Meiotic Recombination in Arabidopsis Is Catalysed by DMC1, with RAD51 Playing a Supporting Role

Abstract

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.

The plant‐specific CDKB 1‐ CYCB 1 complex mediates homologous recombination repair in Arabidopsis

“…Matching the previously reported localization patterns after DNA damage (Da Ines et al , 2013b), we found that RAD51 builds foci in the nuclei of root cells after treatment with cisplatin as seen with an antibody raised against RAD51 (Fig 5D and E). These foci depend on the presence of the checkpoint kinase ATM and to a lesser degree on ATR activity while they are absent, as expected, in the rad51 mutant (Fig 5D).…”

“…Homology search and strand invasion are performed by RAD51 family members (Serra et al , 2013), the eukaryotic homolog of the E. coli recA protein (Mengiste & Paszkowski, 1999). RAD51 has five paralogs in Arabidopsis (RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3), all of which function in HR in somatic or meiotic cells and show fewer homologous recombination events after DNA damage (Abe et al , 2005; Da Ines et al , 2013a,b; Serra et al , 2013), which is in line with studies in other eukaryotes (Hosoya & Miyagawa, 2014). …”

“…Currently, it remains difficult to further test the functional relevance of RAD51 phosphorylation by CDKB1‐CYCB1 complexes since a genomic RAD51 reporter was not functional during DNA damage response (Da Ines et al , 2013b). Nevertheless, the in vitro phosphorylation identified here suggests a direct regulation of RAD51 in vivo by mitotic kinases and corroborates the conclusion that CDKB1 and CYCB1 act together in the control of HR in Arabidopsis .…”

“…A second hint for a role of HR in DSB repair comes from the observation that the cdkb1;1 cdkb1;2 double mutant was also sensitive to BLM (see above, Fig 4F and L). In addition, mutants in the plant RAD51 family are well known to be sensitive to DSB‐inducing drugs such as BLM and MMC (Bleuyard et al , 2005; Da Ines et al , 2013b; Wang et al , 2014). Previous studies have shown that NHEJ is the most prominent repair mechanism in plants and hence a role of CDKB1‐CYCB1 complexes in DSB repair might be largely masked by a very efficient execution of NHEJ (Knoll et al , 2014).…”

BRCA2 regulates DMC1-mediated recombination through the BRC repeats

“…Work in Saccharomyces cerevisiae and Arabidopsis has established that Rad51 is needed for Dmc1 filament formation, and one model posits that Rad51 serves to initiate a Dmc1 filament (4,21,22). In our previous work, we showed that BRC1-4 bind with high affinity to free RAD51, enhancing ssDNA assembly and limiting its nucleation on dsDNA, resulting in a stimulation of DNA strand exchange.…”