Abstract
Gyrase is a type II DNA topoisomerase that introduces negative supercoils into DNA in an ATP-dependent reaction. It consists of a topoisomerase core, formed by the N-terminal domains of the two GyrA subunits and by the two GyrB subunits, that catalyzes double-stranded DNA cleavage and passage of a second double-stranded DNA through the gap in the first. The C-terminal domains (CTDs) of the GyrA subunits form a β-pinwheel and bind DNA around their positively charged perimeter. As a result, DNA is bound as a positive supercoil that is converted into a negative supercoil by strand passage. The CTDs contain a conserved 7-amino acid motif that connects blades 1 and 6 of the β-pinwheel and is a hallmark feature of gyrases. Deletion of this so-called GyrA-box abrogates DNA bending by the CTDs and DNA-induced narrowing of the N-gate, affects T-segment presentation, reduces the coupling of DNA binding to ATP hydrolysis, and leads to supercoiling deficiency. Recently, a severe loss of supercoiling activity of Escherichia coli gyrase upon deletion of the non-conserved acidic C-terminal tail (C-tail) of the CTDs has been reported. We show here that, in contrast to E. coli gyrase, the C-tail is a very moderate negative regulator of Bacillus subtilis gyrase activity. The C-tail reduces the degree of DNA bending by the CTDs but has no effect on DNA-induced conformational changes of gyrase that precede strand passage and reduces DNA-stimulated ATPase and DNA supercoiling activities only 2-fold. Our results are in agreement with species-specific, differential regulatory effects of the C-tail in gyrases from different organisms.
Highlights
DNA gyrase catalyzes ATP-dependent negative DNA supercoiling
The GyrA C-terminal Acidic Tail in B. subtilis Gyrase Does Not Associate with the C-terminal domains (CTDs) or Other Parts of Gyrase—A recent study has identified the C-terminal tail of E. coli gyrase as a regulatory element and has shown that its deletion severely impacts the DNA supercoiling activity (34)
The C terminus of the GyrA subunit, an appendix to the -pinwheel fold of the C-terminal domain that varies in sequence and length, has received little attention until recently when its modulatory effect on DNA wrapping and supercoiling E. coli gyrase activity was discovered (34)
Summary
DNA gyrase catalyzes ATP-dependent negative DNA supercoiling. Results: Deletion of the acidic C-terminal tail causes stronger DNA bending by the B. subtilis gyrase C-terminal domains and accelerated DNA-stimulated ATPase and supercoiling activities of gyrase. It consists of a topoisomerase core, formed by the N-terminal domains of the two GyrA subunits and by the two GyrB subunits, that catalyzes double-stranded DNA cleavage and passage of a second double-stranded DNA through the gap in the first. The CTDs contain a conserved 7-amino acid motif that connects blades 1 and 6 of the -pinwheel and is a hallmark feature of gyrases Deletion of this so-called GyrA-box abrogates DNA bending by the CTDs and DNA-induced narrowing of the N-gate, affects T-segment presentation, reduces the coupling of DNA binding to ATP hydrolysis, and leads to supercoiling deficiency. A highly conserved C-terminal domain (CTD), connected to the N-terminal domain (NTD) of GyrA by a flexible linker (see Fig. 2), is an important determinant for the DNA supercoiling activity of gyrase (21, 22), and deletion of the CTDs converts gyrase into a regular type II topoisomerase that catalyzes ATPdependent relaxation (23).
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