How cells create genomic diversity
French National Research Agency (ANR)  
Funder: French National Research Agency (ANR)Project code: ANR-18-CE12-0018
Funder Contribution: 421,779 EUR
Recombination is essential for the repair of DNA double-strand breaks, whether pathological such as due to clastogenic agents (irradiation, chemicals), or programmed as in the case of meiosis for the formation of gametes. Recombination also serves to restart stalled replication forks, as in phage and bacteria where it is an integral part of genome duplication. In eukaryotes, chromosome replication is organized in a way, with multiple replication origins and checkpoints monitoring fork integrity, to ensure that DNA synthesis is almost always completed before mitotic entry. The latter point is essential for the proper segregation of chromosomes in mitosis, and for the maintenance of genomic integrity. However, over longer time scales, for adaptation to stress or in the genesis of cancer, it is also important that genomes can evolve, creating diversity to better adapt to a changing environment. Interestingly, the chromosomal regions that replicate last during the cell cycle are also those that evolve the fastest, suggesting that mutagenic mechanisms acting in mitosis are at play on these regions. Indeed, it is known that collapsed replication forks can be repaired by BIR (break-induced replication), which is up-regulated in mitosis and responsible for hyper-mutation, loss of heterozygosity and chromosomal rearrangements both in cancer and during neuronal diversification. We have developed an assay generating late-replicating zones in yeast, but, importantly, without activating the surveillance mechanisms that inhibit entry into mitosis. Surprisingly, we found that these cells then enter mitosis with under-replicated chromosomes, to later complete genome duplication using a recombination-based mechanism, just as in phage and bacteria. We better defined these mechanisms and identified a component of the replication fork that, when phosphorylated by mitotic kinases, stimulates BIR but also produces genomic rearrangements. Intriguingly, phosphorylation of this essential replication protein is also required for meiotic recombination, thus identifying a new regulatory step of mitotic and meiotic recombination. The aim of this research proposal is to identify the precise step, common to BIR and meiosis, which is promoted by this phosphorylation. To this end, the two partners’ labs will pool their very complementary skills and tools. We will use engineered yeast strains that undergo meiosis very synchronously, and monitor the formation of recombination intermediates. We will also identify the proteins responsible for meiotic recombination DNA synthesis (MRDS), and factors controlling this key step for the outcome of recombination thanks to a novel assay that detects newly synthesized DNA during recombination. With regard to mitotic recombination by BIR, we will identify its regulation and the complete spectrum of chromosomal rearrangements produced, productive or not, before their selection. For this, we will use the new technique of nanopore sequencing, which is able to read DNA molecules several hundreds of kilobases long, and thus identify complex chromosomal rearrangements, such as those produced by chromothripsis in tumours. We are confident that this project will shed new light on the mechanisms implemented, in mitosis and meiosis, to create the genetic diversity that allows the evolution of genomes.
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