DNA Repair and Transcription in Living OrganismsIn order to allow the analysis of DNA repair and Transcription in living tissues, we have generated two new model systems, a knock-in mouse model (Xpby/y) expressing a fluorescently tagged TFIIH subunit (XPB) to study NER and Transcription and a knock-in mouse model (Fen1y/y) expressing a fluorescently tagged Fen-1 (Flap-endonuclease-1) to study BER and Replication. Both fluorescent proteins are fully functional and both models are bona fide source of knowledge on the spatio-temporal properties of DNA repair in vivo.Thanks to these new model systems, we have pushed live cell analysis and molecular microscopy to an unprecedented level of sophistication, realizing Fluorescence Recovery after Photobleaching (FRAP) analysis, for the very first time, in living mammalian tissues. Our goals for this project are
- 1. Study the structure of the chromatin in different post-mitotic cells
- 2. Measure the DNA repair performance in somatic cells in their natural microenvironment.
- 3. Reconstitute in vitro the chromatin environment found in post-mitotic cells.
Transcription Resumption after DNA RepairWhen DNA lesions are located on a transcribed gene, transcription is blocked at the damaged site and stalled RNAP2 will recruit DNA repair proteins to restore the genetic information via the pathway known as Transcription Coupled Repair (TCR). Repair proteins will excise the damaged DNA and the replication machinery will resynthesize the gap left by the reaction. Once the replication process is completed, transcription is expected to restart to restore proper cellular functions. Despite this important role for maintaining cellular functionality, the mechanism of RNAP2 restart after DNA repair has been poorly studied. Our goals for this project are
- 1. Determine which RNAP2 post-translational modifications are strictly required for restart.
- 2. Investigate whether kinases are implicated in RNAP2 restart.
DNA Repair of Ribosomal DNARibosome biogenesis is the most energetically costly activity of cells, particularly of high-metabolism cells, such as neurons. More than 60% of cellular transcription results from RNAP1. RNAP1 transcription, the first and rate-limiting step of ribosome biogenesis, is specifically dedicated to produce the ribosomal RNAs (rRNA) from the ribosomal DNA (rDNA) located in the nucleolus.
Within DNA Repair, uncharted territories still remain to be explored; repair of rDNA is one of these territories. The need of acquiring knowledge on this field is more and more evident in view of the importance of ribosome biogenesis for cells like neurons and myocytes, which require a high amount of protein production and hence a high amount of ribosomes. More specifically, neurological problems in CS and TTD patients can be due to problems in ribosome biogenesis. We have recently demonstrated that rDNAs are repaired by the TCR machinery and that during this repair reaction, the RNAP1 is displaced at the border of the nucleolus. Our goals for this project are
- 1. Determine which known repair proteins contribute to the repair of the rDNA.
- 2. Discover new repair proteins that contribute to the repair of the rDNA.
- 3. Map in space and time the repositioning of the RNAP1 during DNA repair.
- 4. Investigate which proteins control the displacement of the RNAP1.
- ELL, a novel TFIIH partner, is involved in transcription restart after DNA repair.
Mourgues S., Gautier V., Lagarou A., Bordier C, Mourcet A., Slingerland J., Kaddoum L., Coin F., Vermeulen W., Gonzales de Peredo A., Monsarrat B., Mari PO and Giglia-Mari G. Proc Natl Acad Sci USA (2013) Oct 29; 110(44): 17927-32.
- Mutations in TFIIH causing trichothiodystrophy are responsible for defects in ribosomal RNA production and processing.
Nonnekens J, Perez-Fernandez J, Theil AF, Gadal O, Bonnart C , Giglia-Mari G. Hum Mol Genet (2013) Jul 15;22(14):2881-93.
- Generation of DNA single-strand displacement by compromised nucleotide excision repair.
Godon C#, Mourgues S#, Nonnekens J, Mourcet A, Coin F, Vermeulen W, Giglia-Mari G. EMBO J. (2012) 31: 3550-3563.
- Kinetics of endogenous mouse FEN1 in base excision repair.
Kleppa L, Mari PO, Larsen E, Lien GF, Godon C, Theil AF, Nesse GJ, Wiksen H, Vermeulen W, Giglia-Mari G*, Klungand, A *. Nucleic Acids Res (2012) 40: 9044-9059..
- Differentiation driven changes in the dynamic organization of Basal transcription initiation.
Giglia-Mari, G.*, Theil, A. F., Mari, P. O., Mourgues, S., Nonnekens, J., Andrieux, L. O., de Wit, J., Miquel, C., Wijgers, N., Maas, A., Fousteri, M., Hoeijmakers, J. H., and Vermeulen, W.* PLoS Biol (2009) 7, e1000220..
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- Association pour La Recherche sur le Cancer
- Ligue contre le Cancer