Rad52 forms DNA repair and recombination centers during S phase

Abstract

Maintenance of genomic integrity and stable transmission of genetic information depend on a number of DNA repair processes. Failure to faithfully perform these processes can result in genetic alterations and subsequent development of cancer and other genetic diseases. In the eukaryote Saccharomyces cerevisiae, homologous recombination is the major pathway for repairing DNA double-strand breaks. The key role played by Rad52 in this pathway has been attributed to its ability to seek out and mediate annealing of homologous DNA strands. In this study, we find that S. cerevisiae Rad52 fused to green fluorescent protein (GFP) is fully functional in DNA repair and recombination. After induction of DNA double-strand breaks by gamma-irradiation, meiosis, or the HO endonuclease, Rad52-GFP relocalizes from a diffuse nuclear distribution to distinct foci. Interestingly, Rad52 foci are formed almost exclusively during the S phase of mitotic cells, consistent with coordination between recombinational repair and DNA replication. This notion is further strengthened by the dramatic increase in the frequency of Rad52 focus formation observed in a pol12-100 replication mutant and a mec1 DNA damage checkpoint mutant. Furthermore, our data indicate that each Rad52 focus represents a center of recombinational repair capable of processing multiple DNA lesions.

Figures

Figure 1

GFP-tagging strategy for construction of a RAD52-GFP strain. (A) In the first PCR reactions, two pairs of PCR primers were used to amplify sequences upstream and downstream of the GFP insertion site at the 3′ end of RAD52 (see Materials and Methods). (B) Adaptamer-mediated fusion of PCR products. The resulting upstream and downstream fragments each contained an additional 19 base pairs complementary to GFP sequence to allow the fusion of these two PCR products to DNA sequences consisting of GFP fused to either the 5′- or 3′-two-thirds of the K. lactis URA3 gene. (C) Integration by in vivo recombination. The two DNA fragments produced in the second set of PCR reactions were transformed into S. cerevisiae to integrate a direct repeat of GFP immediately upstream of the endogenous RAD52 stop codon. Subsequently, pop-out recombinants were selected on 5-FOA to remove the URA3 marker and the extra copy of GFP.

Figure 2

Characterization of the RAD52-GFP strain. (A) Western blot analysis of Rad52 and Rad52-GFP. Protein extracts obtained from RAD52 (W2297-12D) and RAD52-GFP (W2297-4A) strains were fractionated by SDS/PAGE. Rad52 and Rad52-GFP were visualized by using anti-Rad52 antibody. (B) γ-Ray survival curves for RAD52 (W2297-12D), RAD52-GFP (W2297-4A), and rad52 null (W2366-6B) strains. Each data point represents the mean of three independent trials.

Figure 3

Induction of nuclear Rad52-GFP foci by γ-irradiation. (A and B) Microscopy of cells (J861) expressing the Rad52-GFP fusion protein. Pictures shown are pseudocolored monochrome images: Rad52-GFP in green and DAPI-stained DNA in blue. (A) Diffuse nuclear localization of Rad52-GFP in nonirradiated cells. Note that one cell contains a spontaneous Rad52-GFP focus. (B) Relocalization of Rad52-GFP to distinct foci in cells after exposure to 20 krad of γ-irradiation. At this dose, most cells contain Rad52-GFP foci. (C–F) Dose dependence of Rad52 focus formation in G1 (open diamonds), S (open triangles), and G2/M (solid squares) phase haploid (W2297–4A) and diploid (W2682) cells. All focal planes were inspected for Rad52 foci. The number of foci observed after γ-irradiation was constant in the time frame from 15 to 90 min after irradiation and all numbers were obtained within this time frame. (C) Percentage of haploid cells with ≥1 focus. (D) The average number of Rad52 foci that are observed in haploid cells with ≥1 focus. (E) Percentage of diploid cells with ≥1 focus. (F) The average number of Rad52 foci that are observed in diploid cells with ≥1 focus.

Figure 4

Formation of Rad52-GFP foci during sporulation. Microscopy of sporulating cells (W2682) expressing the Rad52-GFP fusion. (A) A representative sporulating cell before the first meiotic division after 11 h in SPO medium. Four optical sections separated by 0.4 μm are displayed (see schematic). (B) A four-spored tetrad after 15 h in SPO medium is displayed showing that Rad52 foci have disassembled after the second meiotic division.

Figure 5

Rad52-GFP foci induced by the HO endonuclease. (A) The percentages of cells in G1, S, and G2/M containing foci after induction of HO in strains with no (W2334–1D), one (W2322–5B and W2613–22A), or two (W2322–8A) HO-cut sites, are shown. (B) The average number of Rad52 foci that are observed in G1 (open diamonds), S (open triangles), and G2/M (solid squares) phase cells with ≥1 focus.