabolism* / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D014616:Vaccinia virus / Q000201:enzymology*
D004247:DNA; D004264:DNA Topoisomerases, Type I