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

GCR rates and percent <i>hph</i> retention in mutants affecting <i>de novo</i> telomere addition.

<p>* Rate data from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004277#pgen.1004277-Putnam1" target="_blank">[14]</a>.</p><p>** Rate and <i>hph</i> retention data from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004277#pgen.1004277-Putnam1" target="_blank">[14]</a>.</p><p>*** Rate data from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004277#pgen.1004277-Putnam4" target="_blank">[21]</a>.</p>†<p>Rate of accumulating Can^r 5-FOA^r progeny. The number in parenthesis is the fold increase relative to the wild-type <i>yel068c::CAN1/URA3</i> assay.</p>§<p>Retention of <i>hph</i> in GCR-containing isolates is statistically significantly different than wild type (G-test).</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

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

GCR rates and percent <i>hph</i> retention in checkpoint defective mutants.

<p>* Rate data from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004277#pgen.1004277-Putnam1" target="_blank">[14]</a>.</p><p>** Rate and <i>hph</i> retention data from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004277#pgen.1004277-Putnam1" target="_blank">[14]</a>.</p>†<p>Rate of accumulating Can^r 5-FOA^r progeny. The number in parenthesis is the fold increase relative to the wild-type <i>yel068c::CAN1/URA3</i> assay.</p>‡<p><i>hta-S129X</i> is the genotype <i>hta1-S129X hta2-S129X</i>.</p>§<p>Retention of <i>hph</i> in GCR-containing isolates is statistically significantly different than wild type (G-test).</p

GCR formation in plasmid-complemented strains.

†<p>Rate of accumulating Can^r 5FOA^r progeny. The number in parenthesis is the fold increase relative to the wild-type uGCR assay.</p>§<p>Retention of <i>hph</i> in GCR-containing isolates is statistically significantly different than wild type (G-test).</p

GCR rates and percent <i>hph</i> retention in <i>tel1</i>, <i>sae2</i>, and related mutants.

<p>* Rate data from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004277#pgen.1004277-Putnam1" target="_blank">[14]</a>.</p><p>** Rate and <i>hph</i> retention data from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004277#pgen.1004277-Putnam1" target="_blank">[14]</a>.</p>†<p>Rate of accumulating Can^r 5FOA^r progeny. The number in parenthesis is the fold increase relative to the wild-type uGCR assay.</p>§<p>Retention of <i>hph</i> in GCR-containing isolates is statistically significantly different than wild type (G-test).</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 of <i>hph</i>+ isolates with larger than wild-type-sized chrV. 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>) 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

Biased distribution of GCRs retaining <i>hph</i>.

<p>(<b>A and B</b>) Schematic showing the positions of the <i>CAN1/URA3</i> cassette in the uGCR and dGCR assays relative to the 4.2 kb <i>HXT13-DSF1</i> segmental duplication on chrV. The GCR breakpoint region (horizontal bracket) is the region in which rearrangements must occur to lose <i>CAN1/URA3</i> cassette but not the essential gene <i>PCM1</i>. (<b>C</b>) Plot of the percent retention of <i>hph</i> in the uGCR assay in various mutant backgrounds against the respective p-value for retention (G-test) using the wild-type distribution (2 of 27) as the expected distribution. These data include strains generated and analyzed in this study. Points to the left of the vertical dashed line correspond to mutations with p-values<0.01. The horizontal dashed line is the frequency of <i>hph</i> retention in the wild-type uGCR assay strain.</p

Count of GCR events with and without duplication of the left arm of chromosome V from the uGCR assay.

<p>* The presence of a duplication of the left arm of chromosome V was determined by WGS, aCGH, and/or MLPA.</p><p>** The <i>mre11-3</i> and <i>mre11-H125N</i> alleles were present on plasmids in a strain with a chromosomal deletion of <i>MRE11</i>.</p