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A strategically located serine residue is critical for the mutator activity of DNA polymerase IV from Escherichia coli.

Sharma A, Kottur J, Narayanan N, Nair DT - Nucleic Acids Res. (2013)

Bottom Line: In vitro and in vivo assays show that the fidelity of the PolIV enzyme increases drastically when this Ser residue was mutated to Ala.In addition, the structure of PolIV with the mismatch A:C in the active site shows that the Ser42 residue plays an important role in stabilizing dCTP in a conformation compatible with catalysis.Overall, the structural, biochemical and functional data presented here show that the Ser42 residue is present at a strategic location to stabilize mismatches in the PolIV active site, and thus facilitate the appearance of transition and transversion mutations.

View Article: PubMed Central - PubMed

Affiliation: National Centre for Biological Sciences (NCBS-TIFR), UAS-GKVK Campus, Bellary Road, Bangalore 560065, India.

ABSTRACT
The Y-family DNA polymerase IV or PolIV (Escherichia coli) is the founding member of the DinB family and is known to play an important role in stress-induced mutagenesis. We have determined four crystal structures of this enzyme in its pre-catalytic state in complex with substrate DNA presenting the four possible template nucleotides that are paired with the corresponding incoming nucleotide triphosphates. In all four structures, the Ser42 residue in the active site forms interactions with the base moieties of the incipient Watson-Crick base pair. This residue is located close to the centre of the nascent base pair towards the minor groove. In vitro and in vivo assays show that the fidelity of the PolIV enzyme increases drastically when this Ser residue was mutated to Ala. In addition, the structure of PolIV with the mismatch A:C in the active site shows that the Ser42 residue plays an important role in stabilizing dCTP in a conformation compatible with catalysis. Overall, the structural, biochemical and functional data presented here show that the Ser42 residue is present at a strategic location to stabilize mismatches in the PolIV active site, and thus facilitate the appearance of transition and transversion mutations.

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Fidelity profile of wt PolIV and the mutant Ser42Ala. The results for primer extension assay wherein wtPolIV (A) or the Ser42Ala mutant (B) was incubated with DNA duplexes (TAP*, TTP*, TGP* and TCP*) and different dNTPs are displayed. (B) 3D plot exhibiting the misincorporation profile of wtPolIV and the mutant. Incorporation was quantitated in the form of percentage of primer extended. The level of incorporation of the incorrect nucleotide is plotted on the z-axis for different template nucleotides (dA, dT, dG and dC; y-axis), for all four incoming nucleotides (dATP, dTTP, dGTP and dCTP; x-axis) for the two proteins (wtPolIV and Ser42Ala; x-axis).
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gkt146-F3: Fidelity profile of wt PolIV and the mutant Ser42Ala. The results for primer extension assay wherein wtPolIV (A) or the Ser42Ala mutant (B) was incubated with DNA duplexes (TAP*, TTP*, TGP* and TCP*) and different dNTPs are displayed. (B) 3D plot exhibiting the misincorporation profile of wtPolIV and the mutant. Incorporation was quantitated in the form of percentage of primer extended. The level of incorporation of the incorrect nucleotide is plotted on the z-axis for different template nucleotides (dA, dT, dG and dC; y-axis), for all four incoming nucleotides (dATP, dTTP, dGTP and dCTP; x-axis) for the two proteins (wtPolIV and Ser42Ala; x-axis).

Mentions: To further probe the role of Ser42 in influencing fidelity, we mutated this residue to Ala. Primer extension assays were carried out to assess the ability of the mutant protein to incorporate the correct and incorrect nucleotide opposite all four possible template nucleotides in comparison with native PolIV. In the presence of the correct nucleotide, both wt and mutant enzyme showed maximal extension of the primers. For the reaction conditions used, ∼80% of the primer was extended on addition of the correct nucleotide in case of all the templates (Figure 3A). However, the wild-type (wt) enzyme exhibited significant misincorporation for a number of different combinations of template and incoming nucleotide (Figure 3A). For template nucleotide dA, the wtPolIV exhibited significant incorporation of dCTP. Also, all three incorrect nucleotides dTTP, dGTP and dCTP could be incorporated opposite template dT. Additionally, dATP and dTTP were added opposite template dG. Finally, when the template nucleotide was dC, there was significant misincorporation in the presence of all three incorrect nucleotides (Figure 3A) with maximal extension in case of dTTP (∼20%). In comparison, the Ser42Ala mutant exhibited substantially higher fidelity than the native enzyme with no misincorporation opposite the two template nucleotides dA and dG (Figure 3B). Marginal incorporation of dTTP opposite dT was observed and misincorporation opposite dC was considerably reduced and observed only in case of dTTP (∼7%). Also, in case of wtPolIV, significant incorporation of dTTP, dATP and dCTP was observed opposite nucleotides 5′ of the correct template nucleotide dA, dT and dG, respectively (Figure 3A). However, in case of the Ser42Ala, these additions were not observed (Figure 3B). Overall, the primer extension assays show that the fidelity of the Ser42Ala mutant is substantially higher than the wtPolIV (Figure 3C).Figure 3.


