RAD51 protects against nonconservative DNA double-strand break repair through a nonenzymatic function.

Selection of the appropriate DNA double-strand break (DSB) repair pathway is decisive for genetic stability. It is proposed to act according to two steps: 1-canonical nonhomologous end-joining (C-NHEJ) versus resection that generates single-stranded DNA (ssDNA) stretches; 2-on ssDNA, gene conversion (GC) versus nonconservative single-strand annealing (SSA) or alternative end-joining (A-EJ). Here, we addressed the mechanisms by which RAD51 regulates this second step, preventing nonconservative repair in human cells. Silencing RAD51 or BRCA2 stimulated both SSA and A-EJ, but not C-NHEJ, validating the two-step model. Three different RAD51 dominant-negative forms (DN-RAD51s) repressed GC and stimulated SSA/A-EJ. However, a fourth DN-RAD51 repressed SSA/A-EJ, although it efficiently represses GC. In living cells, the three DN-RAD51s that stimulate SSA/A-EJ failed to load efficiently onto damaged chromatin and inhibited the binding of endogenous RAD51, while the fourth DN-RAD51, which inhibits SSA/A-EJ, efficiently loads on damaged chromatin. Therefore, the binding of RAD51 to DNA, rather than its ability to promote GC, is required for SSA/A-EJ inhibition by RAD51. We showed that RAD51 did not limit resection of endonuclease-induced DSBs, but prevented spontaneous and RAD52-induced annealing of complementary ssDNA in vitro. Therefore, RAD51 controls the selection of the DSB repair pathway, protecting genome integrity from nonconservative DSB repair through ssDNA occupancy, independently of the promotion of CG.

Genomic rearrangements induced by unscheduled DNA double strand breaks in somatic mammalian cells.

DNA double-strand breaks (DSBs) are highly toxic lesions that can lead to profound genome rearrangements and/or cell death. They routinely occur in genomes due to endogenous or exogenous stresses. Efficient repair systems, canonical non-homologous end-joining and homologous recombination exist in the cell and not only ensure the maintenance of genome integrity but also, via specific programmed DNA double-strand breaks, permit its diversity and plasticity. However, these repair systems need to be tightly controlled because they can also generate genomic rearrangements. Thus, when DSB repair is not properly regulated, genome integrity is no longer guaranteed. In this review, we will focus on non-programmed genome rearrangements generated by DSB repair, in somatic cells. We first discuss genome rearrangements induced by homologous recombination and end-joining. We then discuss recently described rearrangement mechanisms, driven by microhomologies, that do not involve the joining of DNA ends but rather initiate DNA synthesis (microhomology-mediated break-induced replication, fork stalling and template switching and microhomology-mediated template switching). Finally, we discuss chromothripsis, which is the shattering of a localized region of the genome followed by erratic rejoining.

Control of alternative end joining by the chromatin remodeler p400 ATPase.

Repair of DNA double-strand breaks occurs in a chromatin context that needs to be modified and remodeled to allow suitable access to the different DNA repair machineries. Of particular importance for the maintenance of genetic stability is the tight control of error-prone pathways, such as the alternative End Joining pathway. Here, we show that the chromatin remodeler p400 ATPase is a brake to the use of alternative End Joining. Using specific intracellular reporter susbstrates we observed that p400 depletion increases the frequency of alternative End Joining events, and generates large deletions following repair of double-strand breaks. This increase of alternative End Joining events is largely dependent on CtIP-mediated resection, indicating that it is probably related to the role of p400 in late steps of homologous recombination. Moreover, p400 depletion leads to the recruitment of poly(ADP) ribose polymerase (PARP) and DNA ligase 3 at DNA double-strand breaks, driving to selective killing by PARP inhibitors. All together these results show that p400 acts as a brake to prevent alternative End Joining-dependent genetic instability and underline its potential value as a clinical marker.