(C) PLOS One
This story was originally published by PLOS One and is unaltered.
. . . . . . . . . .



DMC1 attenuates RAD51-mediated recombination in Arabidopsis [1]

['Olivier Da Ines', 'Institut Génétique Reproduction Et Développement', 'Igred', 'Université Clermont Auvergne', 'Umr Cnrs', 'Inserm', 'Clermont-Ferrand', 'Jeanne Bazile', 'Maria E. Gallego', 'Charles I. White']

Date: 2022-10

Ensuring balanced distribution of chromosomes in gametes, meiotic recombination is essential for fertility in most sexually reproducing organisms. The repair of the programmed DNA double strand breaks that initiate meiotic recombination requires two DNA strand-exchange proteins, RAD51 and DMC1, to search for and invade an intact DNA molecule on the homologous chromosome. DMC1 is meiosis-specific, while RAD51 is essential for both mitotic and meiotic homologous recombination. DMC1 is the main catalytically active strand-exchange protein during meiosis, while this activity of RAD51 is downregulated. RAD51 is however an essential cofactor in meiosis, supporting the function of DMC1. This work presents a study of the mechanism(s) involved in this and our results point to DMC1 being, at least, a major actor in the meiotic suppression of the RAD51 strand-exchange activity in plants. Ectopic expression of DMC1 in somatic cells renders plants hypersensitive to DNA damage and specifically impairs RAD51-dependent homologous recombination. DNA damage-induced RAD51 focus formation in somatic cells is not however suppressed by ectopic expression of DMC1. Interestingly, DMC1 also forms damage-induced foci in these cells and we further show that the ability of DMC1 to prevent RAD51-mediated recombination is associated with local assembly of DMC1 at DNA breaks. In support of our hypothesis, expression of a dominant negative DMC1 protein in meiosis impairs RAD51-mediated DSB repair. We propose that DMC1 acts to prevent RAD51-mediated recombination in Arabidopsis and that this down-regulation requires local assembly of DMC1 nucleofilaments.

Essential for fertility and responsible for a major part of genetic variation in sexually reproducing species, meiotic recombination establishes the physical linkages between homologous chromosomes which ensure their balanced segregation in the production of gametes. These linkages, or chiasmata, result from DNA strand exchange catalyzed by the RAD51 and DMC1 recombinases and their numbers and distribution are tightly regulated. Essential for maintaining chromosomal integrity in mitotic cells, the strand-exchange activity of RAD51 is downregulated in meiosis, where it plays a supporting role to the activity of DMC1. Notwithstanding considerable attention from the genetics community, precisely why this is done and the mechanisms involved are far from being fully understood. We show here in the plant Arabidopsis that DMC1 can downregulate RAD51 strand-exchange activity and propose that this may be a general mechanism for suppression of RAD51-mediated recombination in meiosis.

Our data thus suggest that DMC1 triggers down-regulation of RAD51 strand-exchange activity and that local assembly of DMC1 nucleofilaments—but not their strand-exchange activity–are sufficient to mediate this down-regulation in Arabidopsis. Interestingly, our data in vegetative cells also suggest that DMC1 assembly into damage-induced foci may not strictly require meiosis-specific factors. Of course, post-assembly function of these DMC1 foci in resolving damage does require additional, yet to be discovered, meiotic factor(s) (beyond perhaps HOP2-MND1, which is known to be expressed in somatic tissues of Arabidopsis [ 59 – 64 ]).

Here, we sought further insights into the mechanism by which RAD51 is downregulated during meiosis in plants and our results support the hypothesis that DMC1 ssDNA binding attenuates RAD51 strand invasion activity. Ectopic expression of DMC1 in vegetative cells blocks RAD51-dependent homologous recombination and this involves inhibition of its activity rather than inhibition of nucleofilament formation, reminiscent of the situation in meiosis. This conclusion is further supported by the observation that expression of a dominant negative DMC1-GFP fusion protein in meiosis impairs RAD51-mediated meiotic DSB repair.

Apart from budding yeast, the mechanisms suppressing RAD51 activity during meiosis have not been determined. In plants, bioinformatics searches fail to identify homologs of yeast Hed1 and Mek1. Arabidopsis rad51 mutants are sterile and dmc1 mutants have severely reduced fertility. They both show defects in pairing and synapsis, but rad51 and rad51 dmc1 mutants exhibit extensive, SPO11-dependent chromosome fragmentation in meiotic prophase I, while dmc1 mutants are characterized by the presence of intact univalent chromosomes in meiotic metaphase I [ 53 – 55 ]. Thus, in the absence of DMC1, meiotic DSB are repaired in a RAD51-dependent manner [ 53 , 55 – 57 ] which does not generate chiasmata. This has led to the hypothesis that DMC1 could be involved in down-regulation of RAD51 in Arabidopsis [ 57 , 58 ].

