RAD54 is essential for RAD51-mediated repair of meiotic DSB in Arabidopsis

Abstract

An essential component of the homologous recombination machinery in eukaryotes, the RAD54 protein is a member of the SWI2/SNF2 family of helicases with dsDNA-dependent ATPase, DNA translocase, DNA supercoiling and chromatin remodelling activities. It is a motor protein that translocates along dsDNA and performs multiple functions in homologous recombination. In particular, RAD54 is an essential cofactor for regulating RAD51 activity. It stabilizes the RAD51 nucleofilament, remodels nucleosomes, and stimulates the homology search and strand invasion activities of RAD51. Accordingly, deletion of RAD54 has dramatic consequences on DNA damage repair in mitotic cells. In contrast, its role in meiotic recombination is less clear. RAD54 is essential for meiotic recombination in Drosophila and C. elegans, but plays minor roles in yeast and mammals. We present here characterization of the roles of RAD54 in meiotic recombination in the model plant Arabidopsis thaliana. Absence of RAD54 has no detectable effect on meiotic recombination in otherwise wild-type plants but RAD54 becomes essential for meiotic DSB repair in absence of DMC1. In Arabidopsis, dmc1 mutants have an achiasmate meiosis, in which RAD51 repairs meiotic DSBs. Lack of RAD54 leads to meiotic chromosomal fragmentation in absence of DMC1. The action of RAD54 in meiotic RAD51 activity is thus mainly downstream of the role of RAD51 in supporting the activity of DMC1. Equivalent analyses show no effect on meiosis of combining dmc1 with the mutants of the RAD51-mediators RAD51B, RAD51D and XRCC2. RAD54 is thus required for repair of meiotic DSBs by RAD51 and the absence of meiotic phenotype in rad54 plants is a consequence of RAD51 playing a RAD54-independent supporting role to DMC1 in meiotic recombination.

Fig 1. Characterisation of rad54-2 T-DNA insertion mutant and sensitivity to MMC.

(A) Structure of AtRAD54 (At3g19210) and the rad54-1 and rad54-2 T-DNA insertion mutant alleles. Boxes show exons (unfilled) and 5’ and 3’UTRs (grey fill). The positions of the T-DNA insertions in the two alleles (inverted triangles) is indicated, with arrows above showing orientation of the left borders, and the sequences of the rad54-2 T-DNA/chromosome junctions below. The rad54-2 T-DNA insertion is flanked by two left borders (LB1, LB2) and accompanied by a 11 bp deletion in exon 4. An in-frame TGA STOP codon in rad54-2 is underlined. Numbering under the sequences is relative to the RAD54 start codon. (B) RT-PCR analyses of transcripts of rad54-1 and rad54-2. Amplification of the actin transcript (ACT) was used as a control for RT-PCR. Positions and orientations of the PCR primers are shown on the diagrams. (C-D) Sensitivity of rad54-1 and rad54-2 plants to MMC. (C) Two-week-old seedlings grown without, or with 40 μM MMC are shown. (D) Sensitivity of the seedlings was scored after 2 weeks (see Materials and Methods) and the percentages of sensitive plants (plants with 3 true leaves or less) are shown. Symbols are mean ± SD of at least 3 independent experiments with ≥ 25 seedlings per genotype per experiment. (*p<0.05 and ** p<0.005; paired two-tailed t-test).

Fig 2. Both rad54-1 and rad54-2 mutants have WT meiosis.

Chromosome spreads of male meiocytes in wild type (A-D), rad54-1 (E-H) and rad54-2 (I-L). Pachytene (A,E,I); Metaphase I (B,F,J); Metaphase II (C,G,K); Telophase II (D,H,L). Chromosomes were spread and stained with DAPI. (Scale bar = 10 μm).

Fig 3. Crossing-over is not affected in rad54-2 mutant meiosis.

Genetic distances (in centiMorgans, cM) measured from fluorescent tetrad analyses in marked intervals on (A) chromosome 1 (I1b and I1c) and (B) chromosome 2 (I2f and I2g). Bars indicate mean ± SD. On all intervals, WT and rad54 do not significantly differ (p>0.05; Z-test).

Fig 4. Absence of RAD54 leads to chromosome fragmentation in dmc1 meiosis.

Male meiosis is shown in (A-D) wild-type, (E-H) dmc1, (I-L) dmc1 rad54-1, (M-P) dmc1 rad54-2, and rad51 (Q-T). Chromosome spreads at late prophase I (A,E,I,M,Q), Metaphase I (B,F,J,N,R), Anaphase I (C,G,K,O,S) and Telophase II/Tetrad (D,H,L,P,T). Chromosomes were spread and stained with DAPI. (Scale bar = 10 μm).

Fig 5. Absence of RAD54 does not affect numbers of meiotic RAD51 foci.

(A) Co-immunolocalization of RAD51 (green) and the chromosome axis protein ASY1 (red) on leptotene/zygotene meiotic chromosome spreads. (Scale Bars: 5 μm). (B) Quantification of RAD51 foci per positive cell through early prophase I in wild-type, rad54, dmc1, and dmc1 rad54-2 mutants. Means ± SD are indicated. n.s.: not significantly different (p-value > 0.05, Kruskal-Wallis test).

Fig 6. Absence of RAD51B, RAD51D or XRCC2 does not affect dmc1 meiosis.

Male meiosis is shown in (A-C) dmc1, (D-F) dmc1 rad51b, (G-I) dmc1 rad51d, (J-L) dmc1 xrcc2, and dmc1 xrcc3 (M-O). Chromosome spreads at (A,D,G,J,M) late prophase I, (B,E,H,K,N) Metaphase I, (C,F,I,L,O) Anaphase I. Chromosomes were spread and stained with DAPI. (Scale bar = 10 μm).