In the past decade great advances have been made in understanding the links between DNA repair pathways and transcription. Transcription Coupled Nucleotide Excision Repair (TC-NER) leads to the accelerated repair of RNA Polymerase (RNAP) stalling lesions on the transcribed strand of DNA. Mfd (Mutation Frequency Decline Protein) mediates prokaryotic TC-NER and is held in an autoinhibited state in solution until it is activated by binding stalled RNAP. Mfd then uses ATP-dependent forward DNA translocation to move RNAP away from the damage. Mfd remains associated with both DNA and RNAP, which is no longer productively associated with DNA, to form a long- lived intermediate that can translocate for thousands of base pairs. Next, Mfd is predicted to undergo significant conformational changes to expose its binding interface to recruit UvrA. The timing and requirement of these structural rearrangements remains unclear, and this work examined this using intramolecular crosslinking to restrict Mfd movement. This determined that significant rearrangements are essential to form the long-lived Mfd- RNAP translocating intermediate. Furthermore, examination of alternative Mfd functions implicated it in a novel mechanism for R-loop formation and showed that Mfd provides an advantage for E. coli cells in a competitive growth environment following UV damage. Additionally, the modulation and interplay between different DNA associated processes was investigated. The role of MMR proteins in TC-NER has long been controversial and this study indicates they have no essential function in this process. Furthermore, research into the conflict between transcription and double strand break repair illustrates that core RNAP inhibits RecBCD resection activity and Mfd and UvrD have no role in reversing this. Finally, the inhibitory activity of the T4 bacteriophage protein GP55.1, the only known inhibitor of E. coli UvrA, was investigated. It was demonstrated that GP55.1 partially inhibits ATP hydrolysis and DNA binding activity of UvrA.
|Date of Award||11 May 2021|
- The University of Bristol
|Supervisor||Nigel J Savery (Supervisor) & Mark S Dillingham (Supervisor)|
- DNA Repair