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. 2009 Feb;5(2):e1000364.
doi: 10.1371/journal.pgen.1000364. Epub 2009 Feb 6.

Genome-wide analysis of factors affecting transcription elongation and DNA repair: a new role for PAF and Ccr4-not in transcription-coupled repair

Hélène Gaillard  1 , Cristina Tous, Javier Botet, Cristina González-Aguilera, Maria José Quintero, Laia Viladevall, María L García-Rubio, Alfonso Rodríguez-Gil, Antonio Marín, Joaquín Ariño, José Luis Revuelta, Sebastián Chávez, Andrés Aguilera
Affiliations

Genome-wide analysis of factors affecting transcription elongation and DNA repair: a new role for PAF and Ccr4-not in transcription-coupled repair

Hélène Gaillard et al. PLoS Genet. 2009 Feb.

Abstract

RNA polymerases frequently deal with a number of obstacles during transcription elongation that need to be removed for transcription resumption. One important type of hindrance consists of DNA lesions, which are removed by transcription-coupled repair (TC-NER), a specific sub-pathway of nucleotide excision repair. To improve our knowledge of transcription elongation and its coupling to TC-NER, we used the yeast library of non-essential knock-out mutations to screen for genes conferring resistance to the transcription-elongation inhibitor mycophenolic acid and the DNA-damaging agent 4-nitroquinoline-N-oxide. Our data provide evidence that subunits of the SAGA and Ccr4-Not complexes, Mediator, Bre1, Bur2, and Fun12 affect transcription elongation to different extents. Given the dependency of TC-NER on RNA Polymerase II transcription and the fact that the few proteins known to be involved in TC-NER are related to transcription, we performed an in-depth TC-NER analysis of a selection of mutants. We found that mutants of the PAF and Ccr4-Not complexes are impaired in TC-NER. This study provides evidence that PAF and Ccr4-Not are required for efficient TC-NER in yeast, unraveling a novel function for these transcription complexes and opening new perspectives for the understanding of TC-NER and its functional interconnection with transcription elongation.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Analysis of genes similarly affected by 4-NQO and MPA.
(A) Venn diagram representing the overlap between genes whose expression is changed after treatment with 4-nitroquinoline-N-oxide (4-NQO) and mycophenolic acid (MPA). (B) Fraction of mutant strains sensitive to 4-NQO (left) and MPA (right), classified by GO annotation. The MPA-sensitive set is significantly enriched in genes with the label “RNA elongation” and “chromatin modification” (p<3.38E-03 and p<7.96E-05, respectively), and the 4-NQO-sensitive set in “response to DNA damage” (p<6.3E-19). Enrichment analyses were carried out with the GO Term Finder tool of the SGD. (C) Venn diagram representing the overlap between genes conferring resistance to 4-NQO and MPA. The 25 genes common to both analyses are listed to the right.
Figure 2
Figure 2. Gene expression analyses of selected MPA-sensitive and functionally related mutants.
PHO5-lacZ/PHO5 and PHO5-LAC4/PHO5 ratios were calculated after assaying acid phosphatase activity of mutants lacking the indicated gene. Averages of at least three independent assays are shown. Error bars indicate standard deviation.
Figure 3
Figure 3. In vitro transcription elongation.
In vitro transcription assay of WCEs from BY4741 (wt), med15, bur2, fun12, bre1, spt20, tpd3, and not5 strains. Each reaction was stopped after 30 min, treated with RNaseT1 and run in a 6% PAGE. Two bands from each G-less cassette were obtained, probably due to incomplete action of RNaseT1. Efficiency of transcription elongation was determined as the percentage of transcripts that reach the 376-nt cassette in respect to those that cover the 84-nt cassette. Radioactivity incorporated into the cassettes was quantified in a Fuji FLA3000 and normalized with respect to the C content of each cassette. The mean value of the wt (84%) was normalized to 100%. Mean value and standard deviation of three independent experiments are shown.
Figure 4
Figure 4. RNAPII occupancy analysis.
ChIP analyses in BY4741 (wt), not5, spt20, bre1, med15, bur2, fun12, and tpd3 strains in the LAUR expression system. The scheme of the gene and the PCR-amplified fragments are shown. The DNA ratios in region 5′, 3′ and URA3 were calculated from their signal relative to the signal of the intergenic region. The recruitment data shown are referred to the value of the 5′ region taken as 100%. ChIPs were performed from 3 independent cultures, and quantitative PCRs were repeated three times for each culture. Error bars, SDs.
Figure 5
Figure 5. UV sensitivity in the absence of global genome repair in selected MPA-sensitive mutants.
UV sensitivity analysis of 18 mutant strains in which the RAD7 gene has been disrupted. rad7 and wild-type strains were used as control. Cell dilutions were dropped on YPDA plates, UV irradiated with 15 J/m2 and grown at 30°C in the dark for 3 days.
Figure 6
Figure 6. PAF and Ccr4-Not mutants are sensitive to UV in the absence of global genome repair.
UV sensitivity curves of strains carrying single and double combinations of the (A) rtf1, (B) cdc73, (C) paf1, (D) leo1, (E) not5, (F) not4, (G) not3, (H) caf1, (I) ccr4, and rad7 mutations. Mutants shown in (A–D) are subunits of the PAF and mutants shown in (E–I) of the Ccr4-Not complex. Average values and standard deviations from 3 independent experiments are shown.
Figure 7
Figure 7. The increased UV sensitivity of PAF and Ccr4-Not mutants in the absence of global genome repair is not due to checkpoint activation failure.
(A) UV sensitivity curves of strains carrying single and double combinations of the rad9 or bre1, and rad7 mutations. (B) UV sensitivity curves of strains carrying single, double, and triple combinations of the rtf1, rad9, and rad7 mutations (left), the not5, rad9, and rad7 mutations (middle), or the rpb9, rad9, and rad7 mutations (right). Average values and standard deviations from at least 3 independent experiments are shown.
Figure 8
Figure 8. Transcription coupled repair is impaired in PAF and Ccr4-Not deficient cells.
(A) Southern blot analysis showing repair of a 4.4 kb (NsiI/PvuI) RPB2 fragment in tho2, rft1, not5, and wt cells. Initial damage was on the average 0.31±0.07 CPD/Kb in the transcribed strand (TS, left) and 0.26±0.06 CPD/Kb in the non-transcribed strand (NTS, right). The remaining intact restriction fragment after treatment of damaged DNA with T4endoV (+UV, +T4endoV) corresponds to the fraction of undamaged DNA. Non-irradiated DNA (−UV) and DNA not treated with T4endoV (−T4endoV) were used as controls. (B) Graphical representation of the repair analysis. The CPD content was calculated using the Poisson expression, −ln (RFa/RFb), where RFa and RFb represent the intact restriction fragment signal intensities of the T4endoV- and mock-treated DNA, respectively. Repair curves were calculated as the fraction of CPDs removed vs. repair time. Average values derived from two independent experiments are plotted. Repair curves of rad26 and rad7 (data taken from [14]) are depicted for the TS and the NTS, respectively (dash lines).
Figure 9
Figure 9. Synergistic increase of UV sensitivity phenotypes in rft1 def1 and rft1 rpb9 double mutants.
UV sensitivity curves of strains carrying single, double, and triple combinations of the rft1, def1, and rad7 mutations (left) or the rtf1, rpb9, and rad7 mutations (right). Average values and standard deviations from at least 3 independent experiments are shown.
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