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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 rates of loss of heterozygosity at CHR3 in top1Δ backgrounds.A. The total rates of LOH events (5-FOAR). GTOP, or GBTM indicates pTET-lys2-GTOP or pTET-lys2-BTM allele integrated in each of the strains analyzed. The 95% confidence intervals are indicated by the error bars. B. The rates of LOH initiating at pTET-lys2-GTOP or pTET-lys2-BTM cassette. LOH events are defined as initiating at pTET-lys2-GTOP or pTET-lys2-BTM cassette when URA3 and G418R are lost, only YJM789 sequence is present at StyI, BsrBI, and SpeI SNP sites and both YJM789 and YPH45 sequence is present at NarI and HindIII SNP sites (See Table S2, Table 2 and Table 4). P values were determined using the chi square analysis. *P = 0.014; **P = 0.0035; ***P<0.0001.
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pgen-1004839-g005: The rates of loss of heterozygosity at CHR3 in top1Δ backgrounds.A. The total rates of LOH events (5-FOAR). GTOP, or GBTM indicates pTET-lys2-GTOP or pTET-lys2-BTM allele integrated in each of the strains analyzed. The 95% confidence intervals are indicated by the error bars. B. The rates of LOH initiating at pTET-lys2-GTOP or pTET-lys2-BTM cassette. LOH events are defined as initiating at pTET-lys2-GTOP or pTET-lys2-BTM cassette when URA3 and G418R are lost, only YJM789 sequence is present at StyI, BsrBI, and SpeI SNP sites and both YJM789 and YPH45 sequence is present at NarI and HindIII SNP sites (See Table S2, Table 2 and Table 4). P values were determined using the chi square analysis. *P = 0.014; **P = 0.0035; ***P<0.0001.

Mentions: We observed a dramatic and specific increase in the rates of gene conversion [33] and GCR (see above) associated with highly transcribed Sμ sequences when Top1 was disrupted in a haploid YPH45 background. Importantly, both occurred at significantly higher rates when the guanine-run containing strand was on the NTS where its single stranded nature fosters G4 DNA formation. In order to determine whether LOH on CHR3 is similarly affected by the location of G4-forming sequence on the TS vs. NTS, we compared LOH rates associated with pTET-lys2-GTOP and -BTM constructs in top1Δ/top1Δ backgrounds. There was no significant difference between overall rates of LOH events associated with pTET-lys2-GTOP and -GBTM constructs when replication was in the SAME direction (Fig. 5A). However, when replication was in the OPPO orientation, the overall rate of LOH events was ∼3 fold higher for the pTET-lys2-GTOP than for the pTET-lys2-GBTM construct. The rates of LOH initiating at the pTET-lys2-GTOP/GBTM cassette in the SAME or OPPO orientation in top1Δ/top1Δ background were determined by analyzing 47–93 5-FOAR isolates by the RFLP-SNP assay (Table 4). When transcription from the pTET promoter was in the SAME orientation relative to replication originating at ARS306, the rate of LOH initiating at the G4-containing sequence was similar whether the guanine-runs were on the NTS (pTET-lys2-GTOP) or on the TS (pTET-lys2-GBTM) (Fig. 5B). However, the rate of LOH initiated near pTET-lys2-GTOP in the OPPO orientation was >20 fold higher than at pTET-lys2-GBTM in OPPO orientation and ∼4 fold higher than at pTET-lys2-GTOP in the SAME orientation.


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 rates of loss of heterozygosity at CHR3 in top1Δ backgrounds.A. The total rates of LOH events (5-FOAR). GTOP, or GBTM indicates pTET-lys2-GTOP or pTET-lys2-BTM allele integrated in each of the strains analyzed. The 95% confidence intervals are indicated by the error bars. B. The rates of LOH initiating at pTET-lys2-GTOP or pTET-lys2-BTM cassette. LOH events are defined as initiating at pTET-lys2-GTOP or pTET-lys2-BTM cassette when URA3 and G418R are lost, only YJM789 sequence is present at StyI, BsrBI, and SpeI SNP sites and both YJM789 and YPH45 sequence is present at NarI and HindIII SNP sites (See Table S2, Table 2 and Table 4). P values were determined using the chi square analysis. *P = 0.014; **P = 0.0035; ***P<0.0001.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004839-g005: The rates of loss of heterozygosity at CHR3 in top1Δ backgrounds.A. The total rates of LOH events (5-FOAR). GTOP, or GBTM indicates pTET-lys2-GTOP or pTET-lys2-BTM allele integrated in each of the strains analyzed. The 95% confidence intervals are indicated by the error bars. B. The rates of LOH initiating at pTET-lys2-GTOP or pTET-lys2-BTM cassette. LOH events are defined as initiating at pTET-lys2-GTOP or pTET-lys2-BTM cassette when URA3 and G418R are lost, only YJM789 sequence is present at StyI, BsrBI, and SpeI SNP sites and both YJM789 and YPH45 sequence is present at NarI and HindIII SNP sites (See Table S2, Table 2 and Table 4). P values were determined using the chi square analysis. *P = 0.014; **P = 0.0035; ***P<0.0001.
Mentions: We observed a dramatic and specific increase in the rates of gene conversion [33] and GCR (see above) associated with highly transcribed Sμ sequences when Top1 was disrupted in a haploid YPH45 background. Importantly, both occurred at significantly higher rates when the guanine-run containing strand was on the NTS where its single stranded nature fosters G4 DNA formation. In order to determine whether LOH on CHR3 is similarly affected by the location of G4-forming sequence on the TS vs. NTS, we compared LOH rates associated with pTET-lys2-GTOP and -BTM constructs in top1Δ/top1Δ backgrounds. There was no significant difference between overall rates of LOH events associated with pTET-lys2-GTOP and -GBTM constructs when replication was in the SAME direction (Fig. 5A). However, when replication was in the OPPO orientation, the overall rate of LOH events was ∼3 fold higher for the pTET-lys2-GTOP than for the pTET-lys2-GBTM construct. The rates of LOH initiating at the pTET-lys2-GTOP/GBTM cassette in the SAME or OPPO orientation in top1Δ/top1Δ background were determined by analyzing 47–93 5-FOAR isolates by the RFLP-SNP assay (Table 4). When transcription from the pTET promoter was in the SAME orientation relative to replication originating at ARS306, the rate of LOH initiating at the G4-containing sequence was similar whether the guanine-runs were on the NTS (pTET-lys2-GTOP) or on the TS (pTET-lys2-GBTM) (Fig. 5B). However, the rate of LOH initiated near pTET-lys2-GTOP in the OPPO orientation was >20 fold higher than at pTET-lys2-GBTM in OPPO orientation and ∼4 fold higher than at pTET-lys2-GTOP in the SAME orientation.

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