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Evidence for lesion bypass by yeast replicative DNA polymerases during DNA damage.

Sabouri N, Viberg J, Goyal DK, Johansson E, Chabes A - Nucleic Acids Res. (2008)

Bottom Line: The enzyme ribonucleotide reductase, responsible for the synthesis of deoxyribonucleotides (dNTP), is upregulated in response to DNA damage in all organisms.Here we show that in a yeast strain with all specialized translesion DNA polymerases deleted, 4-nitroquinoline oxide (4-NQO) treatment increases mutation frequency approximately 3-fold, and that an increase in dNTP concentration significantly improves the tolerance of this strain to 4-NQO induced damage.The nucleotide inserted opposite 8-oxoG is dATP.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry and Biophysics, Umeå University, SE 901 87 Umeå, Sweden.

ABSTRACT
The enzyme ribonucleotide reductase, responsible for the synthesis of deoxyribonucleotides (dNTP), is upregulated in response to DNA damage in all organisms. In Saccharomyces cerevisiae, dNTP concentration increases approximately 6- to 8-fold in response to DNA damage. This concentration increase is associated with improved tolerance of DNA damage, suggesting that translesion DNA synthesis is more efficient at elevated dNTP concentration. Here we show that in a yeast strain with all specialized translesion DNA polymerases deleted, 4-nitroquinoline oxide (4-NQO) treatment increases mutation frequency approximately 3-fold, and that an increase in dNTP concentration significantly improves the tolerance of this strain to 4-NQO induced damage. In vitro, under single-hit conditions, the replicative DNA polymerase epsilon does not bypass 7,8-dihydro-8-oxoguanine lesion (8-oxoG, one of the lesions produced by 4-NQO) at S-phase dNTP concentration, but does bypass the same lesion with 19-27% efficiency at DNA-damage-state dNTP concentration. The nucleotide inserted opposite 8-oxoG is dATP. We propose that during DNA damage in S. cerevisiae increased dNTP concentration allows replicative DNA polymerases to bypass certain DNA lesions.

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Related in: MedlinePlus

4-NQO increases mutation frequency in the rev1Δ rad30Δ rev3Δ pol4Δ strain. (a) wild-type and rev1Δ rad30Δ rev3Δ pol4Δ logarithmically growing in YPD were treated with increasing amounts of 4-NQO for 2 h and were after appropriate dilutions spread on YPD plates in triplicates to determine survival. (b) Yeast cells treated as in (a) were after appropriate dilutions spread on synthetic complete medium −arginine +L-canavanine to determine mutation frequencies in CAN1 gene by dividing the number of Can1r mutants by the average number of surviving cells (c) rev1Δ rad30Δ rev3Δ pol4Δ pGAL-RNR1 strain grown in YPRaf was divided into two cultures, one of which was induced by 2% galactose and mutation frequencies were determined after 2 and 4 h induction. Hatched bars: uninduced cells; open bars: galactose-induced cells.
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Figure 3: 4-NQO increases mutation frequency in the rev1Δ rad30Δ rev3Δ pol4Δ strain. (a) wild-type and rev1Δ rad30Δ rev3Δ pol4Δ logarithmically growing in YPD were treated with increasing amounts of 4-NQO for 2 h and were after appropriate dilutions spread on YPD plates in triplicates to determine survival. (b) Yeast cells treated as in (a) were after appropriate dilutions spread on synthetic complete medium −arginine +L-canavanine to determine mutation frequencies in CAN1 gene by dividing the number of Can1r mutants by the average number of surviving cells (c) rev1Δ rad30Δ rev3Δ pol4Δ pGAL-RNR1 strain grown in YPRaf was divided into two cultures, one of which was induced by 2% galactose and mutation frequencies were determined after 2 and 4 h induction. Hatched bars: uninduced cells; open bars: galactose-induced cells.

Mentions: The increased DNA damage tolerance of the rev1Δ rad30Δ rev3Δ pol4Δ strain in the presence of elevated dNTP concentration suggests that the replicative DNA polymerases are able to bypass some lesions produced by 4-NQO. Alternatively, other DNA repair pathways, e.g. nucleotide excision repair (NER) or base excision repair (BER), are somehow stimulated by increased dNTP pools. However, these pathways do not involve a direct bypass of a lesion by a DNA polymerase and should not be mutagenic. Therefore, we measured the induced mutation frequencies in the rev1Δ rad30Δ rev3Δ pol4Δ and wild-type strains after 2 h incubation with increasing concentrations of 4-NQO. The initial increase in the induced mutation frequencies (about 3-fold) and the initial decrease in survival showed the same dynamics in both strains (Figure 3a and b). At 0.04 mg/l 4-NQO the induced mutation frequency in the rev1Δ rad30Δ rev3Δ pol4Δ strain reached a plateau, while the induced mutation frequency in the wild-type strain continued to increase. Since in both strains the treatment with 4-NQO leads to elevation of dNTP pools ∼8-fold (Figure 1b), it is possible that the observed initial increase in mutation frequencies is due to higher error rates of replicative polymerases in the presence of high dNTP concentration and not due to lesion bypass. However, mutation frequencies did not increase in the rev1Δ rad30Δ rev3Δ pol4Δ pGAL-RNR1 strain induced by galactose for 2 or 4 h (Figure 3c), even though the dNTP concentration increases ∼10-fold after the galactose induction in the absence of 4-NQO (Figure 1b). Thus, the increase in the 4-NQO-induced mutation frequency in the rev1Δ rad30Δ rev3Δ pol4Δ strain is most likely due to increased translesion synthesis by the replicative DNA polymerases.Figure 3.


