The Recombinases DMC1 and RAD51 Are Functionally and Spatially Separated during Meiosis in Arabidopsis

Abstract

Meiosis ensures the reduction of the genome before the formation of generative cells and promotes the exchange of genetic information between homologous chromosomes by recombination. Essential for these events are programmed DNA double strand breaks (DSBs) providing single-stranded DNA overhangs after their processing. These overhangs, together with the RADiation sensitive51 (RAD51) and DMC1 Disrupted Meiotic cDNA1 (DMC1) recombinases, mediate the search for homologous sequences. Current models propose that the two ends flanking a meiotic DSB have different fates during DNA repair, but the molecular details remained elusive. Here we present evidence, obtained in the model plant Arabidopsis thaliana, that the two recombinases, RAD51 and DMC1, localize to opposite sides of a meiotic DSB. We further demonstrate that the ATR kinase is involved in regulating DMC1 deposition at meiotic DSB sites, and that its elimination allows DMC1-mediated meiotic DSB repair even in the absence of RAD51. DMC1's ability to promote interhomolog DSB repair is not a property of the protein itself but the consequence of an ASYNAPTIC1 (Hop1)-mediated impediment for intersister repair. Taken together, these results demonstrate that DMC1 functions independently and spatially separated from RAD51 during meiosis and that ATR is an integral part of the regular meiotic program.

Spontaneous self-segregation of Rad51 and Dmc1 DNA recombinases within mixed recombinase filaments

“…We do not rule out the possibility that there may be some intermixing of the recombinases in our assays, but if intermixing is taking place, then it does not represent the majority of the population, and is not detected in our Hed1-GFP binding assays. Importantly, the side-by-side arrangement of homotypic Rad51 and Dmc1 filaments observed in our experiments is strikingly similar to in vivo microscopic observations of Rad51 and Dmc1 foci (19,20), and recent in vivo super-resolution microscopy data that indicate Rad51 and Dmc1 form at discrete regions on meiotic filaments (21). In addition, our data are also consistent with genetic experiments that had previously suggested Rad51 and Dmc1 might not form intermixed filaments (43,44).…”

“…This scenario is not unreasonable given that the two proteins are closely related and share ~45% sequence identity (6,8,9). Alternatively, the two recombinases might not interact with one another, in which case homotypic tracts of Rad51 or Dmc1 might be expected to form on the ssDNA, as has been suggested from microscopic observations of recombination foci or filaments during meiosis ( Figure 1A) (19)(20)(21). We present these two opposing scenarios for simplicity, with the understanding that the actual situation might lie somewhere between these two ends of the spectrum.…”

“…and Dmc1 are both required for timely progression through meiosis (1,6), and the two proteins form closely associated foci at sites of meiotic DSBs in vivo (18)(19)(20)(21). However, there have been a very limited number of in vitro studies designed to test the properties of presynaptic complexes comprised of both Rad51 and Dmc1 (13), and to our knowledge there is no experimental data suggesting how mixed filaments might assemble.…”

“…Indeed, Rad51 and Dmc1 appear as partially offset foci in many organisms (18)(19)(20), which led to the proposal of an asymmetric loading model in which Rad51-only filaments formed on one end of a meiotic DSB, and Dmc1-only filaments formed on the other end of the same DSB (6,19,20). More recently, super-resolution microscopy experiments have shown that S. cerevisiae Rad51 and Dmc1 co-occupy both ends of meiotic DSBs, arguing against the asymmetric loading model, and instead suggesting a model in which Rad51 and Dmc1 form separate filaments on the same ssDNA (21).…”

The Axial Element Protein DESYNAPTIC2 Mediates Meiotic Double-Strand Break Formation and Synaptonemal Complex Assembly in Maize

“…DSBs are then resected by exonucleases to generate 39 single-stranded DNA (ssDNA) overhangs, which further associate with DNA recombinase, DISRUPTED MEIOTIC CDNA1 (DMC1), and RAD51, and their accessory factors (Bishop, 1994;Da Ines et al, 2012;Kurzbauer et al, 2012). These ssDNA nucleoprotein complexes, also called presynaptic filaments, mediate the homology search process and form joint DNA molecule intermediates by invading homologous sequences (Shinohara et al, 1992;Baumann et al, 1996;Pradillo et al, 2014).…”