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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 for hph− 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 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) The log base 2 ratio of aCGH hybridization intensity for all of chrV for isolates 213 and 2976. Red brackets indicate duplicated chromosomal regions. (C) The log base 2 ratio of aCGH hybridization intensity for all of chrIV for isolates 3124 and 3125. Red brackets indicate duplicated chromosomal regions. (D) Proposed mechanism for rearrangement formation (see Discussion). Orange arrows indicate DSBs.
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pgen-1004277-g005: 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 for hph− 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 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) The log base 2 ratio of aCGH hybridization intensity for all of chrV for isolates 213 and 2976. Red brackets indicate duplicated chromosomal regions. (C) The log base 2 ratio of aCGH hybridization intensity for all of chrIV for isolates 3124 and 3125. Red brackets indicate duplicated chromosomal regions. (D) Proposed mechanism for rearrangement formation (see Discussion). Orange arrows indicate DSBs.

Mentions: PFGE analysis of the 13 hph− GCR-containing isolates from the tel1Δ uGCR assay strain revealed that 9 contained a wild-type-sized chrV and 4 contained a large chrV (Figure S6A). PCR mapping [26] revealed that the 9 isolates with wild-type-sized chrV had deletions that included the CAN1/URA3 cassette; sequencing the breakpoints of 4 of these GCRs confirmed that one was a translocation and 3 were de novo telomere additions (Figure S6B). In contrast, aCGH analysis of the 4 isolates with a larger than wild-type chrV (Figure 5A–C) was consistent with a chrV inverted duplication combined with rearrangements targeting homologies unrelated to URA3: these GCRs contained a chrV L deletion from the telomere to the GCR breakpoint region (Figure 5A), a chrV L duplication from the GCR breakpoint region to a Ty-related repetitive element (Figure 5A), and an additional duplication of at least one other additional genomic region bounded by Ty-related elements and telomeres (Figure 5B and C). Isolate 3125 had two duplicated regions (between the inverted Ty pairs YDRWTy2-2/YDRCTy1-2 and YDRWTy2-3/YDRCTy1-3 and between YDRWTy1-5 and TEL04R), which was consistent with a mechanism involving more than one round of HR-mediated rearrangements similar to GCRs obtained using other GCR assays [15], [20]. The inversion junctions were identified and sequenced by analysis of WGS data from isolates 3124 and 3125 (Figure S5; Table S1 and S2). Thus, the hph− inverted duplications differed from the hph+ inverted duplications only with regard to the homologies involved in the resolution of the initial inversion chromosome (Figure 5D).


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 for hph− 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 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) The log base 2 ratio of aCGH hybridization intensity for all of chrV for isolates 213 and 2976. Red brackets indicate duplicated chromosomal regions. (C) The log base 2 ratio of aCGH hybridization intensity for all of chrIV for isolates 3124 and 3125. Red brackets indicate duplicated chromosomal regions. (D) 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-g005: 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 for hph− 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 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) The log base 2 ratio of aCGH hybridization intensity for all of chrV for isolates 213 and 2976. Red brackets indicate duplicated chromosomal regions. (C) The log base 2 ratio of aCGH hybridization intensity for all of chrIV for isolates 3124 and 3125. Red brackets indicate duplicated chromosomal regions. (D) Proposed mechanism for rearrangement formation (see Discussion). Orange arrows indicate DSBs.
Mentions: PFGE analysis of the 13 hph− GCR-containing isolates from the tel1Δ uGCR assay strain revealed that 9 contained a wild-type-sized chrV and 4 contained a large chrV (Figure S6A). PCR mapping [26] revealed that the 9 isolates with wild-type-sized chrV had deletions that included the CAN1/URA3 cassette; sequencing the breakpoints of 4 of these GCRs confirmed that one was a translocation and 3 were de novo telomere additions (Figure S6B). In contrast, aCGH analysis of the 4 isolates with a larger than wild-type chrV (Figure 5A–C) was consistent with a chrV inverted duplication combined with rearrangements targeting homologies unrelated to URA3: these GCRs contained a chrV L deletion from the telomere to the GCR breakpoint region (Figure 5A), a chrV L duplication from the GCR breakpoint region to a Ty-related repetitive element (Figure 5A), and an additional duplication of at least one other additional genomic region bounded by Ty-related elements and telomeres (Figure 5B and C). Isolate 3125 had two duplicated regions (between the inverted Ty pairs YDRWTy2-2/YDRCTy1-2 and YDRWTy2-3/YDRCTy1-3 and between YDRWTy1-5 and TEL04R), which was consistent with a mechanism involving more than one round of HR-mediated rearrangements similar to GCRs obtained using other GCR assays [15], [20]. The inversion junctions were identified and sequenced by analysis of WGS data from isolates 3124 and 3125 (Figure S5; Table S1 and S2). Thus, the hph− inverted duplications differed from the hph+ inverted duplications only with regard to the homologies involved in the resolution of the initial inversion chromosome (Figure 5D).

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