(A) Diagram showing the insertion sites for Ty1/Ty2 families of long terminal repeats (LTR) retrotransposons on S. cerevisiae sixteen chromosomes, aligned by their centromeres (white circle). Insertion sites for full length Ty1 (black flag) and Ty2 (white flag) are shown as perpendicular lines above chromosomes while insertion sites for solo Ty1/Ty2 LTRs (also called deltas) are perpendicular lines below chromosomes. Continuous lines above and below chromosome show the same insertion site for full length Tys and solo LTRs. Ty1 and Ty2 are the most abundant of the Ty families and closely related, sharing almost identical LTR sequences. Note that the diagram is drawn to scale except for chromosome XII where 1–2 Mb rDNA array position is noted. Heat map representing the longest block of uninterrupted identity of pairwise comparisons between 50 Ty1/Ty2 in (B) and 306 LTRs (C) in the S. cerevisiae genome. Length of blocks are binned and colored in intervals of 250 base pairs (bp), as indicated in key below. Binning analysis based on MEPS of ∼250 bp, the minimal length of identity empirically determined for efficient NAHR in yeast [15]. Note that most comparisons are above this MEPS value (blue-shaded), predicting that the majority of Ty1/Ty2 pairings are competent for efficient NAHR. Details for (B) in Table S4 and (C) in Table S5.

Top: NAHR between S. cerevisiae Ty elements may be determined with or without allelic competition in S. cerevisiae purebred and S. cerevisiae/S. bayanus hybrid diploids, respectively. LEU2, HYG, URA3 are heterozygous in both diploids to classify events that occur on the S. cerevisiae chromosome III homolog containing the I-SceI cut site (cs, scissors). For more detail, see Figure 3 below. Bottom: Viability after a DSB in hybrid diploids (MH3360) and purebred diploids (MH3359). Relative viability assayed by colony forming units (CFUs) on –ade +2% galactose plates (continuous induction of I-SceI-induced DSB) compared to CFUs on –ade +2% glucose plates (no induction of DSB). Error bars indicate SD performed on four independent experiments.

(A) Flow chart showing the nonselective characterization of clones after galactose induction of I-SceI endonuclease (see text for details). Top: Map of S. cerevisiae chromosome III showing the I-SceIcs (scissors) at position 163cs (number refers to chromosome III SGD coordinates in kb); centromere, white circle; left telomere, black triangle; right telomere, white triangle. Tys are represented as open rectangles flanked by solid triangles (LTRs). Five Ty insertion loci are highlighted in red, left arm transposition hotspot (LAHS); orange, YCRCdelta6 (∂6); yellow, YCRCdelta7 (∂7); green, right arm transposition hotspot (RAHS); blue, far right arm transposition hotspot (FRAHS) (see Figure S1 for more detail of Ty elements). Bottom: Clones were scored for heterozygous genetic markers (LEU2, HYG, URA3) to determine whether the founding cell had experienced an I-SceI-induced DSB (Hygromycin-sensitive, Hyg^S) followed by repair (class I, Leu⁺Hyg^SUra⁺; class II, Leu⁺Hyg^SUra⁻) or loss of the broken chromosome (class III, Leu⁻Hyg^SUra⁻). Ty-mediated NAHR rearrangement structures from these repair classes (details in Materials and Methods) show recipient-donor partners at the recombination junctions according to Ty locus color in map above. (B) Southern blots showing that the majority of cells initiate the I-SceI site-specific DSB at 163cs after galactose induction in purebred (MH3359) and hybrid (MH3360) diploids. YCR024C probe also hybridizes to the other homolog in purebred diploids (smaller size than uncut band due to the absence of the 1.6 kb I-SceIcs/HYG construct). (C) Frequencies of S. cerevisiae chromosome III repair (class I+II) or loss (class III) after DSB in wild-type (MH3359) and rad52 (MH3475) purebred diploids, and wild-type (MH3360) and rad52 (MH3476) hybrid diploids. Error bars indicate SD. At least two independent experiments assayed for each strain. (D) PFGE/Southern analysis on representative repair clones in purebred (MH3359) and hybrid (MH3360) diploids. Note that, in purebred diploids, the uncut homolog contains leu2Δ1 allele which also hybridizes with the LEU2 probe and, in hybrid diploids, allelic HR occurs at a low frequency between the divergent homologs.

