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Topoisomerase I plays a critical role in suppressing genome instability at a highly transcribed G-quadruplex-forming sequence.

Yadav P, Harcy V, Argueso JL, Dominska M, Jinks-Robertson S, Kim N - PLoS Genet. (2014)

Bottom Line: Transcription confers a critical strand bias since genome rearrangements at the G4-forming Sμ are elevated only when the guanine-runs are located on the non-transcribed strand.The direction of replication and transcription, when in a head-on orientation, further contribute to the elevated genome instability at a potential G4 DNA-forming sequence.The implications of our identification of Top1 as a critical factor in suppression of instability associated with potential G4 DNA-forming sequences are discussed.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, University of Texas Health Science Center at Houston, Houston, Texas, United States of America.

ABSTRACT
G-quadruplex or G4 DNA is a non-B secondary DNA structure that comprises a stacked array of guanine-quartets. Cellular processes such as transcription and replication can be hindered by unresolved DNA secondary structures potentially endangering genome maintenance. As G4-forming sequences are highly frequent throughout eukaryotic genomes, it is important to define what factors contribute to a G4 motif becoming a hotspot of genome instability. Using a genetic assay in Saccharomyces cerevisiae, we previously demonstrated that a potential G4-forming sequence derived from a guanine-run containing immunoglobulin switch Mu (Sμ) region becomes highly unstable when actively transcribed. Here we describe assays designed to survey spontaneous genome rearrangements initiated at the Sμ sequence in the context of large genomic areas. We demonstrate that, in the absence of Top1, a G4 DNA-forming sequence becomes a strong hotspot of gross chromosomal rearrangements and loss of heterozygosity associated with mitotic recombination within the ∼ 20 kb or ∼ 100 kb regions of yeast chromosome V or III, respectively. Transcription confers a critical strand bias since genome rearrangements at the G4-forming Sμ are elevated only when the guanine-runs are located on the non-transcribed strand. The direction of replication and transcription, when in a head-on orientation, further contribute to the elevated genome instability at a potential G4 DNA-forming sequence. The implications of our identification of Top1 as a critical factor in suppression of instability associated with potential G4 DNA-forming sequences are discussed.

No MeSH data available.


Related in: MedlinePlus

Classes of GCRs associated with the highly transcribed G4 DNA motif.A. PFGE analysis of different classes of GCR events. The full-length chromosomes of representative 5-FOAR CanR clone derived from the top1Δ strain containing the pTET-lys2-GTOP cassette are separated out by size. Classes I, II, IIIa and IIIb are described in the text. The chromosomes in the parental strain (P) are indicated to the left and the arrow indicates the normal (unrearranged) CHR5. The locations of novel chromosomal bands in each sample are indicated by red asterisks. B. Summary of the array-CGH analyses. The red and blue bars indicate the loss and the gain of the copy number of the corresponding regions of CHR5, respectively. The coordinates are approximate and correspond to the S288c reference genome sequence according to Saccharomyces Genome Database. The relevant genomic features are indicated at the bottom of the figure. The locations of relevant solo LTRs and full length Ty retrotransposons are indicated. At coordinate ∼115,000, the ura3-52 allele with a Crick-oriented insertion of a Ty1 element is present in this strain background. The quantitative analysis of the PFGE in Fig. 2A and the detailed CHR5 gene dosage plots for the GCR classes are shown in Fig. S2. The detailed breakpoint nucleotide sequence of the segmental deletion in Class II events (*) is shown in Figure S3.
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pgen-1004839-g002: Classes of GCRs associated with the highly transcribed G4 DNA motif.A. PFGE analysis of different classes of GCR events. The full-length chromosomes of representative 5-FOAR CanR clone derived from the top1Δ strain containing the pTET-lys2-GTOP cassette are separated out by size. Classes I, II, IIIa and IIIb are described in the text. The chromosomes in the parental strain (P) are indicated to the left and the arrow indicates the normal (unrearranged) CHR5. The locations of novel chromosomal bands in each sample are indicated by red asterisks. B. Summary of the array-CGH analyses. The red and blue bars indicate the loss and the gain of the copy number of the corresponding regions of CHR5, respectively. The coordinates are approximate and correspond to the S288c reference genome sequence according to Saccharomyces Genome Database. The relevant genomic features are indicated at the bottom of the figure. The locations of relevant solo LTRs and full length Ty retrotransposons are indicated. At coordinate ∼115,000, the ura3-52 allele with a Crick-oriented insertion of a Ty1 element is present in this strain background. The quantitative analysis of the PFGE in Fig. 2A and the detailed CHR5 gene dosage plots for the GCR classes are shown in Fig. S2. The detailed breakpoint nucleotide sequence of the segmental deletion in Class II events (*) is shown in Figure S3.

