pol30 elg1Δ mutants are resistant to inhibition of DNA replication by HU. (A) Western blot analysis of whole cell extracts prepared from the indicated logarithmically growing strains. Immunoblots were probed with anti-PCNA antibody. Ponceau S staining of the blot is shown in the bottom panel as a loading control. (B) Logarithmically growing cultures of the indicated strains were arrested in G1 with alpha factor (αF) and released into 0.2 M HU. At the indicated times, samples were fixed and DNA contents were analysed by flow cytometry. The positions of cells with 1C and 2C DNA contents are indicated. (C) DNA replication was measured in wild type and pol30^felg1Δ by comparative genome hybridization on tiling microarrays after logarithmically growing cells were arrested in G1 with alpha factor and released into 0.2 M HU for 60 min. The log2 ratio of signal from each S phase (HU) sample relative to unreplicated (G1) DNA is shown for chromosome XV. Replicated regions, defined by identifying peaks that overlap replication origins, along chromosome XV are shown as red bars below each histogram. Confirmed replication origins annotated in oriDB are indicated, early-firing origins in green, late-firing origins in blue, and origins without timing data in black (). Late-firing ARS1506.5 is indicated by an asterisk. (D) Replication fork distance distributions after 30 min in HU. The distance from the centre of each ARS to peak edge for 166 replication forks across the genome was measured and the result displayed as a boxplot. The median is indicated by the horizontal bar, the box spans the first through third quartiles, the whiskers extend to the last data points within 1.5 times the interquartile range, and outliers are plotted as circles. Median fork distance significantly greater than wild type (P<0.01, one-tail Wilcoxon rank-sum test) is indicated (^*), as is median fork distance significantly greater than elg1Δ and pol30^f (^**P<0.01). (E) Replication fork distance distributions after 60 min in HU. (F) Replication fork rate distribution, between 30 and 60 min in HU. Fork rate was measured for 88 replication forks as the difference in fork distance between 60 and 30 min in HU, divided by 30 min. Fork rate in pol30^f elg1Δ (260 bp/min) was significantly greater than wild type (84 bp/min; ^*P=3 × 10⁻⁹). (G) PCNA localizes to origin-distal regions in pol30^f elg1Δ cells released from G1 into HU for 90 min. Enrichment of DNA fragments in the PCNA-bound fraction relative to the unbound fraction is shown along 500 kbp of chromosome V for pol30^f (top) and pol30^f elg1Δ cells (bottom). The signal intensity ratio on a log2 scale is shown on the y axis and the position along the chromosome is shown on the x axis. Positive signal represents occupancy by PCNA, and regions where the positive signal is statistically significant () over 300 bp are shown in orange. Replication origins are indicated.

RNR activity is upregulated in pol30^f elg1Δ mutants. (A) Logarithmically growing cells were arrested in G1 with alpha factor (α) and released into 0.2 M HU. At the indicated times, samples were fixed and the level of each dNTP was measured in the strains shown. dNTP levels are expressed as dNTP:NTP ratios. (B) Western blot analysis of whole cell extracts prepared from cells that were arrested in G1 with alpha factor. Immunoblots were probed with anti-Rnr1, Rnr3, and Rnr4 antibodies to detect RNR subunits. Tubulin was used as a loading control and detected using an anti-tubulin antibody. (C) Northern blot analysis of RNA prepared from cells arrested in G1 phase. Blots were hybridized with probes for the RNR1, RNR3, and RNR4 genes. The rRNA is shown as a loading control.

pol30^f elg1Δ mutants exhibit endogenous DNA damage (A) Logarithmically growing cells were arrested in G1 with alpha factor (α) and synchronously released into S phase. Samples were fixed every 20min and extracts were fractionated on a western blot and probed with anti-γH2A, Rnr1, Rnr3, Rnr4, Sml1, and PCNA antibodies. Western blots were also probed with an anti-Rad53 antibody. Phosphorylation of Rad53 causes a shift in electrophoretic mobility (Rad53-P) and is a marker for checkpoint activation as seen in the control sample of asynchronously growing wild-type cells treated with MMS (lane 1). Ponceau S staining of the Pol30 blot is shown as a protein loading control. (B) Cells sampled from (A) were analysed by flow cytometry. The positions of 1C and 2C DNA contents are indicated. (C) The indicated strains were grown asynchronously (ASY), arrested in G1 with alpha factor (αF), or treated with MMS. Cells were fixed with TCA and whole cell extracts were analysed by western blot for phosphorylation-dependent Rad53 mobility shift using an anti-Rad53 antibody. (D) Western blot analysis of whole cell extracts prepared from the indicated strains following arrest in G1 with alpha factor. Immunoblots were probed with anti-Rnr3 and Rnr4 antibodies to detect RNR subunits. Ponceau S staining is shown as a protein loading control.

pol30^felg1Δ mutants exhibit a mutator phenotype. (A) The rates of forward mutation of CAN1 cells to canavanine resistance were determined. The means and standard deviations of at least three independent fluctuation tests for the indicated strains are plotted. (B) DNA contents of wild-type, pol30^f elg1Δ, and pol30^f elg1Δ swi4Δ cells, either in logarithmic phase (log), arrested in G1 (αF), or released synchronously into the cell cycle for the indicated times in the presence of 0.2 M HU were analysed by flow cytometry. The positions of cells with 1C and 2C DNA contents are indicated. (C) Immunoblots of samples from (B) were probed with anti-PCNA and anti-Rnr1, Rnr3, and Rnr4 antibodies to detect RNR subunits. Ponceau S staining of the Pol30 blot is shown as a protein loading control.

Distinct genome integrity mutants exhibit increased dNTP levels and are mutators. (A) DNA contents of the indicated strains, either in logarithmic phase (log), arrested in G1 (αF), or released synchronously into the cell cycle for the indicated times in the presence of 0.2 M HU were analysed by flow cytometry. The positions of cells with 1C and 2C DNA contents are indicated. (B) Replication fork distance distributions after 60 min in HU. The distance from the centre of each ARS to peak edge for 164 replication forks across the genome was measured from aCGH data, and the results displayed as a boxplot. Median fork distance significantly greater than wild type (P<0.01, one-tail Wilcoxon rank-sum test) is indicated (^*). (C) Western blot analysis of whole cell extracts prepared from the indicated strains following arrest in G1 with alpha factor. Immunoblots were probed with anti-Rnr3 and Rnr4 antibodies to detect RNR subunits. Ponceau S staining is shown as a protein loading control. (D) dNTP levels, expressed as dNTP:NTP ratios, were measured for the indicated strains following release of logarithmically growing cells from G1 into 0.2 M HU for 60 min. (E) The rates of forward mutation of CAN1 cells to canavanine resistance were determined for the indicated strains. The means and standard deviations of three independent fluctuation tests are plotted.

Model of genome stability gene action in regulating dNTP levels. Mutation of genome stability genes results in DNA damage during S phase. The response to the DNA damage causes an upregulation of RNR and expansion of cellular dNTP pools, resulting in increased mutation frequency, which contributes to further genome instability.