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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 wild-type-sized chrV are interstitial deletions.(A) Diagram of the uGCR chrV and the features on the first 50 kb containing hph, the CAN1/URA3 cassette and the GCR breakpoint region. (B) Map of the retained (solid bar) and deleted (dotted line) regions for the 8 hph+ GCR isolates with wild-type-sized chrV. Interstitial deletions on chrV entirely (isolates 214, 219, 220, 2975, 3115, and 3118) or partially (isolates 212 and 221) spanned the CAN1/URA3 cassette. All of the isolates are simple deletions, indicated by a Δ symbol, other than 214, which is fused to a fragment of a Ty element. (C) The log base 2 ratio of the aCGH hybridization intensity for a portion of chrV L from isolate 3118 illustrating the agreement between aCGH and sequenced junctions. The coordinates are mapped to the “uGCR Chromosome V” of RDKY6677, which differs somewhat from the database S288c sequence due to modifications introduced onto chrV during strain construction. No data are present for the hph and can1::hisG insertions because these regions were not probed by the aCGH array.
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pgen-1004277-g003: hph+ GCRs associated with wild-type-sized chrV are interstitial deletions.(A) Diagram of the uGCR chrV and the features on the first 50 kb containing hph, the CAN1/URA3 cassette and the GCR breakpoint region. (B) Map of the retained (solid bar) and deleted (dotted line) regions for the 8 hph+ GCR isolates with wild-type-sized chrV. Interstitial deletions on chrV entirely (isolates 214, 219, 220, 2975, 3115, and 3118) or partially (isolates 212 and 221) spanned the CAN1/URA3 cassette. All of the isolates are simple deletions, indicated by a Δ symbol, other than 214, which is fused to a fragment of a Ty element. (C) The log base 2 ratio of the aCGH hybridization intensity for a portion of chrV L from isolate 3118 illustrating the agreement between aCGH and sequenced junctions. The coordinates are mapped to the “uGCR Chromosome V” of RDKY6677, which differs somewhat from the database S288c sequence due to modifications introduced onto chrV during strain construction. No data are present for the hph and can1::hisG insertions because these regions were not probed by the aCGH array.

Mentions: Analysis of the 8 hph+ GCRs with a wild-type-sized chrV revealed that they all contained interstitial deletions. We used PCR to map and amplify the rearrangement breakpoints. Sanger sequencing of the PCR products revealed the presence of interstitial deletions that spanned the CAN1/URA3 cassette (Figure 3A and B) and had short sequence identities at the breakpoint junctions (0–5 basepairs in length; Figure S2), consistent with previous observations [18]. In addition, isolate 214 contained an insertion of a ∼4 kb fragment of a Ty retrotransposon at the breakpoint. Lack of copy number changes other than the interstitial deletion was verified by array comparative genomic hybridization (aCGH) of isolate 3118 (Figure 3C). Paired-end whole genome sequencing (WGS) of isolate 3118 (Table S1 and S2) confirmed the interstitial deletion by the identification of 572 read pairs (‘junction-defining’ read pairs) that had mapped inter-read distances of ∼5.4 kb as compared to the median mapped inter-read distance of 417 bp for all 11,333,616 uniquely mapping read pairs (Figure S3). Additionally, alignment of 114 unmapped reads, which were paired with a read that mapped adjacent to the junction-defining read pairs (‘junction-sequencing’ reads), identified the same junction sequence observed by PCR amplification and Sanger sequencing (Figure S3).


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 wild-type-sized chrV are interstitial deletions.(A) Diagram of the uGCR chrV and the features on the first 50 kb containing hph, the CAN1/URA3 cassette and the GCR breakpoint region. (B) Map of the retained (solid bar) and deleted (dotted line) regions for the 8 hph+ GCR isolates with wild-type-sized chrV. Interstitial deletions on chrV entirely (isolates 214, 219, 220, 2975, 3115, and 3118) or partially (isolates 212 and 221) spanned the CAN1/URA3 cassette. All of the isolates are simple deletions, indicated by a Δ symbol, other than 214, which is fused to a fragment of a Ty element. (C) The log base 2 ratio of the aCGH hybridization intensity for a portion of chrV L from isolate 3118 illustrating the agreement between aCGH and sequenced junctions. The coordinates are mapped to the “uGCR Chromosome V” of RDKY6677, which differs somewhat from the database S288c sequence due to modifications introduced onto chrV during strain construction. No data are present for the hph and can1::hisG insertions because these regions were not probed by the aCGH array.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004277-g003: hph+ GCRs associated with wild-type-sized chrV are interstitial deletions.(A) Diagram of the uGCR chrV and the features on the first 50 kb containing hph, the CAN1/URA3 cassette and the GCR breakpoint region. (B) Map of the retained (solid bar) and deleted (dotted line) regions for the 8 hph+ GCR isolates with wild-type-sized chrV. Interstitial deletions on chrV entirely (isolates 214, 219, 220, 2975, 3115, and 3118) or partially (isolates 212 and 221) spanned the CAN1/URA3 cassette. All of the isolates are simple deletions, indicated by a Δ symbol, other than 214, which is fused to a fragment of a Ty element. (C) The log base 2 ratio of the aCGH hybridization intensity for a portion of chrV L from isolate 3118 illustrating the agreement between aCGH and sequenced junctions. The coordinates are mapped to the “uGCR Chromosome V” of RDKY6677, which differs somewhat from the database S288c sequence due to modifications introduced onto chrV during strain construction. No data are present for the hph and can1::hisG insertions because these regions were not probed by the aCGH array.
Mentions: Analysis of the 8 hph+ GCRs with a wild-type-sized chrV revealed that they all contained interstitial deletions. We used PCR to map and amplify the rearrangement breakpoints. Sanger sequencing of the PCR products revealed the presence of interstitial deletions that spanned the CAN1/URA3 cassette (Figure 3A and B) and had short sequence identities at the breakpoint junctions (0–5 basepairs in length; Figure S2), consistent with previous observations [18]. In addition, isolate 214 contained an insertion of a ∼4 kb fragment of a Ty retrotransposon at the breakpoint. Lack of copy number changes other than the interstitial deletion was verified by array comparative genomic hybridization (aCGH) of isolate 3118 (Figure 3C). Paired-end whole genome sequencing (WGS) of isolate 3118 (Table S1 and S2) confirmed the interstitial deletion by the identification of 572 read pairs (‘junction-defining’ read pairs) that had mapped inter-read distances of ∼5.4 kb as compared to the median mapped inter-read distance of 417 bp for all 11,333,616 uniquely mapping read pairs (Figure S3). Additionally, alignment of 114 unmapped reads, which were paired with a read that mapped adjacent to the junction-defining read pairs (‘junction-sequencing’ reads), identified the same junction sequence observed by PCR amplification and Sanger sequencing (Figure S3).

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