Inducible overexpression, purification, and active site mapping of DNA topoisomerase II from the yeast Saccharomyces cerevisiae.
Overexpression of yeast DNA topoisomerase II was achieved by placing the coding sequences of the gene TOP2 downstream of an inducible promoter PGAL1 on a multicopy plasmid. By using a simple purification procedure, milligram amounts of the enzyme of a high specific activity can be obtained from a few liters of culture. In the presence of a drug VM-26 (teniposide), more than 90% of the enzyme molecules become covalently bound to DNA upon addition of the protein denaturant sodium dodecyl sulfate. The formation of the covalent complex was used to map the tyrosine residue that becomes covalently linked to DNA when the enzyme transiently breaks DNA. After exhaustive digestion of the DNA-protein complex with trypsin, a DNA-linked peptide was purified and sequenced directly to identify Tyr-783 as the active site residue.
Worland
S T
ST
Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, Massachusetts 02138.
Wang
J C
JC
D001665:Binding Sites; D004247:DNA / Q000378:metabolism; D004250:DNA Topoisomerases, Type II / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004278:DNA, Superhelical / Q000378:metabolism; D004790:Enzyme Induction / Q000187:drug effects; D005690:Galactose / Q000494:pharmacology; D005786:Gene Expression Regulation; D005800:Genes, Fungal; D008970:Molecular Weight; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D013713:Teniposide / Q000494:pharmacology; D014170:Transformation, Genetic; D014443:Tyrosine
D004278:DNA, Superhelical; D014443:Tyrosine; D004247:DNA; D013713:Teniposide; D004250:DNA Topoisomerases, Type II; D005690:Galactose