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 formation 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 catalyzed by Sufolobus shibatae DNA topoisomerase VI (topoVI), a member of the recently described type IIB DNA topoisomerase family. A purification procedure 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 conformation able to cleave the DNA backbone. The structure of cleavage complexes on a linear DNA fragment have been analyzed at the nucleotide level. Similarly to other type II DNA topoisomerases, topoVI is covalently attached to the 5'-ends of the broken DNA. However, sequence analysis of the double-strand breaks revealed that they are all characterized by staggered two-nucleotide long 5' overhangs, contrasting with the four-base staggered double-strand breaks catalyzed by type IIA DNA topoisomerases. While no clear consensus sequences surrounding the cleavage sites could be described, interestingly 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 topoisomerase.
Buhler
C
C
Institut de Génétique et Microbiologie, CNRS UMR 8621, Bât 409, Université Paris-Sud, 91405, Orsay Cedex, France. cyril.buhler@igmors.u-psud.fr
Lebbink
J H
JH
Bocs
C
C
Ladenstein
R
R
Forterre
P
P
D000255:Adenosine Triphosphate / Q000378:metabolism*; D019843:Archaeal Proteins; D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D017422:Sequence Analysis, DNA
D019843:Archaeal Proteins; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D004247:DNA; C105141:DNA topoisomerase VI; D004250:DNA Topoisomerases, Type II