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Nucleic acid fragment analysis system; probing mechanisms of nucleic acid replication repair and catalysis

Reference	REI18458
Principal Investigator / Supervisor	Dr David Mark Williams
Co-Investigators / Co-Supervisors	Professor Jane Grasby, Professor Jon Sayers, Dr Michelle Webb, Professor Nicholas Williams
Institution	University of Sheffield
Department	Chemistry
Funding type	Research
Value (£)	44,376
Status	Completed
Type	Research Grant
Start date	01/04/2003
End date	01/04/2006
Duration	36 months

Abstract

We have recently shown that denaturing HPLC (DHPLC) can be invaluable for developing kinetic assays for enzymes involved in DNA replication and repair and in the study of RNA transesterification by catalytic RNA (ribozymes). We have also used the technique for RNA footprinting. Using DHPLC we have achieved the separation of nucleic acid fragments with high resolution and with the appropriate choice of buffer conditions and gradient, have obtained a comparable fractionation to that achievable using polyacrylamide gel electrophoresis (PAGE). Furthermore, when used in combination with fluorescent detection of labelled nucleic acids, we have obtained data of a suitable quality for deriving kinetic constants that show good correlation with traditional radioactive assays. Since the separation and quantitation is performed simultaneously, there is a considerable saving in time, since unlike standard radioactive-based assays, there is no PAGE, no drying of gels and the data is obtained without the need for phosphorimaging techniques. In addition, the hazards of using radioactivity are avoided and regular relabelling of the substrate is not necessary. Assays have been developed to determine the catalytic parameters of DNA polymerase, a 5-nuclease essential in DNA replication and the hairpin and VS ribozymes. The development of these assays has greatly facilitated work on the structure-function relationships and mechanistic studies of these enzymes. It is planned to develop similar assays to enable programmes on the study of RNaseHII, O6-alkylguanine DNA alkylltransferase, in vitro selected deoxyribozymes and ribozyme mimetics. The current DHPLC system that we have is in constant use and there is considerable demand for further access to this equipment. Whilst the application of DHPLC considerably speeds up the determination of rate constants, a typical kinetic run (96 well plate) takes up to 12 hours. This leaves no instrument time for the development of assays associated with new projects with little time to complete all the data analysis generated through ongoing research. We therefore request 50 per cent of the funding required for the purchase of a second DHPLC system.

Summary

unavailable

Committee	Closed Committee - Biomolecular Sciences (BMS)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	Research Equipment Initiative 2002 (REI) [2002]
Funding Scheme	X – not Funded via a specific Funding Scheme

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