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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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Related in: MedlinePlus

Biased distribution of GCRs retaining hph.(A and B) Schematic showing the positions of the CAN1/URA3 cassette in the uGCR and dGCR assays relative to the 4.2 kb HXT13-DSF1 segmental duplication on chrV. The GCR breakpoint region (horizontal bracket) is the region in which rearrangements must occur to lose CAN1/URA3 cassette but not the essential gene PCM1. (C) Plot of the percent retention of hph 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 hph retention in the wild-type uGCR assay strain.
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pgen-1004277-g001: Biased distribution of GCRs retaining hph.(A and B) Schematic showing the positions of the CAN1/URA3 cassette in the uGCR and dGCR assays relative to the 4.2 kb HXT13-DSF1 segmental duplication on chrV. The GCR breakpoint region (horizontal bracket) is the region in which rearrangements must occur to lose CAN1/URA3 cassette but not the essential gene PCM1. (C) Plot of the percent retention of hph 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 hph retention in the wild-type uGCR assay strain.

Mentions: Two GCR assays on chrV that incorporated a telomeric hygromycin resistance marker (hph) (Figure 1A and B) [14] were used to characterize the GCR rate and the frequency of GCRs retaining hph in over 95 mutant strains [14], [21]. The yel072w::CAN1/URA3 GCR (dGCR) assay primarily mediates GCRs by duplication-mediated rearrangements with chromosomes IV, X, and XIV; the GCRs derived using this assay frequently lost the telomeric portion of chrV that includes the hph marker [14], [21]. Consistent with this, 0 of 62 GCRs (0%) derived in the wild-type dGCR assay strain and 15 of 2435 GCRs (0.6%) formed in all tested dGCR assay strains retained hph. In contrast, the frequency of hph retention was higher in the GCRs formed in the yel068c::CAN1/URA3 GCR (uGCR) assay, which mediates GCRs by single copy or “unique” genomic sequences. In the wild-type uGCR assay strain, 2 of the 27 GCR-containing isolates (7%) retained hph, and 367 of 2670 GCRs (14%) formed in all tested uGCR assay strains retained the hph marker. Specific mutations significantly increased the frequency of hph+ GCRs relative to wild type (Figure 1C). These mutations included tel1Δ (58% hph+; p = 3×10−13, G-test), sae2Δ (50% hph+; p = 2×10−9, G-test), rad53Δ sml1Δ (31% hph+; p = 7×10−6, G-test), and mrc1Δ tof1Δ (64% hph+; p = 6×10−8, G-test).


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)

Biased distribution of GCRs retaining hph.(A and B) Schematic showing the positions of the CAN1/URA3 cassette in the uGCR and dGCR assays relative to the 4.2 kb HXT13-DSF1 segmental duplication on chrV. The GCR breakpoint region (horizontal bracket) is the region in which rearrangements must occur to lose CAN1/URA3 cassette but not the essential gene PCM1. (C) Plot of the percent retention of hph 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 hph retention in the wild-type uGCR assay strain.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3974649&req=5

pgen-1004277-g001: Biased distribution of GCRs retaining hph.(A and B) Schematic showing the positions of the CAN1/URA3 cassette in the uGCR and dGCR assays relative to the 4.2 kb HXT13-DSF1 segmental duplication on chrV. The GCR breakpoint region (horizontal bracket) is the region in which rearrangements must occur to lose CAN1/URA3 cassette but not the essential gene PCM1. (C) Plot of the percent retention of hph 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 hph retention in the wild-type uGCR assay strain.
Mentions: Two GCR assays on chrV that incorporated a telomeric hygromycin resistance marker (hph) (Figure 1A and B) [14] were used to characterize the GCR rate and the frequency of GCRs retaining hph in over 95 mutant strains [14], [21]. The yel072w::CAN1/URA3 GCR (dGCR) assay primarily mediates GCRs by duplication-mediated rearrangements with chromosomes IV, X, and XIV; the GCRs derived using this assay frequently lost the telomeric portion of chrV that includes the hph marker [14], [21]. Consistent with this, 0 of 62 GCRs (0%) derived in the wild-type dGCR assay strain and 15 of 2435 GCRs (0.6%) formed in all tested dGCR assay strains retained hph. In contrast, the frequency of hph retention was higher in the GCRs formed in the yel068c::CAN1/URA3 GCR (uGCR) assay, which mediates GCRs by single copy or “unique” genomic sequences. In the wild-type uGCR assay strain, 2 of the 27 GCR-containing isolates (7%) retained hph, and 367 of 2670 GCRs (14%) formed in all tested uGCR assay strains retained the hph marker. Specific mutations significantly increased the frequency of hph+ GCRs relative to wild type (Figure 1C). These mutations included tel1Δ (58% hph+; p = 3×10−13, G-test), sae2Δ (50% hph+; p = 2×10−9, G-test), rad53Δ sml1Δ (31% hph+; p = 7×10−6, G-test), and mrc1Δ tof1Δ (64% hph+; p = 6×10−8, G-test).

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