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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

Polε bypasses an 8-oxoG and MeG lesions at DNA-damage-state, but not at normal S-phase-state, dNTP concentration. (a) Primer extension assays were performed with 4 nM Polε and 2 nM wild-type or 8-oxoG templates at low (L), normal S-phase (N) and DNA-damage-state (H) dNTP concentrations (see Table 1 for details) for 10 min at 30°C. The lesion bypass was calculated by dividing the sum of the products at position 5 (position after G/8-oxoG) or greater by the sum of the products at position 3 or greater. The sequence of the template and the positions of the nucleotides are indicated on the right. (b) Assays under single-hit conditions were performed with 0.17 nM Polε and 2 nM wild-type or 8-oxoG templates at low (L), normal S-phase (N) and DNA-damage-state (H) dNTP concentrations (see Table 1 for details) for 2 min at 30°C. The bypass probability was calculated by dividing the sum of the products at position 5 (position after G/8-oxoG) or greater by the sum of the products at position 3 or greater as previously described (30). The amount of extended primer is the intensity of all products greater than the primer divided by the intensity of the primer and all products greater than the primer. The total amount of primer extended in all reactions was far <20%, demonstrating that the conditions for single completed hits were reached (40). The sequence of the template and the positions of the nucleotides are indicated on the right. (c) Primer extension assays were performed with 4 nM Polε and 2 nM wild-type or MeG templates at low (L), normal S-phase (N) and DNA-damage-state (H) dNTP concentrations (see Table 1 for details) for 10 min at 30°C. The sequence of the template and the positions of the nucleotides are indicated on the right. (d) Assays under single-hit conditions were performed with 0.06 nM Polε and 2 nM wild-type or MeG templates at low (L), normal S-phase (N) and DNA-damage-state (H) dNTP concentrations for 2 min at 30°C. The sequence of the template and the positions of the nucleotides are indicated on the right.
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Figure 4: Polε bypasses an 8-oxoG and MeG lesions at DNA-damage-state, but not at normal S-phase-state, dNTP concentration. (a) Primer extension assays were performed with 4 nM Polε and 2 nM wild-type or 8-oxoG templates at low (L), normal S-phase (N) and DNA-damage-state (H) dNTP concentrations (see Table 1 for details) for 10 min at 30°C. The lesion bypass was calculated by dividing the sum of the products at position 5 (position after G/8-oxoG) or greater by the sum of the products at position 3 or greater. The sequence of the template and the positions of the nucleotides are indicated on the right. (b) Assays under single-hit conditions were performed with 0.17 nM Polε and 2 nM wild-type or 8-oxoG templates at low (L), normal S-phase (N) and DNA-damage-state (H) dNTP concentrations (see Table 1 for details) for 2 min at 30°C. The bypass probability was calculated by dividing the sum of the products at position 5 (position after G/8-oxoG) or greater by the sum of the products at position 3 or greater as previously described (30). The amount of extended primer is the intensity of all products greater than the primer divided by the intensity of the primer and all products greater than the primer. The total amount of primer extended in all reactions was far <20%, demonstrating that the conditions for single completed hits were reached (40). The sequence of the template and the positions of the nucleotides are indicated on the right. (c) Primer extension assays were performed with 4 nM Polε and 2 nM wild-type or MeG templates at low (L), normal S-phase (N) and DNA-damage-state (H) dNTP concentrations (see Table 1 for details) for 10 min at 30°C. The sequence of the template and the positions of the nucleotides are indicated on the right. (d) Assays under single-hit conditions were performed with 0.06 nM Polε and 2 nM wild-type or MeG templates at low (L), normal S-phase (N) and DNA-damage-state (H) dNTP concentrations for 2 min at 30°C. The sequence of the template and the positions of the nucleotides are indicated on the right.

