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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

Gross Chromosomal Rearrangement (GCR) assay.A. A schematic representation of the GCR assay. The location of pTET-lys2-GTOP or –GBTM cassette is indicated by the hashed box with the arrow above indicating the direction of transcription. The distances (in kb) are approximate and not to scale. Telomeres are represented as “tgtgtg”. Blue bars with labels A, B, C, D, and E represents the approximate location of the PCR products used for characterization of GCR products (see Table 1). B. The transcription orientations of the Sμ-containing cassettes. Guanine-runs are on the non-transcribed strand in single stranded state in pTET-lys2-GTOP cassette and on the transcribed strand annealed to the nascent RNA (red line) in the pTET-lys2–GBTM cassette. RNA polymerase holoenzyme is indicated by the blue oval. C. The rates of GCRs occurring at CHR5 containing the pTET-lys2-GTOP (gray bar) or –GBTM (hashed bar) cassette. +DX; Doxycycline was added to the final concentration of 2 µg/ml to repress the transcription from pTET. The relative RNA levels under high and low transcription conditions are shown in Table S1. *; the rates determined by the p0 method. **; the rates determined by the method of median. 95% confidence intervals are indicated by the error bars.
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pgen-1004839-g001: Gross Chromosomal Rearrangement (GCR) assay.A. A schematic representation of the GCR assay. The location of pTET-lys2-GTOP or –GBTM cassette is indicated by the hashed box with the arrow above indicating the direction of transcription. The distances (in kb) are approximate and not to scale. Telomeres are represented as “tgtgtg”. Blue bars with labels A, B, C, D, and E represents the approximate location of the PCR products used for characterization of GCR products (see Table 1). B. The transcription orientations of the Sμ-containing cassettes. Guanine-runs are on the non-transcribed strand in single stranded state in pTET-lys2-GTOP cassette and on the transcribed strand annealed to the nascent RNA (red line) in the pTET-lys2–GBTM cassette. RNA polymerase holoenzyme is indicated by the blue oval. C. The rates of GCRs occurring at CHR5 containing the pTET-lys2-GTOP (gray bar) or –GBTM (hashed bar) cassette. +DX; Doxycycline was added to the final concentration of 2 µg/ml to repress the transcription from pTET. The relative RNA levels under high and low transcription conditions are shown in Table S1. *; the rates determined by the p0 method. **; the rates determined by the method of median. 95% confidence intervals are indicated by the error bars.

Mentions: We previously showed that gene conversion resulting from ectopic recombination is increased due to co-transcriptionally formed G4 DNA. In order to determine whether co-transcriptionally formed G4 DNA can also elevate gross chromosomal rearrangements (GCRs), we modified the GCR reporter system previously described by Chen and Kolodner [34], [35]. In this reporter system, the URA3 gene was integrated into the left arm of chromosome V (CHR5) replacing the HXT13 gene located ∼8.5 kb centromere-distal to the CAN1 gene (Fig. 1A). The loss of functional CAN1 or URA3 results in resistance to the drug canavanine (Can) or 5-Fluoroorotic acid (5-FOA), respectively. Because the first essential gene on the left arm of CHR5, PCM1, is located ∼60 kb from the telomere, the region containing CAN1 and URA3 genes can be lost without affecting viability of haploids. The hypothetical rate of double drug resistance (CanR/5-FOAR) occurring via independent mutations in CAN1 and URA3 is approximately 10−12, which is significantly lower than the observed rate of deletion of the left arm of CHR5 (10−11–10−10 in a wild-type background) [35]. Therefore, by selecting for colonies resistant to both Can and 5-FOA, GCR events resulting in simultaneous loss of CAN1 and URA3 genes are detected.


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)

Gross Chromosomal Rearrangement (GCR) assay.A. A schematic representation of the GCR assay. The location of pTET-lys2-GTOP or –GBTM cassette is indicated by the hashed box with the arrow above indicating the direction of transcription. The distances (in kb) are approximate and not to scale. Telomeres are represented as “tgtgtg”. Blue bars with labels A, B, C, D, and E represents the approximate location of the PCR products used for characterization of GCR products (see Table 1). B. The transcription orientations of the Sμ-containing cassettes. Guanine-runs are on the non-transcribed strand in single stranded state in pTET-lys2-GTOP cassette and on the transcribed strand annealed to the nascent RNA (red line) in the pTET-lys2–GBTM cassette. RNA polymerase holoenzyme is indicated by the blue oval. C. The rates of GCRs occurring at CHR5 containing the pTET-lys2-GTOP (gray bar) or –GBTM (hashed bar) cassette. +DX; Doxycycline was added to the final concentration of 2 µg/ml to repress the transcription from pTET. The relative RNA levels under high and low transcription conditions are shown in Table S1. *; the rates determined by the p0 method. **; the rates determined by the method of median. 95% confidence intervals are indicated by the error bars.
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Related In: Results  -  Collection

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

pgen-1004839-g001: Gross Chromosomal Rearrangement (GCR) assay.A. A schematic representation of the GCR assay. The location of pTET-lys2-GTOP or –GBTM cassette is indicated by the hashed box with the arrow above indicating the direction of transcription. The distances (in kb) are approximate and not to scale. Telomeres are represented as “tgtgtg”. Blue bars with labels A, B, C, D, and E represents the approximate location of the PCR products used for characterization of GCR products (see Table 1). B. The transcription orientations of the Sμ-containing cassettes. Guanine-runs are on the non-transcribed strand in single stranded state in pTET-lys2-GTOP cassette and on the transcribed strand annealed to the nascent RNA (red line) in the pTET-lys2–GBTM cassette. RNA polymerase holoenzyme is indicated by the blue oval. C. The rates of GCRs occurring at CHR5 containing the pTET-lys2-GTOP (gray bar) or –GBTM (hashed bar) cassette. +DX; Doxycycline was added to the final concentration of 2 µg/ml to repress the transcription from pTET. The relative RNA levels under high and low transcription conditions are shown in Table S1. *; the rates determined by the p0 method. **; the rates determined by the method of median. 95% confidence intervals are indicated by the error bars.
Mentions: We previously showed that gene conversion resulting from ectopic recombination is increased due to co-transcriptionally formed G4 DNA. In order to determine whether co-transcriptionally formed G4 DNA can also elevate gross chromosomal rearrangements (GCRs), we modified the GCR reporter system previously described by Chen and Kolodner [34], [35]. In this reporter system, the URA3 gene was integrated into the left arm of chromosome V (CHR5) replacing the HXT13 gene located ∼8.5 kb centromere-distal to the CAN1 gene (Fig. 1A). The loss of functional CAN1 or URA3 results in resistance to the drug canavanine (Can) or 5-Fluoroorotic acid (5-FOA), respectively. Because the first essential gene on the left arm of CHR5, PCM1, is located ∼60 kb from the telomere, the region containing CAN1 and URA3 genes can be lost without affecting viability of haploids. The hypothetical rate of double drug resistance (CanR/5-FOAR) occurring via independent mutations in CAN1 and URA3 is approximately 10−12, which is significantly lower than the observed rate of deletion of the left arm of CHR5 (10−11–10−10 in a wild-type background) [35]. Therefore, by selecting for colonies resistant to both Can and 5-FOA, GCR events resulting in simultaneous loss of CAN1 and URA3 genes are detected.

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