Limits...
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 RNA∶DNA hybrid and gene conversion at the highly transcribed Sμ.A. A model depicting two alternate pathways of G-loop formation. In absence of RNase H activity, RNA∶DNA hybrid accumulation fosters folding of G-runs into G4 DNA. In absence of Top1 activity, G4 DNA, which function as a sink for negative torsional stress, can promote RNA∶DNA hybrid formation. B. The rates of gene conversion at pTET-lys2-GTOP (gray bar) or –GBTM (hashed bar) cassette. Indicated strains were transformed with either pGAL1-RNH1 (+RNH1) or pRS416 as vector control. RNHΔ* indicates rnh1Δ rnh201Δ double mutant strains. The 95% confidence intervals are indicated by the error bars.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4256205&req=5

pgen-1004839-g007: The RNA∶DNA hybrid and gene conversion at the highly transcribed Sμ.A. A model depicting two alternate pathways of G-loop formation. In absence of RNase H activity, RNA∶DNA hybrid accumulation fosters folding of G-runs into G4 DNA. In absence of Top1 activity, G4 DNA, which function as a sink for negative torsional stress, can promote RNA∶DNA hybrid formation. B. The rates of gene conversion at pTET-lys2-GTOP (gray bar) or –GBTM (hashed bar) cassette. Indicated strains were transformed with either pGAL1-RNH1 (+RNH1) or pRS416 as vector control. RNHΔ* indicates rnh1Δ rnh201Δ double mutant strains. The 95% confidence intervals are indicated by the error bars.

Mentions: Activated transcription through G/C rich sequence can lead to formation of R-loops, which comprise of a long and stable hybrid between nascent RNA and template DNA strand [64]. R-loop accumulation and associated hyper-recombination can ensue when the mRNA packaging and export is disturbed in THO-TREX defective strains or when the degradation of RNA in RNA∶DNA hybrid is deficient due to absence of RNase H activity [65]. Accumulation of negative supercoils in Topoisomerase-deficient cells can also lead to R-loop accumulation [66]. Duquette et al reported that the combination of R-loop and G4 DNA, referred to as G-loop, is identifiable by electron microscopy when the Ig switch sequence is highly transcribed either in vitro or in bacteria [13]. At the pTET-lys2-GTOP cassette containing the Sμ sequence, therefore, the elevated genome instability could be the result of RNA∶DNA hybrid and/or G4 DNA. G-loop formation can be instigated by G4 DNA nucleation in the NTS, which lead to the stable annealing of the nascent RNA with the unpaired TS of DNA (Fig. 7). Alternatively, G-loop formation can initiate via the formation of RNA∶DNA hybrid, which leaves the NTS unpaired and free to fold into G4 structure. In this case, the higher stability of rG∶dC base pairing compared to rC∶dG could account for the greater instability we observed when the G-runs are on the NTS (-GTOP) [67]. During in vitro transcription, rG∶dC containing RNA∶DNA hybrid is critical for the formation of G-loop structure by Ig switch sequence [13], and required for the transcription blockage by a guanine-run [19]. We tested whether G4-induced hyper-recombination is dependent on RNA∶DNA hybrid formation by overexpressing RNase H1 in top1Δ background. RNase H1 is an enzyme that degrades RNA hybridized to DNA and was shown to counteract the hyper-recombination phenotype associated with R-loops in THO/TREX mutant background [65]. As shown in Fig. 7B, RNase H1 overexpression did not reduce the elevated recombination at the highly expressed pTET-lys2-GTOP cassette, which suggests that RNA∶DNA hybrid is not required for the elevated recombination occurring at the G4 motif in absence of Top1. We previously reported that, upon disruption of both RNase H1 and RNase H2 (rnh1Δ rnh2Δ), the rates of gene conversion for the pTET-lys2-GTOP and –GBTM constructs were elevated by 28- and 8- fold, respectively [33]. RNase H1 overexpression led to significant decreases in the rates of gene conversion in rnh1Δ rnh2Δ backgrounds indicating that RNA∶DNA hybrid is responsible for the elevated recombination at both the pTET-lys2-GTOP and –GBTM constructs in rnh1Δ rnh2Δ mutant strains (Fig. 7B). We postulate that RNA∶DNA hybrid and G4 DNA can each result in genome instability but that R-loop is not the primary cause of elevated recombination we observed for the pTET-lys2-GTOP upon disruption of Top1. A function of Top1 other than the prevention of R-loop formation is relevant in suppressing genome instability at G4 DNA, which will require further investigation to identify.


