Subventions et des contributions :

Subvention ou bourse octroyée s'appliquant à plus d'un exercice financier. (2017-2018 à 2022-2023)

SUMMARY Cells accumulate DNA damage that must be repaired to maintain genome stability. Nucleotide Excision Repair (NER) removes UV induced DNA damage that, when present on gene transcribed strands, block RNA polymerases. NER is conserved across organisms; we use yeast as genetically tractable model to study how NER operates in vivo .
In cells, DNA is buried in chromatin. Thus, NER must find and remove DNA damage in chromatin. For short regions of the genome, it was shown that chromatin modifying-enzymes that participate in DNA transcription and replication also help NER in chromatin. However, there are indications that the intrinsic mobility of nucleosomes permits exposure of DNA damage to NER, downgrading the participation of chromatin modifying-enzymes.

Supported by NSERC since 2006, our research program is to help describing how NER detects and removes UV induced DNA damage in chromatin.

We propose 3 projects :
1) To investigate how blocked RNA polymerase-I (RNAPI) is released from the damaged transcribed strand . We showed that elongating RNAPI block at UV damage and then are released from the transcribed strand. We will investigate how RNAPI are released from the damaged transcribed strand in yeast mutants that have defects in RNAPI transcription elongation and transcription termination. Investigations will be done by Chromatin Immuno-Precipitation, Chromatin Endogenous-Cleavage and Electron Microscopy.
2) To define if chromatin modifying-enzymes that participate in gene transcription promote NER . We have a unique model, the yeast rDNA locus that is formed by ~150 rRNA genes: half are transcribed and without nucleosomes and half are repressed and with nucleosomes. We will compare NER in the two structures, in wild-type and yeast mutants for chromatin modifying-enzymes. The results will help defining how NER removes DNA damage in chromatin. To measure NER separately in rRNA genes without nucleosomes (internal control) and rRNA genes with nucleosomes, we will employ in nuclei restriction enzyme-digestion and psoralen crosslinking of chromatin. Mapping of UV induced DNA damage will be done by T4endoV and primer extension assays.
3) To search auxiliary-factors associated with NER in chromatin . Yet unknown protein factors could promote NER in chromatin. We will take a proteomic approach, whereby target genomic regions will be tagged with sequences for site-specific recombination. During NER, recombination will be induced to release chromatin rings that will be isolated and analyzed by mass spectrometry, in collaboration with Dr Griesenbeck at the University of Regensburg (Germany).

Theses studies will form graduate students in genetics, biochemistry and proteomics. They will advance our perception of NER in chromatin, will help understanding how genome stability is maintained and what is the impact of failed processes that promote NER in chromatin on DNA repair and cell survival.