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DNA repair pathway selection caused by defects in TEL1, SAE2, and de novo telomere addition generates specific chromosomal rearrangement signatures.

Putnam CD, Pallis K, Hayes TK, Kolodner RD - PLoS Genet. (2014)

Bottom Line: By analyzing over 95 mutant strains of Saccharomyces cerevisiae, we found that the frequency of GCRs that deleted an internal CAN1/URA3 cassette on chrV L while retaining a chrV L telomeric hph marker was significantly higher in tel1Δ, sae2Δ, rad53Δ sml1Δ, and mrc1Δ tof1Δ mutants.Mutants with impaired de novo telomere addition had increased frequencies of hph-containing GCRs, whereas mutants with increased de novo telomere addition had decreased frequencies of hph-containing GCRs.Interestingly, the inverted duplications observed here resemble common GCRs in metastatic pancreatic cancer.

View Article: PubMed Central - PubMed

Affiliation: Ludwig Institute for Cancer Research, University of California School of Medicine, San Diego, La Jolla, California, United States of America; Department of Medicine, University of California School of Medicine, San Diego, La Jolla, California, United States of America.

ABSTRACT
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 Saccharomyces cerevisiae, we found that the frequency of GCRs that deleted an internal CAN1/URA3 cassette on chrV L while retaining a chrV L telomeric hph marker was significantly higher in tel1Δ, sae2Δ, rad53Δ sml1Δ, and mrc1Δ tof1Δ mutants. The hph-retaining GCRs isolated from tel1Δ 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 hph-containing end of chrV L. In contrast, hph-containing GCRs from other mutants were primarily interstitial deletions (mrc1Δ tof1Δ) or inverted duplications (sae2Δ and rad53Δ sml1Δ). Mutants with impaired de novo telomere addition had increased frequencies of hph-containing GCRs, whereas mutants with increased de novo telomere addition had decreased frequencies of hph-containing GCRs. Both types of hph-retaining GCRs occurred in wild-type strains, suggesting that the increased frequencies of hph 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.

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hph+ GCRs associated with chrV larger than wild-type contain duplicated chrV sequences.(A) The log base 2 ratio of the aCGH hybridization intensity for chrV L of hph+ 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 hph, the CAN1/URA3 cassette, the ura3-52 mutation, and the centromere (CEN5) are indicated at top. Red brackets indicate duplicated chromosomal regions that span from the GCR breakpoint region (between the CAN1/URA3 cassette and PCM1) to a Ty-related element, most frequently ura3-52. (B) Proposed mechanism for rearrangement formation (see Discussion). Orange arrows indicate DSBs.
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pgen-1004277-g004: hph+ GCRs associated with chrV larger than wild-type contain duplicated chrV sequences.(A) The log base 2 ratio of the aCGH hybridization intensity for chrV L of hph+ 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 hph, the CAN1/URA3 cassette, the ura3-52 mutation, and the centromere (CEN5) are indicated at top. Red brackets indicate duplicated chromosomal regions that span from the GCR breakpoint region (between the CAN1/URA3 cassette and PCM1) to a Ty-related element, most frequently ura3-52. (B) Proposed mechanism for rearrangement formation (see Discussion). Orange arrows indicate DSBs.

Mentions: The 10 hph+ GCRs with a large chrV were inverted duplications associated with a second homology-mediated rearrangement. aCGH analysis revealed that in the strains containing these GCRs all of the copy number changes detected were restricted to chrV: the changes associated with these GCRs included a ∼4–19 kb chrV L deletion spanning the CAN1/URA3 cassette, and a ∼80–100 kb chrV L duplication extending from the GCR breakpoint region, which is bounded by the CAN1/URA3 cassette and PCM1 (Figure 1A), to a centromeric repetitive element, which was most frequently the Ty-containing ura3-52 (Figure 4A). In each case, the aCGH data also indicated that the GCRs retained the hph-containing region of chrV from TEL05L to the telomeric half of YEL068C, consistent with HR-mediated fusion between ura3-52 and URA3 in the CAN1/URA3 cassette. We verified the ura3-52/URA3 fusion by PCR amplification and Sanger sequencing (Figure S4). WGS of 8 isolates (Table S1) identified and sequenced an inversion junction at the telomeric end of the chrV L duplication (Figure S5; Table S2). If these junctions were formed by folding back and priming of a single strand (Figure 4B), then the homologies for priming were 3–9 bases and the unpaired single-stranded hairpin ranged from 25 to 44 bases (Figure S5). Palindromes are typically difficult to amplify, which may explain the reduced number of junction-defining read pairs recovered for the inversion junctions relative to other rearrangement junctions introduced during strain construction (Table S2). The analyses of these inverted duplication GCRs were consistent with the changes observed by PFGE (Figure 2B and C; Figure S1), because the duplicated regions lacked AscI sites, and the rearranged chromosomes were capped by the AscI-containing left telomeric fragment.


