Congratulations to our new doctor Kevin Ho Wan with the very Honourable grade and congratulations of the Jury on September 30th 2011



Thesis title : Single-Molecule Studies of the Bacterial Transcription Coupled Repair Process

Abstract :

Transcription coupled repair (TCR), a sub-pathway of the nucleotide excision repair mechanism, is activated when a RNA polymerase (RNAP) is arrested during transcription by DNA damage. TCR is a ubiquitous cellular response important for maintenance of DNA integrity. Some human genetic disorders are associated with defect on TCR, like the Cockayne syndrome for instance. In the bacterium Escherichia coli, TRCF, the product of the mfd gene, is the DNA translocase that couples transcription and DNA repair: it recognizes a stalled ternary elongation complex, dissociates it, and recruits the UvrABC repair machinery.
We used a single molecule approach, by means of magnetic tweezers, to study the initiation of TCR and monitored the dissociation of individual RNAPs by individual TRCFs in real time. We have identified that TRCF acts on stalled RNAP by remodeling the transcription bubble in two successive ATP-dependent steps separated by a novel intermediate denoted RD*.
Statistical analysis of the time required to dissociate the stalled RNAP at different concentrations of TRCF has shown that the displacement of the ternary elongation complex is a three limiting-steps process: (i) recruitment of TRCF to the stalled RNAP, (ii) activation of TRCF and initiation of the dissociation of RNAP by rewinding of 2/3 of the "transcription bubble", and (iii) complete dissociation of the elongation complex. The intermediate complex RD* formed after the first step of rewinding is characterized by a long lifetime, which suggests that it would behave as a temporally reliable marker, enabling the recruitment of the UvrABC repair machinery to the damaged site.