The role of DNA double-strand breaks in spontaneous homologous recombination in S. cerevisiae

Abstract

Homologous recombination (HR) is a source of genomic instability and the loss of heterozygosity in mitotic cells. Since these events pose a severe health risk, it is important to understand the molecular events that cause spontaneous HR. In eukaryotes, high levels of HR are a normal feature of meiosis and result from the induction of a large number of DNA double-strand breaks (DSBs). By analogy, it is generally believed that the rare spontaneous mitotic HR events are due to repair of DNA DSBs that accidentally occur during mitotic growth. Here we provide the first direct evidence that most spontaneous mitotic HR in Saccharomyces cerevisiae is initiated by DNA lesions other than DSBs. Specifically, we describe a class of rad52 mutants that are fully proficient in inter- and intra-chromosomal mitotic HR, yet at the same time fail to repair DNA DSBs. The conclusions are drawn from genetic analyses, evaluation of the consequences of DSB repair failure at the DNA level, and examination of the cellular re-localization of Rad51 and mutant Rad52 proteins after introduction of specific DSBs. In further support of our conclusions, we show that, as in wild-type strains, UV-irradiation induces HR in these rad52 mutants, supporting the view that DNA nicks and single-stranded gaps, rather than DSBs, are major sources of spontaneous HR in mitotic yeast cells.

Figure 1. γ-Ray Sensitivity of rad52-C180A, rad52Δ, and Wild-Type Strains

Figure 2. Gap Repair Is Impaired in rad52 Class C Mutant Strains

(A) Graphical representation of the assay used to address the nature of gap closure events. Gapped pRS413-TRP1 repaired by HR. This event results in transfer of the trp1–1 mutation in the genome to the plasmid. The position of the trp1–1 mutation relative to the gapped TRP1 plasmid-borne sequence is indicated by an asterisk. Repair by simple NHEJ (without any further rearrangement/deletion of plasmid DNA) results in a 273-bp deletion in TRP1. The two types of events were distinguished by PCR using a plasmid specific primer pair, indicated as small arrows. The PCR product sizes expected from transformants resulting from a gapped plasmid that has been repaired by HR and from one that has been closed by NHEJ are shown. (B) Gel electrophoresis analysis of PCR fragments obtained from strains transformed with gapped pRS413-TRP1. Arrows point to the band sizes expected if the gapped plasmid has been repaired by HR or by NHEJ. Representative analyses of ten transformants obtained with wild-type strains (lanes 1–10), ten with rad52-C180A strains (lanes 12–21), and ten with rad52Δ strains (lanes 23–32). Sizes of relevant bands in the DNA marker (lane 11, 22, and 33) are indicated.

Figure 3. Kinetics of the Repair of a DSB Produced between Directly Repeated leu2 Heteroalleles

(A) Graphical representation of the assay used to follow HO-endonuclease-induced direct-repeat HR. The positions of the leu2-ΔBstEII and leu2-ΔEcoRI heteroalleles are indicated by black wedges. The position of the HO cut-site, HOcs, is indicated by an arrow. The product resulting from DSB repair by SSA is shown below the direct-repeat assay. Note that the product is given as a wild-type sequence, but it could also contain any combination of the leu2-ΔBstEII and leu2-ΔEcoRI alleles, as the analysis performed here does not discriminate between these possibilities. Arrows labeled SpeI indicate the positions of the SpeI cut-sites used to release the region from its chromosomal context for genomic blot analysis. Horizontal bars represent the location of the probe used for genomic blot analysis. (B) The DSB was produced by induction of the HO-endonuclease and repair was analyzed in three different strain backgrounds, wild-type, rad52Δ, and rad52-C180A, as indicated. In each strain, the kinetics of three DNA species in the process was followed: uncut DNA, cut DNA, and product. The positions of these species are indicated by arrows. A DNA fragment serving as loading control (see Materials and Methods) is also visualized. The number above each lane indicates the time point after induction of the HO-endonuclease in hours.

