Proteolysis patterns of epitopically labeled yeast DNA topoisomerase II suggest an allosteric transition in the enzyme induced by ATP binding.
A cloned yeast TOP2 gene was modified to produce yeast DNA topoisomerase II (EC 5.99.1.3) epitopically labeled at its amino or carboxyl terminus. Limited digestion with SV8 endoprotease shows three distinct protease-sensitive sites in each polypeptide of the dimeric enzyme. These sites were mapped by immunostaining of the end-labeled proteolytic fragments resolved by SDS/polyacrylamide gel electrophoresis; two of the mapped locations were confirmed by sequencing the amino ends of two unlabeled peptic fragments. Proteolytic cleavage by SV8 endoprotease at a pair of sites corresponding to the carboxyl sides of Glu-411 and Glu-680 is modulated by the binding of the nonhydrolyzable ATP analogs adenosine 5'-[beta, gamma-imido]triphosphate (5'-adenylyl imidodiphosphate) and adenosine 5'-[gamma-thio]triphosphate: in their absence cleavage occurs predominantly at Glu-411; in the presence of either analog, cleavage occurs predominantly at Glu-680. These results are interpreted in terms of allosteric interdomainal movements in the type II DNA topoisomerase following the binding of ATP.
Lindsley
J E
JE
Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, MA 02138.
Wang
J C
JC
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004250:DNA Topoisomerases, Type II / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D010450:Endopeptidases; D000939:Epitopes / Q000032:analysis; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
D000939:Epitopes; D046911:Macromolecular Substances; D010446:Peptide Fragments; D000255:Adenosine Triphosphate; D010450:Endopeptidases; D004250:DNA Topoisomerases, Type II