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The mechanism of DNA cleavage by DNA gyrase

Reference	BBS/E/J/00004020
Principal Investigator / Supervisor	Professor Anthony Maxwell
Co-Investigators / Co-Supervisors
Institution	John Innes Centre
Department	John Innes Centre Department
Funding type	Research
Value (£)	15,379
Status	Completed
Type	Institute Project
Start date	01/10/1999
End date	30/09/2002
Duration	36 months

Abstract

DNA gyrase introduces supercoils into DNA using the free energy of ATP hydrolysis. It is a member of a class of enzymes called DNA topoisomerases which control the topological state of DNA in cells. Many of these enzymes are drug targets: gyrase is the target for the quinolone and coumarin antibacterial agents. Gyrase consists of 2 proteins, A and B (97 and 90 kDa); the active enzyme being an A2B2 complex. The mechanism of DNA supercoiling by gyrase involves the breakage of double-stranded DNA and the passage of another DNA segment through the break. The gyrase proteins consist of domains whose function has been determined. GyrA consists of 2 domains: a ~60 kDa N-terminal domain responsible for the DNA breakage-reunion reaction and containing the binding site for quinolone drugs, and a 33 kDa C-terminal domain involved in DNA wrapping. GyrB comprises a 43 kDa N-terminal domain responsible for ATP hydrolysis and containing the coumarin-binding site, and a 47 kDa C-terminal domain which interacts with GyrA and DNA. We have recently solved the crystal structure to high resolution of a 59 kDa N- terminal fragment of GyrA. This now permits the investigation of the gyrase cleavage reaction in detail. The cleavage of DNA by gyrase (and other topoisomerases) and the ability to reseal the break is the essence of the topoisomerase reaction. The cleavage-religation process has to be carried out with high fidelity otherwise the reaction would be aborted. Indeed the action of the quinolone drugs interrupts the cleavage-reaction process and thus arrests DNA supercoiling. A detailed study of the cleavage reaction will reveal mechanistic features of this process and the mode of action of the quinolones. We will use the following approaches: Site-directed mutagenesis. The crystal structure of the 59 kDa GyrA fragment now gives insight into the residues involved in cleavage-religation. Tyr122 is the residue which attacks the phosphodiester bond in DNA and forms a covalent intermediate with the 5has-phosphate, but we need to know the roles of other residues. For example it is important that, following cleavage, the 3has-OH group is held non-covalently by the enzyme. We will use SDM to determine the residue(s) involved in this process. If a residue involved in constraining the 3has-OH group is mutated this may lead to an enzyme which cleaves without religating and would allow study of the cleavage reaction independently, e.g. it would then be possible to assess the effects of quinolones on the cleavage reaction alone. Residues involved in other aspects of the reaction, e.g. aligning and polarising the active-site tyrosine, will also be investigated by SDM. Candidate residues have already been identified from the crystal structure. Construction of novel DNA substrates. A complementary approach will be to manipulate the DNA rather than the enzyme. One way in which this can be done is to introduce thiophosphates into DNA. For example, it is possible to synthesise oligonucleotides with 3has-bridging phosphothioate linkages. When such an oligo is incorporated into a DNA cleavage substrate for gyrase it may well constitute a suicide" substrate, i.e. one which can be cleaved but not religated, due to the reduced nucleophilicity of the SH group. Again this would allow separation of the cleavage and religation processes. Other types of thio- substituted oligos will be synthesised to explore the specificty of the cleavage reaction and to isolate reaction intermediates.

Summary

unavailable

Committee	Closed Committee - Biochemistry & Cell Biology (BCB)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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