Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.690774
Title: Damage detection during transcription coupled DNA repair in Escherichia coli
Author: Haines, Nia
ISNI:       0000 0004 5915 3490
Awarding Body: University of Bristol
Current Institution: University of Bristol
Date of Award: 2015
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Abstract:
Transcription coupled DNA repair (TCR) is a sub-pathway of nucleotide excision repair (NER), which enhances the repair of damage on the template strand of active genes. In E. coli, this is initiated by stalling of the transcribing RNA polymerase (RNAP) at the lesion. This results in recruitment of the Mfd helicase which displaces RNAP, allowing repair to be carried out by Uvr proteins through the NER pathway. In this work, in order to better understand damage recognition in this pathway, single lesion-containing plasmid templates were created and analysed for repair. In these experiments the RNAP stalling step was separated from damage recognition by artificially stalling the RNAP upstream of the lesion. The results demonstrated that RNAP does not need to be stalled directly at a lesion in order to stimulate enhanced repair of a downstream lesion on the transcribed strand. This occurs both for the classical cyclopyrimidine dimer TCR substrate and the non-RNAP-stalling biotin and abasic site lesions. The TCR pathway was also shown to be inherently strand specific, as even lesions far downstream of the stalled RNAP were only repaired by TCR if they were on the transcribed strand. Introduction of a backtracking OpS transcriptional pause instead of a complete RNAP stall was also shown to induce an increase in downstream repair ofnon-RNAPstalling lesions. This downstream repair ability is likely facilitated by the observed translocation ability of the Mfd protein after RNAP displacement, and suggests that Mfd is part of a translocating damage search complex which is able to detect damage away from the initial RNAP stall site. The possible role of UvrD in backtracking RNAP to facilitate an Mfd-independent TCR pathway was investigated, however no evidence for this proposed ability of UvrD was observed. To try and understand the complexity of TCR and the possibly overlapping pathways, the initial steps in developing a high throughput genome wide in vivo assay for TCR were carried out.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.690774  DOI: Not available
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