Genetic analysis of BIR intermediates processing in SSE mutants.

<p>(A) (B) Frequency of translocants of the different strains tested. See Material and Methods for details. ** and *, differences with the WT statistically significant (p<0.01 and p<0.05, respectively, χ² with Yates' correction) °, statistically different from <i>mus81Δ</i> (p<0.01, χ² with Yates' correction). Error bars represent standard deviations. (C) Appearance of BIR products as monitored by Southern analysis in <i>HMR-inc</i> WT, <i>HMR-inc pol32Δ</i> and <i>HMR-inc mus81Δ yen1Δ</i> strains. Experiments were performed as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002979#pgen-1002979-g002" target="_blank">Figure 2C</a>. A representative Southern analysis is shown for each genotype analyzed. Positions of the bands corresponding to 7/3-<i>MAT</i>, 7/3-<i>HMR</i> and 7/3-<i>HML</i> intermediates, and to the uncut and cut chromosome VII (Chr. VII) are indicated. GAL, galactose; h, hours. (D) Quantification of BIR product accumulation. Quantification results of chromosome VII cleavage (DSB), 7/3<i>-HMR</i>, 7/3<i>-HML</i> and 7/3<i>-MAT</i> BIR intermediates are shown as percentage. Mean values and standard deviations for 2–3 independent experiments are shown.</p

Normal CPD repair and DNA-damage response alteration in transcription termination mutants.

<p>(A) Southern analysis showing repair of a 4.4-kb (<i>Nsi</i>I/<i>Pvu</i>I) <i>RPB2</i> fragment in wild-type, <i>rna14-1, rna15-1</i>, <i>hrp1-5, rad7Δ and rna14-1 rad7Δ</i> cells. Initial damage was on the average 0.247±0.091 CPD/Kb in the transcribed strand (TS, left) and 0.245±0.098 CPD/Kb in the non-transcribed strand (NTS, right). The remaining intact restriction fragment after treatment 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 quantified results. The CPD content was calculated using the Poisson expression, -ln (RF_a/RF_b), where RF_a and RF_b 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 <i>vs</i>. repair time. Average values derived from three independent experiments are plotted with their standard deviation. Repair curve of <i>rad26</i> (data taken from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004203#pgen.1004203-Gaillard2" target="_blank">[9]</a>) is depicted for the TS. (C) Western analysis of Rpb1 and Rad53 upon UV irradiation in <i>rna14-1</i> and wild-type cells. β-actin is shown as loading control. (D) Graphical representation of the quantified results from Rpb1 and Rad53 Western analyses. The amount of Rpb1 is shown as the percentage of Rpb1 in the non-irradiated sample. The percentage of hyper-phosphorylated Rad53 is plotted for each condition. Average values derived from three independent experiments are plotted with their standard deviation. (E) Genetic interaction analysis between the <i>rna14-1</i> and the <i>def1Δ</i> mutants. Serial dilutions (10-fold) of exponentially growing cultures are shown. This panel complemented with the data of the <i>rna15-1 def1Δ</i>, <i>hrp1-5 def1Δ</i>, and <i>rat1-1 def1Δ</i> mutants are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004203#pgen.1004203.s002" target="_blank">Figure S2</a>.</p

Absence of functional G1/S checkpoint leads to DNA damage and genomic instability in <i>rna14-1</i> cells.

<p>(A) Analysis of phosphorylated histone H2A (H2A-P) accumulation during release from α-factor-mediated G1-arrest in wild-type (WT), <i>rna14-1</i>, <i>sic1Δ</i> and <i>rna14-1 sic1Δ</i> strains. Asynchronous (async.), α-factor synchronized (sync.) and released cells were analysed. β-actin is shown as loading control. FACS analysis of all samples is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004203#pgen.1004203.s007" target="_blank">Figure S7</a>. (B) Recombination analysis using a direct-repeat (LY<i>Δ</i>NS) and an inverted-repeat (TINV) plasmid-borne system. A scheme of each system is shown on the right of the corresponding panel. Recombination frequencies were obtained as the median value of six independent colonies. The average and standard deviation of at least three independent fluctuation tests are shown for each genotype. Statistical analyses were performed with a two-tailed unpaired student t-test compared with the wild type. Where indicated, statistical analyses between two mutants were also performed. *p<0.01, **p<0.005, ***p<0.001. (C) Percentage of S/G2 cells containing Rad52-YFP foci. Average of numbers obtained from at least three independent transformants and the corresponding standard deviation are shown. Statistical analyses as in B.</p

CFI mutations lead to UV sensitivity in the absence of global genome repair.

