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Energy coupling in type II topoisomerases: why do they hydrolyse ATP?

Reference	BB/C517376/1
Principal Investigator / Supervisor	Professor Anthony Maxwell
Co-Investigators / Co-Supervisors	Dr Andrew Bates
Institution	John Innes Centre
Department	Biological Chemistry
Funding type	Research
Value (£)	218,193
Status	Completed
Type	Research Grant
Start date	15/11/2005
End date	14/04/2009
Duration	41 months

Abstract

A key question in biology is how enzymes achieve their exquisite specificity. This is usually considered in terms of enzyme-substrate (or enzyme-transition state) interactions. However, there are some instances where the specificity apparently exceeds that expected from thermodynamic considerations. Examples include DNA polymerises, aminoacyl-tRNA synthetases and the ribosome. In these cases additional mechanisms, which must be energy requiring, exist to attain the high-level specificity of these processes. DNA topoisomerases are involved in the inter-conversion of different topological forms of DNA and participate in DNA replication, transcription and the control of gene expression. With the exception of DNA gyrase, these enzymes catalyse reactions that are not obviously energy-requiring. However, type II topoisomerases require ATP hydrolysis. The question is why? Work by Rybenkov et al. (1997) has suggested that ATP hydrolysis is utilised to drive topo II reactions beyond thermodynamic equilibrium, towards simplification of DNA topology. In this proposal we aim to elucidate the mechanism of this phenomenon. Specifically we will investigate the Rybenkov observations and determine how general they are by examining a number of type II enzymes and three different reactions; preliminary work from our labs has suggested that the phenomenon may only apply to bacterial topo IV. Using a number of approaches we will explore the energy requirements of the reaction and determine whether ATP binding and/or hydrolysis is required in both monomers of the enzyme dimer. There have been three models suggested to explain this phenomenon. We will use small circle DNA substrates as a probe to distinguish two of these models, and we will examine the cooperatively between DNA and nucleotide binding to explore the third (kinetic proofreading). Single-molecule experiments, time-resolved CD and Monte-Carlo simulations will be used to further test these models.

Summary

unavailable

Committee	Closed Committee - Biomolecular Sciences (BMS)
Research Topics	Microbiology
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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