Figure 4. rad52 Class C Mutant Strains Are Sensitive to Stable Topoisomerase-Induced DNA Nicks

(A) Serial 10-fold dilutions of wild-type, rad52-C180A, and rad52 null strains were spotted on solid medium containing no camptothecin or 0.5 μg/ml camptothecin as indicated. (B) pWJ1439 (TOP1), pWJ1440 (top1-T722A), and pRS415 were transferred to wild-type, rad52Δ, and all rad52 class C mutant strains by plasmoduction as described in Materials and Methods. The positions of wild-type, rad52Δ, rad52-Y66A, -R70A, -W84A, -R85A, -Y96A, -R156A, -T163A, -C180A, and -F186A strains on selective plates are indicated by numbers: 1, (2 and 3), 4, 5, 6, 7, 8, 9, 10, 11, and 12, respectively.

Figure 5. Duration of Spontaneous Rad52-YFP and Rad52-C180A-YFP Foci

(A) Rad52-YFP and Rad52-C180A-YFP foci are formed in small-budded cells in mitotically growing cultures. Arrowheads point to Rad52-YFP foci. Scale bar, 3 μm. (B) The percentage of cells that do not develop a Rad52 focus during a cell cycle is shown in the left side of the histogram. The percentage of cells that do form a Rad52 focus is shown in the right side of the histogram as a distribution arranged according to the duration of the Rad52 focus observed in individual cells. Each column represents the percentage of cells that have turned the Rad52 focus over in the time frame indicated. Results from RAD52 and rad52-C180A strains are shown as indicated. Median duration of Rad52 foci is 8 min for the wild-type and 57 min for rad52-C180A.

Figure 6. Rad52-C180A-CFP Is Recruited to a Specific DNA Double-Strand Break

(A) Assay in Chromosome III for visualizing an HO-endonuclease inducible DSB. Yellow boxes: lacO sites. Cyan triangle: HO cut-site (HOcs). Solid circle: centromere. (B) Localization of a Rad52–CFP focus to an HO-endonuclease-induced DSB. The panels show YFP, CFP, CFP/YFP-merged, and DIC images of representative cells of a strain expressing Rad52-C180A-CFP in a strain with a lacO tandem array next to an HOcs on Chromosome III (left, strain W4021-20A) and in a strain with an HOcs on Chromosome III and a lacO tandem array on Chromosome IV (right, strain W4341-16A). The lacO tandem array is visualized by LacI-YFP as a yellow focus. The marked foci (arrowheads) in the left panels are examples of Rad52/LacI co-localization and the arrowheads in the example on the right indicate the absence of co-localization when the lacO elements and the HOcs are on different chromosomes. Scale bar, 3 μm. (C) Quantitative analysis of co-localization between Rad52-CFP and YFP-LacI foci. Chromosomal locations of the HOcs and the lacO tandem array are given below the histogram columns. As a control, strain W4341-6D with no HOcs was analyzed. The wild-type dataset shown for comparison is from [39].

Figure 7. Rad51-CFP and Rad52-C180A-YFP Are Recruited to a Specific DNA DSB

(A) Assay in Chromosome V for visualizing an I-SceI-endonuclease inducible DSB. Red boxes: tetO sites. Cyan triangle: I-SceI cut-site (I-SceIcs). Solid circle: centromere. (B) Localization of Rad51-CFP and Rad52-C180A-YFP foci to an I-SceI-endonuclease-induced DSB. The panels show YFP, CFP, RFP, RFP/YFP-merged, and CFP/RFP/YFP-merged, as well as a bright field image of representative cells containing Rad51-CFP and Rad52-C180A-YFP in a strain with a tetO tandem array next to an I-SceIcs on Chromosome V. The tetO tandem array is visualized by TetI-RFP as a red focus. The Rad51-CFP, Rad52-YFP, and TetI-RFP foci are marked by arrowheads. Scale bar, 3 μm. (C) Quantitative analysis of co-localization between Rad52-C180A-YFP/RFP–TetI foci and Rad51-CFP foci. The wild-type dataset is shown for comparison.