Reverse gyrase of Sulfolobus: purification to homogeneity and characterization.
By using hydrophobic interaction as the first chromatographic stage, we purified to homogeneity reverse gyrase, an ATP-dependent DNA topoisomerase I, isolated from the thermoacidophilic archaebacterium Sulfolobus acidocaldrius. This procedure allowed quick and complete separation of reverse gyrase from nucleases and DNA binding proteins present in Sulfolobus. The final product was revealed, by SDS-PAGE, as a unique band with an apparent molecular mass of 128 kDa, and the amino acid composition was determined. Western blotting experiments with antibodies raised against reverse gyrase indicate that no proteolysis occurred during the purification course. Gel filtration and sedimentation data gave a Stokes radius of 42 A and a sedimentation coefficient of 5.7 S, suggesting a monomeric structure for the native enzyme which was confirmed by electron microscopy. Finally, pure reverse gyrase in a monomeric state was still able to promote positive supercoiling of the DNA.
Nadal
M
M
UA 554 CNRS, Université Pierre et Marie Curie, Paris, France.
Jaxel
C
C
Portemer
C
C
Forterre
P
P
Mirambeau
G
G
Duguet
M
M
D000255:Adenosine Triphosphate / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D001105:Archaea / Q000201:enzymology*; D001419:Bacteria / Q000201:enzymology*; D001426:Bacterial Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002847:Chromatography, Agarose; D004264:DNA Topoisomerases, Type I; D004250:DNA Topoisomerases, Type II / Q000302:isolation & purification* / Q000378:metabolism; D004269:DNA, Bacterial / Q000378:metabolism; D004278:DNA, Superhelical / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D008970:Molecular Weight
D000596:Amino Acids; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004278:DNA, Superhelical; D000255:Adenosine Triphosphate; C041813:DNA reverse gyrase; D004264:DNA Topoisomerases, Type I; D004250:DNA Topoisomerases, Type II