Koordination von Konformationsänderungen bei der DNA-Superspiralisierung durch DNA-Gyrase
Zusammenfassung der Projektergebnisse
DNA gyrase is a type II DNA topoisomerase that introduces negative supercoils into DNA in an ATP-dependent reaction. In single molecule FRET studies, we have delineated a cascade of DNA- and nucleotide-induced conformational changes in gyrase up to the step where strand passage would occur. We have shown that gyrase with only one catalytic tyrosine that can only cleave on DNA strand can catalyze DNA supercoiling, and introduces supercoils by changing the linking number in steps of two. These findings cannot be explained by the strand passage mechanism, and demonstrate that gyrase can catalyze DNA supercoiling by an alternative mechanism, using nicking/closing. It is currently unclear if this mechanism is an alternative mechanism or if gyrase generally catalyzes DNA supercoiling without strand passage. In contrast to DNA supercoiling, reactions catalyzed by topo II and topo IV, namely ATP-dependent relaxation and decatenation, require double-strand cleavage and strand passage. Using asymmetric gyrase with only one catalytic tyrosine, we have shown that the communication between DNA cleavage by the catalytic tyrosine and the ATPase site is required in a defined, cross-over-like geometry. Furthermore, the CTD on the cleavage-deficient GyrA subunit has a key role in DNA supercoiling. Supercoiling by gyrase with only one catalytic tyrosine is thus not a halfsite reactivity, but involves cooperation of all four subunits. Gyrase is an attractive drug target in the treatment of bacterial infections. Although our results are not of direct economic use, an in-depth understanding of the mechanism of DNA supercoiling by gyrase is instrumental for the targeted design of mechanism-based inhibitors. The discovery of DNA supercoiling in steps of two by a nicking/closing mechanism has been received with great skepticism in the field. Because of this unexpected finding and the difficulties in convincing researchers in the field, we had to focus on aim 3 (Symmetry, asymmetry and subunit coordination) for most of the past funding period, and had to assign lower priorities to aims 1 (Kinetics of conformational changes and their coordination) and 2 (Monitoring strand passage) because of limited resources. Our findings have generated a number of novel hypothesis that we (and others) will study in the future, and have undoubtedly provided a valuable, new impetus to the topoisomerase field.
Projektbezogene Publikationen (Auswahl)
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(2013) Mapping the spectrum of conformational states of the DNA- and C-gates in Bacillus subtilis gyrase, J. Mol. Biol. 425(15):2632-40
Rudolph, M.G. & Klostermeier, D.
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(2014) Single molecule FRET reveals DNA- and nucleotide-induced conformational changes in DNA gyrase preceeding the strand passage reaction, for the DNA repair Special Issue “Single molecule approaches: watching DNA repair one molecule at a time”, DNA repair 16: 23-34
Gubaev, A. & Klostermeier, D.
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(2014) The acidic C-terminal tail of GyrA down-regulates the DNA supercoiling activity of B. subtilis gyrase, J. Biol. Chem. 289(18):12275-12285
Lanz, M.A., Farhat, M., Klostermeier, D.
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(2016) Gyrase with a single catalytic tyrosine can catalyze negative DNA supercoiling by a nicking-closing mechanism, Nucleic Acids Res. 44(21):10354-10366
Gubaev, A., Weidlich, D., Klostermeier, D.
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(2016) Investigating the mechanism of negative DNA supercoiling by gyrase using single molecule FRET and confocal microscopy, in “Single molecule Enzymology”, Methods in Enzymology 581:317-351
Hartmann, S., Weidlich, D. & Klostermeier, D.
