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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

Overexpression of RNR1 efficiently elevates dNTP concentration in yeast strains lacking TLS polymerases. (a) rev1Δ rad30Δ rev3Δ pol4Δ and rev1Δ rad30Δ rev3Δ pol4Δ pGAL-RNR1 strains were incubated in liquid YP media with 2% galactose and treated with 0.2 mg/L 4-NQO as shown in the diagram. (b) Samples (indicated by numbers 1–4) treated as outlined in (a) were used for determination of dNTP pools. The numbers above the bars indicate the amount of the individual dNTP expressed in pmols/108 cells. Four overlaid HPLC chromatograms (raw data, not normalized by the number of cells) are shown on the inset. (c) Samples (indicated by numbers 1–4) treated as outlined in (a) were used for analysis of Rnr1 protein levels by 6% SDS–PAGE. M indicates protein marker lane. (d) The cell cycle progression is not altered in the strains lacking TLS polymerases. wild-type, pGAL-RNR1, rev1Δ rad30Δ rev3Δ pol4Δ and rev1Δ rad30Δ rev3Δ pol4Δ pGAL-RNR1 strains were inoculated in liquid YPD and incubated overnight at 30°C. Next morning cultures were diluted in fresh YPD to an OD600 of 0.1 and grown at 30°C. Samples were collected after 4.5 h and prepared for flow-cytometric analysis.
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Figure 1: Overexpression of RNR1 efficiently elevates dNTP concentration in yeast strains lacking TLS polymerases. (a) rev1Δ rad30Δ rev3Δ pol4Δ and rev1Δ rad30Δ rev3Δ pol4Δ pGAL-RNR1 strains were incubated in liquid YP media with 2% galactose and treated with 0.2 mg/L 4-NQO as shown in the diagram. (b) Samples (indicated by numbers 1–4) treated as outlined in (a) were used for determination of dNTP pools. The numbers above the bars indicate the amount of the individual dNTP expressed in pmols/108 cells. Four overlaid HPLC chromatograms (raw data, not normalized by the number of cells) are shown on the inset. (c) Samples (indicated by numbers 1–4) treated as outlined in (a) were used for analysis of Rnr1 protein levels by 6% SDS–PAGE. M indicates protein marker lane. (d) The cell cycle progression is not altered in the strains lacking TLS polymerases. wild-type, pGAL-RNR1, rev1Δ rad30Δ rev3Δ pol4Δ and rev1Δ rad30Δ rev3Δ pol4Δ pGAL-RNR1 strains were inoculated in liquid YPD and incubated overnight at 30°C. Next morning cultures were diluted in fresh YPD to an OD600 of 0.1 and grown at 30°C. Samples were collected after 4.5 h and prepared for flow-cytometric analysis.

Mentions: To establish strains, in which dNTP concentration could be experimentally controlled, we utilized the GAL1-driven wild-type RNR1 gene introduced into the URA3 locus of the yeast genome. We measured dNTP pools in the rev1Δ rad30Δ rev3Δ pol4Δ and rev1Δ rad30Δ rev3Δ pol4Δ pGAL-RNR1 strains grown in galactose-containing media before and after DNA damage induced by 4-NQO (Figure 1a). Induction of the RNR1 gene by galactose in the rev1Δ rad30Δ rev3Δ pol4Δ pGAL-RNR1 strain resulted in overexpression of the Rnr1 protein and a 9- to 13-fold elevation of dNTP concentration compared to rev1Δ rad30Δ rev3Δ pol4Δ strain (Figure 1b and c). Addition of 4-NQO to the rev1Δ rad30Δ rev3Δ pol4Δ pGAL-RNR1 strain induced by galactose further increased dNTP concentration 3- to 4-fold (Figure 1b). This further increase can be explained by the induction of the RNR2-4 genes, degradation of Sml1 and a decreased utilization of dNTP during DNA damage. Addition of 4-NQO to the rev1Δ rad30Δ rev3Δ pol4Δ strain elevated the dNTP concentration 5- to 8-fold (Figure 1b). The same fold increase in dNTP concentration occurs in wild-type yeast during DNA damage (18). Simultaneous deletion of all non-replicative polymerases had no effect on cell proliferation or cell division cycle under normal growth conditions (i.e. in the absence of 4-NQO) (Figure 1d). Overexpression of RNR1 in all strains did not affect proliferation rates and viability as judged by the number and the size of colonies (Figure 2a).Figure 1.


