Cells expressing murine RAD52 splice variants favor sister chromatid repair

Abstract

The RAD52 gene is essential for homologous recombination in the yeast Saccharomyces cerevisiae. RAD52 is the archetype in an epistasis group of genes essential for DNA damage repair. By catalyzing the replacement of replication protein A with Rad51 on single-stranded DNA, Rad52 likely promotes strand invasion of a double-stranded DNA molecule by single-stranded DNA. Although the sequence and in vitro functions of mammalian RAD52 are conserved with those of yeast, one difference is the presence of introns and consequent splicing of the mammalian RAD52 pre-mRNA. We identified two novel splice variants from the RAD52 gene that are expressed in adult mouse tissues. Expression of these splice variants in tissue culture cells elevates the frequency of recombination that uses a sister chromatid template. To characterize this dominant phenotype further, the RAD52 gene from the yeast Saccharomyces cerevisiae was truncated to model the mammalian splice variants. The same dominant sister chromatid recombination phenotype seen in mammalian cells was also observed in yeast. Furthermore, repair from a homologous chromatid is reduced in yeast, implying that the choice of alternative repair pathways may be controlled by these variants. In addition, a dominant DNA repair defect induced by one of the variants in yeast is suppressed by overexpression of RAD51, suggesting that the Rad51-Rad52 interaction is impaired.

FIG. 1.

(A) Twelve exons of the mouse RAD52 gene are shown as numbered boxes approximately to scale; the position of each intron is shown as a short gap, but the introns are not shown to scale. A gray box above the gene indicates the open reading frame of the full-length RAD52 peptide, which extends from the start of exon 2 to the start of exon 12. The three primer pairs for RT-PCR across the RAD52 transcript are indicated by converging arrows (exons 1 to 7, 6 to 11, and 10 to 12). (B, C, and D) Autoradiographs of the blots derived from the mouse RT-PCR are shown. A limited number of PCR cycles (30) was used, followed by genomic blotting, to ensure that all products detected are specific for the mouse RAD52 gene. Each autoradiograph has 18 lanes, 8 with samples derived from the lung and 8 with samples derived from liver, plus 2 control lanes (H₂O, no template added). Two separate tissue samples were prepared from each of the two mice, and in each case the cDNA was prepared with and without reverse transcriptase (rt, indicated by + or − above the lanes) to ensure that all products are derived from RNA. The sizes of the bands are indicated based upon the position of size markers from the original TBE-agarose gels. The top autoradiograph (B) shows the results using primers designed to amplify RAD52 exons 1 to 7, and the middle autoradiograph (C) shows the results using primers designed to amplify RAD52 exons 6 to 11. The bottom autoradiograph (D) shows the results using primers designed to amplify GAPDH exons 5 to 8, which serves as a control for RNA preparation.

FIG. 2.

RAD52 peptide is aligned with the predicted product of the two variant transcripts, RAD52Δexon4 and RAD52+intron8. The RAD52Δexon4 sequence includes the first 63 amino acids of RAD52 followed by 18 “out of frame” amino acids (underlined and italicized), encoded by exon 5. The RAD52+intron8 sequence includes the first 248 amino acids of RAD52, followed by 42 amino acids encoded by intron 8 (underlined and italicized).

FIG. 3.

Two autoradiographs are shown of blots of RT-PCRs specific for RAD52Δexon4 (primers amplify exons 1 to 7, top) and RAD52+intron8 (primers amplify exons 6 to 11, bottom). Each blot contains 10 lanes, representing the 10 fractions from the sucrose gradient, 1 to 10 in the order they came off the gradient. The lane to the right (1) represents products obtained from the bottom of the gradient; this may include some residual nuclei and high-molecular-mass complexes. The lanes to the left (8 to 10) contain low-molecular-mass complexes including free RNA molecules. Lanes 2 to 7 represent samples obtained from the polyribosome-containing fraction of the gradient. The UV absorbance profile of the polyribosome gradient is shown separately in Fig. SC of the supplemental material.

FIG. 4.

(A) The FACS profile shows cells stained with propidium iodide at seven different time points after release from nocodazole arrest and mitotic shake off. At time zero the cells are arrested in G₂, by 6 h most of the cells have entered G₁, and by 12 h many of the cells have entered S phase. The percentages for 2n and 4n indicate the percentage of the cell population that fall within the limits of the black bars shown above the FACS profile. (B) Autoradiographs (as in Fig. 1B, C, and D) for the RT-PCR products used to identify RAD52Δexon4 (top), RAD52+intron8 (middle), and GAPDH control (bottom) from the time points shown above.

FIG. 5.

(A) Schematic representation of the reporter system developed by Pierce and colleagues (31). puro, puromycin-N-acetyltransferase gene. A double-strand break can be introduced into this reporter using the I-SceI endonuclease, and subsequent HDR from a downstream GFP fragment enables the repair of a functional GFP sequence. These HDR events can be scored by FACS analysis. Four expression vectors were created from a derivative of the pIRES-EGFP vector (Clontech) as described in Materials and Methods (B). The four vectors include the empty vector control pCMV, full-length mouse RAD52 (pRAD52), or the two splice variants, pRAD52Δexon4 and pRAD52+intron8, as indicated in panel B. (C) The frequency of HDR events in CHO cells (scored as % GFP⁺ cells using FACS analysis) is shown. For each transfection the plasmid is indicated below. In addition, each transfection included an I-SceI-expressing plasmid; the mean of four experiments is indicated (±standard deviation). Analysis of variance indicates significant differences between the groups (data not shown). Both of the variant plasmids (pRAD52Δexon4 and pRAD52+intron8) have a higher frequency of HDR events than the wild-type RAD52 plasmid (one-tailed t test, P = 4.9 × 10⁻⁵ and P = 7.5 × 10⁻⁴, respectively). A CHO cell line, deficient in XRCC3, was also tested in the same way (D). The difference between the wild-type RAD52 plasmid and the pRAD52Δexon4 plasmid is not significant (one-tailed t test, P = 0.094). The difference between the wild-type RAD52 plasmid and the pRAD52+intron8 plasmid is significant (one-tailed t test, P = 0.044).

