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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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In vitro assays for the dCMP transferase of Rev1 and the Rev1D467A/E468A mutant protein. (A) Purified mutant Rev1D467A/E468A protein, which was visualized by staining the 10% polyacrylamide gel with Coomassie blue. The full-length mutant Rev1 is indicated by the arrowhead. (B) Standard translesion synthesis assays were performed with purified wild-type (lanes 1–6) or mutant Rev1D467A/E468A (lanes 7–12) protein using either a G template or an AP template as indicated. The AP site in the template sequence is indicated by the X. The 17-mer DNA band is indicative of the dCMP transferase activity.
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Figure 3: In vitro assays for the dCMP transferase of Rev1 and the Rev1D467A/E468A mutant protein. (A) Purified mutant Rev1D467A/E468A protein, which was visualized by staining the 10% polyacrylamide gel with Coomassie blue. The full-length mutant Rev1 is indicated by the arrowhead. (B) Standard translesion synthesis assays were performed with purified wild-type (lanes 1–6) or mutant Rev1D467A/E468A (lanes 7–12) protein using either a G template or an AP template as indicated. The AP site in the template sequence is indicated by the X. The 17-mer DNA band is indicative of the dCMP transferase activity.

Mentions: In order to separate the dCMP transferase activity of Rev1 from its non-catalytic function, we mutated two conserved amino acid residues in the catalytic domain, i.e. D467A/E468A. Whereas the dCMP transferase was readily detected with the wild-type Rev1 protein in vitro (Figure 3B, lanes 2, 3, 5 and 6), this activity was not detectable with the purified mutant Rev1 (Rev1D467A/E468A) (Figure 3B, lanes 8, 9, 11 and 12). To determine whether the mutant Rev1D467A/E468A retains its non-catalytic function in translesion synthesis, we transformed the mutant rev1 gene on a plasmid vector into the rev1 deletion mutant strain and assayed for translesion synthesis and mutagenesis following UV radiation. Loss of translesion synthesis in yeast cells as in the case of rev1 deletion mutant cells results in moderate UV sensitivity (10,13). Thus, cellular UV sensitivity was measured as an indication for the translesion synthesis function of various yeast strains. As shown in Figure 4, UV sensitivity of the rev1 deletion mutant strain was complemented by the mutant rev1D467A/E468A gene on a plasmid vector, as efficient as complementation by the wild-type REV1 gene on the same vector. Thus, the mutant Rev1D467A/E468A has lost its dCMP transferase activity, but retained its non-catalytic activity for translesion synthesis. These results show that the Rev1 dCMP transferase activity is separable from its non-catalytic function in translesion synthesis.Figure 3.


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)

In vitro assays for the dCMP transferase of Rev1 and the Rev1D467A/E468A mutant protein. (A) Purified mutant Rev1D467A/E468A protein, which was visualized by staining the 10% polyacrylamide gel with Coomassie blue. The full-length mutant Rev1 is indicated by the arrowhead. (B) Standard translesion synthesis assays were performed with purified wild-type (lanes 1–6) or mutant Rev1D467A/E468A (lanes 7–12) protein using either a G template or an AP template as indicated. The AP site in the template sequence is indicated by the X. The 17-mer DNA band is indicative of the dCMP transferase activity.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 3: In vitro assays for the dCMP transferase of Rev1 and the Rev1D467A/E468A mutant protein. (A) Purified mutant Rev1D467A/E468A protein, which was visualized by staining the 10% polyacrylamide gel with Coomassie blue. The full-length mutant Rev1 is indicated by the arrowhead. (B) Standard translesion synthesis assays were performed with purified wild-type (lanes 1–6) or mutant Rev1D467A/E468A (lanes 7–12) protein using either a G template or an AP template as indicated. The AP site in the template sequence is indicated by the X. The 17-mer DNA band is indicative of the dCMP transferase activity.
Mentions: In order to separate the dCMP transferase activity of Rev1 from its non-catalytic function, we mutated two conserved amino acid residues in the catalytic domain, i.e. D467A/E468A. Whereas the dCMP transferase was readily detected with the wild-type Rev1 protein in vitro (Figure 3B, lanes 2, 3, 5 and 6), this activity was not detectable with the purified mutant Rev1 (Rev1D467A/E468A) (Figure 3B, lanes 8, 9, 11 and 12). To determine whether the mutant Rev1D467A/E468A retains its non-catalytic function in translesion synthesis, we transformed the mutant rev1 gene on a plasmid vector into the rev1 deletion mutant strain and assayed for translesion synthesis and mutagenesis following UV radiation. Loss of translesion synthesis in yeast cells as in the case of rev1 deletion mutant cells results in moderate UV sensitivity (10,13). Thus, cellular UV sensitivity was measured as an indication for the translesion synthesis function of various yeast strains. As shown in Figure 4, UV sensitivity of the rev1 deletion mutant strain was complemented by the mutant rev1D467A/E468A gene on a plasmid vector, as efficient as complementation by the wild-type REV1 gene on the same vector. Thus, the mutant Rev1D467A/E468A has lost its dCMP transferase activity, but retained its non-catalytic activity for translesion synthesis. These results show that the Rev1 dCMP transferase activity is separable from its non-catalytic function in translesion synthesis.Figure 3.

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