Mentions: To further characterize the chromosome breakpoints in GCR events associated with the pTET-lys2-GTOP cassette, we carried out PCR with a degenerate primer annealing to generic yeast telomere sequence (CA16; 5′ CACCACACCCACACAC 3′) and a primer annealing to the 5′ untranslated region of the LYS2 gene. Out of 27 samples where the disruption of the pTET-lys2-GTOP cassette was confirmed by PCR mapping, telomere-anchored PCR products of 700 to 1900 bp were obtained for 16 samples. Subsequent sequencing of these fragments showed that, in 15 CanR/5-FOAR clones, de novo telomere additions occurred at various locations within the G4-forming Sμ fragment (Class I events in Fig. 2 and Fig. S1). Due to the high G/C content and repetitiveness of the Sμ sequence, sequencing analysis failed to identify the site of telomere addition in one of the PCR fragments. In concurrence with preferential telomere addition sites previously identified [41], the junctions of de novo telomere addition were located at GT dinucleotides and frequently at 5 to 6 nt clusters of GT-rich sequence. Additionally, mutations, deletions/insertions, and duplications within the Sμ fragment were detected in seven of the 15 CanR/5-FOAR clones with telomere additions.


Topoisomerase I plays a critical role in suppressing genome instability at a highly transcribed G-quadruplex-forming sequence.

Yadav P, Harcy V, Argueso JL, Dominska M, Jinks-Robertson S, Kim N - PLoS Genet. (2014)

Classes of GCRs associated with the highly transcribed G4 DNA motif.A. PFGE analysis of different classes of GCR events. The full-length chromosomes of representative 5-FOAR CanR clone derived from the top1Δ strain containing the pTET-lys2-GTOP cassette are separated out by size. Classes I, II, IIIa and IIIb are described in the text. The chromosomes in the parental strain (P) are indicated to the left and the arrow indicates the normal (unrearranged) CHR5. The locations of novel chromosomal bands in each sample are indicated by red asterisks. B. Summary of the array-CGH analyses. The red and blue bars indicate the loss and the gain of the copy number of the corresponding regions of CHR5, respectively. The coordinates are approximate and correspond to the S288c reference genome sequence according to Saccharomyces Genome Database. The relevant genomic features are indicated at the bottom of the figure. The locations of relevant solo LTRs and full length Ty retrotransposons are indicated. At coordinate ∼115,000, the ura3-52 allele with a Crick-oriented insertion of a Ty1 element is present in this strain background. The quantitative analysis of the PFGE in Fig. 2A and the detailed CHR5 gene dosage plots for the GCR classes are shown in Fig. S2. The detailed breakpoint nucleotide sequence of the segmental deletion in Class II events (*) is shown in Figure S3.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4256205&req=5