Mentions: dNTP concentrations used in primer extension assays shown in Figure 4


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)

Polε bypasses an 8-oxoG and MeG lesions at DNA-damage-state, but not at normal S-phase-state, dNTP concentration. (a) Primer extension assays were performed with 4 nM Polε and 2 nM wild-type or 8-oxoG templates at low (L), normal S-phase (N) and DNA-damage-state (H) dNTP concentrations (see Table 1 for details) for 10 min at 30°C. The lesion bypass was calculated by dividing the sum of the products at position 5 (position after G/8-oxoG) or greater by the sum of the products at position 3 or greater. The sequence of the template and the positions of the nucleotides are indicated on the right. (b) Assays under single-hit conditions were performed with 0.17 nM Polε and 2 nM wild-type or 8-oxoG templates at low (L), normal S-phase (N) and DNA-damage-state (H) dNTP concentrations (see Table 1 for details) for 2 min at 30°C. The bypass probability was calculated by dividing the sum of the products at position 5 (position after G/8-oxoG) or greater by the sum of the products at position 3 or greater as previously described (30). The amount of extended primer is the intensity of all products greater than the primer divided by the intensity of the primer and all products greater than the primer. The total amount of primer extended in all reactions was far <20%, demonstrating that the conditions for single completed hits were reached (40). The sequence of the template and the positions of the nucleotides are indicated on the right. (c) Primer extension assays were performed with 4 nM Polε and 2 nM wild-type or MeG templates at low (L), normal S-phase (N) and DNA-damage-state (H) dNTP concentrations (see Table 1 for details) for 10 min at 30°C. The sequence of the template and the positions of the nucleotides are indicated on the right. (d) Assays under single-hit conditions were performed with 0.06 nM Polε and 2 nM wild-type or MeG templates at low (L), normal S-phase (N) and DNA-damage-state (H) dNTP concentrations for 2 min at 30°C. The sequence of the template and the positions of the nucleotides are indicated on the right.
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Related In: Results  -  Collection

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Show All Figures
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Figure 4: Polε bypasses an 8-oxoG and MeG lesions at DNA-damage-state, but not at normal S-phase-state, dNTP concentration. (a) Primer extension assays were performed with 4 nM Polε and 2 nM wild-type or 8-oxoG templates at low (L), normal S-phase (N) and DNA-damage-state (H) dNTP concentrations (see Table 1 for details) for 10 min at 30°C. The lesion bypass was calculated by dividing the sum of the products at position 5 (position after G/8-oxoG) or greater by the sum of the products at position 3 or greater. The sequence of the template and the positions of the nucleotides are indicated on the right. (b) Assays under single-hit conditions were performed with 0.17 nM Polε and 2 nM wild-type or 8-oxoG templates at low (L), normal S-phase (N) and DNA-damage-state (H) dNTP concentrations (see Table 1 for details) for 2 min at 30°C. The bypass probability was calculated by dividing the sum of the products at position 5 (position after G/8-oxoG) or greater by the sum of the products at position 3 or greater as previously described (30). The amount of extended primer is the intensity of all products greater than the primer divided by the intensity of the primer and all products greater than the primer. The total amount of primer extended in all reactions was far <20%, demonstrating that the conditions for single completed hits were reached (40). The sequence of the template and the positions of the nucleotides are indicated on the right. (c) Primer extension assays were performed with 4 nM Polε and 2 nM wild-type or MeG templates at low (L), normal S-phase (N) and DNA-damage-state (H) dNTP concentrations (see Table 1 for details) for 10 min at 30°C. The sequence of the template and the positions of the nucleotides are indicated on the right. (d) Assays under single-hit conditions were performed with 0.06 nM Polε and 2 nM wild-type or MeG templates at low (L), normal S-phase (N) and DNA-damage-state (H) dNTP concentrations for 2 min at 30°C. The sequence of the template and the positions of the nucleotides are indicated on the right.
Mentions: dNTP concentrations used in primer extension assays shown in Figure 4

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