Identification of active site residues in the "GyrA" half of yeast DNA topoisomerase II.
Site-directed mutagenesis was carried out at 10 highly conserved polar residues within the C-terminal half of yeast DNA topoisomerase II, which corresponds to the A subunit of bacterial DNA gyrase, to identify amino acid side chains that augment the active site tyrosine Tyr-782 in the breakage and rejoining of DNA strands. Complementation tests show that alanine substitution at Arg-690, Asp-697, Lys-700, Arg-704, or Arg-781, but not at His-735, His-736, Glu-738, Gln-750, or Asn-828, inactivates the enzyme in vivo. Measurements of DNA relaxation and cleavage by purified mutant enzymes show that these activities are abolished in the R690A mutant and are much reduced in the mutants D697A, K700A, R704A, and R781A. When a Y782F polypeptide with a phenylalanine substituting for the active site tyrosine was expressed in cells that also express the R690A polypeptide, the resulting heterodimeric yeast DNA topoisomerase II was found to nick plasmid DNA. Thus in a dimeric wild-type enzyme, Tyr-782 in one protomer and Arg-690 in the other cooperate in trans in the catalysis of DNA cleavage. For the residues D697A, K700A, R704A, and R781A, their locations in the crystal structures of type II DNA topoisomerase fragments suggest that Arg-781 and Lys-700 might be involved in anchoring the 5' and 3' sides of the broken DNA, respectively, and the roles of Asp-697 and Arg-704 are probably less direct.
Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA.
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D001665:Binding Sites / Q000235:genetics; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II / Q000737:chemistry*; D004278:DNA, Superhelical / Q000378:metabolism; D019281:Dimerization; D005656:Fungal Proteins / Q000737:chemistry; D005658:Fungi / Q000201:enzymology; D005816:Genetic Complementation Test; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009690:Nucleic Acid Conformation; D011487:Protein Conformation
D004278:DNA, Superhelical; D005656:Fungal Proteins; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II