Fig 1.
Alternative hypotheses for the transcription-associated elevation in uracil-dependent mutations.
A) Higher number of uracil residues are present at highly transcribed loci resulting in the higher rate of uracil-dependent mutations. B) Uniform number of uracil residues are present regardless of the transcription level; the uracil DNA glycosylase activity is enhanced at highly transcribed genomic loci. In both instances, the uracil residue (U) is recognized and excised by Ung1 to create AP sites (o). The resulting abasic site is bypassed by translesion synthesis (TLS) polymerases in a mutagenic manner.
Fig 2.
The effect of CDG expression on uracil- and cytosine-derived mutations.
Schematic representations of the mutations originating from A) the uracil and B) the cytosine excised by the CDG glycosylase. Uracil or cytosine residue is removed by CDG creating an abasic site. The AP site (o) is bypassed by TLS polymerases inserting predominantly C across the AP site. *NTS; non-transcribed strand, **TS; transcribed strand. C) Overall mutation rates in yeast strains with CDG glycosylase-expressing plasmid under high-transcription (no doxycycline) or low-transcription (doxycyline added) conditions. Error bars represents 95% confidence intervals. D) and E) Rates of the uracil-dependent A>C and T>G mutations and the cytosine-dependent G>C mutations, respectively. See MATERIALS AND METHODS for the calculation of these rates. The numerical values of the median mutation frequencies and the confidence intervals represented as graphs in this figure are listed in S2 Table. All mutation rates are calculated from 24 independent cultures.
Fig 3.
The effect of CDG expression on uracil- and cytosine-derived mutations in the absence of Topoisomerase 1 or RNase Hs.
A) and D) Overall mutation rates in yeast strains with CDG glycosylase-expressing plasmid in under high-transcription (no doxycycline) or low-transcription (doxycyline added) conditions. Error bars represents 95% confidence intervals. B), C), E) and F) Rates of the uracil-dependent A>C and T>G mutations and the cytosine-dependent G>C mutations. The numerical values of the median mutation frequencies and the confidence intervals represented as graphs in this figure are listed in S2 Table. The mutation rates in A), B), and C) are calculated from 24 independent cultures and the mutation rates in D), E), and F) are calculated from 12 independent cultures.
Fig 4.
Uracil-density measured by the long amplicon qPCR approach.
A) Relative percent amplification of the genomic DNA samples from the yeast cells treated with the indicated concentration of 5-FU for 24 hrs. B) The uracil-density of the genomic DNA samples calculated from the relative percent amplification shown in A). See MATERIALS AND METHODS for the equation used for the calculation. C)–F), the genomic DNA samples isolated from the indicated yeast strains grown in the absence (-dox) or presence (+dox) of 2 μg/mL doxycycline in the media were used for qPCR and the calculation of uracil-density. C) Uracil density at LYS2 calculated from qPCR using “LYS2 3 kb” primers. D) Uracil density at LYS2 calculated from qPCR using “LYS2 4 kb” primers. E) Uracil density at CAN1 calculated from qPCR using “CAN1 3 kb” primers. F) Uracil density at TDH3 calculated from qPCR using “TDH3 3 kb” primers. For A) to F), error bars represent standard deviation (p-values were calculated using the unpaired student t-test; n.s—not significant) and all measurements are from N = 6. The numerical data represented as graphs in this figure are listed in S3 Table.
Fig 5.
Mutation frequencies following the 5-FU-, 4NQO-, and CPT-treatment.
A), C), and E) The frequencies of overall Lys+ mutations following treatments with 5-FU (10 μM), CPT (100 μM), or 4NQO (0.2 μg/mL), respectively, for 24 hrs. B), D), and F) The frequencies of the uracil-dependent A>C and T>G mutations following treatments with 5-FU, CPT and 4NQO, respectively. Error bars indicate 95% confidence intervals. The number of cultures used to determine the frequencies of mutations and the numerical values of the median mutation frequencies and the confidence intervals represented as graphs in this figure are listed in S4 Table.
Fig 6.
Uracil residues in the DNA following the 4NQO-treatment.
A) Quantification of uracil residues in DNA from ung1Δ yeast cells treated with the indicated concentrations of 5-FU for 24 hrs. The genomic DNA samples from untreated Hela and Daudi are used as negative and positive controls, respectively. All measurements are from N = 6. B) Quantification of uracil residues in DNA from yeast cells treated with the indicated concentrations of 4NQO. Three replicates are shown in the figure for each 4NQO concentrations. Left- Cy5 signal from AA3-labeled DNA dot-blotted on a nylon membrane. Right- Quantification of the cy5 signal shown on the left. Cy5 quantity is represented as relative to untreated (0μg/mL) sample. The numerical data represented as graphs are listed in S5 Table. Error bars indicate standard deviations and all measurements are from N = 6. Survival after growth in C) the 5-FU- or D) 4NQO-supplemented liquid culture, represented as the relative percentage compared to the untreated cells (p-values were calculated using the unpaired student t-test). N = 10.
Fig 7.
The rates of mutation in cells overexpressing Dut1 from the cell-cycle regulated promoters.
The rates of Lys+ reversion mutations of the apn1Δ ntg1Δ ntg2Δ strain transformed with plasmids expressing DUT1 from pGAL, pCLN2 (G1), pHHO1 (S), or pCLB2 (G2). The growth conditions were in rich media supplemented with galactose and raffinose (A), glycerol and ethanol (B), galactose and raffinose plus doxycycline (C), or glycerol and ethanol plus doxycycline (D). The error bars indicate 95% confidence intervals. (p-values were calculated using the unpaired student t-test). The number of cultures used to determine the rates of mutations and the numerical values of the median mutation rates and the confidence intervals represented as graphs in this figure are listed in S6 Table.
Fig 8.
The model of uracil-incorporation into DNA during G1, S, or G2 phases of the cell cycle.
Dut1 level is higher and the ratio of [dUTP]/[dTTP] is lower in S phase compared to G1 or G2. A) Uracil-incorporation during replication in S phase. The replicative synthesis occurs in S phase; the extent of uracil-incorporation and the ensuing uracil-associated mutagenesis is minor due to the low [dUTP]/[dTTP]. B) Uracil-incorporation during the repair-associated DNA synthesis. In S-phase (middle), the repair synthesis induced by transcription-associated endogenous DNA damage is subject to the low [dUTP]/[dTTP]; the extent of uracil-incorporation and the ensuing uracil-associated mutagenesis is minor due to the low [dUTP]/[dTTP]. In G1 or G2 phase, the repair synthesis induced by transcription-associated endogenous DNA damage is subject to the relatively high [dUTP]/[dTTP]; the extent of uracil-incorporation and the ensuing uracil-associated mutagenesis is significant.