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eSymposia | Genomic Stability and DNA Repair


The Use of Microhomologies by RAD51 Generates Genomic Rearrangements Between Non-homologous Sequences


Sep 21, 2020 12:00am ‐ Sep 21, 2020 12:00am

Description

Function of human DNA polymerase lambda The repair of DNA double-strand breaks (DSBs) is essential for genomic stability but also generates genomic instability: gross chromosome rearrangements (GCR) and/or mutagenesis at the repair junction lead(s) to sequence alterations like, mutations, translocations, deletions or insertions. Analyzing GCR junctions is very informative on the molecular mechanisms that actively generate GCRs. Using intrachromosomal reporters in immortalized fibroblasts, we have shown that repair of distant DSBs, but not close DSBs, favor insertion of ectopic chromosomal sequences at the scar (Guirouilh-Barbat et al. PLOS genetics 2016). These Templated Sequence Insertions (TSIs) are coupled to CtIP-mediated resection and are inhibited by 53BP1. Here we show that insertions of TSIs are observed in a physio-pathological context at junctions of rearrangements implicating distant ends, but not close ends, in breast tumors. In addition, we found TSIs to be less frequent in BRCA2 deficient tumors and we confirmed the implication of BRCA2 and RAD51 in the capture of TSIs using our intrachromosomal reporter for distant DSB repair in fibroblasts. High Throughput Genome wide Translocations Sequencing (HTGTS) revealed that i) RAD51 mediates complex genomic rearrangements that are observed upon 53BP1 depletion, ii) RAD51 is implicated in bipartite rearrangements, i.e. translocations. Mimicking the reciprocal hNMP1-ALK translocation with CRIPR-Cas9 in RPE1 cells confirmed that RAD51 mediates the increased frequency of translocation in 53BP1 depleted cells. None of these RAD51-mediated genomic rearrangements (translocations, complex rearrangements, capture of TSIs) involve sequence homologies, but only microhomologies. Using transmission electron microscopy (TEM) we found that RAD51 can foster the pairing of heterologous molecules using one or two microhomology patch(es). Our data show that RAD51 and BRCA2 have the intrinsic capacity to generate genetic instability through a mechanism independent of sequence homology and that this capacity is counteracted by 53BP1. We propose that these events arise by microhomology-mediated template switch when RAD51 samples DNA in the course of the search of homology .

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