FIG. 6.

(A) Frequency of direct-repeat recombination was assayed as indicated. Two leu2 alleles are schematically represented (top), with dark triangles indicating the two mutations that inactivate the gene. Recombination between the two repeated sequences can restore a functional LEU2 gene, and two possible LEU⁺ recombinant structures are shown. First, a replacement event restores the wild-type LEU2 sequence to one of the leu2 alleles (e.g., the upstream sequence is restored). Second, a pop-out event is shown where single-strand annealing recombines the two repeated leu2 alleles, leading to loss of the URA3 gene and both mutations within LEU2. Other possible events such as triplications are discussed elsewhere (41). (B) The median leu2 recombination frequency m, as defined by Lea and Coulson (22), is shown, where the error bars indicate ± standard deviation of m. The wild-type RAD52 allele is abbreviated wt. Both variant alleles (rad52Δ77/null and rad52Δ284/null) have a higher frequency of direct-repeat recombination than the heterozygous wild-type strain (wt/null), (one-tailed t tests, P = 8.5 × 10⁻³ and 4.4 × 10⁻⁶, respectively). The frequency of recombination in the heterozygous wild-type strain (wt/null) versus a wild-type diploid strain (wt/wt) is not significantly different (one-tailed t test, P = 0.41). Similarly, the recombination frequency in the rad52Δ77/null strain is not significantly different from that of the rad52Δ284/null strain (one-tailed t test, P = 0.089).

FIG. 7.

(A) Heteroallelic recombination is measured using two ade2 alleles as shown schematically. The dark triangles indicate the two mutations that inactivate the gene: ade2-a is a fill-in mutation of the AatII site in the 5′ end of the gene, and ade2-n is a fill-in mutation of the NdeI site in the 3′ end of the gene (12). Recombination between the two homologs restores a functional ADE2 gene, as indicated. The gene conversion event depicted has lost the 3′ ade2-n mutation, leading to restoration of a functional ADE2 gene. The frequency of heteroallelic recombination is calculated using the methods described by Lea and Coulson (22). (B) The median ade2 recombination frequency m as defined by Lea and Coulson; the error bars indicate ± standard deviation of m. The wild-type allele is abbreviated wt. Both variant alleles (rad52Δ77/null and rad52Δ284/null) have a lower frequency of heteroallelic recombination than the heterozygous wild-type strain (wt/null) (one-tailed t tests, P < 10⁻⁴). The frequency of recombination in the diploid wild-type strain (wt/wt) does not differ significantly from the heterozygous wild-type strain (wt/null) (two-tailed t test, P = 0.87). The rad52Δ77/null strain has a significantly lower frequency of recombination than the rad52Δ284/null strain (one-tailed t test, P < 2 × 10⁻⁴).

FIG. 8.

(A) Microscopy of a diploid yeast strain containing both Rad52Δ284-YFP and Rad52-CFP, indicating colocalization. A spontaneously occurring Rad52 focus is seen in one cell, and both the YFP and CFP signals colocalize, as seen in the merged fluorescence image, indicated by a white arrowhead. The length of the scale bar in the bottom left of the bright-field image represents 5 μm. (B) The proportion of S/G₂-phase yeast cells (defined as mononuclear cells with a bud) that contain at least one Rad52 foci was calculated. The yellow bars indicate the proportion of cells with Rad52-YFP foci, and the blue bars indicate the proportion of cells with Rad52-CFP foci (CFP foci are brighter and more easily counted by microscopy, hence the higher proportion of CFP foci in the wild-type strain). The error bars indicate the 95% confidence intervals of the binomial proportions. The two diploid strains, containing novel variant alleles (rad52Δ77-YFP/RAD52-CFP and rad52Δ284-YFP/RAD52-CFP), have a significantly higher proportion of wild-type (CFP) foci than the wild-type diploid strain (RAD52-YFP/RAD52-CFP) (Fishers exact test, P = 1.3 × 10⁻⁵ and 2.3 × 10⁻⁴, respectively).

FIG. 9.

(A) Two plates of yeast cultures are shown. On each plate seven strains are represented from top to bottom, and six successive 10-fold dilutions of each strain are plated from left to right. Two plates for each experiment were seeded identically, and one plate was exposed to 200 Gy of γ-irradiation. The white arrow at the bottom of the irradiated plate shows increased sensitivity for the heterozygous rad52Δ284/RAD52 diploid strain compared to the top strain (wild-type/null), indicating the dominant-negative phenotype of rad52Δ284. The strains in both the top and bottom of panel B are the same as those described in panel A, except that they each contain plasmid pYES-S10-51 that expresses RAD51 under the control of a galactose-inducible promoter. The top portion of panel B shows cells grown in the presence of glucose, which does not allow expression of RAD51. The lower portion of panel B shows galactose-induced cells expressing RAD51. Partial suppression of the γ-ray sensitivity of the RAD52-CFP/rad52Δ284-YFP strain, when grown on galactose, is indicated with a white arrow.