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, 23 (12), 4266-79

Identification of Plant RAD52 Homologs and Characterization of the Arabidopsis Thaliana RAD52-like Genes

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Identification of Plant RAD52 Homologs and Characterization of the Arabidopsis Thaliana RAD52-like Genes

Aviva Samach et al. Plant Cell.

Abstract

RADiation sensitive52 (RAD52) mediates RAD51 loading onto single-stranded DNA ends, thereby initiating homologous recombination and catalyzing DNA annealing. RAD52 is highly conserved among eukaryotes, including animals and fungi. This article reports that RAD52 homologs are present in all plants whose genomes have undergone extensive sequencing. Computational analyses suggest a very early RAD52 gene duplication, followed by later lineage-specific duplications, during the evolution of higher plants. Plant RAD52 proteins have high sequence similarity to the oligomerization and DNA binding N-terminal domain of RAD52 proteins. Remarkably, the two identified Arabidopsis thaliana RAD52 genes encode four open reading frames (ORFs) through differential splicing, each of which specifically localized to the nucleus, mitochondria, or chloroplast. The A. thaliana RAD52-1A ORF provided partial complementation to the yeast rad52 mutant. A. thaliana mutants and RNA interference lines defective in the expression of RAD52-1 or RAD52-2 showed reduced fertility, sensitivity to mitomycin C, and decreased levels of intrachromosomal recombination compared with the wild type. In summary, computational and experimental analyses provide clear evidence for the presence of functional RAD52 DNA-repair homologs in plants.

