Single molecule investigation of lesion hand-off during transcription coupled repair
- Molecular Horizons Institute and School of Chemistry, University of Wollongong, Wollongong, Australia.
- Illawarra Health and Medical Research Institute, Wollongong, Australia.
During transcription elongation, bacterial RNA polymerase (RNAP) can pause, backtrack or stall on template DNA. Stalled transcription elongation complexes (TECs) at sites of lesions can be rescued by the transcription terminator Mfd. In this transcription-coupled repair (TCR) reaction, Mfd recognizes stalled TECs and recruits the nucleotide excision repair machinery (NER) to the site. Extensive biochemical, structural, genetic and single-molecule investigations have shed insight into various aspects of TCR. Despite these studies, the molecular mechanisms of stalled RNAP recognition by Mfd and recruitment of the NER machinery in vivo largely remain unknown. Single-molecule live-cell imaging of fluorescently labelled Mfd revealed that Mfd associates with TECs even in the absence of exogenous genotoxic stresses. This interaction requires an intact RNAP-interacting domain of Mfd, and is stabilized under conditions in which RNAP is stalled on template DNA. Binding of Mfd is highly regulated in cells, and mutations in its ATPase domain or the UvrB-homology module result in deregulated and non-specific DNA binding. Using an interval imaging approach on live-cells, we determined the lifetime of Mfd to be 18s during TCR in wild-type cells. We found that this lifetime is influenced by the presence of the NER machinery components UvrA and UvrB. Whereas absence of UvrA led to a longer lifetime of 30s, cells lacking functional UvrB exhibited Mfd foci that were arrested on DNA for 60-80s. Our findings shed insight into the recruitment and regulation of the transcription-repair coupling factor, and the execution of the TCR in cells.