Inverted DNA repeats channel repair of distant double-strand breaks into chromatid fusions and chromosomal rearrangements

Abstract

Inverted DNA repeats are known to cause genomic instabilities. Here we demonstrate that double-strand DNA breaks (DSBs) introduced a large distance from inverted repeats in the yeast (Saccharomyces cerevisiae) chromosome lead to a burst of genomic instability. Inverted repeats located as far as 21 kb from each other caused chromosome rearrangements in response to a single DSB. We demonstrate that the DSB initiates a pairing interaction between inverted repeats, resulting in the formation of large dicentric inverted dimers. Furthermore, we observed that propagation of cells containing inverted dimers led to gross chromosomal rearrangements, including translocations, truncations, and amplifications. Finally, our data suggest that break-induced replication is responsible for the formation of translocations resulting from anaphase breakage of inverted dimers. We propose a model explaining the formation of inverted dicentric dimers by intermolecular single-strand annealing (SSA) between inverted DNA repeats. According to this model, anaphase breakage of inverted dicentric dimers leads to gross chromosomal rearrangements (GCR). This "SSA-GCR" pathway is likely to be important in the repair of isochromatid breaks resulting from collapsed replication forks, certain types of radiation, or telomere aberrations that mimic isochromatid breaks.

FIG. 1.

Arrangement of chromosome III markers in strains used to study effect of inverted repeats on DSB repair. (A) RAD51 haploid strain AM919. A DSB (black vertical arrow) is induced at MATa by a galactose-inducible HO gene. HML and HMR are replaced by ADE1. URA3 is inserted 133 kb from the left telomere, about 67 kb proximal to MATa. FS2 consists of two Ty1 elements (labeled Ty1α and Ty1β) in inverted orientation (24) and located 30 kb proximal to MAT. FS1 consists of two Ty1 elements (labeled Ty1γ and Ty1δ) in direct orientation located 57 kb centromere proximal to MATa. (B) RAD51 haploid strain AM964. This strain is similar to AM919 but with an inverted repeat of FEN2 replacing FS2. The distance between the two inverted copies of FEN2 is 1 kb. (C) rad51Δ/rad51Δ diploid strain AM960. In this strain, one copy of chromosome III (shown at the top) is the same as the chromosome in AM919. The other homologue contains MATα-inc, which cannot be cut by HO, and also has a mutant copy of the thr4 gene. This chromosome is about 20 kb longer than the homologue from AM919. (D) RAD51 diploid strain AM792. In this strain, the MATa-containing chromosome is truncated by insertion of the LEU2 gene fused to telomere (tel) sequences (29). The other homologue is similar to that of AM960, except that the HMR gene is replaced by the NAT (nourseothricin [Clonat] resistance) gene.

FIG. 2.

Interaction between inverted Ty1 elements leads to chromosome rearrangements. In this figure, the repair of DSBs in AM919 and related RAD51 strains was examined by PFGE. (A) Formation of chromosome (Chr) rearrangements depends on the presence of an intact FS2. DNA was prepared for PFGE at intervals (0, 3, and 22 h) after the induction of a DSB at MATa in the RAD51 haploid strains AM919, AM903 (ΔTy1α), and AM936 (ΔTy1α ΔTy1β). Southern blots were probed with URA3, which hybridizes with its normal locus on chromosome V and to a URA3 insertion on chromosome III (Fig. 1A). In AM919, two repair products were detected, one of about 370 kb (inverted dimer [Inv.dim] 1) and another of 340 kb. The smaller product was shown subsequently to represent two products of similar size (inverted dimers 2 and 3). The fragment labeled CF is the chromosome fragment resulting from deletion of the sequences centromere distal to the HO cleavage site. An additional CF band, observed 3 h after the addition of galactose, corresponds to the processed (partially single-stranded) form of cut fragment (K. VanHulle and A. Malkova, unpublished observation). (B) Formation of chromosome rearrangements is stimulated by an inverted repeat of the FEN2 gene. Experiments similar to those described for panel A were performed with the haploid strains AM915 (containing a deletion of FS2) and AM964 (containing an inverted repeat of FEN2 replacing FS2). Southern blots were probed with ADE1. A repair product of approximately 370 kb was observed in AM964 but not in AM915. wt, wild type.

FIG. 3.