DMC1 is the predominant strand-exchange protein during meiosis, catalyzing most strand-exchange events, while RAD51 acts only to support DMC1 and to repair residual DSBs after IH recombination and synapsis are complete [ 38 , 39 , 42 – 44 ]. Accordingly, data from budding yeast indicate that RAD51 activity is downregulated during meiosis so that DMC1 catalyzes DNA strand-exchange using the homologous chromosome as a template [ 38 , 45 – 47 ]. In budding yeast, down-regulation of Rad51 activity is mediated through inhibition of Rad51-Rad54 complex formation by the coordinated phosphorylation of Hed1 and the Rad51 accessory factor Rad54 by the meiosis-specific kinase Mek1 [ 18 , 45 , 46 , 48 – 51 ]. Hed1 is a meiosis-specific factor that binds to Rad51, blocking access of Rad54 and thereby restricting activity of the Rad51 nucleofilaments in meiosis [ 48 , 49 , 51 , 52 ]. Second, phosphorylation of threonine 132 of Rad54 by Mek1 reduces the affinity of Rad54 for Rad51 [ 47 , 50 ]. Both mechanisms affect Rad51-Rad54 complex formation and this in turn attenuates the activity of Rad51, presumably to minimize the use of sister chromatid templates and hence favor DMC1-dependent inter-homolog recombination.

Only CO between non-sister chromatids physically link homologous chromosome pairs and the choice of the sister or non-sister chromatid template for repair of meiotic DSB is thus a key determinant for the outcome of recombination. This must be tightly regulated to favor interhomolog recombination for coordinated chromosomal disjunction at the first meiotic anaphase [ 29 ]. With a few exceptions, this crucial step of meiotic recombination requires the co-operation of the two strand exchange proteins, RAD51 and DMC1 (reviewed by [ 16 , 17 , 30 ]). RAD51 and DMC1 emerged following a gene duplication event that occurred in the common ancestor of all eukaryotes [ 31 ]. They retain roughly 50% amino acid identity and closely related structural and biochemical properties [ 16 , 26 , 32 – 34 ]. Both enzymes assemble on ssDNA at sites of breaks and promote break repair by searching for and invading an intact homologous DNA template molecule. However, while RAD51 is essential for both mitotic and meiotic homologous recombination, DMC1 is meiosis-specific [ 16 , 27 , 35 , 36 ]. Why meiotic recombination requires two strand-exchange proteins is not well understood, but given the similar activities of the two proteins it has been generally assumed that they play complementary roles in catalyzing meiotic DSB repair—with DMC1 promoting interhomolog recombination (IH) and crossing-over, while RAD51 favors sister-chromatid repair of the DSB [ 18 , 37 ]. This assumption has however been called into question by the characterization of the yeast rad51-II3A [ 38 ] and Arabidopsis RAD51-GFP proteins [ 39 ]. While they retain the ability to form nucleoprotein filaments at DSB, these proteins are unable to catalyze invasion of the homologous template. They do however fully complement the absence of RAD51 in meiosis [ 38 – 41 ]. It is thus the presence of the RAD51 protein and not its strand-exchange activity that is needed in meiosis.

Meiotic recombination is initiated by the programmed induction of DNA double strand breaks (DSB) by the SPO11 DNA topoisomerase VI-like complex [ 3 – 5 ] in the chromosomes and their repair by homologous recombination [ 6 – 11 ]. The pair of DNA ends formed are resected by nucleases [ 12 ], which remove SPO11 and generate long 3′ single-stranded DNA overhangs (ssDNA), which are bound by the heterotrimeric protein RPA to protect the ssDNA and prevent formation of secondary structures [ 13 – 15 ]. The specialized RAD51 and DMC1 recombinases are then loaded onto the ssDNA to form right-handed helical nucleoprotein filaments [ 16 , 17 ]. Earlier studies have proposed that recombinase loading occurs asymmetrically, with RAD51 and DMC1 making separate homofilaments on opposite ends of a break [ 18 – 20 ]. However, more recent work in budding yeast and mouse suggested that RAD51 and DMC1 homofilaments do load on both DSB ends resulting in adjacent recombinase homofilaments [ 21 – 23 ]. This "mixed" recombinase filament organization has been reconstituted in vitro [ 24 ] and has thus become the favored model for recombinase loading [ 16 , 17 , 23 – 27 ]. The RAD51/DMC1-DNA nucleoprotein filament is the active molecular species in searching for and invading a homologous DNA template by the 3’-ended DNA strand(s) [ 16 , 17 , 26 , 27 ]. This process is the core of the meiotic recombination pathway and results in the formation of a joint recombination intermediate, which can be resolved to yield non-crossover (NCO) or crossover (CO) products [ 1 , 2 , 28 ]. CO between non-sister-chromatids, in combination with sister chromatid cohesion, establish the physical linkages (chiasmata) necessary to ensure proper chromosomal segregation at the first meiotic anaphase [ 1 , 2 , 28 ].

