Identifying Lys359 as a critical residue for the ATP-dependent reactions of Drosophila DNA topoisomerase II.
Substituting Lys359 with either Gln or Glu in the highly conserved QTK-loop in the DNA gyrase B protein homologous domain of Drosophila topoisomerase II inactivates its catalytic activities. Although strand passage and DNA-dependent ATPase activities are affected in these mutant proteins, their DNA cleavage activity is comparable with the wild-type enzyme and can be stimulated to the same level by topoisomerase-targeting anticancer drugs. The sequence specificity in the DNA cleavage reaction remains unaltered for the mutant proteins. We have used both glass fiber filter binding assay and CsCl density gradient ultracentrifugation to monitor the formation of a salt-stable, protein-clamp complex. Both Gln and Glu mutant proteins can form a clamp complex in the presence of 5'-adenylyl-beta,gamma-imidodiphosphate, albeit with a lower efficiency than the wild-type enzyme. However, the mutant proteins can form a stable complex either in the presence of ATP or in the absence of any cofactors. These results are in an interesting contrast with the wild-type enzyme, which cannot form a stable complex under similar conditions. Our data suggest that Lys359 is critical for the catalytic activity of topoisomerase II and may have an important function in the ATP signaling process.
Hu
T
T
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
Chang
S
S
Hsieh
T
T
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000266:Adenylyl Imidodiphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004250:DNA Topoisomerases, Type II / Q000737:chemistry* / Q000378:metabolism*; D004270:DNA, Circular / Q000737:chemistry / Q000378:metabolism; D004330:Drosophila / Q000201:enzymology*; D018698:Glutamic Acid; D005973:Glutamine; D007700:Kinetics; D008239:Lysine; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011487:Protein Conformation; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D004270:DNA, Circular; D005973:Glutamine; D000266:Adenylyl Imidodiphosphate; D018698:Glutamic Acid; D000255:Adenosine Triphosphate; D004247:DNA; D004250:DNA Topoisomerases, Type II; D008239:Lysine