The HO-stimulated translocation assay monitors single-strand annealing following the creation of DNA double-strand breaks at multiple loci in diploid Saccharomyces cerevisiae. This mechanism may model genome rearrangements in somatic cells of higher eukaryotes following exposure to high doses of ionizing radiation.
Genetic variation is frequently mediated by genomic rearrangements that arise through interaction between dispersed repetitive elements present in every eukaryotic genome. This process is an important mechanism for generating diversity between and within organisms1-3. The human genome consists of approximately 40% repetitive sequence of retrotransposon origin, including a variety of LINEs and SINEs4. Exchange events between these repetitive elements can lead to genome rearrangements, including translocations, that can disrupt gene dosage and expression that can result in autoimmune and cardiovascular diseases5, as well as cancer in humans6-9.
Exchange between repetitive elements occurs in a variety of ways. Exchange between sequences that share perfect (or near-perfect) homology occurs by a process called homologous recombination (HR). By contrast, non-homologous end joining (NHEJ) uses little-or-no sequence homology for exchange10,11. The primary purpose of HR, in mitotic cells, is to repair double-strand breaks (DSBs) generated endogenously by aberrant DNA replication and oxidative lesions, or by exposure to ionizing radiation (IR), and other exogenous DNA damaging agents.
In the assay described here, DSBs are simultaneously created bordering recombination substrates at two different chromosomal loci in diploid cells by a galactose-inducible HO-endonuclease (Figure 1). The repair of the broken chromosomes generates chromosomal translocations by single strand annealing (SSA), a process where homologous sequences adjacent to the chromosome ends are covalently joined subsequent to annealing. One of the substrates, his3-Δ3′, contains a 3′ truncated HIS3 allele and is located on one copy of chromosome XV at the native HIS3 locus. The second substrate, his3-Δ5′, is located at the LEU2 locus on one copy of chromosome III, and contains a 5′ truncated HIS3 allele. Both substrates are flanked by a HO endonuclease recognition site that can be targeted for incision by HO-endonuclease. HO endonuclease recognition sites native to the MAT locus, on both copies of chromosome III, have been deleted in all strains. This prevents interaction between the recombination substrates and other broken chromosome ends from interfering in the assay. The KAN-MX-marked galactose-inducible HO endonuclease expression cassette is inserted at the TRP1 locus on chromosome IV. The substrates share 311 bp or 60 bp of the HIS3 coding sequence that can be used by the HR machinery for repair by SSA. Cells that use these substrates to repair broken chromosomes by HR form an intact HIS3 allele and a tXV::III chromosomal translocation that can be selected for by the ability to grow on medium lacking histidine (Figure 2A). Translocation frequency by HR is calculated by dividing the number of histidine prototrophic colonies that arise on selective medium by the total number of viable cells that arise after plating appropriate dilutions onto non-selective medium (Figure 2B). A variety of DNA repair mutants have been used to study the genetic control of translocation formation by SSA using this system12-14.
High doses of ionizing radiation present an inherent risk of genome instability through the generation of a large number of DSBs19. Eukaryotic genomes are replete with repetitive sequences that are excellent substrates for generating translocations and other genomic rearrangements20,21. Chromosomal translocations by HR are frequently observed when DSBs are introduced between repetitive sequences12,21,22. Overwhelming evidence suggests that much of the genomic instability associated with leukemias and lymphomas can be attributed to chromosomal translocations, highlighting the importance of understanding how this mechanism occurs in eukaryotes22,23. We have developed a system in budding yeast for examining the formation of translocation chromosomes by DSB-induced HR between short regions of homology on different chromosomes that are similar in size to repetitive elements dispersed throughout the yeast and human genomes.
In the assay, the his3-Δ3′ translocation substrate is located on one copy of chromosome XV. The other his3 allele (his3-Δ200) has a ~1kb deletion of the HIS3 promoter and coding sequence that prevents this sequence from being used as a template for repair24. The his3-Δ5′ substrate is located at the LEU2 locus on one copy of chromosome III, with the other copy of chromosome III containing an unaltered LEU2 allele (Figure 1). A galactose-inducible HO endonuclease expression cassette marked with KAN-MX was inserted into the TRP1 locus of chromosome IV (trp1::GAL-HO-KAN-MX). Each translocation substrate is flanked by a HO-endonuclease recognition sequence that can be targeted for cleavage by inducing expression of the HO gene through the addition of galactose to the medium. After HO-endonuclease induced cleavage at the his3-Δ3′ and his3-Δ5′ substrates, cells can efficiently use the shared short tract of HIS3 sequence homology (311 bp or 60 bp) to repair the broken chromosomes by HR, generating a translocation chromosome with an intact HIS3 allele12-14,25.
Because the parent cells lack an intact copy of the HIS3 gene, they are unable to grow on –His medium. Only cells that have undergone the translocation event can be selected for on medium lacking histidine. Therefore, the frequency of chromosomal translocations can be calculated by dividing the number of histidine prototrophic colonies by the total number of viable cells plated, determined by plating onto YPD. Genomic DNA and intact chromosomes can then be isolated from representative His+ colonies, and the presence of a translocation chromosome verified by genomic Southern and chromosome blot analyses.
Careful analysis has allowed us to gather additional important information about the assay12. Genomic Southern blot analysis has provided evidence that there is virtually complete cutting of chromosomes XV and III after 30 minutes of HO-endonuclease induction, and thus there is no significant background of uncut chromosomal substrates in the population (G. Manthey & A. Bailis, unpublished results). Genomic Southern and chromosome blot analyses of His– survivors indicates that cells frequently lose one, the other, or both cut chromosomes and remain viable (L. Liddell & A. Bailis, unpublished results). Importantly, the nearly equivalent plating efficiencies on non-selective medium before and after induction of expression of HO-endonuclease indicates that neither the failure to repair broken chromosomes, nor the failure to retain translocation chromosomes affects the ability to survive DSB formation. Consistent with this, the tXV:III translocation chromosome has been shown to be unstable in mitotic cells in the absence of selection. This was demonstrated by growing tXV::III containing His+ recombinants overnight non-selectively, plating single colonies onto non-selective plates, and replica plating onto selective medium lacking histidine. Ten to 70% of the colonies arising on these plates had lost the tXV::III translocation chromosome (N. Pannunzio & A. Bailis, unpublished results).
Translocation chromosomes generated by IR exposure in humans exhibit a similar instability26. This suggests that translocation formation may contribute to early events in tumorigenesis by promoting loss of heterozygosity. Second, extensive genetic and molecular analyses suggest that SSA, an efficient and obligatorily non-conservative mechanism of HR, is the primary mechanism of translocation formation by HR following the simultaneous creation of DSBs on two chromosomes12,27,28. This is consistent with the finding that large doses of IR result in a density of DSBs sufficient to create breaks adjacent to multiple repetitive sequences in the yeast genome, and a high frequency of translocation formation by HR. Together, these observations suggest that the oncogenic effect of IR exposure in humans may, in part, result from the repair of DSBs by an efficient mechanism of HR that generates translocations that, through their inherent instability, promote genetic changes that launch tumorigenesis. Because radiation is often used to treat cancer, genome rearrangements that result from repair of radiation-induced DSBs may contribute to the generation of secondary cancers that arise frequently in patients. Thus, this model may help us gain a better understanding of the genetic and molecular basis of an important clinical response to IR treatment.
The authors have nothing to disclose.
This work was supported by funds from the National Institutes of Health and the Beckman Research Institute of the City of Hope. We would like to thank the reviewers for their constructive comments that added clarity to the manuscript.