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Mechanism of transcription complex displacement by a transcription-repair coupling factor

Reference	BB/C507053/1
Principal Investigator / Supervisor	Professor Nigel Savery
Co-Investigators / Co-Supervisors
Institution	University of Bristol
Department	Biochemistry
Funding type	Research
Value (£)	192,082
Status	Completed
Type	Research Grant
Start date	01/10/2004
End date	30/09/2007
Duration	36 months

Abstract

The Escherichia coli Mfd protein is a transcription-repair coupling factor, responsible for promoting the preferential repair of DNA lesions in the transcribed strand of active genes. Its role in the repair of lesions that present an absolute block to transcription is well established, but recent experiments suggest that it also has a role in the repair of lesions that do not prevent transcription. The objectives of this proposal are: (1) To determine the effect of the transcription-repair coupling factor, Mfd, on RNA polymerase at DNA lesions that do not block transcription. We will generate templates containing single defined lesions in the transcribed strand, and measure the effect of Mfd on the frequency of transcription termination at the lesion and the rate of lesion bypass using conventional and rapid-mix in vitro transcription assays. (2) To determine the DNA template requirements for Mfd-catalysed displacement of RNA polymerase from DNA. Using purified proteins and nucleic acids we will assemble stalled transcription complexes carrying modifications in the handle of DNA required by Mfd. We will determine the nature of modifications that prevent Mfd displacing RNA polymerase and will interpret this data to refine current modules of DNA manipulation by Mfd. We will also use rapid reaction footprinting techniques to detect short-lived intermediates in the displacement process. (3) To determine the DNA template requirements for optimal DNA-binding by Mfd. We will define the minimum DNA template length for Mfd binding and define the optimal binding sequence for Mfd using a SELEX in vitro selection procedure.

Summary

unavailable

Committee	Closed Committee - Genes & Developmental Biology (GDB)
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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