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Nucleic Acids Res. 2000 Jul 15;28(14):2658-63.

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

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  • 1Molecular Biology Program, Sloan-Kettering Institute, 1275 York Avenue, New York, NY 10021, USA.


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.

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