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The catalytic function of the Rev1 dCMP transferase is required in a lesion-specific manner for translesion synthesis and base damage-induced mutagenesis.

Zhou Y, Wang J, Zhang Y, Wang Z - Nucleic Acids Res. (2010)

Bottom Line: The Rev1-Polzeta pathway is believed to be the major mechanism of translesion DNA synthesis and base damage-induced mutagenesis in eukaryotes.This was achieved by mutating two conserved amino acid residues in the catalytic domain of Rev1, i.e. D467A/E468A, where its catalytic function was abolished but its non-catalytic function remained intact.Specifically, the predominant A-->G mutations resulting from C insertion opposite the lesion were abolished.

View Article: PubMed Central - PubMed

Affiliation: Graduate Center for Toxicology, University of Kentucky, Lexington, KY 40536, USA.

ABSTRACT
The Rev1-Polzeta pathway is believed to be the major mechanism of translesion DNA synthesis and base damage-induced mutagenesis in eukaryotes. While it is widely believed that Rev1 plays a non-catalytic function in translesion synthesis, the role of its dCMP transferase activity remains uncertain. To determine the relevance of its catalytic function in translesion synthesis, we separated the Rev1 dCMP transferase activity from its non-catalytic function in yeast. This was achieved by mutating two conserved amino acid residues in the catalytic domain of Rev1, i.e. D467A/E468A, where its catalytic function was abolished but its non-catalytic function remained intact. In this mutant strain, whereas translesion synthesis and mutagenesis of UV radiation were fully functional, those of a site-specific 1,N(6)-ethenoadenine were severely deficient. Specifically, the predominant A-->G mutations resulting from C insertion opposite the lesion were abolished. Therefore, translesion synthesis and mutagenesis of 1,N(6)-ethenoadenine require the catalytic function of the Rev1 dCMP transferase, in contrast to those of UV lesions, which only require the non-catalytic function of Rev1. These results show that the catalytic function of the Rev1 dCMP transferase is required in a lesion-specific manner for translesion synthesis and base damage-induced mutagenesis.

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Translesion synthesis of 1,N6-ethenoadenine DNA adducts by human Polη, Polκ, and Polι. (A) The DNA template for translesion synthesis. A 20-mer primer was labeled with 32P at its 5′-end (*) and annealed right before a template 1,N6-ethenoadenine. (B) Translesion synthesis reactions were performed with purified human Polη (lanes 1–5), Polκ (lanes 6–10), and Polι (lanes 11–15) as indicated in the presence of a single deoxyribonucleoside triphosphate dATP (A), dCTP (C), dTTP (T) or dGTP (G), or all four dNTPs (N4). Reaction products were separated by electrophoresis on denaturing polyacrylamide gel and visualized by autoradiography. Quantitation of extended primers is shown at the bottom of the gel. DNA size markers in nucleotides are indicated on the left.
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Figure 1: Translesion synthesis of 1,N6-ethenoadenine DNA adducts by human Polη, Polκ, and Polι. (A) The DNA template for translesion synthesis. A 20-mer primer was labeled with 32P at its 5′-end (*) and annealed right before a template 1,N6-ethenoadenine. (B) Translesion synthesis reactions were performed with purified human Polη (lanes 1–5), Polκ (lanes 6–10), and Polι (lanes 11–15) as indicated in the presence of a single deoxyribonucleoside triphosphate dATP (A), dCTP (C), dTTP (T) or dGTP (G), or all four dNTPs (N4). Reaction products were separated by electrophoresis on denaturing polyacrylamide gel and visualized by autoradiography. Quantitation of extended primers is shown at the bottom of the gel. DNA size markers in nucleotides are indicated on the left.

Mentions: The 1,N6-ethenoadenine is an important type of exocyclic DNA adduct. To understand molecular mechanisms of bypass and mutagenesis of this lesion, we examined its translesion synthesis in vitro by the human Y family DNA polymerases REV1, Polη, Polι and Polκ. Translesion synthesis was performed with the purified polymerases on a 29-mer DNA template containing a 32P-labeled 20-mer primer annealed right before the lesion (Figure 1A). As shown in Figure 1B (lane 1), human Polη efficiently bypassed 1,N6-ethenoadenine. To identify the base incorporated opposite the lesion, we performed translesion synthesis assays with only one deoxyribonucleoside triphosphate: dATP, dCTP, dGTP or dTTP individually. As shown in Figure 1B (lanes 2–5), human Polη preferred A insertion opposite the lesion, although the other three nucleotides were also inserted. To more accurately compare the efficiency of nucleotide insertion, we performed kinetic analysis using increasing concentrations of a single dNTP. From these assays, the kinetic parameters Vmax and Km were obtained (Table 1). While the efficiency of nucleotide insertion is indicated by the Vmax/Km value, the accuracy of nucleotide insertion is indicated by the finc value, i.e. (Vmax/Km)incorrect/(Vmax/Km)correct. These kinetic measurements show that human Polη is error-prone in catalyzing translesion synthesis of 1,N6-ethenoadenine in vitro, with nucleotide insertion efficiency in the order of A > T > C ∼ G (Table 1).Figure 1.


