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. 2013 Jun 7;8(6):e65541.
doi: 10.1371/journal.pone.0065541. Print 2013.

R-loop Mediated Transcription-Associated Recombination in trf4Δ Mutants Reveals New Links Between RNA Surveillance and Genome Integrity

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Free PMC article

R-loop Mediated Transcription-Associated Recombination in trf4Δ Mutants Reveals New Links Between RNA Surveillance and Genome Integrity

Sandra Gavaldá et al. PLoS One. .
Free PMC article

Abstract

To get further insight into the factors involved in the maintenance of genome integrity we performed a screening of Saccharomyces cerevisiae deletion strains inducing hyperrecombination. We have identified trf4, a gene encoding a non-canonical polyA-polymerase involved in RNA surveillance, as a factor that prevents recombination between DNA repeats. We show that trf4Δ confers a transcription-associated recombination phenotype that is mediated by the nascent mRNA. In addition, trf4Δ also leads to an increase in the mutation frequency. Both genetic instability phenotypes can be suppressed by overexpression of RNase H and are exacerbated by overexpression of the human cytidine deaminase AID. These results suggest that in the absence of Trf4 R-loops accumulate co-transcriptionally increasing the recombination and mutation frequencies. Altogether our data indicate that Trf4 is necessary for both mRNA surveillance and maintenance of genome integrity, serving as a link between RNA and DNA metabolism in S. cerevisiae.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Recombination analyses of med2 , lsg1, trf4, rpl13A, tos3, art1 and apc9 mutants.
A diagram of each recombination system (not drawn to scale) is shown at the top. Repeats are shown as gray boxes. Arrows indicate relevant transcripts produced by the constructs. For the L, LY and SU systems, recombination frequencies were determined in wild-type (FY1679) and mutant strains transformed with plasmids pRS314-L and pRS314-LY carrying the leu2 direct-repeat systems, and pRS314-SU carrying an inverted repeats system. Recombinants were selected as Leu+. The average median value and SD of 3–4 fluctuation tests are shown. Recombination frequencies of med2Δ (MGY1-2D), lsg1Δ (AFGL-7D), trf4Δ (AWT4-1C), rpl13AΔ (WFDL-1D), tos3Δ (AFGL-2D), art1Δ (AFOR-1A), apc9Δ (WFLR-2B) and wild-type (MGY6-1A) congenic strains carrying the chromosomal leu2-k::ADE2-URA3::leu2-k system are shown. For recombination analyses, independent colonies were obtained from SC and recombinants were selected in SC+FOA.
Figure 2
Figure 2. Effect of the level of transcription on the trf4Δ hyperrecombination phenotype.
Isogenic strains W303-1A (WT) and TRF4D-C5 (trf4Δ) were transformed with plasmids pSCH204 (L-lacZ recombination system) or pRS314GL-lacZ (GL-lacZ) in which transcription is under the control of LEU2 and GAL1-10 promoters, respectively. Gray boxes represent LEU2 repeats that flank the lacZ sequence. Arrow indicates the transcript produced. P. Promoter. Recombination frequencies are plotted as a function of the transcription levels. Low transcription refers to the GL-lacZ systems in strains cultured in 2% glucose; medium refers to L-lacZ in 2% glucose, and high to GL-lacZ in 2% galactose. The average median value and SD of 3-4 fluctuation tests are shown. Asterisks indicate statistically significant differences between the strains indicated, according to Student's t-tests (*, Ρ<0.05; ***, Ρ<0.0005).
Figure 3
Figure 3
Transcription analysis of the trf4Δ strain. (A) Analysis of the ability of W303-1A and TRF4D-C5 (trf4Δ) strains carrying the Ptet::lacZ-URA3 (LAUR) fusion construct (plasmid pCM184-LAUR) to form colonies on SC-trp-ura medium and to form blue colonies on SC-Trp complemented with X-Gal. (B) Northern analysis of the expression of the Ptet::lacZ-URA3. RNA was isolated from two different mid-log phase cultures from each strain, grown in SC-trp. We used the 3-kb BamHI lacZ fragment and an internal 589-bp 25S rDNA fragment obtained by PCR, as probes.
Figure 4
Figure 4. Nascent mRNA-dependency of the hyperrecombination phenotype of trf4
Δ mutants. (A) Direct-repeat recombination systems GL-Rib+ and GL-ribm containing the PHO5-Rib+ or PHO5- ribm sequences flanked by two truncated copies of LEU2 in direct orientation under the GAL1 promoter. These systems contain respectively an active or inactive 52-bp ribozyme (Rib). The ribm system (inactive ribozyme) yields a long transcript, whereas in the Rib+ system (active ribozyme) self-cleavage of the PHO5-Rib transcript leads to a shorter mRNA (represented by arrows). (B) Recombination frequencies in W303-1A (WT) and TRF4D-C5 (trf4Δ) cells containing the recombination systems GL-Rib+ and GL-ribm. Experiments were performed in 2% galactose to allow expression of the direct repeats. The average median value and SD of 3–4 fluctuation tests are shown. Asterisk indicates statistically significant differences, according to Student's t-tests (*, Ρ<0.05).
Figure 5
Figure 5. The hyperrecombination phenotype of trf4
Δ mutants is not dependent of its poly-adenylation catalytic domain. Recombination frequencies in AWT4-1C cells carrying the chromosomal leu2-k::ADE2-URA3::leu2-k system and transformed with the pNOPPATA1L vector either empty (trf4Δ) or carrying the wild-type TRF4 (WT) or the mutant TRF4-DADA alleles (TRF4-DADA). The average median value and SD of 3–4 fluctuation tests are shown. Asterisks indicate statistically significant differences, according to Student's t-tests (*, P<0.05).
Figure 6
Figure 6. Genetic evidence for R-loop formation in trf4
Δ mutants. Effect of RNaseH1 and AID over-expression on the mutation frequency in trf4 mutants. Upper panel shows the analysis of recombination frequencies in W303-1A (WT) and TRF4D-C5 (trf4Δ) cells containing the recombination system LYΔNS, without RNaseH1 overexpression (-RNH1) or with over-expression of RNaseH1 (+RNH1). The latter was achivied with the multicopy plasmid pGAL-RNH1 carrying RNH1 under the GAL1 promoter. Lower panel shows recombination frequencies as in the upper one, but over-expressing AID from plasmid p413GALAID. The average median value and SD of 3–4 fluctuation tests are shown. Other details as in Figure 2. Asterisks indicate statistically significant differences, according to Student's t tests (***, Ρ<0.0005).
Figure 7
Figure 7
Spontaneous and AID-induced mutation frequencies in wild-type and trf4Δ strains. Mutation frequency of W303-1A (WT), TRF4D-C5 (trf4Δ) strains, using the LAUR fusion construct. Ura- mutants are selected in SC+FOA. The human AID gene was overexpressed in 2% galactose medium using plasmid p413GALAID. The median values of mutation frequencies and SD of 3–4 different fluctuation tests are shown. Asterisks indicate statistically significant differences, according to Student's t tests (*, P<0.05; **, Ρ<0.005).

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Grant support

This work was supported by grants from the Spanish Ministry of Science and Research (BFU2010-16372 and Consolider Ingenio 2010 CSD2007-00015), Junta de Andalucía (CVI-4567) and European Union (FEDER). SG was the recipient of a predoctoral training grant from the Spanish Ministry of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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