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 RNA∶DNA hybrid and gene conversion at the highly transcribed Sμ.A. A model depicting two alternate pathways of G-loop formation. In absence of RNase H activity, RNA∶DNA hybrid accumulation fosters folding of G-runs into G4 DNA. In absence of Top1 activity, G4 DNA, which function as a sink for negative torsional stress, can promote RNA∶DNA hybrid formation. B. The rates of gene conversion at pTET-lys2-GTOP (gray bar) or –GBTM (hashed bar) cassette. Indicated strains were transformed with either pGAL1-RNH1 (+RNH1) or pRS416 as vector control. RNHΔ* indicates rnh1Δ rnh201Δ double mutant strains. 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-g007: The RNA∶DNA hybrid and gene conversion at the highly transcribed Sμ.A. A model depicting two alternate pathways of G-loop formation. In absence of RNase H activity, RNA∶DNA hybrid accumulation fosters folding of G-runs into G4 DNA. In absence of Top1 activity, G4 DNA, which function as a sink for negative torsional stress, can promote RNA∶DNA hybrid formation. B. The rates of gene conversion at pTET-lys2-GTOP (gray bar) or –GBTM (hashed bar) cassette. Indicated strains were transformed with either pGAL1-RNH1 (+RNH1) or pRS416 as vector control. RNHΔ* indicates rnh1Δ rnh201Δ double mutant strains. The 95% confidence intervals are indicated by the error bars.
Mentions: Activated transcription through G/C rich sequence can lead to formation of R-loops, which comprise of a long and stable hybrid between nascent RNA and template DNA strand [64]. R-loop accumulation and associated hyper-recombination can ensue when the mRNA packaging and export is disturbed in THO-TREX defective strains or when the degradation of RNA in RNA∶DNA hybrid is deficient due to absence of RNase H activity [65]. Accumulation of negative supercoils in Topoisomerase-deficient cells can also lead to R-loop accumulation [66]. Duquette et al reported that the combination of R-loop and G4 DNA, referred to as G-loop, is identifiable by electron microscopy when the Ig switch sequence is highly transcribed either in vitro or in bacteria [13]. At the pTET-lys2-GTOP cassette containing the Sμ sequence, therefore, the elevated genome instability could be the result of RNA∶DNA hybrid and/or G4 DNA. G-loop formation can be instigated by G4 DNA nucleation in the NTS, which lead to the stable annealing of the nascent RNA with the unpaired TS of DNA (Fig. 7). Alternatively, G-loop formation can initiate via the formation of RNA∶DNA hybrid, which leaves the NTS unpaired and free to fold into G4 structure. In this case, the higher stability of rG∶dC base pairing compared to rC∶dG could account for the greater instability we observed when the G-runs are on the NTS (-GTOP) [67]. During in vitro transcription, rG∶dC containing RNA∶DNA hybrid is critical for the formation of G-loop structure by Ig switch sequence [13], and required for the transcription blockage by a guanine-run [19]. We tested whether G4-induced hyper-recombination is dependent on RNA∶DNA hybrid formation by overexpressing RNase H1 in top1Δ background. RNase H1 is an enzyme that degrades RNA hybridized to DNA and was shown to counteract the hyper-recombination phenotype associated with R-loops in THO/TREX mutant background [65]. As shown in Fig. 7B, RNase H1 overexpression did not reduce the elevated recombination at the highly expressed pTET-lys2-GTOP cassette, which suggests that RNA∶DNA hybrid is not required for the elevated recombination occurring at the G4 motif in absence of Top1. We previously reported that, upon disruption of both RNase H1 and RNase H2 (rnh1Δ rnh2Δ), the rates of gene conversion for the pTET-lys2-GTOP and –GBTM constructs were elevated by 28- and 8- fold, respectively [33]. RNase H1 overexpression led to significant decreases in the rates of gene conversion in rnh1Δ rnh2Δ backgrounds indicating that RNA∶DNA hybrid is responsible for the elevated recombination at both the pTET-lys2-GTOP and –GBTM constructs in rnh1Δ rnh2Δ mutant strains (Fig. 7B). We postulate that RNA∶DNA hybrid and G4 DNA can each result in genome instability but that R-loop is not the primary cause of elevated recombination we observed for the pTET-lys2-GTOP upon disruption of Top1. A function of Top1 other than the prevention of R-loop formation is relevant in suppressing genome instability at G4 DNA, which will require further investigation to identify.

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