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Protein gates in DNA gyrase

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posted on 2014-12-15, 10:31 authored by Nicola Louise. Williams
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.

History

Date of award

1999-01-01

Author affiliation

Biochemistry

Awarding institution

University of Leicester

Qualification level

  • Doctoral

Qualification name

  • PhD

Language

en

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