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Stabilization of dicentric translocations through secondary rearrangements mediated by multiple mechanisms in S. cerevisiae.

Pennaneach V, Kolodner RD - PLoS ONE (2009)

Bottom Line: The dicentric GCRs were found to be unstable and to have undergone secondary rearrangements to produce stable monocentric GCRs.We also observed examples of chromosomes with extensive ongoing end decay in mec1 tlc1 mutants, suggesting that Mec1 protects chromosome ends from degradation and contributes to telomere maintenance by HR.HR between repeated sequences resulting in secondary rearrangements was the most prevalent pathway for resolution of dicentric GCRs regardless of the structure of the initial dicentric GCR, although at least three other resolution mechanisms were observed.

View Article: PubMed Central - PubMed

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

ABSTRACT

Background: The gross chromosomal rearrangements (GCRs) observed in S. cerevisiae mutants with increased rates of accumulating GCRs include predicted dicentric GCRs such as translocations, chromosome fusions and isoduplications. These GCRs resemble the genome rearrangements found as mutations underlying inherited diseases as well as in the karyotypes of many cancers exhibiting ongoing genome instability

Methodology/principal findings: The structures of predicted dicentric GCRs were analyzed using multiple strategies including array-comparative genomic hybridization, pulse field gel electrophoresis, PCR amplification of predicted breakpoints and sequencing. The dicentric GCRs were found to be unstable and to have undergone secondary rearrangements to produce stable monocentric GCRs. The types of secondary rearrangements observed included: non-homologous end joining (NHEJ)-dependent intramolecular deletion of centromeres; chromosome breakage followed by NHEJ-mediated circularization or broken-end fusion to another chromosome telomere; and homologous recombination (HR)-dependent non-reciprocal translocations apparently mediated by break-induced replication. A number of these GCRs appeared to have undergone multiple bridge-fusion-breakage cycles. We also observed examples of chromosomes with extensive ongoing end decay in mec1 tlc1 mutants, suggesting that Mec1 protects chromosome ends from degradation and contributes to telomere maintenance by HR.

Conclusions/significance: HR between repeated sequences resulting in secondary rearrangements was the most prevalent pathway for resolution of dicentric GCRs regardless of the structure of the initial dicentric GCR, although at least three other resolution mechanisms were observed. The resolution of dicentric GCRs to stable rearranged chromosomes could in part account for the complex karyotypes seen in some cancers.

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HR defects are associated with decreased frequencies of predicted dicentric GCRs.A total of 366 events were analyzed including 225 predicted monocentric GCRs (de novo telomere addition GCRs where excluded from this analysis) and 141 predicted dicentric GCRs; these GCRs are described in [30]. The percentage of predicted monocentric GCRs and dicentric GCRs were determined for each indicated group of strains. Telomerase deficient includes all strains that contain tlc1 or est2 mutations. CHEK includes strains that contain chk1, dun1, mec1, mec3, pds1, rad9, rad53 and/or tel1 mutations, REC includes strains that contain rad51, rad52, rad54, rad55, rad59 and/or rdh54 mutations and NHEJ includes strains that contain lig4, ku70, ku80 or mre11 mutations. Strains containing other mutations that might affect these different pathways were not included in this analysis. Numbers above the histogram indicate the actual number of GCRs in each group.
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pone-0006389-g006: HR defects are associated with decreased frequencies of predicted dicentric GCRs.A total of 366 events were analyzed including 225 predicted monocentric GCRs (de novo telomere addition GCRs where excluded from this analysis) and 141 predicted dicentric GCRs; these GCRs are described in [30]. The percentage of predicted monocentric GCRs and dicentric GCRs were determined for each indicated group of strains. Telomerase deficient includes all strains that contain tlc1 or est2 mutations. CHEK includes strains that contain chk1, dun1, mec1, mec3, pds1, rad9, rad53 and/or tel1 mutations, REC includes strains that contain rad51, rad52, rad54, rad55, rad59 and/or rdh54 mutations and NHEJ includes strains that contain lig4, ku70, ku80 or mre11 mutations. Strains containing other mutations that might affect these different pathways were not included in this analysis. Numbers above the histogram indicate the actual number of GCRs in each group.

