Blanc SVSE 5 - Blanc - SVSE 5 - Physique, chimie du vivant et innovations biotechnologiques

Single-molecule reconstruction of transcription-coupled DNA repair pathways. – RepOne

We use novel and ultra-sophisticated microscopies enabling us to simultaneously manipulate and observe single molecules and their interactions.

We have reconstituted the main steps of the transcription-coupled repair pathway and monitored the order of assembly and disassembly of components on damaged DNA

We wish to tease apart the final steps of the process whereby repair proteins are cleared from DNA after repair. We are also working to extend these results to the yeast system.

We have published these results in a high-impact journal, and a second article is currently under review. We have a patent application pending.

Submission summary

This project proposes to use single-molecule methods to reconstruct from the bottom up a complete DNA repair pathway, that of transcription-coupled repair (TCR).

Transcription-coupled repair, or TCR, is a key DNA repair pathway which employs components of the transcription machinery to identify DNA lesions and initiate their repair. TCR is a highly conserved process, found in all living organisms; in humans it has recently been implicated in the protection of DNA from damage that may arise in skin tissue exposed to ultraviolet (UV) light, or epithelial lung tissue exposed to tobacco smoke. TCR begins when transcribing RNA polymerase (RNAp) stalls on a DNA lesion such as the canonical thymine-thymine dimer caused by exposure to ultraviolet light. As it is extremely stably bound to DNA the RNAp thus stays stuck on the lesion, preventing repair factors from accessing it. The stalled RNAp then recruits a superfamily 2 DNA helicase which drives RNAp off of DNA while at the same time recruiting downstream repair factors such as exonucleases and helicases to the damage site made accessible by clearing the RNAp. Thus after incision of the damaged DNA strand on either side of the damage, helicase unwinding removes the damaged fragment, allowing for resynthesis of fresh DNA.

As is clear from this succinct description, TCR is a complex, multi-step and multi-component pathway, and this has made its mechanistic and kinetic analysis particularly challenging. Recent work from our laboratory (“Initiation of transcription-coupled repair characterized at single-molecule resolution”, by K. Howan et al., Nature 2012 490: 431—434) has provided new experimental methods from the single-molecule toolkit to begin molecular reconstruction of the TCR pathway from E. coli. Using single-molecule DNA nanomanipulation, we have been able to observe single RNAp molecules initiate transcription and then stall on DNA damage. We are then able to introduce the superfamily 2 TCR helicase, Mfd, and measure the rate at which it drives stalled RNAp off of DNA. Most interestingly, we found that Mfd remains on the DNA for a long, reliable amount of time (~5 +/- 2 minutes (SD)). These results are consistent with the role that Mfd has downstream of RNAp displacement, namely that of recruitment of the UvrABC excisime. Indeed it makes sense that Mfd, having removed the most long-lived and stable marker for DNA damage – the RNAp itself – would in turn remain on the DNA for a long and reliable amount of time so as to ensure that downstream repair components will indeed be recruited to the damage site to direct its repair.

In this grant we therefore propose to pursue reconstruction of downstream steps of DNA repair using these and other single-molecule approaches we have implemented and developed over the past few years in the lab (magnetic trapping; optical trapping; combined single-molecule fluorescence + magnetic trapping). The proposed research described in this grant will thus characterize 1) structural and stochiometric features of the Mfd-DNA damage-signaling complex, 2) assembly and action of the UvrA/UvrB/UvrC proteins on the Mfd-DNA damage-signaling complex, 3) subsequent kinetics of DNA incision and 4) post-incision and post-repair clearance of remnant repair components. Both bacterial and yeast systems will be studied.These experiments will provide us with both a detailed yet integrative view of the determinants behind the remarkable efficiency of DNA repair.

Project coordination

Terence Strick (Organisme de recherche)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.


Help of the ANR 350,000 euros
Beginning and duration of the scientific project: December 2013 - 42 Months

Useful links

Explorez notre base de projets financés



ANR makes available its datasets on funded projects, click here to find more.

Sign up for the latest news:
Subscribe to our newsletter