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)

Overexpression of RNR1 efficiently elevates dNTP concentration in yeast strains lacking TLS polymerases. (a) rev1Δ rad30Δ rev3Δ pol4Δ and rev1Δ rad30Δ rev3Δ pol4Δ pGAL-RNR1 strains were incubated in liquid YP media with 2% galactose and treated with 0.2 mg/L 4-NQO as shown in the diagram. (b) Samples (indicated by numbers 1–4) treated as outlined in (a) were used for determination of dNTP pools. The numbers above the bars indicate the amount of the individual dNTP expressed in pmols/108 cells. Four overlaid HPLC chromatograms (raw data, not normalized by the number of cells) are shown on the inset. (c) Samples (indicated by numbers 1–4) treated as outlined in (a) were used for analysis of Rnr1 protein levels by 6% SDS–PAGE. M indicates protein marker lane. (d) The cell cycle progression is not altered in the strains lacking TLS polymerases. wild-type, pGAL-RNR1, rev1Δ rad30Δ rev3Δ pol4Δ and rev1Δ rad30Δ rev3Δ pol4Δ pGAL-RNR1 strains were inoculated in liquid YPD and incubated overnight at 30°C. Next morning cultures were diluted in fresh YPD to an OD600 of 0.1 and grown at 30°C. Samples were collected after 4.5 h and prepared for flow-cytometric analysis.
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Figure 1: Overexpression of RNR1 efficiently elevates dNTP concentration in yeast strains lacking TLS polymerases. (a) rev1Δ rad30Δ rev3Δ pol4Δ and rev1Δ rad30Δ rev3Δ pol4Δ pGAL-RNR1 strains were incubated in liquid YP media with 2% galactose and treated with 0.2 mg/L 4-NQO as shown in the diagram. (b) Samples (indicated by numbers 1–4) treated as outlined in (a) were used for determination of dNTP pools. The numbers above the bars indicate the amount of the individual dNTP expressed in pmols/108 cells. Four overlaid HPLC chromatograms (raw data, not normalized by the number of cells) are shown on the inset. (c) Samples (indicated by numbers 1–4) treated as outlined in (a) were used for analysis of Rnr1 protein levels by 6% SDS–PAGE. M indicates protein marker lane. (d) The cell cycle progression is not altered in the strains lacking TLS polymerases. wild-type, pGAL-RNR1, rev1Δ rad30Δ rev3Δ pol4Δ and rev1Δ rad30Δ rev3Δ pol4Δ pGAL-RNR1 strains were inoculated in liquid YPD and incubated overnight at 30°C. Next morning cultures were diluted in fresh YPD to an OD600 of 0.1 and grown at 30°C. Samples were collected after 4.5 h and prepared for flow-cytometric analysis.
Mentions: To establish strains, in which dNTP concentration could be experimentally controlled, we utilized the GAL1-driven wild-type RNR1 gene introduced into the URA3 locus of the yeast genome. We measured dNTP pools in the rev1Δ rad30Δ rev3Δ pol4Δ and rev1Δ rad30Δ rev3Δ pol4Δ pGAL-RNR1 strains grown in galactose-containing media before and after DNA damage induced by 4-NQO (Figure 1a). Induction of the RNR1 gene by galactose in the rev1Δ rad30Δ rev3Δ pol4Δ pGAL-RNR1 strain resulted in overexpression of the Rnr1 protein and a 9- to 13-fold elevation of dNTP concentration compared to rev1Δ rad30Δ rev3Δ pol4Δ strain (Figure 1b and c). Addition of 4-NQO to the rev1Δ rad30Δ rev3Δ pol4Δ pGAL-RNR1 strain induced by galactose further increased dNTP concentration 3- to 4-fold (Figure 1b). This further increase can be explained by the induction of the RNR2-4 genes, degradation of Sml1 and a decreased utilization of dNTP during DNA damage. Addition of 4-NQO to the rev1Δ rad30Δ rev3Δ pol4Δ strain elevated the dNTP concentration 5- to 8-fold (Figure 1b). The same fold increase in dNTP concentration occurs in wild-type yeast during DNA damage (18). Simultaneous deletion of all non-replicative polymerases had no effect on cell proliferation or cell division cycle under normal growth conditions (i.e. in the absence of 4-NQO) (Figure 1d). Overexpression of RNR1 in all strains did not affect proliferation rates and viability as judged by the number and the size of colonies (Figure 2a).Figure 1.

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