Chromosome arm duplication distributions for wild-type and <i>msh2Δ</i> strains versus the distribution of Ty1 and delta elements in the S288C reference genome.

<p>A. Distribution of Ty1 and solo delta elements on each chromosome arm. B. Distribution of Ty1 elements on each chromosome arm. C. Observed chromosome arm duplication distribution from 112 wild-type isolates. * indicates significantly overrepresented duplicated chromosome arms compared to the distribution of Ty1 and solo delta elements. D. Log2 ratios of observed versus expected chromosome arm duplication rates from an <i>msh2Δ</i> strain compared to wild type. Labeled chromosome arms were duplicated significantly more frequently in the <i>msh2Δ</i> mutant than predicted from the bulk increase in GCR rate in the <i>msh2Δ</i> mutant compared to the wild-type strain.</p

GCR rates.

a<p>Numbers in brackets represent the 95% confidence interval of the median; numbers in parenthesis represent fold over wild-type.</p>b<p>See Ref. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002539#pgen.1002539-Chan1" target="_blank">[12]</a>.</p><p>*MAT<b>a</b><i>rtt109Δ</i> +8 mutants were generated by sporulating from 2n+1 diploid strains RDKY7709-7711.</p

Localization of translocation hotspots by MLPA in a wild-type strain.

a<p>The most centromeric probe with increased copy number.</p>b<p>Number of isolates identified with the given recombination target.</p>c<p>Total is defined as the number of isolates with the indicated chromosome arm duplication.</p

Assay model, design of MLPA telomeric and centromeric probe sets, and verification of results obtained using MLPA.

<p>A. Overview of the chromosome V +Ty912 assay strain. The <i>Ty912</i> was inserted between <i>NPR2</i> and <i>CIN8</i> oriented so that it was transcribed towards the telomere. <i>Ty912</i> and other genes are not drawn to scale. B. Schematic of the MLPA telomeric and centromeric probes. Telomeric and centromeric probes are designated by solid black and grey lines above the chromosomes, respectively. Ty delta elements are represented as hollow blue triangles, and Ty1 and Ty2 elements are designated as pairs of hollow blue triangles connected by solid blue lines. The orientation of the triangles represent the transcriptional orientation of the elements. Tys and chromosome arms are not drawn to scale. C. Schematic of the MLPA process (1: chromosomal DNA, 2: MLPA probes, 3: universal PCR primers). MLPA probes (2) are hybridized to the chromosomal DNA (1), ligated, amplified by PCR using universal primers (3) and analyzed using capillary electrophoresis. D. Graphical display of MLPA data generated using telomeric MLPA probes. Chromosome arms are represented by peaks in either a wild-type isolate or GCR-containing isolate. Ratios of the normalized peak areas compared to a relative set of wild-type control isolates are given above the indicated peaks of interest. Black vertical arrows indicate either a chromosome V-L deletion (left arrow) or a chromosome XIII-L duplication (right arrow) in the GCR-containing isolate and normal chromosome arm complements in the wild-type isolate.</p

MLPA, Southern blot, and sequencing analyses of GCRs derived from a <i>rad52Δ</i> recombination-deficient mutant strain.

<p>A. MLPA data reveals duplicated chromosome arms associated with GCRs derived in a <i>rad52Δ</i> mutant strain. B. Southern blot of a Pulse Field Gel with separated chromosomes from GCR-containing isolates from a <i>rad52Δ</i> mutant strain also reveals larger than wild-type chromosome Vs (I16, I17, and I19–I21). C. Pseudo multiple sequence alignment of sequenced breakpoint junctions from <i>rad52Δ</i> derived GCRs. Italicized base pairs flanking <i>Ty912</i> on chromosome V represent nonreference-sequence restriction sites used for cloning and inserting <i>Ty912</i> on chromosome V. * - sequence upstream of the 5′ end of the Ty- element mediating the translocation matches part of the reference sequence supposedly deleted by FS2; this suggests that the FS2 loci is not completely identical to that described originally in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002539#pgen.1002539-Lemoine2" target="_blank">[15]</a>. † Amplified fragment is consistent with two tandem Tys in FS1; however, the 5′ flanking sequence displayed is part of the tandem Ty1 construct and was what was read when the 5′ primer used to amplify the fragment was used to sequence the fragment.</p

Schematic of MLPA probes designed for hotspot chromosome arms and MLPA data generated from GCR–containing isolates derived from a wild-type strain.

<p>Filled squares represent telomeres and filled circles represent centromeres. Hollow triangles represent delta sequences and pairs of hollow triangles connected by blue lines represent Ty1 or Ty2 sequences. The transcriptional orientation of the elements is represented by the direction of the triangles. Solid orange lines above the chromosome arms represent chromosome arm duplications predicted to be mediated by a Ty element; dotted green lines represent duplications predicted to be mediated by a non-Ty element. A. MLPA data for chromosome III-R. Hotspots exist at FS1 and FS2. FS1 represents a tandem pair of Tys replacing the sequence between the 5′ end of <i>SRD1</i> to <i>YCRWdelta10</i>. FS2 replaces <i>YCRWdelta11</i> with an inverted pair of Tys separated by a short spacer sequence. See Ref <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002539#pgen.1002539-Lemoine2" target="_blank">[15]</a>. B. MLPA data for chromosome V-R. Hotspots exist at or near <i>YERWdelta17</i>/<i>YERWdelta21</i>/<i>YERCTy1-1</i> and the YERCdelta14/<i>YERCdelta15</i>/<i>YERCdelta16</i> loci. C. MLPA data for chromosome IXV-R. A hotspot exists at the <i>YNLCTy2-1</i> Ty2 locus. D. MLPA data for chromosome X-R. A hotspot occurs at the <i>YJRWTy1-1/YJRWTy1-2</i> locus.</p

MLPA analysis of GCRs derived in a <i>rad51Δ</i> strain.

