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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

The effect of replication direction on the gene conversion rate.A. A schematic representation of the gene conversion assay. The pTET-lys2-GTOP cassette (indicated by the hashed box) is located in “Head-on” orientation relative to ARS306. In the ars306Δ strain, the closest origin of replication is ARS305 from which replication proceeds in a co-directional orientation with the transcription from pTET. The distances (in kb) are approximate and not to scale. B. The rates of gene conversion at pTET-lys2-GTOP or –GBTM cassette. Transcription and replication are in the “Head-on” orientation when ARS306 (gray bar) is present and in the “Co-directional” orientation in ars306Δ strains (hashed bar). The 95% confidence intervals are indicated by the error bars.
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pgen-1004839-g006: The effect of replication direction on the gene conversion rate.A. A schematic representation of the gene conversion assay. The pTET-lys2-GTOP cassette (indicated by the hashed box) is located in “Head-on” orientation relative to ARS306. In the ars306Δ strain, the closest origin of replication is ARS305 from which replication proceeds in a co-directional orientation with the transcription from pTET. The distances (in kb) are approximate and not to scale. B. The rates of gene conversion at pTET-lys2-GTOP or –GBTM cassette. Transcription and replication are in the “Head-on” orientation when ARS306 (gray bar) is present and in the “Co-directional” orientation in ars306Δ strains (hashed bar). The 95% confidence intervals are indicated by the error bars.

Mentions: In our previous report regarding the rate of gene conversion events induced by the highly transcribed Sμ sequence, the pTET-lys2-GTOP cassette was integrated in the orientation and location identical to the “OPPO” construct described above for the LOH assay (Fig. 6A). In this orientation, transcription from pTET promoter and replication originating at ARS306 are in the convergent or “head-on” orientation. In order to determine whether the rate of gene conversion is dependent on the relative orientation of transcription and replication, we deleted ARS306 by replacing the ARS consensus sequence (5′-WTTTAYRTTTW-3′) [50] with the gene encoding hygromycin B phosphotransferase (Hph). In the resulting ars306Δ strain, replication through the pTET-lys2-GTOP cassette originates from the ARS305 located about 27 kb away and is in co-directional orientation relative to transcription [51]. As previously reported, in the gene conversion assay, recombination initiating at the pTET-lys2 cassette can be completed using a truncated lys2 gene fragment integrated on CHR15 resulting in lysine prototrophy (Lys+) (Fig. 6A) [33]. In a top1Δ strain containing the pTET-lys2-GTOP cassette, reversing the replication orientation by deletion of ARS306 resulted in a three-fold decrease in the Lys+ rate indicating that replication-transcription conflict is a factor in elevated gene conversion initiating at co-transcriptionally formed G4 DNA (Fig. 6B). The deletion of ARS306 did not significantly affect the gene conversion rates at pTET-lys2-GBTM in top1Δ background or at pTET-lys2-GTOP or –GBTM cassette in WT backgrounds.


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)

The effect of replication direction on the gene conversion rate.A. A schematic representation of the gene conversion assay. The pTET-lys2-GTOP cassette (indicated by the hashed box) is located in “Head-on” orientation relative to ARS306. In the ars306Δ strain, the closest origin of replication is ARS305 from which replication proceeds in a co-directional orientation with the transcription from pTET. The distances (in kb) are approximate and not to scale. B. The rates of gene conversion at pTET-lys2-GTOP or –GBTM cassette. Transcription and replication are in the “Head-on” orientation when ARS306 (gray bar) is present and in the “Co-directional” orientation in ars306Δ strains (hashed bar). The 95% confidence intervals are indicated by the error bars.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004839-g006: The effect of replication direction on the gene conversion rate.A. A schematic representation of the gene conversion assay. The pTET-lys2-GTOP cassette (indicated by the hashed box) is located in “Head-on” orientation relative to ARS306. In the ars306Δ strain, the closest origin of replication is ARS305 from which replication proceeds in a co-directional orientation with the transcription from pTET. The distances (in kb) are approximate and not to scale. B. The rates of gene conversion at pTET-lys2-GTOP or –GBTM cassette. Transcription and replication are in the “Head-on” orientation when ARS306 (gray bar) is present and in the “Co-directional” orientation in ars306Δ strains (hashed bar). The 95% confidence intervals are indicated by the error bars.
Mentions: In our previous report regarding the rate of gene conversion events induced by the highly transcribed Sμ sequence, the pTET-lys2-GTOP cassette was integrated in the orientation and location identical to the “OPPO” construct described above for the LOH assay (Fig. 6A). In this orientation, transcription from pTET promoter and replication originating at ARS306 are in the convergent or “head-on” orientation. In order to determine whether the rate of gene conversion is dependent on the relative orientation of transcription and replication, we deleted ARS306 by replacing the ARS consensus sequence (5′-WTTTAYRTTTW-3′) [50] with the gene encoding hygromycin B phosphotransferase (Hph). In the resulting ars306Δ strain, replication through the pTET-lys2-GTOP cassette originates from the ARS305 located about 27 kb away and is in co-directional orientation relative to transcription [51]. As previously reported, in the gene conversion assay, recombination initiating at the pTET-lys2 cassette can be completed using a truncated lys2 gene fragment integrated on CHR15 resulting in lysine prototrophy (Lys+) (Fig. 6A) [33]. In a top1Δ strain containing the pTET-lys2-GTOP cassette, reversing the replication orientation by deletion of ARS306 resulted in a three-fold decrease in the Lys+ rate indicating that replication-transcription conflict is a factor in elevated gene conversion initiating at co-transcriptionally formed G4 DNA (Fig. 6B). The deletion of ARS306 did not significantly affect the gene conversion rates at pTET-lys2-GBTM in top1Δ background or at pTET-lys2-GTOP or –GBTM cassette in WT backgrounds.

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