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Substitution of a residue contacting the triphosphate moiety of the incoming nucleotide increases the fidelity of yeast DNA polymerase zeta.

Howell CA, Kondratick CM, Washington MT - Nucleic Acids Res. (2008)

Bottom Line: The R1057A and K1086A proteins do not complement the rev3Delta mutation, and these proteins have significantly reduced polymerase activity relative to the wild-type protein.In contrast, the K1061A protein partially complements the rev3Delta mutation and has nearly normal polymerase activity.Interestingly, the K1061A protein has increased fidelity relative to wild-type pol zeta and is somewhat less efficient at extending from mismatched primer-terminal base pairs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Iowa College of Medicine, Iowa City, IA 52242-1109, USA.

ABSTRACT
DNA polymerase zeta (pol zeta), which is required for DNA damage-induced mutagenesis, functions in the error-prone replication of a wide range of DNA lesions. During this process, pol zeta extends from nucleotides incorporated opposite template lesions by other polymerases. Unlike classical polymerases, pol zeta efficiently extends from primer-terminal base pairs containing mismatches or lesions, and it synthesizes DNA with moderate fidelity. Here we describe genetic and biochemical studies of three yeast pol zeta mutant proteins containing substitutions of highly conserved amino acid residues that contact the triphosphate moiety of the incoming nucleotide. The R1057A and K1086A proteins do not complement the rev3Delta mutation, and these proteins have significantly reduced polymerase activity relative to the wild-type protein. In contrast, the K1061A protein partially complements the rev3Delta mutation and has nearly normal polymerase activity. Interestingly, the K1061A protein has increased fidelity relative to wild-type pol zeta and is somewhat less efficient at extending from mismatched primer-terminal base pairs. These findings have important implications both for the evolutionary divergence of pol zeta from classical polymerases and for the mechanism by which this enzyme accommodates distortions in the DNA caused by mismatches and lesions.

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Genetic complementation analysis of the pol ζ mutant proteins. (A) The UV sensitivity of yeast strains lacking pol ζ (rev3Δ) harboring plasmids producing either no pol ζ (open circle), wild-type pol ζ (filled circle), the R1057A mutant protein (inverted filled triangle), the K1061A mutant protein (inverted open triangle) or the K1086A mutant protein (filled square). The percentage of cells surviving each UV dose is shown. (B) The UV-induced mutagenesis of these same yeast strains. The number of CAN1S to can1r forward mutations per 107 surviving cells for each UV dose is shown.
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Figure 2: Genetic complementation analysis of the pol ζ mutant proteins. (A) The UV sensitivity of yeast strains lacking pol ζ (rev3Δ) harboring plasmids producing either no pol ζ (open circle), wild-type pol ζ (filled circle), the R1057A mutant protein (inverted filled triangle), the K1061A mutant protein (inverted open triangle) or the K1086A mutant protein (filled square). The percentage of cells surviving each UV dose is shown. (B) The UV-induced mutagenesis of these same yeast strains. The number of CAN1S to can1r forward mutations per 107 surviving cells for each UV dose is shown.

Mentions: We carried out genetic analyses to assay the function of the pol ζ mutant proteins in vivo. We first assayed them for the ability to complement the sensitivity of the rev3Δ yeast strain (Figure 2A). Yeast expressing the wild-type pol ζ are more resistant to UV radiation than yeast lacking pol ζ. Yeast expressing either the R1057A or the K1086A mutant proteins were as sensitive to UV-radiation as the strain lacking pol ζ. Yeast expressing the K1061A mutant protein were less sensitive to UV-radiation than yeast lacking pol ζ, but were not as resistant as yeast expressing wild-type pol ζ.Figure 2.


Substitution of a residue contacting the triphosphate moiety of the incoming nucleotide increases the fidelity of yeast DNA polymerase zeta.

Howell CA, Kondratick CM, Washington MT - Nucleic Acids Res. (2008)

Genetic complementation analysis of the pol ζ mutant proteins. (A) The UV sensitivity of yeast strains lacking pol ζ (rev3Δ) harboring plasmids producing either no pol ζ (open circle), wild-type pol ζ (filled circle), the R1057A mutant protein (inverted filled triangle), the K1061A mutant protein (inverted open triangle) or the K1086A mutant protein (filled square). The percentage of cells surviving each UV dose is shown. (B) The UV-induced mutagenesis of these same yeast strains. The number of CAN1S to can1r forward mutations per 107 surviving cells for each UV dose is shown.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2275142&req=5

Figure 2: Genetic complementation analysis of the pol ζ mutant proteins. (A) The UV sensitivity of yeast strains lacking pol ζ (rev3Δ) harboring plasmids producing either no pol ζ (open circle), wild-type pol ζ (filled circle), the R1057A mutant protein (inverted filled triangle), the K1061A mutant protein (inverted open triangle) or the K1086A mutant protein (filled square). The percentage of cells surviving each UV dose is shown. (B) The UV-induced mutagenesis of these same yeast strains. The number of CAN1S to can1r forward mutations per 107 surviving cells for each UV dose is shown.
Mentions: We carried out genetic analyses to assay the function of the pol ζ mutant proteins in vivo. We first assayed them for the ability to complement the sensitivity of the rev3Δ yeast strain (Figure 2A). Yeast expressing the wild-type pol ζ are more resistant to UV radiation than yeast lacking pol ζ. Yeast expressing either the R1057A or the K1086A mutant proteins were as sensitive to UV-radiation as the strain lacking pol ζ. Yeast expressing the K1061A mutant protein were less sensitive to UV-radiation than yeast lacking pol ζ, but were not as resistant as yeast expressing wild-type pol ζ.Figure 2.

Bottom Line: The R1057A and K1086A proteins do not complement the rev3Delta mutation, and these proteins have significantly reduced polymerase activity relative to the wild-type protein.In contrast, the K1061A protein partially complements the rev3Delta mutation and has nearly normal polymerase activity.Interestingly, the K1061A protein has increased fidelity relative to wild-type pol zeta and is somewhat less efficient at extending from mismatched primer-terminal base pairs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Iowa College of Medicine, Iowa City, IA 52242-1109, USA.

ABSTRACT
DNA polymerase zeta (pol zeta), which is required for DNA damage-induced mutagenesis, functions in the error-prone replication of a wide range of DNA lesions. During this process, pol zeta extends from nucleotides incorporated opposite template lesions by other polymerases. Unlike classical polymerases, pol zeta efficiently extends from primer-terminal base pairs containing mismatches or lesions, and it synthesizes DNA with moderate fidelity. Here we describe genetic and biochemical studies of three yeast pol zeta mutant proteins containing substitutions of highly conserved amino acid residues that contact the triphosphate moiety of the incoming nucleotide. The R1057A and K1086A proteins do not complement the rev3Delta mutation, and these proteins have significantly reduced polymerase activity relative to the wild-type protein. In contrast, the K1061A protein partially complements the rev3Delta mutation and has nearly normal polymerase activity. Interestingly, the K1061A protein has increased fidelity relative to wild-type pol zeta and is somewhat less efficient at extending from mismatched primer-terminal base pairs. These findings have important implications both for the evolutionary divergence of pol zeta from classical polymerases and for the mechanism by which this enzyme accommodates distortions in the DNA caused by mismatches and lesions.

Show MeSH
Related in: MedlinePlus