Evidence for lesion bypass by yeast replicative DNA polymerases during DNA damage.

Sabouri N, Viberg J, Goyal DK, Johansson E, Chabes A - Nucleic Acids Res. (2008)

4-NQO increases mutation frequency in the rev1Δ rad30Δ rev3Δ pol4Δ strain. (a) wild-type and rev1Δ rad30Δ rev3Δ pol4Δ logarithmically growing in YPD were treated with increasing amounts of 4-NQO for 2 h and were after appropriate dilutions spread on YPD plates in triplicates to determine survival. (b) Yeast cells treated as in (a) were after appropriate dilutions spread on synthetic complete medium −arginine +L-canavanine to determine mutation frequencies in CAN1 gene by dividing the number of Can1r mutants by the average number of surviving cells (c) rev1Δ rad30Δ rev3Δ pol4Δ pGAL-RNR1 strain grown in YPRaf was divided into two cultures, one of which was induced by 2% galactose and mutation frequencies were determined after 2 and 4 h induction. Hatched bars: uninduced cells; open bars: galactose-induced cells.
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Related In: Results  -  Collection

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Figure 3: 4-NQO increases mutation frequency in the rev1Δ rad30Δ rev3Δ pol4Δ strain. (a) wild-type and rev1Δ rad30Δ rev3Δ pol4Δ logarithmically growing in YPD were treated with increasing amounts of 4-NQO for 2 h and were after appropriate dilutions spread on YPD plates in triplicates to determine survival. (b) Yeast cells treated as in (a) were after appropriate dilutions spread on synthetic complete medium −arginine +L-canavanine to determine mutation frequencies in CAN1 gene by dividing the number of Can1r mutants by the average number of surviving cells (c) rev1Δ rad30Δ rev3Δ pol4Δ pGAL-RNR1 strain grown in YPRaf was divided into two cultures, one of which was induced by 2% galactose and mutation frequencies were determined after 2 and 4 h induction. Hatched bars: uninduced cells; open bars: galactose-induced cells.
Mentions: The increased DNA damage tolerance of the rev1Δ rad30Δ rev3Δ pol4Δ strain in the presence of elevated dNTP concentration suggests that the replicative DNA polymerases are able to bypass some lesions produced by 4-NQO. Alternatively, other DNA repair pathways, e.g. nucleotide excision repair (NER) or base excision repair (BER), are somehow stimulated by increased dNTP pools. However, these pathways do not involve a direct bypass of a lesion by a DNA polymerase and should not be mutagenic. Therefore, we measured the induced mutation frequencies in the rev1Δ rad30Δ rev3Δ pol4Δ and wild-type strains after 2 h incubation with increasing concentrations of 4-NQO. The initial increase in the induced mutation frequencies (about 3-fold) and the initial decrease in survival showed the same dynamics in both strains (Figure 3a and b). At 0.04 mg/l 4-NQO the induced mutation frequency in the rev1Δ rad30Δ rev3Δ pol4Δ strain reached a plateau, while the induced mutation frequency in the wild-type strain continued to increase. Since in both strains the treatment with 4-NQO leads to elevation of dNTP pools ∼8-fold (Figure 1b), it is possible that the observed initial increase in mutation frequencies is due to higher error rates of replicative polymerases in the presence of high dNTP concentration and not due to lesion bypass. However, mutation frequencies did not increase in the rev1Δ rad30Δ rev3Δ pol4Δ pGAL-RNR1 strain induced by galactose for 2 or 4 h (Figure 3c), even though the dNTP concentration increases ∼10-fold after the galactose induction in the absence of 4-NQO (Figure 1b). Thus, the increase in the 4-NQO-induced mutation frequency in the rev1Δ rad30Δ rev3Δ pol4Δ strain is most likely due to increased translesion synthesis by the replicative DNA polymerases.Figure 3.

Bottom Line: The enzyme ribonucleotide reductase, responsible for the synthesis of deoxyribonucleotides (dNTP), is upregulated in response to DNA damage in all organisms.Here we show that in a yeast strain with all specialized translesion DNA polymerases deleted, 4-nitroquinoline oxide (4-NQO) treatment increases mutation frequency approximately 3-fold, and that an increase in dNTP concentration significantly improves the tolerance of this strain to 4-NQO induced damage.The nucleotide inserted opposite 8-oxoG is dATP.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry and Biophysics, Umeå University, SE 901 87 Umeå, Sweden.

ABSTRACT
The enzyme ribonucleotide reductase, responsible for the synthesis of deoxyribonucleotides (dNTP), is upregulated in response to DNA damage in all organisms. In Saccharomyces cerevisiae, dNTP concentration increases approximately 6- to 8-fold in response to DNA damage. This concentration increase is associated with improved tolerance of DNA damage, suggesting that translesion DNA synthesis is more efficient at elevated dNTP concentration. Here we show that in a yeast strain with all specialized translesion DNA polymerases deleted, 4-nitroquinoline oxide (4-NQO) treatment increases mutation frequency approximately 3-fold, and that an increase in dNTP concentration significantly improves the tolerance of this strain to 4-NQO induced damage. In vitro, under single-hit conditions, the replicative DNA polymerase epsilon does not bypass 7,8-dihydro-8-oxoguanine lesion (8-oxoG, one of the lesions produced by 4-NQO) at S-phase dNTP concentration, but does bypass the same lesion with 19-27% efficiency at DNA-damage-state dNTP concentration. The nucleotide inserted opposite 8-oxoG is dATP. We propose that during DNA damage in S. cerevisiae increased dNTP concentration allows replicative DNA polymerases to bypass certain DNA lesions.

Show MeSH
Related in: MedlinePlus