To maintain stable ploidy across generations, sexually reproducing eukaryotes must halve the number of chromosomes in gametes with respect to their mother cells. This is accomplished by meiosis, a specialized cellular division in which DNA replication is followed by two sequential rounds of chromosome segregation [ 1 , 2 ]. Proper chromosome segregation in mitosis, in which a single division follows DNA replication, is ensured by sister-chromatid cohesion established during S-Phase. In contrast, balanced segregation of chromosomes at the first meiotic division relies upon both sister-chromatid cohesion and the establishment of physical connections between the two homologous chromosomes. In most species, these connections are realized by reciprocal recombination events called cross-overs (COs). This process also produces novel combinations of alleles from the two chromosomes, recombining the parental genetic contributions in the production of the new generation. Meiotic recombination is thus both essential for fertility and to reshuffle genetic information, with important consequences for genome evolution [ 1 , 2 ].

Results

Ectopic expression of DMC1 in vegetative cells We expressed DMC1 in vegetative cells, in which RAD51 is normally the only active recombinase, to test the intrinsic ability of DMC1 to downregulate RAD51. The Arabidopsis DMC1 genomic sequence (DMC1g) was cloned and placed under the control of the RAD51 promoter to drive its expression in somatic cells (Fig 1A). The complementation of the meiotic defects of dmc1 mutants by this transgene confirms correct expression of functional DMC1 protein (Fig 1B and 1C). We selected 3 independent dmc1-/- pRAD51::DMC1g transgenic lines and show that all were fertile with a number of seeds per silique very similar to that of wild-type plants, although slightly reduced (Fig 1C and S1 Data). Chromosome spreads of pollen mother cells confirm that the pRAD51::DMC1g transgene restores normal meiosis in dmc1 mutants (S1 Fig). We note that a similar experiment using the DMC1 coding sequence (DMC1 CDS ) instead of the genomic sequence, did not rescue the fertility of dmc1 mutants, presumably indicating the importance of an intron sequence and/or correct splicing for proper DMC1 expression (Fig 1B). Knowing that the chimeric pRAD51::DMC1g transgene allows high expression of DMC1 we then tested whether it could drive expression of DMC1 in somatic cells and whether or not it showed DNA damage-inducible expression, as does RAD51. We generated wild-type plants homozygous for the transgene insertion and performed RT-PCR on RNA from one-week-old seedlings. RAD51 expression is relatively low in wild-type seedlings grown under standard conditions but its expression is strongly induced in seedlings treated with 20 μM Mitomycin C (MMC) for 8 hours (Fig 1D). In contrast (and in accordance with its meiosis-specific function), DMC1 expression is not induced after DNA damage treatment in WT plants. As expected, we observed strong induction of DMC1 expression in all 3 independent pRAD51::DMC1g transgenic lines treated with 20 μM MMC for 8 hours (Fig 1D). We also tested expression of HOP2, MND1 and RAD54, which are HR genes involved in the strand invasion process and known to be induced in somatic cells after treatment with DNA damaging agents [59–64]. Expression of all three genes was induced in both the transgenic plants and the wild-type controls (Fig 1D). Similar results were obtained when seedlings were treated with γ-rays (S2 Fig). Together, these data show that the pRAD51::DMC1g transgene allows high expression and induction of DMC1 in both meiosis and mitosis and that this ectopic expression of DMC1 does not appear to affect the expression of the other HR genes. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 1. pRAD51::DMC1g restores fertility of the Arabidopsis dmc1 mutant and induces expression of DMC1 in somatic cells. (A) Schematic representation of the pRAD51::DMC1g construct. DMC1g indicates DMC1 genomic sequence. Exons are shown as blue rectangles. (B) Wild-type plants have long siliques full of seeds (mean ± S.D: 54.1 ± 0.6 seeds per silique), while dmc1 mutants have very low fertility (mean ± S.D: 3.5 ± 0.25 seeds per silique). Expression of the pRAD51::DMC1g genomic sequence in dmc1 mutants restores fertility. (C) number of seeds per silique in DMC1, dmc1-/-, and three dmc1-/- + pRAD51::DMC1g independent transformants (T1-1, T1-2, and T1-3) showing restored fertility. Mean numbers are indicated ± S.D. n = 4 plants, N = 10 siliques per plant. (D) RT-PCR expression analysis of RAD51, DMC1, RAD54, HOP2 and MND1 in 7-day-old, untreated and MMC-treated (8 h, 20 μM) seedlings expressing or not the pRAD51::DMC1g transgene. Expression was analyzed in wild-type plants and three independent transgenic lines (T1-1, T1-2, and T1-3). Actin is used as a loading control. https://doi.org/10.1371/journal.pgen.1010322.g001

