Limits...
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.

Show MeSH

Related in: MedlinePlus

A mechanistic model for translesion synthesis of the 1,N6-ethenoadenine DNA adduct in yeast cells. The replication complex (represented by the filled blue oval) is blocked by the lesion, signaling translesion synthesis. Translesion synthesis is mediated predominantly by C insertion opposite the lesion catalyzed by the Rev1 dCMP transferase. Extension synthesis by Polζ completes the lesion bypass. This major mechanism of translesion synthesis results in A→G transition mutations.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2926598&req=5

Figure 6: A mechanistic model for translesion synthesis of the 1,N6-ethenoadenine DNA adduct in yeast cells. The replication complex (represented by the filled blue oval) is blocked by the lesion, signaling translesion synthesis. Translesion synthesis is mediated predominantly by C insertion opposite the lesion catalyzed by the Rev1 dCMP transferase. Extension synthesis by Polζ completes the lesion bypass. This major mechanism of translesion synthesis results in A→G transition mutations.

Mentions: In yeast cells, 1,N6-ethenoadenine induces A→G transition mutations as the major event and A→T tansversion mutations as a minor event. Such a mutagenesis specificity is similar to that in the mammalian system (42). In the absence of the Rev1-Polζ translesion synthesis pathway, as in the rev3 or rev1 deletion mutant cells, replication of the damaged plasmid was dramatically reduced and the predominant A→G transition mutations were abolished. Thus, mutations induced by 1,N6-ethenoadenine DNA adducts result from replication of the damaged site via translesion synthesis by the Rev1-Polζ pathway. In a unique strain where the Rev1 protein had been mutated (Rev1D467A/E468A) inactivating its dCMP transferase but retaining its non-catalytic function in lesion bypass, translesion synthesis of 1,N6-ethenoadenine DNA adducts was dramatically reduced and A→G transition mutations were abolished, just like the rev1 deletion mutant strain. Therefore, the Rev1 dCMP transferase activity is exclusively responsible for inserting C opposite the lesion as the major mechanism of translesion synthesis and mutagenesis of 1,N6-ethenoadenine DNA adducts in yeast cells. Since Rev1 is unable to perform extension synthesis from opposite 1,N6-ethenoadenine (25), another bypass polymerase is needed to complete translesion synthesis. Without an extension polymerase, translesion synthesis and A→G transition mutations would not occur. This was indeed the case in rev3 deletion mutant cells that lack Polζ function, suggesting that Polζ acts as the extension polymerase following C insertion by Rev1 during translesion synthesis and mutagenesis of 1,N6-ethenoadenine DNA adducts. Therefore, 1,N6-ethenoadenine DNA adducts are predominantly replicated in yeast cells by Rev1-catalyzed C insertion followed by Polζ-catalyzed extension, a two-polymerase two-step mechanism of translesion synthesis (Figure 6).Figure 6.


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)

A mechanistic model for translesion synthesis of the 1,N6-ethenoadenine DNA adduct in yeast cells. The replication complex (represented by the filled blue oval) is blocked by the lesion, signaling translesion synthesis. Translesion synthesis is mediated predominantly by C insertion opposite the lesion catalyzed by the Rev1 dCMP transferase. Extension synthesis by Polζ completes the lesion bypass. This major mechanism of translesion synthesis results in A→G transition mutations.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 6: A mechanistic model for translesion synthesis of the 1,N6-ethenoadenine DNA adduct in yeast cells. The replication complex (represented by the filled blue oval) is blocked by the lesion, signaling translesion synthesis. Translesion synthesis is mediated predominantly by C insertion opposite the lesion catalyzed by the Rev1 dCMP transferase. Extension synthesis by Polζ completes the lesion bypass. This major mechanism of translesion synthesis results in A→G transition mutations.
Mentions: In yeast cells, 1,N6-ethenoadenine induces A→G transition mutations as the major event and A→T tansversion mutations as a minor event. Such a mutagenesis specificity is similar to that in the mammalian system (42). In the absence of the Rev1-Polζ translesion synthesis pathway, as in the rev3 or rev1 deletion mutant cells, replication of the damaged plasmid was dramatically reduced and the predominant A→G transition mutations were abolished. Thus, mutations induced by 1,N6-ethenoadenine DNA adducts result from replication of the damaged site via translesion synthesis by the Rev1-Polζ pathway. In a unique strain where the Rev1 protein had been mutated (Rev1D467A/E468A) inactivating its dCMP transferase but retaining its non-catalytic function in lesion bypass, translesion synthesis of 1,N6-ethenoadenine DNA adducts was dramatically reduced and A→G transition mutations were abolished, just like the rev1 deletion mutant strain. Therefore, the Rev1 dCMP transferase activity is exclusively responsible for inserting C opposite the lesion as the major mechanism of translesion synthesis and mutagenesis of 1,N6-ethenoadenine DNA adducts in yeast cells. Since Rev1 is unable to perform extension synthesis from opposite 1,N6-ethenoadenine (25), another bypass polymerase is needed to complete translesion synthesis. Without an extension polymerase, translesion synthesis and A→G transition mutations would not occur. This was indeed the case in rev3 deletion mutant cells that lack Polζ function, suggesting that Polζ acts as the extension polymerase following C insertion by Rev1 during translesion synthesis and mutagenesis of 1,N6-ethenoadenine DNA adducts. Therefore, 1,N6-ethenoadenine DNA adducts are predominantly replicated in yeast cells by Rev1-catalyzed C insertion followed by Polζ-catalyzed extension, a two-polymerase two-step mechanism of translesion synthesis (Figure 6).Figure 6.

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