Figures

Figure 1.
Figure 1.
Sequence Conservation of Plant RAD52 Protein Family and Its Similarity to Other RAD52 Proteins. Multiple alignments of the plant RAD52 protein family and of the animal, fungi, and protist RAD52 protein families are displayed as sequence logos. + indicates positions with similar amino acids compositions. The plant RAD52 family alignment positions are numbered according to A. thaliana Rad52-1B (At1g71310.1 and At1g71310.2), and the animal, fungi, and protist RAD52 family alignment positions are numbered according to human RAD52. Positions marked by dashes indicate insertions relative to the numbering protein (e.g., one or more plant RAD52 proteins have an amino acid insertion between the positions corresponding to Arabidopsis Rad52-1B positions 65 and 66). ^ indicates gaps introduced to align the two families. Magenta circles mark known DNA binding site residues in human RAD52.
Figure 2.
Figure 2.
Plant RAD52 Homolog Phylogeny. A dendogram of plant RAD52 homologs was calculated from the MSA shown in Figure 1 and Supplemental Data Set 2 online. The dendogram was outgroup-rooted from the position of animal, fungal, and protist (human, Saccharomyces cerevisiae, Caligus rogercresseyi [crustacean], Trichoplax, and Entamoeba) RAD52 sequences that were added to the alignment. All the added species clustered together on the dendogram. Bootstrap support values from 1000 replicates are shown in each node. The scale bar shows the number of amino acid substitutions per branch length. Branches are colored by major systematic divisions and labeled accordingly. Prefixes “1” or “2” denote the RAD52 subgroup type, and “a” or “b” indicate that more than a single gene of a particular homolog type was present in a given species. A. thaliana proteins are marked by asterisks. chavu, C. vulgaris; spipr, S. pratensis; marpo, M. polymorpha; zamfi, Zamia fischeri; picsi, Picea sitchensis; pinta, Pinus taeda; welmi, Welwitschia mirabilis; soltu, Solanum tuberosum; aquco, Aquilegia coerulea; arath, A. thaliana; araly, A. lyrata; rapsa, Raphanus sativus; brana, Brassica napus; vitvi, Vitis vinifera; poptr, Populus trichocarpa; carpa, Carica papaya; ricco, Ricinus communis; goshi, Gossypium hirsutum; glyma, Glycine max; vigun, Vigna unguiculata; medtr, Medicago trunculata; phoda, Phoenix dactylifera; horvu, Hordeum vulgare; orysa-J, Oryza sativa japonica cultivar; bradi, Brachypodium distachyon; zeama, Z. mays; sorbi, Sorghum bicolor; phypa, P. patens; synru, S. ruralis; selmo, Selaginella moellendorffii; hupse, Huperzia serrata; cycru, Cycas rumphii; musac, Musa acuminata. The dendogram was drawn using the FigTree program (http://tree.bio.ed.ac.uk/software/figtree).
Figure 3.
Figure 3.
A. thaliana RAD52 Gene Transcripts. There are three known splice variants for RAD52-1 and two for RAD52-2. RAD52-1B.1 and RAD52-1B.2 are the two RAD52-1 (At1g71310) cDNAs, with 531-bp-long ORFs; RAD52-1A has a 498-bp-long ORF. T-DNA mutant SAIL_25_H08 insertion is in the 5′ UTR, 83 bp 3′ to the putative cDNA start, and 97 bp 5′ to ATG. Dashed lines indicate the location of the 385 bp RNAi for silencing of RAD52-1 transcripts through targeting of the first exon, starting at 134 bp 5′ to ATG and ending at 251 bp 3′ to ATG. The NLS of RAD52-1A is marked by an open box. RAD52-2 (At5g47870) cDNAs are RAD52-2A with a 531-bp-long ORF and RAD52-2B with a 600-bp-long ORF. T-DNA mutant WiscDsLox303H06 insertion is in the 5′ UTR 25 bp 3′ to the putative cDNA start and 36 bp 5′ to ATG. Dashed lines indicate the location of the 383 bp RNAi for silencing of RAD52-2 transcripts through targeting of the first exon, starting at 61 bp 5′ to ATG, and ending at 322 bp 3′ to ATG. Full transcript lengths are 1435 bp, 2280 bp, 1559 bp, and 1502 bp for RAD52-1A, RAD52-1B.1, RAD52-1B.2, and RAD52-2B, respectively. Drawings of introns, exons, and UTRs are approximately to scale.
Figure 4.
Figure 4.
Cellular Localization of RAD52 Proteins in A. thaliana Seedlings. Four-day-old A. thaliana seedlings were transformed with agrobacteria carrying the fusion protein constructs (A) 52-1A-EGFP and VirE2-NLS-mRFP; (B) 52-1B-EGFP and Sc-COX4-Mito-mCherry; (C) 52-2A-EGFP and VirE2-NLS-mRFP; (D) 52-2B-EGFP; and (E) 52-1A-EX3-specific and VirE2-NLS-mRFP. VirE2-NLS-mRFP is a nuclear marker. Sc-COX4-Mito-mCherry is a mitochondrial marker. 52-1A-EX3-specific is a short sequence that is unique to RAD52-1A. In each panel, the first picture is of EGFP, the second is of the localization marker, the third is an overlay of the first two, and the last is an image captured under visible light (differential interference contrast [DIC]). Bars = 10 μm.
Figure 5.
Figure 5.
RAD52 RNA Expression in Knockout Lines. RNA was extracted from 8-d-old seedlings. Expression levels of RAD52-1 (Top) and RAD52-2 (Bottom) were determined by real-time PCR and normalized relative to ubiquitin expression. RAD52-1 primers were selected from a region common to all splice variants. RAD52-2 primers were selected from a region specific to the RAD52-2 B splice variant (see Methods). Values represent the average of three independent repeats of the experiment. Error bars represent se. WT, wild type.
Figure 6.
Figure 6.
Reduced Fertility in RAD52 Knockout Lines. Plants of each genotype were grown in soil (n = 8 to 18). Seeds of each plant were collected, scored, and divided by the number of siliques per plant. Error bars represent se. ** indicates P < 0.01 using analysis of variance to compare the wild type (WT) with each genotype.
Figure 7.
Figure 7.
Sensitivity to MMC in Plants with Reduced RAD52 Gene Expression. For each genotype, dry weight per seedling (mg) was compared in the absence of MMC or at 6 d after treatment with 1, 2, 5, 10, 20, 30 μg/mL MMC. The wild type (WT) was compared with each genotype using two-way analysis of variance. * indicates P < 0.05, ** indicates P < 0.01. Each data point is an average of three to four repeat experiments measuring five seedlings each. Error bars represent se.
Figure 8.
Figure 8.
DNA Damage Response and Homologous Recombination in Plants with Reduced RAD52 Gene Expression. Somatic recombination rates were evaluated using the GUS ICR. Results are presented per plant for untreated plants [(A) and (B) n = 128 for each line; (C) and (D) n = 47 wild type (WT) and 40 52-2 RNAi] and per leaf for 2 μg/mL MMC-treated plants [(A) and (B) n = 230 for each line; (C) and (D) n = 235 wild type and 302 52-2 RNAi), because of higher recombination rates. The wild type was compared with each genotype using the Wilcoxon nonparametric test. ** indicates P < 0.01.

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