Ectopic expression of DMC1 in somatic cells blocks RAD51-mediated DSB repair We next analyzed whether RAD51-mediated DNA repair in somatic cells is affected by the presence of DMC1. We tested sensitivity of transgenic plants expressing DMC1 in vegetative cells to the DNA damaging agent Mitomycin C (MMC). MMC forms DNA interstrand cross-link adducts, thereby producing DNA strand breaks that must be repaired by homologous recombination. In Arabidopsis, this is seen in the hypersensitivity of rad51 mutants to MMC [39,65]. Progeny of wild-type plants and rad51 heterozygotes (RAD51+/-) carrying the pRAD51::DMC1g transgene were grown on solid media containing, or not, 20 μM Mitomycin C and growth was scored after 2 weeks (Figs 2 and S3). We tested the progeny of RAD51+/- heterozygous mutant plants as this permits analysis of the effect of vegetative expression of DMC1 in both presence (RAD51+/+ and RAD51+/-) or absence (rad51-/-) of RAD51. Plants grown under standard conditions did not exhibit any visible phenotypical differences indicating that ectopic expression of DMC1 does not affect normal plant growth (Fig 2A). However, when grown in medium supplemented with MMC, transgenic plants showed strong hypersensitivity (Fig 2B and 2C), with 87% to 98% sensitive plants in the progeny of 3 independent RAD51+/- transformants (named T1-1, T1-2, and T1-3) carrying the pRAD51::DMC1g transgene (Fig 2B and 2C). This is comparable to the full sensitivity of RAD51-GFP plants (which behave as rad51 mutants, [39,66]) and in strong contrast to the 29% sensitive plants (corresponding to the segregating rad51 homozygous mutants) observed for the progeny of RAD51+/- plants that do not carry the pRAD51::DMC1g transgene (Fig 2C). To confirm this, we selected RAD51+/+ plants expressing the pRAD51::DMC1g transgene from the progeny of the three independent RAD51+/- transgenic lines tested above and examined their sensitivity to MMC. As expected, the three lines exhibited strong sensitivity (S3A–S3E Fig). Ectopic expression of DMC1 in somatic cells thus has a dominant negative effect and we hypothesize that this occurs through precluding DSB repair mediated by RAD51. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 2. Mitomycin C hypersensitivity of transgenic seedlings expressing pRAD51::DMC1g. (A) No growth difference is observed between WT, RAD51-GFP, RAD51+/- heterozygous and RAD51+/- expressing pRAD51::DMC1g seedlings (two-week-old seedlings grown without MMC). (B) Pictures of two-week-old seedlings grown with 20 μM MMC. WT seedlings (depicted with red square) do not exhibit MMC sensitivity, in contrast to RAD51-GFP seedlings (blue square) and progeny of RAD51+/- transgenic lines expressing pRAD51::DMC1g. Pictures of progeny of two independent RAD51+/- transgenic lines expressing pRAD51::DMC1g are shown (T1-1 and T1-2). As control, WT (red square) and RAD51-GFP plants (blue square) were also grown on each plate. (C) Sensitivity of the seedlings was scored after 2 weeks and the fractions of sensitive plants (plants with less than 4 true leaves) are shown (3 biological repeats, each with N > 45 seedlings). Three independent RAD51+/- heterozygous lines expressing pRAD51::DMC1g (T1-1,T1-2 and T1-3) were tested and all showed strong hypersensitivity to MMC. https://doi.org/10.1371/journal.pgen.1010322.g002

