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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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Increased dNTP concentration improves DNA damage tolerance in the absence of TLS polymerases. (a) Stationary phase cultures grown in YPD were spotted at 10-fold serial dilutions on YPGal (control) and YPGal with 0.24 mg/l 4-NQO plates, and incubated for 4 days at 30°C. (b) rev1Δ rad30Δ rev3Δ pol4Δ and rev1Δ rad30Δ rev3Δ pol4Δ pGAL-RNR1 strains were grown overnight in YPD; appropriate dilutions were plated on YPGal plates containing indicated amounts of 4-NQO, and on YPGal plates to calculate the number of viable cells. Colonies were counted after 4 days of incubation at 30°C.
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Figure 2: Increased dNTP concentration improves DNA damage tolerance in the absence of TLS polymerases. (a) Stationary phase cultures grown in YPD were spotted at 10-fold serial dilutions on YPGal (control) and YPGal with 0.24 mg/l 4-NQO plates, and incubated for 4 days at 30°C. (b) rev1Δ rad30Δ rev3Δ pol4Δ and rev1Δ rad30Δ rev3Δ pol4Δ pGAL-RNR1 strains were grown overnight in YPD; appropriate dilutions were plated on YPGal plates containing indicated amounts of 4-NQO, and on YPGal plates to calculate the number of viable cells. Colonies were counted after 4 days of incubation at 30°C.

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)

Increased dNTP concentration improves DNA damage tolerance in the absence of TLS polymerases. (a) Stationary phase cultures grown in YPD were spotted at 10-fold serial dilutions on YPGal (control) and YPGal with 0.24 mg/l 4-NQO plates, and incubated for 4 days at 30°C. (b) rev1Δ rad30Δ rev3Δ pol4Δ and rev1Δ rad30Δ rev3Δ pol4Δ pGAL-RNR1 strains were grown overnight in YPD; appropriate dilutions were plated on YPGal plates containing indicated amounts of 4-NQO, and on YPGal plates to calculate the number of viable cells. Colonies were counted after 4 days of incubation at 30°C.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2553575&req=5

Figure 2: Increased dNTP concentration improves DNA damage tolerance in the absence of TLS polymerases. (a) Stationary phase cultures grown in YPD were spotted at 10-fold serial dilutions on YPGal (control) and YPGal with 0.24 mg/l 4-NQO plates, and incubated for 4 days at 30°C. (b) rev1Δ rad30Δ rev3Δ pol4Δ and rev1Δ rad30Δ rev3Δ pol4Δ pGAL-RNR1 strains were grown overnight in YPD; appropriate dilutions were plated on YPGal plates containing indicated amounts of 4-NQO, and on YPGal plates to calculate the number of viable cells. Colonies were counted after 4 days of incubation at 30°C.
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