A reverse gyrase with an unusual structure. A type I DNA topoisomerase from the hyperthermophile Methanopyrus kandleri is a two-subunit protein.
Reverse gyrase, an ATP-dependent topoisomerase that positively supercoils DNA, has been purified to near-homogeneity from the hyperthermophile Methanopyrus kandleri. It migrates on SDS-polyacrylamide gel electrophoresis as two principal bands with apparent molecular masses of 150 and 50 kDa. Both proteins remain associated throughout all chromatographic steps. Transfer of a radioactive phosphate from DNA to the 50-kDa protein and gel retardation experiments indicate that this protein forms the covalent complex with DNA. A blot overlay assay identifies the 150-kDa protein as the potential ATPase. This is the first evidence that a reverse gyrase can be a topoisomerase consisting of two protomers. In analogy with the DNA gyrase A subunit (DNA breakage and reunion activity) and the B subunit (ATPase), the 50- and 150-kDa components of Mka reverse gyrase have been designated the A and B subunits, respectively. Methanopyrus reverse gyrase changes DNA linking number in steps of one and its A subunit covalently binds to the 5'-DNA phosphoryl group. It nicks DNA at sites that predominantly have a cytosine at the -4-position. The same rule was derived previously for monomeric reverse gyrase from sulfur-metabolizing hyperthermophiles and for topoisomerase I from mesophilic bacteria. Based on these results, Mka reverse gyrase is classified as belonging to group A of type I topoisomerases. The structural diversity of type I group A topoisomerases parallels the diversity of type II enzymes and suggests the evolution of an essential function by gene fusion.
Kozyavkin
S A
SA
Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Krah
R
R
Gellert
M
M
Stetter
K O
KO
Lake
J A
JA
Slesarev
A I
AI
D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004264:DNA Topoisomerases, Type I / Q000302:isolation & purification* / Q000378:metabolism*; D004250:DNA Topoisomerases, Type II / Q000302:isolation & purification* / Q000378:metabolism*; D004278:DNA, Superhelical / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D019605:Euryarchaeota / Q000201:enzymology*; D006358:Hot Temperature; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D010957:Plasmids / Q000378:metabolism
D004278:DNA, Superhelical; D046911:Macromolecular Substances; C041813:DNA reverse gyrase; D004264:DNA Topoisomerases, Type I; D004250:DNA Topoisomerases, Type II