Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.696585
Title: Protein gates in DNA gyrase
Author: Williams, Nicola Louise
Awarding Body: University of Leicester
Current Institution: University of Leicester
Date of Award: 1999
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Abstract:
DNA gyrase is a molecular machine comprising a series of protein gates. The opening and closing of these gates enables the passage of one segment of double-stranded DNA (the T segment) through a transient break in another (the G segment). We have blocked the passage of DNA through each of three dimer interfaces within gyrase and investigated the effects on gyrase mechanism. This has been achieved by cross-linking novel cysteine residues on either side of the dimer interface, or trapping the dimer interface in a closed conformation using a non-hydrolysable ATP analogue. Cross-linking a pair of novel cysteine residues on either side of the bottom dimer interface of DNA gyrase blocks catalytic supercoiling. Limited strand passage is allowed, but T-segment release is prevented. In contrast, ATP-independent relaxation of negatively supercoiled DNA is completely abolished, suggesting that T-segment entry via the bottom gate is blocked. These findings support a two-gate model for supercoiling in by DNA gyrase and suggest that relaxation by gyrase is the reversal of supercoiling. Cross-linking a truncated version of gyrase, (A642B2) that lacks the DNA wrapping domains, does not block ATP-dependent relaxation. This indicates that passage of DNA through the bottom dimer interface is not essential for this reaction. Using a similar approach, we have locked the DNA gate of gyrase using cysteine cross-linking. We show that this locked-gate mutant can bind quinolone drugs and perform DNA cleavage. However, locking the DNA gate prevents strand passage and the ability of DNA to stimulate ATP hydrolysis.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.696585  DOI: Not available
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