Purification of a DNA supercoiling factor from the posterior silk gland of Bombyx mori.
A protein factor with an estimated molecular mass of 50 kDa has been purified to homogeneity from the silk gland of Bombyx mori. In the presence of a molar excess of this factor and eukaryotic DNA topoisomerase II, relaxed circular DNA is converted to the negatively supercoiled form. Eukaryotic DNA topoisomerase I cannot substitute for eukaryotic DNA topoisomerase II in the supercoiling reaction. The reaction is dependent on ATP and is inhibited by VP-16, a specific inhibitor of eukaryotic DNA topoisomerase II. When DNA topoisomerase I is subsequently added to the supercoiling reaction mixture, the supercoiled DNA becomes relaxed. These results suggest that when both the 50-kDa protein and eukaryotic DNA topoisomerase II are present in excess, unconstrained negative supercoils are introduced into DNA.
Ohta
T
T
DNA Research Center, National Institute of Genetics, Mishima, Japan.
Hirose
S
S
D000818:Animals; D012831:Bombyx / Q000378:metabolism*; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004250:DNA Topoisomerases, Type II / Q000378:metabolism; D004278:DNA, Superhelical / Q000378:metabolism*; D005047:Etoposide / Q000494:pharmacology; D007700:Kinetics; D012627:Sebaceous Glands / Q000378:metabolism; D014157:Transcription Factors / Q000302:isolation & purification / Q000378:metabolism*
D004278:DNA, Superhelical; D014157:Transcription Factors; D005047:Etoposide; D004250:DNA Topoisomerases, Type II