(A) Top: Map indicating the distance of Ty recipients (orange, δ6; yellow, δ7; green, RAHS) from the I-SceIcs at two different positions, 147cs and 163cs. The distance indicates the minimal distance from a DSB that recipient sequences are used for recombination. Bottom: Frequencies of δ6, δ7, and RAHS recipients localized to recombination junctions of Ty-mediated rearrangements (BDR events) in purebred diploids with a DSB at 147cs (MH3469) and 163cs (MH3359). (B) Frequencies of outcomes after a DSB at 163cs in purebred (MH3359) and hybrid (MH3360) diploids. (C) Frequencies of δ6, δ7, and RAHS recipients localized to recombination junctions of Ty-mediated rearrangements (BDR events) in hybrid diploids with a DSB at 147cs (MH3471) and 163cs (MH3360). Note that RAHS and FRAHS generate internal deletions through single-strand annealing (SSA, see text below) and the RAHS assignment of “recipient” for these rearrangements aids in comparisons. Error bars indicate SEM.

(A) Frequencies of intra- and interchromosomal NAHR events after a DSB at 163cs in wild-type (MH3360), msh2 (MH3699), msh6 (MH3692), sgs1 (MH3455) hybrid diploids. Internal deletions, isochromosomes, and rings are ‘Intra-chr. NAHR’, translocations are ‘Inter-chr. NAHR’, and remaining outcomes (allelic and loss) are ‘Other’. Percentages for Inter-chr. NAHR and Intra-chr. NAHR indicated in white and black, respectively. (B) Top: Ranking of sequence identity of chromosome III recipients (δ6, δ7, and RAHS) with intrachromosomal donors (LAHS and FRAHS) out of all donors in the S. cerevisiae genome (out of 305 LTRs for δ6 and δ7, out of 49 Ty1/Ty2 for RAHS). Since multiple Ty elements are present at RAHS, LAHS, and FRAHS, only the highest ranking through local identity (BLAST) comparisons are indicated. *#3 donor ranking for RAHS recipient is attributed to the oppositely oriented YCRCTy1-4 at FRAHS. However, YCRCTy1-5 (#15 out of 49) at FRAHS likely mediates internal deletions due to its direct orientation with RAHS. Bottom: Position of the top three potential interchromosomal donors (#1–3 based on local identity) with chromosome III recipients (orange, yellow, and green correspond to δ6, δ7, and RAHS recipients, respectively). Since YCRWTy1-2 and YCRWTy1-3 are both present at RAHS, top three potential interchromosomal donors for each are indicated as #1a–#3a (green triangle) and #1b–#3b (green square), respectively. Symbols are as Figure 1A. Details of the ranking lists are in Table S7 and Table S8.

Top: Schematic of three S. cerevisiae chromosome III configurations (A–C) analyzed in hybrid diploids. A = 163cs (MH3360), B = 163cs with FRAHSΔ (MH3524/MH3572/MH3573), C = 151cs with FRAHSΔ (MH3551). Ty-mediated BDR products for configuration A are shown below map for A. BDR recipients that mediate each rearrangement are connected with a dashed grey line to the BDR product, with intrachromosomal recipient (left of dashed grey line) and intrachromosomal donor (right of dashed grey line) partners at the recombination junctions indicated by color. Bottom: Frequencies of NAHR pathways (SSA and Rad51-dependent) and chromosome loss after a DSB in hybrid diploids strains with configuration A–C. Inter-Ty deletions are ‘SSA’; rings, translocations, and isochromosomes are ‘Rad51-dependent NAHR’; chromosome loss is ‘Loss’, and remaining outcomes (other and allelic in Table 2) are ‘Other’.

Top: Schematic of S. cerevisiae chromosome III with I-SceIcs inside YCRWTy1-2 of RAHS (referred to as RAHScs). Two main repair products resulting from a DSB at RAHScs are shown below. (1) Intra-Ty deletion likely occurs through SSA within RAHS, indicated by the presence of only one black arrow at RAHS (referred to as ‘intra-Ty’ to distinguish from ‘inter-Ty’ deletions that occur between RAHS and FRAHS). (2) Ty GC likely occurs through a Rad51-dependent pathway and maintains RAHS size, indicated by two black arrows present at RAHS and grey Ty repair patch. Bottom: Frequencies of Intra-Ty deletion and Ty GC events after DSB at RAHScs in hybrid (MH3768) and purebred (MH3764) diploids. ‘Other’ refers to inter-Ty deletions, isochromosomes, rings, translocations, other NAHR, allelic, and loss (see Table 2).

Frequencies of outcomes after DSBs in repetitive DNA (at RAHScs) in hybrid (MH3768) and purebred (MH3764) diploids versus DSBs in unique DNA (at 163cs) in hybrid (MH3360) and purebred (MH3359) diploids. Outcomes are categorized into two classes: (1) ‘Change in gene copy number’ (black) are inter-Ty deletions, translocations, chromosome rings, isochromosomes, other NAHR, and chromosome loss (percentage indicated in white text), (2) ‘No change in gene copy number’ (white) are intra-Ty deletion, Ty GC, and allelic HR.