DMC1 does not substitute for RAD51 in DSB repair in somatic cells An interesting observation from the above data is that DMC1 is not able to substitute for RAD51 in somatic cell DSB repair. Segregating rad51 homozygotes are sensitive to MMC, even though they express DMC1 (Fig 2B and 2C). This result might be surprising given the similar biochemical activities of the two proteins [16,26,32–34,67]. Given that meiotic DMC1 activity requires the presence of a RAD51 nucleofilament [38,39], a possible explanation for this is that if DMC1 also requires a RAD51 filament to be functional in somatic cells, then DMC1 will not repair DSB induced by MMC when expressed in the rad51 mutant. To check this, we tested whether DMC1 would repair MMC-induced DSB when expressed in RAD51-GFP plants. The RAD51-GFP fusion protein forms a nucleofilament which supports the activity of DMC1 in meiosis but is not proficient for DSB repair in somatic cells [39]. All three RAD51-GFP_pRAD51::DMC1g independent lines tested were strongly sensitive to MMC (S3F–S3G Fig). Thus, in contrast to meiosis, the presence of the (inactive) RAD51-GFP filament is not sufficient to support DMC1 activity in somatic cells. This suggests that DMC1 activity requires the presence of meiosis-specific co-factors or, alternatively, that DMC1 activity is blocked in somatic cells by unknown factors.

DMC1 expression in somatic cells does not block RAD51 focus formation We next tested whether expression of DMC1 in somatic cells blocks RAD51 nucleofilament formation at DNA damage sites. The nucleofilament is the active molecular species that performs the homology search and strand exchange. Accordingly, defects in nucleofilament formation affect RAD51 activity. We analyzed RAD51 focus formation upon DNA damage in somatic cells as a proxy for RAD51 nucleofilament formation. We performed RAD51 immunofluorescence staining in root tip nuclei of 5-day-old seedlings treated or not with Mitomycin C (30 μM; Fig 4). As expected, no or very few foci were detected in root tip nuclei of non-treated seedlings (Fig 4A and 4D). In contrast, numerous RAD51 foci were detected in nuclei from root tips fixed 2 h or 8h after treatment with MMC (Fig 4B–4D). In wild-type plants we observed an average of 2.3 foci (± 0.3; n = 228) 2h after MMC treatment and this increases up to 15.8 foci (± 1.3; n = 229) after 8h of MMC treatment (Fig 4 and S1 Data). Strikingly, similar distributions of RAD51 foci were observed in the root tips of two independent transgenic lines expressing pRAD51::DMC1g (T1-1 and T1-2; Fig 4B–4D and S1 Data). Thus, DMC1 expression in somatic cells does not affect RAD51 focus formation and thus probably RAD51 nucleofilament formation. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 4. RAD51 foci in somatic cells of DMC1 overexpressing seedlings. (A-C) Immunolocalization of RAD51 in root tip nuclei of wild-type plants expressing pRAD51::DMC1g, fixed just before (A), and 2 (B) or 8 (C) hours after treatment with 30 μM MMC. T1-1 and T1-2 are two independent transgenic lines expressing pRAD51::DMC1g. MMC-induced RAD51 foci are clearly visible in nuclei of the treated plants. DNA is stained with DAPI (blue) and RAD51 foci (detected using an antibody against RAD51) are colored in red. Images are collapsed Z-stack projections of a deconvoluted 3D image stack. (Scale Bars: 5 μm). (D) Quantification of RAD51 foci in root tip nuclei of wild-type and two independent transgenic lines expressing pRAD51::DMC1g (T1-1 and T1-2) before and after MMC treatment. Means ± s.e.m are indicated for each genotype. More than 200 nuclei from at least 3 seedlings were analyzed per genotype. https://doi.org/10.1371/journal.pgen.1010322.g004

[END]
---
[1] Url: https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1010322

Published and (C) by PLOS One
Content appears here under this condition or license: Creative Commons - Attribution BY 4.0.

via Magical.Fish Gopher News Feeds:
gopher://magical.fish/1/feeds/news/plosone/

You are viewing proxied material from magical.fish. The copyright of proxied material belongs to its original authors. Any comments or complaints in relation to proxied material should be directed to the original authors of the content concerned. Please see the disclaimer for more details.