ATPase domain of eukaryotic DNA topoisomerase II. Inhibition of ATPase activity by the anti-cancer drug bisdioxopiperazine and ATP/ADP-induced dimerization.
We have prepared full-length Drosophila and human topoisomerase II and truncation constructs containing the amino-terminal ATPase domain, and we have analyzed their biochemical properties. The ATPase activity of the truncation proteins, similar to that of the full-length proteins, is greatly stimulated by the presence of DNA. This activity of the truncation proteins is also sensitive to the inhibition by the drug bisdioxopiperazine, ICRF-193, albeit at a much lower level than the full-length protein. Therefore, bisdioxopiperazine can directly interact with the NH(2)-terminal ATPase domain, but the drug-enzyme interaction may involve other domains as well. The ATPase activity of the ATPase domain protein showed a quadratic dependence on enzyme concentration, suggesting that dimerization of the NH(2)-terminal domain is a rate-limiting step. Using both protein cross-linking and sedimentation equilibrium analysis, we showed that the ATPase domain exists as a monomer in the absence of cofactors but can readily dimerize in the presence of a nonhydrolyzable analog of ATP, 5'-adenylyl-beta,gamma-imidodiphosphate. More interestingly, both ATP and ADP can also promote protein dimerization. This result thus suggests that the protein clamp, mediated through the dimerization of ATPase domain, remains closed after ATP hydrolysis and opens upon the dissociation of ADP.
Hu
Tao
T
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
Sage
Harvey
H
Hsieh
Tao-shih
TS
D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D003432:Cross-Linking Reagents; D027081:DNA Gyrase / Q000378:metabolism; D017931:DNA Primers; D004250:DNA Topoisomerases, Type II / Q000737:chemistry* / Q000378:metabolism*; D054659:Diketopiperazines; D019281:Dimerization; D004330:Drosophila / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D010879:Piperazines / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D059005:Topoisomerase II Inhibitors
D003432:Cross-Linking Reagents; D017931:DNA Primers; D054659:Diketopiperazines; D004791:Enzyme Inhibitors; D010879:Piperazines; D059005:Topoisomerase II Inhibitors; C070899:4,4'-(1,2-dimethyl-1,2-ethanediyl)bis-2,6-piperazinedione; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II