Mutation rate analysis of strains overexpressing Pms1, Mlh2 or Mlh3.

a<p>Median rates of <i>hom3-10</i> and <i>lys2-10A</i> reversion and inactivation of the <i>CAN1</i> gene with the 95% C.I. in square brackets and fold increase relative to the wild-type in parentheses.</p

Mutations that perturb the abundance of Pms1 foci also perturb the abundance of Mlh2 foci.

<p>(<b>A</b>) The percentages of nuclei containing Mlh2 foci were quantified in strains containing mutations in MMR genes or (<b>B</b>) genes encoding the catalytic subunits of the DNA polymerases Pol2 and Pol3. (<b>C</b>) Quantification of the percentages of nuclei containing Pms1 foci in an <i>mlh2Δ</i> strain. (<b>D</b>) Quantification of the percentages of nuclei containing Mlh2 foci in strains containing the <i>pms1Δ</i> mutation, the <i>pms1Δ</i> mutation in combination with an <i>msh2Δ</i> mutation or the endonuclease active site <i>pms1</i> mutation <i>pms1-E707K</i>. Error bars indicate the SEM, and “n” indicates the numbers of cells examined.</p

Mlh2 forms foci that partially colocalize with Pms1.

<p>(<b>A</b>) Images of cells expressing Pms1-4×GFP or Mlh2-4×GFP and Nic96-mCherry as a marker of the nuclear pore complex reveal the presence of nuclear Pms1 and Mlh2 foci. (<b>B</b>) Quantitation of Mlh2-4×GFP, Pms1-4×GFP, and Mlh3-4×GFP foci. Error bars indicate the standard error of the mean (SEM), and “n” indicates the number of cells examined. (<b>C</b>) Distribution of Mlh2-4×GFP foci according to bud size: no bud or small (<1.5 µm), medium (1.5–3 µm), or large (>3 µm) budded cells. “n” indicates the number of cells examined. (<b>D</b>) Images of cells expressing Mlh2-tdTomato and Pms1-4×GFP reveal foci that contain both proteins as well as foci containing only one of them. (<b>E</b>) Quantitation of Pms1 foci and Pms1 foci that colocalize with Mlh2. (<b>F</b>) Time-lapse images of cells with colocalized Mlh2-tdTomato and Pms1-4×GFP foci at one-minute intervals. White arrowheads indicate the start of colocalization and yellow arrowheads indicate Mlh2-tdTomato foci that persist after loss of the Pms1-4×GFP signal.</p

Mutation rate analysis of <i>mlh2Δ</i> in combination with mismatch repair mutations.

a<p>Median rates of <i>hom3-10</i> and <i>lys2-10A</i> reversion and inactivation of the <i>CAN1</i> gene with the 95% C.I. in square brackets and fold increase relative to the wild-type in parentheses.</p><p><i>tetO₂-PMS1, PMS1</i> driven by the <i>tetO₂</i> promoter; Dox, doxycycline.</p

Mutation rate analysis of strains overexpressing Pms1, Mlh2 or Mlh3.

a<p>Median rates of <i>hom3-10</i> and <i>lys2-10A</i> reversion and inactivation of the <i>CAN1</i> gene with the 95% C.I. in square brackets and fold increase relative to the wild-type in parentheses.</p

Expression of Pms1, Mlh2 and Mlh3 under control of the endogenous and <i>pGPD</i> promoters.

<p>(<b>A</b>) Expression level of Mlh2-9MYC, Mlh3-9MYC, and Pms1-9MYC driven from their endogenous promoters monitored by immunoblotting with an anti-MYC antibody. Immunoblotting with an anti-Pgk1 antibody was used a loading control. (<b>B</b>) Comparison of the levels of Mlh2-9MYC, Mlh3-9MYC, and Pms1-9MYC by immunoblotting with an anti-MYC antibody when expressed from the endogenous promoter or the <i>pGPD</i> promoter. Bottom panel, comparison after 50-fold dilution of the <i>pGPD</i> samples.</p

Evolutionary conservation of MutL homologs.

<p>The presence of a conserved <i>MLH1</i>, <i>S. cerevisiae PMS1</i>/human <i>PMS2</i>, <i>MLH3</i>, and <i>S. cerevisiae MLH2</i>/human PMS1 homolog in sequenced unikont genomes is indicated by a ‘Y’. ‘N’ indicates the lack of an identifiable homolog. <i>MLH1</i> and <i>ScPMS1</i>/<i>hPMS2</i> are extensively conserved, whereas <i>MLH3</i> and <i>ScMLH2/hPMS1</i> are less well conserved. Alternating light and dark grey backgrounds indicate separations between major unikont groups (Fungi, Nucleariidae and Fonticula, Ichthyosporea, Choanoflagellata, Metazoa, Apusozoa, and Amoebozoa).</p

Pms1 and Mlh2 foci are induced upon treatment with phleomycin but not hydroxyurea and do not colocalize with DNA double-strand breaks.