Mentions: The aCGH analysis presented above revealed that in 11 of the 16 predicted dicentric GCRs, the primary rearrangement was associated with at least one additional rearrangement for which the second breakpoint was in a genomic region containing a repeated sequence. In a collection of 366 translocations identified by breakpoint sequence analysis ([30], and unpublished data), 141 GCRs were predicted to be dicentric with 40% being found in telomerase proficient strains and 60% being found in telomerase deficient strains (Figure 6). Defects in HR pathways were associated with a significant reduction of the frequency of predicted dicentric GCRs to 5% (p = 0.002; Fisher Exact test) and to 40% (p = 0.016; Fisher Exact test) in telomerase proficient and telomerase-deficient strains, respectively. Similarly, checkpoint defects were associated with a significant reduction in the frequency of dicentric GCRs to 12% (p = 0.022; Fisher Exact test) in telomerase proficient strains. The other apparent changes in the distribution of predicted dicentric GCRs were not significant. It was previously suggested that most dicentric GCRs (with the exception of some isoduplications) were formed by end joining mechanisms because the primary breakpoints were at regions of non-homology or microhomology [30]. A possible explanation for these observations is that NHEJ or some type of microhomology-mediated recombination [48]–[50] often plays a role in forming the initial monocentric and dicentric translocation breakpoint and HR-pathways more efficiently promotes the secondary rearrangements that stabilize the GCRs. In the absence of HR, dicentric translocations can form, however breakage of the dicentric GCRs in the absence of efficient secondary rearrangement mechanisms might lead to a high frequency of cell death explaining the decrease of predicted dicentric GCRs observed in HR-deficient strains.


Stabilization of dicentric translocations through secondary rearrangements mediated by multiple mechanisms in S. cerevisiae.

Pennaneach V, Kolodner RD - PLoS ONE (2009)

HR defects are associated with decreased frequencies of predicted dicentric GCRs.A total of 366 events were analyzed including 225 predicted monocentric GCRs (de novo telomere addition GCRs where excluded from this analysis) and 141 predicted dicentric GCRs; these GCRs are described in [30]. The percentage of predicted monocentric GCRs and dicentric GCRs were determined for each indicated group of strains. Telomerase deficient includes all strains that contain tlc1 or est2 mutations. CHEK includes strains that contain chk1, dun1, mec1, mec3, pds1, rad9, rad53 and/or tel1 mutations, REC includes strains that contain rad51, rad52, rad54, rad55, rad59 and/or rdh54 mutations and NHEJ includes strains that contain lig4, ku70, ku80 or mre11 mutations. Strains containing other mutations that might affect these different pathways were not included in this analysis. Numbers above the histogram indicate the actual number of GCRs in each group.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2712687&req=5