<p>A. Distribution of chromosome arm duplications observed in the <i>rad51Δ</i> strain. B. Duplication rates of different chromosome arms from a <i>rad51Δ</i> strain compared to that of wild type. Log2 ratios of the observed duplication rates compared to the expected duplication rates are plotted for each chromosome arm. Labeled chromosome arms had significantly increased or decreased rates of duplication in the <i>rad51Δ</i> mutant than the bulk increase in GCR rate in the <i>rad51Δ</i> mutant compared to the wild type GCR rate. Chromosome III-R appears significantly reduced in the <i>rad51Δ</i> mutant strain. C. Comparison of <i>rad51Δ rad59Δ</i> and <i>rad52Δ</i> mutant strains relative to a <i>rad51Δ</i> mutant strain. * represents chromosome arms with significantly increased or decreased rates of duplication for the given strain compared to a <i>rad51Δ</i> strain. D. Southern blot of a Pulse Field Gel with separated chromosomes from GCR-containing isolates derived from <i>rad51Δ rad59Δ</i> and <i>rad51Δ rad59Δ rad52Δ</i> mutant strains reveals chromosome Vs that are larger than wild type (I1–I7 and I10–I13). <i>MCM3</i>, an essential gene on chromosome V, was used as the probe.</p

DNA Repair Pathway Selection Caused by Defects in <i>TEL1</i>, <i>SAE2</i>, and <i>De Novo</i> Telomere Addition Generates Specific Chromosomal Rearrangement Signatures

<div><p>Whole genome sequencing of cancer genomes has revealed a diversity of recurrent gross chromosomal rearrangements (GCRs) that are likely signatures of specific defects in DNA damage response pathways. However, inferring the underlying defects has been difficult due to insufficient information relating defects in DNA metabolism to GCR signatures. By analyzing over 95 mutant strains of <i>Saccharomyces cerevisiae</i>, we found that the frequency of GCRs that deleted an internal <i>CAN1/URA3</i> cassette on chrV L while retaining a chrV L telomeric <i>hph</i> marker was significantly higher in <i>tel1Δ</i>, <i>sae2Δ</i>, <i>rad53Δ sml1Δ</i>, and <i>mrc1Δ tof1Δ</i> mutants. The <i>hph</i>-retaining GCRs isolated from <i>tel1Δ</i> mutants contained either an interstitial deletion dependent on non-homologous end-joining or an inverted duplication that appeared to be initiated from a double strand break (DSB) on chrV L followed by hairpin formation, copying of chrV L from the DSB toward the centromere, and homologous recombination to capture the <i>hph</i>-containing end of chrV L. In contrast, <i>hph</i>-containing GCRs from other mutants were primarily interstitial deletions (<i>mrc1Δ tof1Δ</i>) or inverted duplications (<i>sae2Δ</i> and <i>rad53Δ sml1Δ</i>). Mutants with impaired <i>de novo</i> telomere addition had increased frequencies of <i>hph</i>-containing GCRs, whereas mutants with increased <i>de novo</i> telomere addition had decreased frequencies of <i>hph</i>-containing GCRs. Both types of <i>hph</i>-retaining GCRs occurred in wild-type strains, suggesting that the increased frequencies of <i>hph</i> retention were due to the relative efficiencies of competing DNA repair pathways. Interestingly, the inverted duplications observed here resemble common GCRs in metastatic pancreatic cancer.</p></div

GCRs retaining <i>hph</i> belong to two size classes.

<p>(<b>A</b>) Digestion of the uGCR chrV divides the uGCR chrV into left telomeric, internal, and right telomeric fragments. Vertical arrows indicate the <i>Asc</i>I cleavage sites and relevant chromosomal features are labeled. (<b>B</b>) Southern blot using an <i>hph</i> probe of a pulsed-field gel (PFG) with DNA from the wild-type strain (RDKY6677) and 6 GCR-containing isolates (212, 214, 215, 217, 218, and 219) with and without <i>Asc</i>I digestion. The <i>hph</i> probe hybridizes to the intact chromosome and the internal and left telomeric fragments. (<b>C</b>) Southern blot of a second PFG with the same samples as in panel B using an <i>MCM3</i> probe. The <i>MCM3</i> probe hybridizes to the intact chromosome and the internal fragment.</p

<i>hph</i>− GCRs associated with chrV larger than wild-type contain duplicated chrV sequences.

<p>(<b>A</b>) The log base 2 ratio of the aCGH hybridization intensity for chrV L for <i>hph</i>− isolates with chrV larger than wild-type. The solid horizontal bar is at 0 and dashed lines are at −1 and 1 (2-fold decreased and increased, respectively). Probes were mapped onto the “uGCR Chromosome V” coordinate system. Chromosomal features such as <i>hph</i>, the <i>CAN1/URA3</i> cassette, the <i>ura3-52</i> mutation, and the centromere (<i>CEN5</i>) are indicated at top. Red brackets indicate duplicated chromosomal regions that span from the GCR breakpoint region (between the <i>CAN1/URA3</i> cassette and <i>PCM1</i>) to a Ty-related element, most frequently <i>ura3-52</i>. (<b>B</b>) The log base 2 ratio of aCGH hybridization intensity for all of chrV for isolates 213 and 2976. Red brackets indicate duplicated chromosomal regions. (<b>C</b>) The log base 2 ratio of aCGH hybridization intensity for all of chrIV for isolates 3124 and 3125. Red brackets indicate duplicated chromosomal regions. (<b>D</b>) Proposed mechanism for rearrangement formation (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004277#s3" target="_blank">Discussion</a>). Orange arrows indicate DSBs.</p