<p>(<b>A</b>) Phleomycin, but not hydroxyurea, increases the number of Pms1-4×GFP and Mlh2-4×GFP foci in an <i>MSH2</i>-dependent manner. “n” indicates the numbers of live cells examined. (<b>B</b>) Images of cells expressing Mre11-GFP, Pms1-4×GFP, or Mlh2-4×GFP in a strain expressing TetR-mRFP and containing a tandem array of <i>tetO</i> sequences adjacent to an I-<i>Sce</i>I restriction site after induction of I-<i>Sce</i>I reveal that Mre11, but not Pms1 or Mlh2, colocalizes with the double-strand break. (<b>C</b>) Quantitation of cells containing Mre11, Pms1, and Mlh2 foci and their colocalization with the <i>tetO</i> array with and without I-<i>Sce</i>I induction.</p

Cdc73 suppresses genome instability by mediating telomere homeostasis

<div><p>Defects in the genes encoding the Paf1 complex can cause increased genome instability. Loss of Paf1, Cdc73, and Ctr9, but not Rtf1 or Leo1, caused increased accumulation of gross chromosomal rearrangements (GCRs). Combining the <i>cdc73Δ</i> mutation with individual deletions of 43 other genes, including <i>TEL1</i> and <i>YKU80</i>, which are involved in telomere maintenance, resulted in synergistic increases in GCR rates. Whole genome sequence analysis of GCRs indicated that there were reduced relative rates of GCRs mediated by <i>de novo</i> telomere additions and increased rates of translocations and inverted duplications in <i>cdc73Δ</i> single and double mutants. Analysis of telomere lengths and telomeric gene silencing in strains containing different combinations of <i>cdc73Δ</i>, <i>tel1Δ</i> and <i>yku80Δ</i> mutations suggested that combinations of these mutations caused increased defects in telomere maintenance. A deletion analysis of Cdc73 revealed that a central 105 amino acid region was necessary and sufficient for suppressing the defects observed in <i>cdc73Δ</i> strains; this region was required for the binding of Cdc73 to the Paf1 complex through Ctr9 and for nuclear localization of Cdc73. Taken together, these data suggest that the increased GCR rate of <i>cdc73Δ</i> single and double mutants is due to partial telomere dysfunction and that Ctr9 and Paf1 play a central role in the Paf1 complex potentially by scaffolding the Paf1 complex subunits or by mediating recruitment of the Paf1 complex to the different processes it functions in.</p></div

Systematic analysis of <i>CDC73</i> as a suppressor of GCRs selected in the dGCR assay.

<p><b>a.</b> Sample patches from each of two biological replicates with single and double mutants for genes with mutations that show a synergistic interaction with the <i>cdc73Δ</i> mutation. Each papilla corresponds to a GCR event and the greater the number of papillae per patch the greater the GCR patch score, which correlate with increased GCR rates. <b>b.</b> dGCR strain scores, which are the average of 3 independent patch scores for mutations that cause increased patch scores when combined with the <i>cdc73Δ</i> mutation. The semi-quantitative scoring strategy assigns a number between 0 and 5 to each patch depending on the number of papillae (0: no papillae, 1: 1 to 5 papillae, 2: 6 to 15 papillae, 3: 16 to ~150–200 papillae, 4: distinct papillae that were too many or too close together to count, 5: a lawn of papillae covering the entire patch). A score of 1 corresponds to the wild-type level of GCRs. Interactions with <i>cdc73Δ</i> or <i>rtf1Δ</i> mutations that resulted in significantly increased patch scores using previously established criteria [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007170#pgen.1007170.ref006" target="_blank">6</a>] are displayed in bold. Single mutations causing decreased telomere lengths are underlined [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007170#pgen.1007170.ref046" target="_blank">46</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007170#pgen.1007170.ref047" target="_blank">47</a>]. <b>c.</b> The number of GCRs in the dGCR assay mediated by recombination between <i>DSF1-HXT13</i> and <i>MAN2-HXT17</i>, t(V;XIV), or between <i>HXT13</i> and <i>HXT15</i> or <i>HXT16</i>, t(V;IV or X), were determined by PCR analysis.</p