Recombination establishes the chiasmata that physically link pairs of homologous chromosomes in meiosis, ensuring their balanced segregation at the first meiotic division and generating genetic variation. The visible manifestation of genetic crossing-overs, chiasmata are the result of an intricate and tightly regulated process involving induction of DNA double-strand breaks and their repair through invasion of a homologous template DNA duplex, catalysed by RAD51 and DMC1 in most eukaryotes. We describe here a RAD51-GFP fusion protein that retains the ability to assemble at DNA breaks but has lost its DNA break repair capacity. This protein fully complements the meiotic chromosomal fragmentation and sterility of Arabidopsis rad51, but not rad51 dmc1 mutants. Even though DMC1 is the only active meiotic strand transfer protein in the absence of RAD51 catalytic activity, no effect on genetic map distance was observed in complemented rad51 plants. The presence of inactive RAD51 nucleofilaments is thus able to fully support meiotic DSB repair and normal levels of crossing-over by DMC1. Our data demonstrate that RAD51 plays a supporting role for DMC1 in meiotic recombination in the flowering plant, Arabidopsis. Recombination ensures coordinated disjunction of pairs of homologous chromosomes and generates genetic exchanges in meiosis and, with some exceptions, involves the co-operation of the RAD51 and DMC1 strand-exchange proteins. We describe here a RAD51-GFP fusion protein that has lost its DNA break repair capacity but retains the ability to assemble at DNA breaks in the plant, Arabidopsis - fully complementing the meiotic chromosomal fragmentation and sterility of rad51 mutants, and this depends upon DMC1. No effect on genetic map distance was observed in complemented rad51 plants even though DMC1 is the only active strand transfer protein. The inactive RAD51 nucleofilaments are thus able to fully support meiotic DSB repair and normal levels of crossing-over by DMC1 in Arabidopsis. The RAD51-GFP protein confers a dominant-negative inhibition of RAD51-dependent mitotic recombination, while remaining fully fertile - a novel and valuable tool for research in this domain. These phenotypes are equivalent to those of the recently reported yeast rad51-II3A mutant, (Cloud et al. 2012), carrying the implication of their probable generality in other eukaryotes and extending them to a species with a very different relation between numbers of meiotic DNA double-strand breaks and crossing-overs (∼2 DSB/CO in yeast; ∼25–30 DSB/CO in Arabidopsis; ∼15 DSB/CO in mice).

Signaling of chromosomal DNA breaks is of primary importance for initiation of repair and, thus, for global genomic stability. Although the Mre11-Rad50-Nbs1 (MRN) complex is the first sensor of double-strand breaks, its role in double-strand break (DSB) signaling is not fully understood. We report the absence of γ-ray-induced, ATM/ATR-dependent histone H2AX phosphorylation in Arabidopsis thaliana rad50 and mre11 mutants, confirming that the MRN complex is required for H2AX phosphorylation by the ATM and ATR kinases in response to irradiation-induced DSB in Arabidopsis. rad50 and mre11 mutants spontaneously activate a DNA damage response, as shown by the presence of γ-H2AX foci and activation of cell cycle arrest in nonirradiated plants. This response is ATR dependent as shown both by the absence of these spontaneous foci and by the wild-type mitotic indices of double rad50 atr and mre11 atr plants. EdU S-phase labeling and fluorescence in situ hybridization analysis using specific subtelomeric probes point to a replicative S-phase origin of this chromosome damage in the double mutants and not to telomere destabilization. Thus, the data presented here show the exclusive involvement of ATR in DNA damage signaling in MRN mutants and provide evidence for a role for ATR in the avoidance of S-phase DNA damage.