<p>(A) UV and 4-NQO sensitivity of six different transcription termination mutants alone and in combination with the <i>rad7Δ</i> mutation. (B) UV sensitivity curves of strains carrying single, double and triple combinations of <i>rna14-1</i> (top) and <i>hrp1-5</i> (bottom) together with <i>rad7Δ</i> and <i>rad26Δ</i> mutations. Average values from at least three independent experiments and corresponding standard deviations are plotted.</p

BIR model showing SSEs involvement in template switching and half-crossovers.

<p>After invasion of the homologous template by one end of the DSB, a D-loop is formed and extended by the priming of DNA synthesis from the invading strand 3′ end. The D-loop can be specifically cleaved by Mus81 or branch migrated to create a HJ cleavable by Yen1, to establish a replication fork. Extensive replication, which requires Pol32, would complete BIR and generate a non-reciprocal translocation (NRT). Alternatively, [a] cleavage of the replication fork by Mus81-Slx4-Yen1 would allow re-invasion of the same template or template switching. [b] cleavage of the replication fork by Mus81-Slx4-Yen1 would terminate the BIR event, causing a half-crossover (NRT). RF, replication fork, HJ, Holliday junction.</p

PFGE analysis of T7/3 translocations in SSE mutants.

<p>(A) PFGE followed by Southern analysis using the 3R1 probe (See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002979#pgen-1002979-g001" target="_blank">Figure 1G</a>) of Ura⁻ 5-FOAr survivors in WT, <i>pol32Δ</i>, <i>rad1Δ</i> and <i>mus81Δ slx4Δ yen1Δ</i> strains. Black arrows indicate the positions of the T7/3-<i>MAT</i> and T7/3-<i>HMR</i> translocations and of the linear chromosome III (Chr. III). PFGE, pulse-field gel electrophoresis. (B) Graphical plotting of percent of translocants containing T7/3-<i>MAT</i> translocations of each strain tested. <b>**</b> and <b>*</b>, differences with the WT statistically significant (p<0.01 and p<0.05, respectively, χ² with Yates' correction). See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002979#pgen.1002979.s011" target="_blank">Table S1</a> for complete statistical analysis.</p

Cell cycle progression is compromised in <i>rna14-1</i> cells.

<p>(A) Sensitivity to hydroxyurea (HU) of six transcription-termination mutants. Serial dilutions (10-fold) of exponentially growing cultures are shown. (B) Analysis of plasmid loss in <i>rna14-1</i>, monitored as the percentage of cells that lost the pRS315 centromeric plasmid after ∼10 divisions in non-selective media. Average and standard deviation of six independent transformants are plotted for each genotype. Statistical analysis was performed with a two-tailed unpaired student t-test compared with the wild type. ***p<0.001. (C) Cell cycle progression analysis in wild-type (WT) and <i>rna14-1</i> strains. Asynchronous (async.), α-factor synchronized (sync.) and released cells were analysed by FACS. (D) Analysis of replication in <i>rna14-1</i> cells. BrdU incorporation upon release of G1-arrested cells was analysed at early replicating origins <i>ARS508</i>, <i>ARS305</i>, and <i>ARS416</i> by immunoprecipitation and RT qPCR. A schematic drawing of each <i>ARS</i> and localization of the amplified regions are depicted (top). Quantification of BrdU incorporation relative to a late replicating locus is plotted for each region. Average from two independent experiments and corresponding standard deviations are shown.</p

Template switching is affected in <i>mus81Δ slx4Δ yen1Δ</i> but not in <i>rad1Δ</i> mutants.

