Caractérisation des mutations et des cibles des gènes mutateurs à l'échelle du génome – MUTome

Organisms are constantly in evolutionary conflicts to maintain and diversify their genome, since a sufficient stability is required to copy the blueprint of the initial genetic information during development but, on long term, a low degree of instability is beneficial for the species to create the genetic diversity on which the selection will act. Over the last years, considerable advances have been made in the mutagenesis and DNA damage response fields. Several key mechanisms regarding cellular responses to DNA damage have been detailed at the molecular level and their role in genetic disease and cancer has been well established. To further elucidate these complex and connected repair pathways, we propose to re-investigate mutational profiles at the genome-wide level, taking advantage of the recent advances in high-throughput Sequencing (HT-SEQ) technologies. This novel approach will have the unprecedented advantage to overcome the intrinsic limitation of the current specialized and artificial mutation 'reporter assays', providing unbiased and large-scale information on the complex features of all natural targets. To interrogate mutational genome-wide profiles in the model yeast S. cerevisiae, we will take advantage of our access to the Applied-SOLiD sequencer in the Institut Curie platform, which allows generating up 4GB of 35bp length fragments in a week experiment, (i.e. >107 fragments). This is the equivalent to approximately 200 S. cerevisiae genomes. HT-SEQ of fragments and paired-end libraries which allow to sequence both extremities of a 1-15 kb size-selected fragment will permit to detect all kind of mutagenic events: punctual, indel and large chromosomal rearrangements including translocations, insertions and inversions and change in chromosome copy number. In this project, we wish to establish (i) the spontaneous mutation spectrum and rate of the wild-type haploid and homozygous diploid strains per generation per cell, then used as the reference genome, (ii) the frequencies and the nature of mutations occurring in the rad27', pif1', mre11', mh2', tsa1', tho2', cac1' cac3' and mec1' tel1' sml1' mutator strains. These genes play a role in distinct DNA metabolism processes. Their inactivation has a strong mutagenic effect that we wish to compare qualitatively and quantitatively. We will perform pedigree analysis in WT and mutant strains by sequencing up to 10 pair of mother and daughter cells in order to detect mutational events that occurred in one generation. (iii) Finally, we wish to determine whether of not the passage of diploid cells through meiosis is unusually mutagenic due to the high level of double-strand break formation. Beside mapping the chromosomal regions of preferential susceptibility, probing the entire genome with ' mutagenesis' as a signature assay will allow to look for the underlying sequence and region-specific determinants using bioinformatics methods and thus provide a yet uncovered picture of the relative contribution of the genome architecture (coding vs non-coding regions, chromatin domains, potential DNA secondary structure formation) in protecting or enhancing mutational events. Total, at least 20 HT-SEQ runs will be performed, corresponding to the survey of the equivalent of 4000 yeast genomes.