RAD59 is required for efficient repair of simultaneous double-strand breaks resulting in translocations in Saccharomyces cerevisiae

Abstract

Exposure to ionizing radiation results in a variety of genome rearrangements that have been linked to tumor formation. Many of these rearrangements are thought to arise from the repair of double-strand breaks (DSBs) by several mechanisms, including homologous recombination (HR) between repetitive sequences dispersed throughout the genome. Doses of radiation sufficient to create DSBs in or near multiple repetitive elements simultaneously could initiate single-strand annealing (SSA), a highly efficient, though mutagenic, mode of DSB repair. We have investigated the genetic control of the formation of translocations that occur spontaneously and those that form after the generation of DSBs adjacent to homologous sequences on two, non-homologous chromosomes in Saccharomyces cerevisiae. We found that mutations in a variety of DNA repair genes have distinct effects on break-stimulated translocation. Furthermore, the genetic requirements for repair using 300bp and 60bp recombination substrates were different, suggesting that the SSA apparatus may be altered in response to changing substrate lengths. Notably, RAD59 was found to play a particularly significant role in recombination between the short substrates that was partially independent of that of RAD52. The high frequency of these events suggests that SSA may be an important mechanism of genome rearrangement following acute radiation exposure.

Figure 1. Assays for translocation formation by HR in diploids strains

(A) Both copies of chromosomes XV (black) and III (gray) are depicted (drawings not to scale). One copy of chromosome XV contains his3-Δ3′ with an adjacent HO endonuclease recognition site, the other copy contains the his3-Δ200 allele, shown here as a dotted line. One copy of chromosome III is unaltered at the LEU2 locus, the other contains his3-Δ5′ at that locus. The coding sequence of his3-Δ5′ is directed away from the centromere to prevent the formation of a dicentric chromosome. Chromosome III also contains the MAT locus that has a native HO cut site. Homologous his3 sequences (either 300 bp or 60 bp) are shown as shaded boxes. Arrows represent telomeres, circles represent centromeres. The “Spontaneous” assay system lacks the HO endonuclease expression cassette so no breaks are generated at the recombination substrates or at the MAT loci. (B) The “Single-Break” assay contains a mutated HO recognition sequence at the his3-Δ3′ substrate on chromosome XV, so that HO expression only generates a DSB at one recombination substrate, as well as at the MAT loci. (C) The “Double-Break” assay has HO recognition sequences at both his3– 3′ and his3– 5′ on chromosome III, so that HO expression generates DSBs at each of the recombination substrates as well as at the MAT loci. (D) In the “MATa::LEU2” assay the HO recognition sequence is present at each of the recombination substrates, but the MAT locus on one copy of chromosome III is deleted, allowing for one intact copy of chromosome III to be present throughout HO induction.

Figure 2. Analysis of DNA from His⁺ products through genomic Southern blotting and chromosome blot analysis reveals distinct patterns of substrate utilization depending upon the number of DSBs generated

(A) BamHI fragments predicted to be detected by the 1.8 kb HIS3 probe (black bar) before (top) and after (bottom) translocation formation by HR are shown. The fragment containing his3-Δ3′ on one copy of chromosome XV is 1.7 kb, while the fragment containing the his3-Δ200 allele on the other copy of chromosome XV is 0.7 kb. The fragment containing his3-Δ5′ on one copy of chromosome III is 7.8 kb. Following DSB-induction, the HIS3 product will be formed by a translocation (tXV:III) that joins the left arm of chromosome XV and the right arm of chromosome III, which will be revealed by a 5.0 kb fragment. Joining of the remaining arms of these chromosomes by translocation (tIII:XV) will be revealed by either a 4.0 kb (DSB-induced) or a 4.9 kb (spontaneous) fragment will be generated. Sizes of the parent chromosomes and the translocation products are shown to the right in mega base-pairs (Mb). (B) A HIS3-probed Southern blot of BamHI digested genomic DNA from representative His⁺ products obtained from the Spontaneous (Sp, lanes 2–5), Single-Break (SB, lanes 6–8), or Double-Break (DB, lanes 9 and 10) assays in wild-type cells. Lane one is DNA from a non-recombinant parent strain. Marker sizes are shown on the left side of the blot in kilobase pairs. Substrates or products contained on each fragment are indicated on the right. (C) Agarose plugs containing whole yeast chromosomes were prepared concurrently with genomic DNA in (B) (Materials and Methods). Chromosomes were separated by CHEF. The ethidium-stained gel is shown on the left. The HIS3 probed blot is shown on the right. Relevant chromosomes are indicated. Class designation is based upon the pattern revealed from the HIS3-probed blot of 15 independent His⁺ colonies followed by comparison with the pattern observed for the same recombinant on the genomic Southern blot.

Figure 3. DSBs generated near sequences that share homology can be repaired by SSA

(A) Following the creation of DSBs on chromosomes XV and III (see figure 1 for a complete description of the diagram, the MAT loci have been omitted for simplicity), enzymes process the breaks to generate 3′ ssDNA overhangs. The sequence homology shared between the two his3 substrates (either 300 bp or 60 bp) is shown as a shaded region. (B) Processing of broken ends reveals the homology, allowing for the formation of a stable annealing intermediate consisting of one arm of chromosome XV and one arm of chromosome III. (C) After removal of non-homologous sequences and ligation, a stable tXV:III translocation product is formed that is selectable by the requirement for histidine prototrophy. While our data supports that the remaining arms of chromosome XV and III can form a tIII:XV reciprocal translocation product (not shown) it is typically lost since the diploid nature of the cell does not necessitate its retention.