A strategically located serine residue is critical for the mutator activity of DNA polymerase IV from Escherichia coli.

Sharma A, Kottur J, Narayanan N, Nair DT - Nucleic Acids Res. (2013)

Fidelity profile of wt PolIV and the mutant Ser42Ala. The results for primer extension assay wherein wtPolIV (A) or the Ser42Ala mutant (B) was incubated with DNA duplexes (TAP*, TTP*, TGP* and TCP*) and different dNTPs are displayed. (B) 3D plot exhibiting the misincorporation profile of wtPolIV and the mutant. Incorporation was quantitated in the form of percentage of primer extended. The level of incorporation of the incorrect nucleotide is plotted on the z-axis for different template nucleotides (dA, dT, dG and dC; y-axis), for all four incoming nucleotides (dATP, dTTP, dGTP and dCTP; x-axis) for the two proteins (wtPolIV and Ser42Ala; x-axis).
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3643571&req=5

gkt146-F3: Fidelity profile of wt PolIV and the mutant Ser42Ala. The results for primer extension assay wherein wtPolIV (A) or the Ser42Ala mutant (B) was incubated with DNA duplexes (TAP*, TTP*, TGP* and TCP*) and different dNTPs are displayed. (B) 3D plot exhibiting the misincorporation profile of wtPolIV and the mutant. Incorporation was quantitated in the form of percentage of primer extended. The level of incorporation of the incorrect nucleotide is plotted on the z-axis for different template nucleotides (dA, dT, dG and dC; y-axis), for all four incoming nucleotides (dATP, dTTP, dGTP and dCTP; x-axis) for the two proteins (wtPolIV and Ser42Ala; x-axis).
Mentions: To further probe the role of Ser42 in influencing fidelity, we mutated this residue to Ala. Primer extension assays were carried out to assess the ability of the mutant protein to incorporate the correct and incorrect nucleotide opposite all four possible template nucleotides in comparison with native PolIV. In the presence of the correct nucleotide, both wt and mutant enzyme showed maximal extension of the primers. For the reaction conditions used, ∼80% of the primer was extended on addition of the correct nucleotide in case of all the templates (Figure 3A). However, the wild-type (wt) enzyme exhibited significant misincorporation for a number of different combinations of template and incoming nucleotide (Figure 3A). For template nucleotide dA, the wtPolIV exhibited significant incorporation of dCTP. Also, all three incorrect nucleotides dTTP, dGTP and dCTP could be incorporated opposite template dT. Additionally, dATP and dTTP were added opposite template dG. Finally, when the template nucleotide was dC, there was significant misincorporation in the presence of all three incorrect nucleotides (Figure 3A) with maximal extension in case of dTTP (∼20%). In comparison, the Ser42Ala mutant exhibited substantially higher fidelity than the native enzyme with no misincorporation opposite the two template nucleotides dA and dG (Figure 3B). Marginal incorporation of dTTP opposite dT was observed and misincorporation opposite dC was considerably reduced and observed only in case of dTTP (∼7%). Also, in case of wtPolIV, significant incorporation of dTTP, dATP and dCTP was observed opposite nucleotides 5′ of the correct template nucleotide dA, dT and dG, respectively (Figure 3A). However, in case of the Ser42Ala, these additions were not observed (Figure 3B). Overall, the primer extension assays show that the fidelity of the Ser42Ala mutant is substantially higher than the wtPolIV (Figure 3C).Figure 3.

Bottom Line: In vitro and in vivo assays show that the fidelity of the PolIV enzyme increases drastically when this Ser residue was mutated to Ala.In addition, the structure of PolIV with the mismatch A:C in the active site shows that the Ser42 residue plays an important role in stabilizing dCTP in a conformation compatible with catalysis.Overall, the structural, biochemical and functional data presented here show that the Ser42 residue is present at a strategic location to stabilize mismatches in the PolIV active site, and thus facilitate the appearance of transition and transversion mutations.

View Article: PubMed Central - PubMed

Affiliation: National Centre for Biological Sciences (NCBS-TIFR), UAS-GKVK Campus, Bellary Road, Bangalore 560065, India.

ABSTRACT
The Y-family DNA polymerase IV or PolIV (Escherichia coli) is the founding member of the DinB family and is known to play an important role in stress-induced mutagenesis. We have determined four crystal structures of this enzyme in its pre-catalytic state in complex with substrate DNA presenting the four possible template nucleotides that are paired with the corresponding incoming nucleotide triphosphates. In all four structures, the Ser42 residue in the active site forms interactions with the base moieties of the incipient Watson-Crick base pair. This residue is located close to the centre of the nascent base pair towards the minor groove. In vitro and in vivo assays show that the fidelity of the PolIV enzyme increases drastically when this Ser residue was mutated to Ala. In addition, the structure of PolIV with the mismatch A:C in the active site shows that the Ser42 residue plays an important role in stabilizing dCTP in a conformation compatible with catalysis. Overall, the structural, biochemical and functional data presented here show that the Ser42 residue is present at a strategic location to stabilize mismatches in the PolIV active site, and thus facilitate the appearance of transition and transversion mutations.

Show MeSH
Related in: MedlinePlus