pgen-1004839-g002: Classes of GCRs associated with the highly transcribed G4 DNA motif.A. PFGE analysis of different classes of GCR events. The full-length chromosomes of representative 5-FOAR CanR clone derived from the top1Δ strain containing the pTET-lys2-GTOP cassette are separated out by size. Classes I, II, IIIa and IIIb are described in the text. The chromosomes in the parental strain (P) are indicated to the left and the arrow indicates the normal (unrearranged) CHR5. The locations of novel chromosomal bands in each sample are indicated by red asterisks. B. Summary of the array-CGH analyses. The red and blue bars indicate the loss and the gain of the copy number of the corresponding regions of CHR5, respectively. The coordinates are approximate and correspond to the S288c reference genome sequence according to Saccharomyces Genome Database. The relevant genomic features are indicated at the bottom of the figure. The locations of relevant solo LTRs and full length Ty retrotransposons are indicated. At coordinate ∼115,000, the ura3-52 allele with a Crick-oriented insertion of a Ty1 element is present in this strain background. The quantitative analysis of the PFGE in Fig. 2A and the detailed CHR5 gene dosage plots for the GCR classes are shown in Fig. S2. The detailed breakpoint nucleotide sequence of the segmental deletion in Class II events (*) is shown in Figure S3.
Mentions: To further characterize the chromosome breakpoints in GCR events associated with the pTET-lys2-GTOP cassette, we carried out PCR with a degenerate primer annealing to generic yeast telomere sequence (CA16; 5′ CACCACACCCACACAC 3′) and a primer annealing to the 5′ untranslated region of the LYS2 gene. Out of 27 samples where the disruption of the pTET-lys2-GTOP cassette was confirmed by PCR mapping, telomere-anchored PCR products of 700 to 1900 bp were obtained for 16 samples. Subsequent sequencing of these fragments showed that, in 15 CanR/5-FOAR clones, de novo telomere additions occurred at various locations within the G4-forming Sμ fragment (Class I events in Fig. 2 and Fig. S1). Due to the high G/C content and repetitiveness of the Sμ sequence, sequencing analysis failed to identify the site of telomere addition in one of the PCR fragments. In concurrence with preferential telomere addition sites previously identified [41], the junctions of de novo telomere addition were located at GT dinucleotides and frequently at 5 to 6 nt clusters of GT-rich sequence. Additionally, mutations, deletions/insertions, and duplications within the Sμ fragment were detected in seven of the 15 CanR/5-FOAR clones with telomere additions.

Bottom Line: Transcription confers a critical strand bias since genome rearrangements at the G4-forming Sμ are elevated only when the guanine-runs are located on the non-transcribed strand.The direction of replication and transcription, when in a head-on orientation, further contribute to the elevated genome instability at a potential G4 DNA-forming sequence.The implications of our identification of Top1 as a critical factor in suppression of instability associated with potential G4 DNA-forming sequences are discussed.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, University of Texas Health Science Center at Houston, Houston, Texas, United States of America.

ABSTRACT
G-quadruplex or G4 DNA is a non-B secondary DNA structure that comprises a stacked array of guanine-quartets. Cellular processes such as transcription and replication can be hindered by unresolved DNA secondary structures potentially endangering genome maintenance. As G4-forming sequences are highly frequent throughout eukaryotic genomes, it is important to define what factors contribute to a G4 motif becoming a hotspot of genome instability. Using a genetic assay in Saccharomyces cerevisiae, we previously demonstrated that a potential G4-forming sequence derived from a guanine-run containing immunoglobulin switch Mu (Sμ) region becomes highly unstable when actively transcribed. Here we describe assays designed to survey spontaneous genome rearrangements initiated at the Sμ sequence in the context of large genomic areas. We demonstrate that, in the absence of Top1, a G4 DNA-forming sequence becomes a strong hotspot of gross chromosomal rearrangements and loss of heterozygosity associated with mitotic recombination within the ∼ 20 kb or ∼ 100 kb regions of yeast chromosome V or III, respectively. Transcription confers a critical strand bias since genome rearrangements at the G4-forming Sμ are elevated only when the guanine-runs are located on the non-transcribed strand. The direction of replication and transcription, when in a head-on orientation, further contribute to the elevated genome instability at a potential G4 DNA-forming sequence. The implications of our identification of Top1 as a critical factor in suppression of instability associated with potential G4 DNA-forming sequences are discussed.

No MeSH data available.


Related in: MedlinePlus