Kinetic and Thermodynamic Analysis of Mutant Type II DNA Topoisomerases That Cannot Covalently Cleave DNA

• Published in: The Journal of biological chemistry Vol. 274; no. 6; pp. 3446 - 3452
• Main Authors: Morris, Shayne K., Harkins, Timothy T., Tennyson, Rachel B., Lindsley, Janet E.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 05.02.1999; American Society for Biochemistry and Molecular Biology
• Subjects: Adenosine Triphosphatases - metabolism; Base Sequence; DNA - metabolism; DNA Primers; DNA Topoisomerases, Type II - chemistry; DNA Topoisomerases, Type II - metabolism; Hydrolysis; Kinetics; Saccharomyces cerevisiae; Thermodynamics
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.274.6.3446

DNA topoisomerase II catalyzes two different chemical reactions as part of its DNA transport cycle: ATP hydrolysis and DNA breakage/religation. The coordination between these reactions was studied using mutants of yeast topoisomerase II that are unable to covalently cleave DNA. In the absence of DNA, the ATPase activities of these mutant enzymes are identical to the wild type activity. DNA binding stimulates the ATPase activity of the mutant enzymes, but with steady-state parameters different from those of the wild type enzyme. These differences were examined through DNA binding experiments and pre-steady-state ATPase assays. One mutant protein, Y782F, binds DNA with the same affinity as wild type protein. This mutant topologically traps one DNA circle in the presence of a nonhydrolyzable ATP analog under the same conditions that the wild type protein catenates two circles. Rapid chemical quench and pulse-chase ATPase experiments reveal that the mutant proteins bound to DNA have the same sequential hydrolysis reaction cycle as the wild type enzyme. Binding of ATP to the mutants is not notably impaired, but hydrolysis of the first ATP is slower than for the wild type enzyme. Models to explain these results in the context of the entire DNA topoisomerase II reaction cycle are discussed.