The catalytic function of the Rev1 dCMP transferase is required in a lesion-specific manner for translesion synthesis and base damage-induced mutagenesis.

Zhou Y, Wang J, Zhang Y, Wang Z - Nucleic Acids Res. (2010)

Translesion synthesis of 1,N6-ethenoadenine DNA adducts by human Polη, Polκ, and Polι. (A) The DNA template for translesion synthesis. A 20-mer primer was labeled with 32P at its 5′-end (*) and annealed right before a template 1,N6-ethenoadenine. (B) Translesion synthesis reactions were performed with purified human Polη (lanes 1–5), Polκ (lanes 6–10), and Polι (lanes 11–15) as indicated in the presence of a single deoxyribonucleoside triphosphate dATP (A), dCTP (C), dTTP (T) or dGTP (G), or all four dNTPs (N4). Reaction products were separated by electrophoresis on denaturing polyacrylamide gel and visualized by autoradiography. Quantitation of extended primers is shown at the bottom of the gel. DNA size markers in nucleotides are indicated on the left.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2926598&req=5

Figure 1: Translesion synthesis of 1,N6-ethenoadenine DNA adducts by human Polη, Polκ, and Polι. (A) The DNA template for translesion synthesis. A 20-mer primer was labeled with 32P at its 5′-end (*) and annealed right before a template 1,N6-ethenoadenine. (B) Translesion synthesis reactions were performed with purified human Polη (lanes 1–5), Polκ (lanes 6–10), and Polι (lanes 11–15) as indicated in the presence of a single deoxyribonucleoside triphosphate dATP (A), dCTP (C), dTTP (T) or dGTP (G), or all four dNTPs (N4). Reaction products were separated by electrophoresis on denaturing polyacrylamide gel and visualized by autoradiography. Quantitation of extended primers is shown at the bottom of the gel. DNA size markers in nucleotides are indicated on the left.
Mentions: The 1,N6-ethenoadenine is an important type of exocyclic DNA adduct. To understand molecular mechanisms of bypass and mutagenesis of this lesion, we examined its translesion synthesis in vitro by the human Y family DNA polymerases REV1, Polη, Polι and Polκ. Translesion synthesis was performed with the purified polymerases on a 29-mer DNA template containing a 32P-labeled 20-mer primer annealed right before the lesion (Figure 1A). As shown in Figure 1B (lane 1), human Polη efficiently bypassed 1,N6-ethenoadenine. To identify the base incorporated opposite the lesion, we performed translesion synthesis assays with only one deoxyribonucleoside triphosphate: dATP, dCTP, dGTP or dTTP individually. As shown in Figure 1B (lanes 2–5), human Polη preferred A insertion opposite the lesion, although the other three nucleotides were also inserted. To more accurately compare the efficiency of nucleotide insertion, we performed kinetic analysis using increasing concentrations of a single dNTP. From these assays, the kinetic parameters Vmax and Km were obtained (Table 1). While the efficiency of nucleotide insertion is indicated by the Vmax/Km value, the accuracy of nucleotide insertion is indicated by the finc value, i.e. (Vmax/Km)incorrect/(Vmax/Km)correct. These kinetic measurements show that human Polη is error-prone in catalyzing translesion synthesis of 1,N6-ethenoadenine in vitro, with nucleotide insertion efficiency in the order of A > T > C ∼ G (Table 1).Figure 1.

Bottom Line: The Rev1-Polzeta pathway is believed to be the major mechanism of translesion DNA synthesis and base damage-induced mutagenesis in eukaryotes.This was achieved by mutating two conserved amino acid residues in the catalytic domain of Rev1, i.e. D467A/E468A, where its catalytic function was abolished but its non-catalytic function remained intact.Specifically, the predominant A-->G mutations resulting from C insertion opposite the lesion were abolished.

View Article: PubMed Central - PubMed

Affiliation: Graduate Center for Toxicology, University of Kentucky, Lexington, KY 40536, USA.

ABSTRACT
The Rev1-Polzeta pathway is believed to be the major mechanism of translesion DNA synthesis and base damage-induced mutagenesis in eukaryotes. While it is widely believed that Rev1 plays a non-catalytic function in translesion synthesis, the role of its dCMP transferase activity remains uncertain. To determine the relevance of its catalytic function in translesion synthesis, we separated the Rev1 dCMP transferase activity from its non-catalytic function in yeast. This was achieved by mutating two conserved amino acid residues in the catalytic domain of Rev1, i.e. D467A/E468A, where its catalytic function was abolished but its non-catalytic function remained intact. In this mutant strain, whereas translesion synthesis and mutagenesis of UV radiation were fully functional, those of a site-specific 1,N(6)-ethenoadenine were severely deficient. Specifically, the predominant A-->G mutations resulting from C insertion opposite the lesion were abolished. Therefore, translesion synthesis and mutagenesis of 1,N(6)-ethenoadenine require the catalytic function of the Rev1 dCMP transferase, in contrast to those of UV lesions, which only require the non-catalytic function of Rev1. These results show that the catalytic function of the Rev1 dCMP transferase is required in a lesion-specific manner for translesion synthesis and base damage-induced mutagenesis.

Show MeSH
Related in: MedlinePlus