DNA topoisomerase VI generates ATP-dependent double-strand breaks with two-nucleotide overhangs

A key step in the DNA transport by type II DNA topoisomerase is the formati on of a double-strand break with the enzyme being covalently linked to the broken DNA ends (referred to as the cleavage complex). In the present study , we have analyzed the formation and structure of the cleavage complex cata lyzed by Sufolobus shibatae DNA topoisomerase VI (topoVI), a member of the recently described type IIB DNA topoisomerase family. A purification proced ure of a fully soluble recombinant topoVI was developed by expressing both subunits simultaneously in Escherichia coli. Using this recombinant enzyme, we observed that the formation of the double-strand breaks on supercoiled or linear DNA is strictly dependent on the presence of ATP or AMP-PNP. This result suggests that ATP binding is required to stabilize an enzyme confor mation able to cleave the DNA backbone. The structure of cleavage complexes on a linear DNA fragment have been analyzed at the nucleotide level. Simil arly to other type II DNA topoisomerases, topoVI is covalently attached to the 5'-ends of the broken DNA. However, sequence analysis of the double-str and breaks revealed that they are all characterized by staggered two-nucleo tide long 5' overhangs, contrasting with the four-base staggered double-str and breaks catalyzed by type IIA DNA topoisomerases. While no clear consens us sequences surrounding the cleavage sites could be described, interesting ly A and T nucleotides are highly represented on the 5' extensions, giving a first insight on the preferred sequences recognized by this type II DNA t opoisomerase.