Essential Role and Mechanism of Transcription-coupled DNA Repair in Bacteria

Transcription-coupled DNA repair (TCR) is presumed to be a minor sub-pathway of nucleotide excision repair (NER) in bacteria. Global genomic repair (GGR) is thought to perform the bulk of repair independently of transcription. TCR is also believed to be mediated exclusively by Mfd – a DNA translocase of a marginal NER phenotype. Here, we combined in cellulo crosslinking mass spectrometry with structural, biochemical, and genetic approaches to map the interactions within the TCR complex (TCRC) and to determine the actual sequence of events leading to NER in vivo. We demonstrate that RNA polymerase (RNAP) serves as the primary DNA damage sensor and platform for the recruitment of NER enzymes. UvrA and UvrD associate with RNAP continuously, forming a surveillance pre-TCRC. In response to DNA damage, pre-TCRC recruits a second UvrD monomer to form a helicase-competent UvrD dimer that promotes TCRC backtracking. The weakening of UvrD-RNAP interactions renders cells sensitive to genotoxic stress. TCRC then recruits a second UvrA molecule and UvrB to initiate the repair process. Contrary to the conventional dogma, we show that TCR accounts for a vast majority of chromosomal repair events, i.e. TCR thoroughly dominates over GGR. We also show that TCR is largely independent of Mfd. We propose that Mfd plays an indirect role in this process: it participates in removing obstructive RNAPs in front of TCRCs and also in recovering TCRCs from backtracking after repair has been completed.