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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

Loss of Heterozygosity (LOH) assay.A. A schematic representation of the LOH assay in heterozygous diploid yeasts (YPH45×YJM789). YPH45-CHR3 and YJM789-CHR3 are represented by black and red lines, respectively. Location of the pTET-lys2-GTOP or –GBTM cassette on the left arm of CHR3 is indicated by the hashed box. Green boxes indicate the approximate locations of the heterozygous SNP markers listed in Table S1. Telomeres are represented as “tgtgtg”. Hemizygous URA3 and G418R markers present only on the YPH45-CHR3 homolog are indicated by blue boxes. The top panel and the bottom two panels represent the parental 5-FOAS diploid and LOH Class E events as described in the text and in Table 2, respectively. LOH Class E events initiating at the pTET-lys2-GTOP or –GBTM cassette either by recombination or deletion will result in the same RFLP-SNP pattern but in the difference in size alterations of the YPH45 CHR3. B. The orientation of pTET-lys2-GTOP or –GBTM cassette insertion relative to ARS306 in SAME or OPPO configuration.
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pgen-1004839-g003: Loss of Heterozygosity (LOH) assay.A. A schematic representation of the LOH assay in heterozygous diploid yeasts (YPH45×YJM789). YPH45-CHR3 and YJM789-CHR3 are represented by black and red lines, respectively. Location of the pTET-lys2-GTOP or –GBTM cassette on the left arm of CHR3 is indicated by the hashed box. Green boxes indicate the approximate locations of the heterozygous SNP markers listed in Table S1. Telomeres are represented as “tgtgtg”. Hemizygous URA3 and G418R markers present only on the YPH45-CHR3 homolog are indicated by blue boxes. The top panel and the bottom two panels represent the parental 5-FOAS diploid and LOH Class E events as described in the text and in Table 2, respectively. LOH Class E events initiating at the pTET-lys2-GTOP or –GBTM cassette either by recombination or deletion will result in the same RFLP-SNP pattern but in the difference in size alterations of the YPH45 CHR3. B. The orientation of pTET-lys2-GTOP or –GBTM cassette insertion relative to ARS306 in SAME or OPPO configuration.

Mentions: Spontaneous DNA breaks in diploid cells are frequently repaired by allelic mitotic recombination using as template either a sister chromatid or a homologous chromosome. We designed an assay that can measure G4 DNA-induced mitotic recombination between chromosome III (CHR3) homologs in diploids (Fig. 3A). First, we integrated the URA3 gene near the telomere of the left arm of CHR3 in a haploid strain derived from YPH45 (a S288c derivative). On the same arm of CHR3, about 44 kb centromere-proximal to the URA3 integration site, pTET-lys2-GTOP or -GBTM was integrated replacing HIS4. As described above for the CHR5 GCR assay, the pTET-lys2-GTOP and -GBTM cassettes contained the 760-bp fragment of Sμ sequence and were adjacent to an aminoglycoside phosphotransferase gene conferring resistance to the drug G418 (G418R). Because the direction of replication fork movement relative to the direction of transcription can affect recombination at highly transcribed regions [46], [47], each cassette was integrated in two orientations relative to the nearby replication origin ARS306. This yielded constructs in which the transcription and replication forks are co-directional (SAME) or in head-on orientation (OPPO) (Fig. 3B). The direction of replication fork movement through this region of CHR3 was previously confirmed by 2D-gel analysis [36].


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)

Loss of Heterozygosity (LOH) assay.A. A schematic representation of the LOH assay in heterozygous diploid yeasts (YPH45×YJM789). YPH45-CHR3 and YJM789-CHR3 are represented by black and red lines, respectively. Location of the pTET-lys2-GTOP or –GBTM cassette on the left arm of CHR3 is indicated by the hashed box. Green boxes indicate the approximate locations of the heterozygous SNP markers listed in Table S1. Telomeres are represented as “tgtgtg”. Hemizygous URA3 and G418R markers present only on the YPH45-CHR3 homolog are indicated by blue boxes. The top panel and the bottom two panels represent the parental 5-FOAS diploid and LOH Class E events as described in the text and in Table 2, respectively. LOH Class E events initiating at the pTET-lys2-GTOP or –GBTM cassette either by recombination or deletion will result in the same RFLP-SNP pattern but in the difference in size alterations of the YPH45 CHR3. B. The orientation of pTET-lys2-GTOP or –GBTM cassette insertion relative to ARS306 in SAME or OPPO configuration.
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Related In: Results  -  Collection

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

pgen-1004839-g003: Loss of Heterozygosity (LOH) assay.A. A schematic representation of the LOH assay in heterozygous diploid yeasts (YPH45×YJM789). YPH45-CHR3 and YJM789-CHR3 are represented by black and red lines, respectively. Location of the pTET-lys2-GTOP or –GBTM cassette on the left arm of CHR3 is indicated by the hashed box. Green boxes indicate the approximate locations of the heterozygous SNP markers listed in Table S1. Telomeres are represented as “tgtgtg”. Hemizygous URA3 and G418R markers present only on the YPH45-CHR3 homolog are indicated by blue boxes. The top panel and the bottom two panels represent the parental 5-FOAS diploid and LOH Class E events as described in the text and in Table 2, respectively. LOH Class E events initiating at the pTET-lys2-GTOP or –GBTM cassette either by recombination or deletion will result in the same RFLP-SNP pattern but in the difference in size alterations of the YPH45 CHR3. B. The orientation of pTET-lys2-GTOP or –GBTM cassette insertion relative to ARS306 in SAME or OPPO configuration.
Mentions: Spontaneous DNA breaks in diploid cells are frequently repaired by allelic mitotic recombination using as template either a sister chromatid or a homologous chromosome. We designed an assay that can measure G4 DNA-induced mitotic recombination between chromosome III (CHR3) homologs in diploids (Fig. 3A). First, we integrated the URA3 gene near the telomere of the left arm of CHR3 in a haploid strain derived from YPH45 (a S288c derivative). On the same arm of CHR3, about 44 kb centromere-proximal to the URA3 integration site, pTET-lys2-GTOP or -GBTM was integrated replacing HIS4. As described above for the CHR5 GCR assay, the pTET-lys2-GTOP and -GBTM cassettes contained the 760-bp fragment of Sμ sequence and were adjacent to an aminoglycoside phosphotransferase gene conferring resistance to the drug G418 (G418R). Because the direction of replication fork movement relative to the direction of transcription can affect recombination at highly transcribed regions [46], [47], each cassette was integrated in two orientations relative to the nearby replication origin ARS306. This yielded constructs in which the transcription and replication forks are co-directional (SAME) or in head-on orientation (OPPO) (Fig. 3B). The direction of replication fork movement through this region of CHR3 was previously confirmed by 2D-gel analysis [36].

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