Purification and characterization of reverse gyrase from Sulfolobus shibatae. Its proteolytic product appears as an ATP-independent topoisomerase.
Sulfolobus shibatae B12 is a thermophilic archaebacterium that contains an inducible virus named SSV1. The viral DNA has been shown to be positively supercoiled before encapsidation. We have previously purified an archaebacterial DNA topoisomerase from Sulfolobus acidocaldarius DSM 639, reverse gyrase, likely responsible for this positive supercoiling reaction. In order to study an homogeneous system containing both reverse gyrase and one of its preferential substrate, SSV1 DNA, we have purified this enzyme from S. shibatae. During the course of the purification, we have detected another topoisomerase activity. In order to separate and purify these two topoisomerases, we have devised a new purification procedure. Purified S. shibatae reverse gyrase is a 124-kDa monomer, with a Stokes radius of 43 A and a sedimentation coefficient of 6.2 S. It is able to perform a DNA reverse gyration per se at 10 mM NaCl in a Mg- and ATP-dependent manner. The other topoisomerase is a monomer of about 40 kDa, with a Stokes radius of 25 A and a sedimentation coefficient of 4 S. This additional topoisomerase activity is Mg-dependent and ATP-independent and catalyzes only a relaxation reaction of negatively supercoiled DNA at 150 mM NaCl. This new ATP-independent topoisomerase activity seems to be a proteolysis product of reverse gyrase.
Nadal
M
M
Laboratoire d'Enzymologie des Acides Nucléiques, Universite Paris Sud, Orsay, France.
Couderc
E
E
Duguet
M
M
Jaxel
C
C
D000255:Adenosine Triphosphate / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D001435:Bacteriophages / Q000378:metabolism; D015153:Blotting, Western; D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004264:DNA Topoisomerases, Type I / Q000302:isolation & purification* / Q000378:metabolism*; D004250:DNA Topoisomerases, Type II / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D004279:DNA, Viral / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases; D007700:Kinetics; D008970:Molecular Weight; D011487:Protein Conformation; D013379:Substrate Specificity; D016944:Sulfolobus / Q000201:enzymology*; D014461:Ultracentrifugation
D000596:Amino Acids; D004279:DNA, Viral; D000255:Adenosine Triphosphate; D010450:Endopeptidases; C041813:DNA reverse gyrase; D004264:DNA Topoisomerases, Type I; D004250:DNA Topoisomerases, Type II