pone-0006389-g006: HR defects are associated with decreased frequencies of predicted dicentric GCRs.A total of 366 events were analyzed including 225 predicted monocentric GCRs (de novo telomere addition GCRs where excluded from this analysis) and 141 predicted dicentric GCRs; these GCRs are described in [30]. The percentage of predicted monocentric GCRs and dicentric GCRs were determined for each indicated group of strains. Telomerase deficient includes all strains that contain tlc1 or est2 mutations. CHEK includes strains that contain chk1, dun1, mec1, mec3, pds1, rad9, rad53 and/or tel1 mutations, REC includes strains that contain rad51, rad52, rad54, rad55, rad59 and/or rdh54 mutations and NHEJ includes strains that contain lig4, ku70, ku80 or mre11 mutations. Strains containing other mutations that might affect these different pathways were not included in this analysis. Numbers above the histogram indicate the actual number of GCRs in each group.
Mentions: The aCGH analysis presented above revealed that in 11 of the 16 predicted dicentric GCRs, the primary rearrangement was associated with at least one additional rearrangement for which the second breakpoint was in a genomic region containing a repeated sequence. In a collection of 366 translocations identified by breakpoint sequence analysis ([30], and unpublished data), 141 GCRs were predicted to be dicentric with 40% being found in telomerase proficient strains and 60% being found in telomerase deficient strains (Figure 6). Defects in HR pathways were associated with a significant reduction of the frequency of predicted dicentric GCRs to 5% (p = 0.002; Fisher Exact test) and to 40% (p = 0.016; Fisher Exact test) in telomerase proficient and telomerase-deficient strains, respectively. Similarly, checkpoint defects were associated with a significant reduction in the frequency of dicentric GCRs to 12% (p = 0.022; Fisher Exact test) in telomerase proficient strains. The other apparent changes in the distribution of predicted dicentric GCRs were not significant. It was previously suggested that most dicentric GCRs (with the exception of some isoduplications) were formed by end joining mechanisms because the primary breakpoints were at regions of non-homology or microhomology [30]. A possible explanation for these observations is that NHEJ or some type of microhomology-mediated recombination [48]–[50] often plays a role in forming the initial monocentric and dicentric translocation breakpoint and HR-pathways more efficiently promotes the secondary rearrangements that stabilize the GCRs. In the absence of HR, dicentric translocations can form, however breakage of the dicentric GCRs in the absence of efficient secondary rearrangement mechanisms might lead to a high frequency of cell death explaining the decrease of predicted dicentric GCRs observed in HR-deficient strains.

Bottom Line: The dicentric GCRs were found to be unstable and to have undergone secondary rearrangements to produce stable monocentric GCRs.We also observed examples of chromosomes with extensive ongoing end decay in mec1 tlc1 mutants, suggesting that Mec1 protects chromosome ends from degradation and contributes to telomere maintenance by HR.HR between repeated sequences resulting in secondary rearrangements was the most prevalent pathway for resolution of dicentric GCRs regardless of the structure of the initial dicentric GCR, although at least three other resolution mechanisms were observed.

View Article: PubMed Central - PubMed

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

ABSTRACT

Background: The gross chromosomal rearrangements (GCRs) observed in S. cerevisiae mutants with increased rates of accumulating GCRs include predicted dicentric GCRs such as translocations, chromosome fusions and isoduplications. These GCRs resemble the genome rearrangements found as mutations underlying inherited diseases as well as in the karyotypes of many cancers exhibiting ongoing genome instability

Methodology/principal findings: The structures of predicted dicentric GCRs were analyzed using multiple strategies including array-comparative genomic hybridization, pulse field gel electrophoresis, PCR amplification of predicted breakpoints and sequencing. The dicentric GCRs were found to be unstable and to have undergone secondary rearrangements to produce stable monocentric GCRs. The types of secondary rearrangements observed included: non-homologous end joining (NHEJ)-dependent intramolecular deletion of centromeres; chromosome breakage followed by NHEJ-mediated circularization or broken-end fusion to another chromosome telomere; and homologous recombination (HR)-dependent non-reciprocal translocations apparently mediated by break-induced replication. A number of these GCRs appeared to have undergone multiple bridge-fusion-breakage cycles. We also observed examples of chromosomes with extensive ongoing end decay in mec1 tlc1 mutants, suggesting that Mec1 protects chromosome ends from degradation and contributes to telomere maintenance by HR.

Conclusions/significance: HR between repeated sequences resulting in secondary rearrangements was the most prevalent pathway for resolution of dicentric GCRs regardless of the structure of the initial dicentric GCR, although at least three other resolution mechanisms were observed. The resolution of dicentric GCRs to stable rearranged chromosomes could in part account for the complex karyotypes seen in some cancers.

Show MeSH
Related in: MedlinePlus