Analysis of inverted dicentric dimers formed by interaction between inverted repeats of Ty1. (A) Expected structures of chromosome III of AM919 and repair intermediates derived from this chromosome. (i) The structure of chromosome III of AM919 before the formation of inverted dicentric dimers. Underlined numbers (1, 2, 4, 7, 8, 9, 10, 11, and 12) indicate the positions of probes used to analyze the region (see the supplemental material for a detailed description of all probes). The positions of AvrII (black A) and BspEI (red B) restriction sites are shown; only those sites relevant to the Southern analysis described below are depicted. (ii) The structure of ID1 (370-kb chromosome), resulting from recombination between Ty1α and Ty1β. Probe 3 (shown as a green arrow) is a 14-kb-long probe specific to the region between FS1 and FS2. Probe 6 (shown as a red arrow) is a 14.3-kb-long probe specific to the region centromere proximal to FS1. (iii) The structure of ID2, resulting from recombination between Ty1α and Ty1δ. (iv) The structure of ID3, resulting from recombination between Ty1α and Ty1γ. (B) Analysis of large inverted dimers (Inv.dim) by gel electrophoresis. DNA was extracted from preinduction (0-h) and postinduction (12-h) samples of AM919. The samples were digested with AvrII, and the resulting fragments were separated by gel electrophoresis and examined by Southern analysis with probe 2. As expected from the maps shown in panel A, the DNA fragment in the uninduced sample was about 34 kb. The expected fragment size from the 370-kb ID1 is 14 kb. The expected fragments for ID2 and ID3 are about 33 and 27 kb, respectively. Since the appearance of the 340-kb intermediates is delayed compared to that of the 370-kb intermediate, only small amounts of the 33- and 27-kb fragments are present 12 h after DSB induction. (C) Analysis of large inverted dimers by DNA combing. DNA was extracted from AM919 cells 12 h after the addition of galactose. This DNA was stretched and hybridized to the fluorescent probe 3 (green arrow) and probe 6 (red arrow). The relative lengths of hybridizing regions and the gaps between them were calculated as a fraction of the length between the termini of the red arrows (96 kb). (D) Southern analysis of ID2 and ID3. DNA was extracted from preinduction (0-h) and postinduction (15-h) samples of AM919. The samples were digested with BspEI, and the resulting fragments were examined by Southern analysis using probe 1. Fragments of the expected sizes were found for the wild-type, ID2, and ID3 chromosomes (19, 12, and 6 kb, respectively).

FIG. 4.

Model to explain the generation of inverted dicentric dimers and their subsequent processing into translocations. The designations AB and A'B' indicate the orientations of Ty elements; the A' strand is complementary to A and the B' strand is complementary to B. In this diagram, we depict the formation of only one type of chromosome aberration, a translocation. Other types of aberrations can be generated by similar mechanisms, as discussed in the text. Ty1α and Ty1β, blue and purple arrows.

FIG. 5.

Kinetics and genetic requirements of Ty-mediated formation of inverted dimers. (A) Kinetics of formation of inverted dimers in AM919. DNA was prepared at intervals after the induction of a DSB at MATa in the RAD51 haploid strain AM919. Nocodazole was added 2 h following the addition of galactose. Southern blots were probed with URA3, which hybridizes to chromosome (Chr) V (its native locus) and to URA3 inserted about 15 kb from CEN3. The repair products (inverted dimers [Inv.dim] 1, 2, and 3) are indicated. Repair products of the same size were also observed after probing the same blots with an ADE1-specific probe located at the left end of chromosome III (data not shown). The nature of the band marked with asterisk is yet to be identified. (B) An experiment similar to the one shown in panel A was performed using AM919 cells that had not been treated with nocodazole. (C) Genetic requirements for formation of inverted dicentric dimers in haploid strains. The formation of inverted dimers was compared in AM919 (wild-type [wt]), EI517 (rad52Δ), and YLS73 (rad51Δ) strains arrested with nocodazole. Southern blots were probed with ADE1, which hybridized to ADE1 inserted at HML and HMR, and to its native locus on chromosome I. Dimer formation is independent of Rad51p but dependent on Rad52p. (D) Formation of inverted dimers in the rad51Δ/rad51Δ diploid strain AM960 treated with nocodazole. DNA was prepared for PFGE at intervals after the induction of DSBs at MATa in AM960 cells (Fig. 1C) arrested with nocodazole. Southern blots were hybridized to the URA3-specific probe. (E) Formation of inverted dimers in the rad51Δ/rad51Δ diploid strain AM960 that was not treated with nocodazole. DNA was prepared at intervals after induction of DSBs in AM960 cells that were not treated with nocodazole. Bf indicates DNA fragments that are likely to reflect breakage of dicentrics; Cf indicates chromosome fragments resulting from deletion of the sequences centromere distal to the HO cleavage site.

