On the coupling between ATP usage and DNA transport by yeast DNA topoisomerase II.
The initial rates of ATP hydrolysis and relaxation of negatively supercoiled DNA by highly purified wild-type and mutant yeast DNA topoisomerase II were measured under identical conditions to study the coupling between the ATPase activity of a type II DNA topoisomerase and its catalysis of the transport of one DNA segment through another. The results indicate that the binding of the enzyme to DNA stimulates its intrinsic ATPase activity by about 20-fold, and ATP binding to the pair of ATPase sites in a DNA-bound dimeric enzyme appears to be cooperative. The cooperativity in ATP binding may be significant in the coordination of the two halves of a DNA-bound enzyme dimer. At low ATP concentrations, the rate-limiting step in ATP usage appears to be slower than that in DNA transport, and DNA transport is relatively efficient in terms of ATP consumption: 1.9 +/- 0.5 ATP molecules are hydrolyzed/DNA transport event. At a saturating ATP concentration, however, there appears to be a reversal of these rate-limiting steps, and DNA transport is less efficient: 7.4 +/- 1.0 ATP molecules are hydrolyzed/DNA transport event. These data are interpreted in terms of a model in which a DNA-bound enzyme acts as an ATP-operated clamp for the capture and transport of a second DNA segment.
Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, Massachusetts 02138.
J E
JE
Lindsley
J C
JC
Wang
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000251:Adenosine Triphosphatases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism* / Q000494:pharmacology; D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D001483:Base Sequence; D004250:DNA Topoisomerases, Type II / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism; D004278:DNA, Superhelical / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
D004271:DNA, Fungal; D004278:DNA, Superhelical; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D000266:Adenylyl Imidodiphosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D004250:DNA Topoisomerases, Type II