PROJECTS

DNA REPAIR AND TRANSCRIPTION IN LIVING ORGANISMS

In 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 (Xpb^y/y) expressing a fluorescently tagged TFIIH subunit (XPB) to study NER and Transcription and a knock-in mouse model (Fen1^y/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 REPAIR

When 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 DNA

Ribosome 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.

SELECTED PUBLICATIONS

• XAB2 Dynamics during DNA Damage-Dependent Transcription Inhibition
  Donnio LM, Cerutti E, Magnani C, Neuillet D, Mari PO, Giglia-Mari G.
  
  BioRxiv
  doi: https://doi.org/10.1101/2021.12.23.473962
• β-Actin and Nuclear Myosin I are responsible for nucleolar reorganization during DNA Repair
  Cerutti E , Daniel L, Donnio LM, Neuillet D, Magnani C, Mari PO, Giglia-Mari G
  BioRxiV
  doi: https://doi.org/10.1101/646471
• Active DNA damage eviction by HLTF stimulates nucleotide excision repair.
  van Toorn M, Turkyilmaz Y, Han S, Zhou D, Kim HS, Salas-Armenteros I, Kim M, Akita M, Wienholz F, Raams A, Ryu E, Kang S, Theil AF, Bezstarosti K, Tresini M, Giglia-Mari G, Demmers JA, Schärer OD, Choi JH, Vermeulen W, Marteijn JA.

  Molecular Cell. 2022 Apr 7;82(7):1343-1358.e8.
  doi: 10.1016/j.molcel.2022.02.020.
  PMID: 35271816

• Cell-type specific concentration regulation of the basal transcription factor TFIIH in XPBy/y mice model.
  Donnio LM, Miquel C, Vermeulen W, Giglia-Mari G, Mari PO.
  Cancer Cell International. 2019 Sep 10;19:237.
  doi: 10.1186/s12935-019-0945-4.
  PMID : 31516394
• CSB-Dependent Cyclin-Dependent Kinase 9 Degradation and RNA Polymerase II Phosphorylation during Transcription-Coupled Repair.
  Donnio LM, Lagarou A, Sueur G, Mari PO, Giglia-Mari G.
  Molecular and Cellular Bioliolgy. 2019 Mar 1;39(6). pii: e00225-18.
  doi: 10.1128/MCB.00225-18.
  PMID: 30602496
• Mechanistic insights in transcription-coupled nucleotide excision repair of ribosomal DNA.
  Daniel L, Cerutti E, Donnio LM, Nonnekens J, Carrat C, Zahova S, Mari PO, Giglia-Mari G.
  Proceedings of the National Academy of Sciences U S A.  2018 Jul 17;115(29):E6770-E6779. doi:10.1073/pnas.1716581115.
  PMID: 29967171

FUNDING

• AFM Myoneuralp (2022-2027)
• INCA (2019-2023)
• Ligue contre le Cancer (2020-2022)