<p>(A) Appearance of BIR repair products, as monitored by Southern analysis, in WT, <i>rad1Δ</i> and <i>mus81Δ slx4Δ yen1Δ</i> strains. Experiments were performed as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002979#pgen-1002979-g002" target="_blank">Figure 2C</a>. (B) Quantification of BIR product accumulation. Quantification results for chromosome VII cleavage (DSB), 7/3<i>-HMR</i> and 7/3<i>-HML</i> BIR intermediate accumulation are shown in percent. (C) Quantification of 7/3<i>-MAT</i>/7/3<i>-HMR</i> BIR intermediate ratios 24 h after HO induction. Mean values and standard deviations for 2–3 independent experiments are shown. (D) Appearance of BIR repair products, as monitored by Southern analysis, in <i>HMR-inc rad1Δ</i>, <i>HMR-inc mus81Δ slx4Δ yen1Δ</i> and <i>HMR-inc slx4Δ</i> strains. (E) Quantification of BIR product accumulation. Quantification results for chromosome VII cleavage (DSB), 7/3<i>-HMR</i>, 7/3<i>-HML</i> and 7/3<i>-MAT</i> BIR intermediate accumulation are shown in percent. Mean values and standard deviations for 2–3 independent experiments are shown. A representative Southern analysis is shown for each genotype analyzed. Positions of the bands corresponding to 7/3-<i>MAT</i>, 7/3-<i>HMR</i> and 7/3-<i>HML</i> intermediates, and to the uncut and cut chromosome VII (Chr. VII) are indicated. Bands marked with a red asterisk likely result from partial digestion. GAL, galactose; h, hours.</p

Template switching occurs between <i>MAT</i>a<i>-inc</i>, <i>HMR</i>, and <i>HML</i> loci.

<p>(A) Schematic representation of chromosomes VII and III showing the positions of the PCR primers, <i>Eco</i>RV sites (<i>E</i>V) and the 7L probe used for the analysis. Distances between the primers and <i>Eco</i>RV sites and the HO cut sites are indicated. (B) Appearance of BIR repair product, as monitored by PCR, in the WT strain. DNA samples used for PCR were extracted at intervals after HO induction with galactose. PCR reactions were performed with the p7 primer and either the p3-M, p3-R or p3-L primer (see panel A). A primer pair corresponding to <i>ACT1</i> locus on chromosome VI was used to control the amount of genomic DNA used for PCR at each time point. 35 cycles of PCR amplifications were performed. (C) Appearance of BIR repair products, as monitored by Southern analysis with the 7L probe, in the WT strain. DNA samples were extracted as for the PCR assay. Positions of the bands corresponding to 7/3-<i>MAT</i>, 7/3-<i>HMR</i> and 7/3-<i>HML</i> intermediates, and to the uncut and cut chromosome VII (chr. VII) are indicated. Two genomic DNA samples coming from survivor colonies containing T7/3-<i>MAT</i> and T7/3-<i>HMR</i> translocations were included in the experiment. GAL, galactose; h, hours. (D) Schematic representation of BIR events involving template switching likely occurring in our assay. Black arrows indicate the first invasion events while red arrows indicate the secondary template switching events. The different possible final outcomes are depicted. The red cross indicates the presence of <i>inc</i> non-cleavable sequences. M; <i>MAT</i>; R, <i>HMR</i>; L, <i>HML</i>. See text for more details.</p

Transcription termination mutants do not tolerate compromised DNA repair.

<p>(A) Sensitivity to Phleomycin (Phleo), methyl methanesulfonate (MMS), and camptothecin (CPT) of six transcription-termination mutants. 10-fold serial dilutions of exponentially growing cultures are shown. (B) Analysis of genetic interactions between <i>rna14-1</i> and mutants impaired in homologous recombination (HR), non-homologous end joining (NHEJ), post-replicative repair (PPR), mismatch repair (MMR), base excision repair (BER), and nucleotide excision repair (NER). 10-fold serial dilutions of exponentially growing cultures are shown. * indicates that the UV dose was 2 J/m².</p