FIG. 6.

Chromosome rearrangements in strains with HO-induced DSBs. (A) Chromosome (Chr) alterations in derivatives of the RAD51 diploid strains AM1000a and AM1000b. DSBs were induced in the nocodazole-arrested RAD51 haploid AM919. The nocodazole was removed, and the haploids were mated to a RAD51 strain of the opposite mating type. We performed PFGE analysis of several independent diploids that were phenotypically Ade⁺ Thr⁻ and expressed MATα information. The chromosomal DNA was analyzed with the URA3 probe (which hybridizes to both chromosomes III and V), although similar results were obtained with the ADE1-specific probe (data not shown). In many of the strains, the repair products were different in size from the normal-length chromosome IIIs, indicating a chromosome rearrangement. (B) Chromosome alterations in nocodazole-treated derivatives of the rad51 diploid strain AM960. DSBs were induced in the nocodazole-arrested rad51Δ diploid strain AM960. The nocodazole was removed, the cells were plated on YEP-plus-galactose medium, and the survivors were examined by PFGE. The chromosome III with the HO site was detected using URA3 as the hybridization probe, although similar results were obtained with the ADE1 probe (data not shown). The majority of the repair products were different in size from the two “normal” chromosome IIIs in AM960 (330 and 350 kb). (C) Chromosome alterations in derivatives of the rad51Δ diploid strain AM960 that were not treated with nocodazole. DSBs were induced in AM960 by plating on a galactose-containing medium, and we examined the consequences of these breaks in the surviving cells by PFGE. The chromosome III with the HO site was detected using ADE1 as the hybridization probe, although similar results were obtained with the URA3 probe (data not shown). The majority of the repair products were different in size from the two “normal” chromosome IIIs in AM960 (330 and 350 kb). The lanes labeled “C” in panels A, B, and C contained DNA from AM960 cells in which the HO site was not cleaved. (D) Genomic microarray analysis of chromosomal rearrangements. We depict gene dosages (CGH Miner format) for chromosomes in strains representing various classes of repair outcomes (see Table S1 in the supplemental material for details). Only chromosome III and rearranged chromosomes (containing chromosomal regions with changed gene dosage) are shown. Genomic DNA was isolated from strains with potential chromosome rearrangements and labeled with a Cy5-labeled fluorescent nucleotide; DNA from a reference strain (AM960) was labeled with Cy3 nucleotide. The two samples were mixed and hybridized to DNA microarrays that contained all yeast genes. Green indicates approximately twofold less DNA in the experimental strain relative to that in the control, and red indicates about 1.5-fold more DNA in the experimental strain. Small regions of red or green color (less than three open reading frames) were not considered significant. Also, regions of red or green color that were disrupted by gray-colored regions were not considered significant.

FIG. 7.

Repair intermediates and chromosome alterations in the RAD51/RAD51 diploid AM792. (A) Formation of inverted dimers (Inv.dim) in AM792. DNA was prepared for PFGE at intervals after induction of DSBs at MATa in nocodazole-arrested AM792. Southern blots were hybridized with ADE1-specific probe, which hybridizes to chromosome (Chr) I, to the truncated (trunc.) chromosome III, and to the repair products. The appearance of two repair products is shown. Inverted dimer 1 corresponds to the 370-kb dicentric dimer described for AM919. As expected, it does not hybridize to a THR4 probe (data not shown). The expected positions of the smaller repair intermediates (inverted dimers 2 and 3) are at the same positions expected for a BIR event, in which the truncated copy of chromosome III is repaired using the other homologue as a template. Thus, we cannot distinguish among these events. (B) DSB repair in AM792 leads to chromosome aberrations. DSBs were induced in nocodazole-arrested AM792 cells. The nocodazole was removed, and the cells were then plated to medium lacking adenine. DNA isolated from the resulting Ade⁺ colonies was analyzed by PFGE, followed by hybridization with an ADE1-specific probe. These strains had a chromosome III that was about 350 kb in length (suggestive of repair by BIR) or a chromosome III that was distinctly larger or smaller than 350 kb, indicating a chromosome rearrangement.