Homologous recombination, but not DNA repair, is reduced in vertebrate cells deficient in RAD52

Abstract

Rad52 plays a pivotal role in double-strand break (DSB) repair and genetic recombination in Saccharomyces cerevisiae, where mutation of this gene leads to extreme X-ray sensitivity and defective recombination. Yeast Rad51 and Rad52 interact, as do their human homologues, which stimulates Rad51-mediated DNA strand exchange in vitro, suggesting that Rad51 and Rad52 act cooperatively. To define the role of Rad52 in vertebrates, we generated RAD52(-/-) mutants of the chicken B-cell line DT40. Surprisingly, RAD52(-/-) cells were not hypersensitive to DNA damages induced by gamma-irradiation, methyl methanesulfonate, or cis-platinum(II)diammine dichloride (cisplatin). Intrachromosomal recombination, measured by immunoglobulin gene conversion, and radiation-induced Rad51 nuclear focus formation, which is a putative intermediate step during recombinational repair, occurred as frequently in RAD52(-/-) cells as in wild-type cells. Targeted integration frequencies, however, were consistently reduced in RAD52(-/-) cells, showing a clear role for Rad52 in genetic recombination. These findings reveal striking differences between S. cerevisiae and vertebrates in the functions of RAD51 and RAD52.

FIG. 1

Strategy of disruption of the RAD52 gene. (A) Schematic representation of part of the RAD52 locus, the two gene disruption constructs, and the configuration of the targeted loci. Solid boxes indicate the positions of exons; numbers show the 3′ nucleotide of each exon relative to the start codon (4). Relevant EcoRI recognition sites are indicated by RI. (B) Southern blot analysis of EcoRI-digested DNA from the indicated genotypes with the probe shown in panel A. The positions and sizes of the hybridizing fragments of the wild-type and targeted loci are indicated. (C) Northern blot analysis of total RNA with the full-length chicken RAD52 cDNA as a probe. The same filter was rehybridized with a chicken β-Actin probe (7).

FIG. 2

Sensitivity of the indicated clones to DNA-damaging agents. The fractions of colonies surviving after the indicated treatment of cells compared to nontreated controls of the same genotype are shown on the y axis on a logarithmic scale. (A) Ionizing radiation; (B) MMS; (C) cisplatin. The radiation doses and the MMS and cisplatin concentrations are displayed on the x axis on a linear scale in each graph. Data shown are the means ± standard deviations of at least three separate experiments.

FIG. 3

Immunofluorescent visualization of Rad51. At the time indicated after 8-Gy γ-irradiation, wild-type and RAD52^−/− cells were analyzed. Controls were stained with normal rabbit serum followed by FITC-conjugated anti-rabbit IgG and are overexposed relative to the experimental frames.

FIG. 4

Measurement of targeted integration frequencies at the Igλ locus. (A) Schematic representation of part of the rearranged Igλ locus and the disruption construct (Igλ-neo). ΨV1, first pseudogene; L, leader sequence; Vλ, variable gene segment; Jλ, joining gene segment; Cλ, constant gene segment. BglI (BgI), BglII (BgII), and XbaI (X) restriction sites are indicated. Not all BglI sites in this region are shown. (B) Histograms of sIgM expression of wild-type (a and b), RAD52^−/− (c and d), and RAD52 cDNA-reconstituted RAD52^−/− (RAD52R) (e and f) clones after no transfection (a) or transfection of Igλ-neo and G418 selection of transfectants of each clone (b to f). The x and y axes show the fluorescence intensity from an FITC-conjugated anti-IgM polyclonal antibody on a logarithmic scale and the cell number on a linear scale in each graph, respectively. The percentage of cells losing sIgM expression is shown in each panel.