DNA repair pathway selection caused by defects in TEL1, SAE2, and de novo telomere addition generates specific chromosomal rearrangement signatures.

Putnam CD, Pallis K, Hayes TK, Kolodner RD - PLoS Genet. (2014)

hph+ GCRs associated with chrV larger than wild-type contain duplicated chrV sequences.(A) The log base 2 ratio of the aCGH hybridization intensity for chrV L of hph+ 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 hph, the CAN1/URA3 cassette, the ura3-52 mutation, and the centromere (CEN5) are indicated at top. Red brackets indicate duplicated chromosomal regions that span from the GCR breakpoint region (between the CAN1/URA3 cassette and PCM1) to a Ty-related element, most frequently ura3-52. (B) Proposed mechanism for rearrangement formation (see Discussion). Orange arrows indicate DSBs.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3974649&req=5

pgen-1004277-g004: hph+ GCRs associated with chrV larger than wild-type contain duplicated chrV sequences.(A) The log base 2 ratio of the aCGH hybridization intensity for chrV L of hph+ 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 hph, the CAN1/URA3 cassette, the ura3-52 mutation, and the centromere (CEN5) are indicated at top. Red brackets indicate duplicated chromosomal regions that span from the GCR breakpoint region (between the CAN1/URA3 cassette and PCM1) to a Ty-related element, most frequently ura3-52. (B) Proposed mechanism for rearrangement formation (see Discussion). Orange arrows indicate DSBs.
Mentions: The 10 hph+ GCRs with a large chrV were inverted duplications associated with a second homology-mediated rearrangement. aCGH analysis revealed that in the strains containing these GCRs all of the copy number changes detected were restricted to chrV: the changes associated with these GCRs included a ∼4–19 kb chrV L deletion spanning the CAN1/URA3 cassette, and a ∼80–100 kb chrV L duplication extending from the GCR breakpoint region, which is bounded by the CAN1/URA3 cassette and PCM1 (Figure 1A), to a centromeric repetitive element, which was most frequently the Ty-containing ura3-52 (Figure 4A). In each case, the aCGH data also indicated that the GCRs retained the hph-containing region of chrV from TEL05L to the telomeric half of YEL068C, consistent with HR-mediated fusion between ura3-52 and URA3 in the CAN1/URA3 cassette. We verified the ura3-52/URA3 fusion by PCR amplification and Sanger sequencing (Figure S4). WGS of 8 isolates (Table S1) identified and sequenced an inversion junction at the telomeric end of the chrV L duplication (Figure S5; Table S2). If these junctions were formed by folding back and priming of a single strand (Figure 4B), then the homologies for priming were 3–9 bases and the unpaired single-stranded hairpin ranged from 25 to 44 bases (Figure S5). Palindromes are typically difficult to amplify, which may explain the reduced number of junction-defining read pairs recovered for the inversion junctions relative to other rearrangement junctions introduced during strain construction (Table S2). The analyses of these inverted duplication GCRs were consistent with the changes observed by PFGE (Figure 2B and C; Figure S1), because the duplicated regions lacked AscI sites, and the rearranged chromosomes were capped by the AscI-containing left telomeric fragment.

Bottom Line: By analyzing over 95 mutant strains of Saccharomyces cerevisiae, we found that the frequency of GCRs that deleted an internal CAN1/URA3 cassette on chrV L while retaining a chrV L telomeric hph marker was significantly higher in tel1Δ, sae2Δ, rad53Δ sml1Δ, and mrc1Δ tof1Δ mutants.Mutants with impaired de novo telomere addition had increased frequencies of hph-containing GCRs, whereas mutants with increased de novo telomere addition had decreased frequencies of hph-containing GCRs.Interestingly, the inverted duplications observed here resemble common GCRs in metastatic pancreatic cancer.

View Article: PubMed Central - PubMed

Affiliation: Ludwig Institute for Cancer Research, University of California School of Medicine, San Diego, La Jolla, California, United States of America; Department of Medicine, University of California School of Medicine, San Diego, La Jolla, California, United States of America.

ABSTRACT
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 Saccharomyces cerevisiae, we found that the frequency of GCRs that deleted an internal CAN1/URA3 cassette on chrV L while retaining a chrV L telomeric hph marker was significantly higher in tel1Δ, sae2Δ, rad53Δ sml1Δ, and mrc1Δ tof1Δ mutants. The hph-retaining GCRs isolated from tel1Δ 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 hph-containing end of chrV L. In contrast, hph-containing GCRs from other mutants were primarily interstitial deletions (mrc1Δ tof1Δ) or inverted duplications (sae2Δ and rad53Δ sml1Δ). Mutants with impaired de novo telomere addition had increased frequencies of hph-containing GCRs, whereas mutants with increased de novo telomere addition had decreased frequencies of hph-containing GCRs. Both types of hph-retaining GCRs occurred in wild-type strains, suggesting that the increased frequencies of hph 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.

Show MeSH
Related in: MedlinePlus