Mechanism of genomic rearrangements driven by NAHR in response to replication stress


Repeated sequences, which are very common in genomes, provide additional opportunities for non-allelic homologous recombination (NAHR). These events can lead to genetic alterations such as deletions, inversions, translocations or copy number variation. As a consequence, many human disorders result from genomic rearrangements involving repeated sequences. For example, the Alu repeats, which are present in ~1 million copies in the human genome, have been found at the deletion breakpoints of a considerable number of genes involved in human pathologies. Despite extensive observations that repeated sequences are predisposed to rearrangements, the events leading to these reactions are not fully understood. Here, we explored the possibility that replication stress, a typical threat to cell survival, could trigger recombination between repeats, and identified the genes regulating this process.

In order to detect and study recombination between repeated sequences, we examined ADE2 recombinants in the yeast genome, which are generated by recombination between inverted repeats of two ade2 mutant alleles separated by a 1.3kb TRP1 gene. It was published previously that spontaneous recombination between the two ade2 copies is highly dependent on Rad52, acting in two independent recombination pathways. First, Rad52 acts as a loader of Rad51 which mediates gene conversion events. In the second pathway, Rad52, in association with Rad59, promotes inversion events which reverse the intervening TRP1 sequence.

We show that NAHR between the two ade2 copies is greatly stimulated by a localized replication barrier (Tus/Ter system) at the precise site of the inverted repeats. Thus, our results strongly suggest that fork stalling near repeated sequences can stimulate NAHR. Unlike spontaneous events, our genetic study reveals that replication associated NAHR reactions rely on a unique and particular pathway dependent on Rad51 catalytic activity, Rad52 and its single-strand annealing partner Rad59. We propose a model implicating a template switch event between the repeated sequences on the two sister chromatids at a stalled replication fork. Our genetic observations suggest that following fork stalling, the fork reversal proteins Rad5 and Mph1, together with the DNA end resection nucleases Mre11 and Exo1, create a ssDNA substrate onto which the Rad57-Rad55 paralogs and the Shu complex facilitate Rad51 filament assembly. The recombinase then catalyses strand invasion of the parental duplex on one of the two repeats, which initiates DNA synthesis by the replicative DNA polymerase Pol δ. Depending on how long DNA synthesis is, the recombination product after resolution of the reversed fork can be gene conversion or inversion.



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