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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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Related in: MedlinePlus

Mutator activity of wtPolIV and Ser42Ala mutant. The bar graph displays the number of colonies obtained on plates containing rifampicin (100 µg/ml) after overnight incubation (37°C) of a dinB deleted strain [dinB749(del)::kan] transformed with (i) pDJNNwtdb, (ii) pDJNNSER42ALAdb and (iii) empty vector.
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gkt146-F4: Mutator activity of wtPolIV and Ser42Ala mutant. The bar graph displays the number of colonies obtained on plates containing rifampicin (100 µg/ml) after overnight incubation (37°C) of a dinB deleted strain [dinB749(del)::kan] transformed with (i) pDJNNwtdb, (ii) pDJNNSER42ALAdb and (iii) empty vector.

Mentions: We carried out an in vivo assay to estimate the mutator activity of Ser42Ala mutant in comparison with wtPolIV. The assay probed for the appearance of rifampicin-resistant colonies when PolIV was expressed ectopically from a plasmid in a strain wherein the dinB gene had been deleted. The bactericidal activity of rifampicin has been attributed to its ability to bind close to the active site of the β-subunit of RNA polymerase (Rpo) and prevent elongation of the nascent RNA transcripts (46). It has been seen earlier that substitution mutations at key locations in the rpoB gene render resistance to E. coli against this antibiotic. Consequently, the designed assay is an appropriate tool to evaluate the ability of wtPolIV and the Ser42Ala mutant to generate substitution mutations. The results clearly show that a plasmid bearing the wtPolIV gene gave rise to nearly five-fold the number of colonies observed for a plasmid with the Ser42Ala mutant or for empty vector (Figure 4). Also, in this assay, the number of colonies obtained with the plasmid bearing the mutated dinB gene and empty vector were nearly equal. These observations show that the Ser42 residue plays a critical role in the ability of PolIV to create substitution mutations.Figure 4.


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)

Mutator activity of wtPolIV and Ser42Ala mutant. The bar graph displays the number of colonies obtained on plates containing rifampicin (100 µg/ml) after overnight incubation (37°C) of a dinB deleted strain [dinB749(del)::kan] transformed with (i) pDJNNwtdb, (ii) pDJNNSER42ALAdb and (iii) empty vector.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt146-F4: Mutator activity of wtPolIV and Ser42Ala mutant. The bar graph displays the number of colonies obtained on plates containing rifampicin (100 µg/ml) after overnight incubation (37°C) of a dinB deleted strain [dinB749(del)::kan] transformed with (i) pDJNNwtdb, (ii) pDJNNSER42ALAdb and (iii) empty vector.
Mentions: We carried out an in vivo assay to estimate the mutator activity of Ser42Ala mutant in comparison with wtPolIV. The assay probed for the appearance of rifampicin-resistant colonies when PolIV was expressed ectopically from a plasmid in a strain wherein the dinB gene had been deleted. The bactericidal activity of rifampicin has been attributed to its ability to bind close to the active site of the β-subunit of RNA polymerase (Rpo) and prevent elongation of the nascent RNA transcripts (46). It has been seen earlier that substitution mutations at key locations in the rpoB gene render resistance to E. coli against this antibiotic. Consequently, the designed assay is an appropriate tool to evaluate the ability of wtPolIV and the Ser42Ala mutant to generate substitution mutations. The results clearly show that a plasmid bearing the wtPolIV gene gave rise to nearly five-fold the number of colonies observed for a plasmid with the Ser42Ala mutant or for empty vector (Figure 4). Also, in this assay, the number of colonies obtained with the plasmid bearing the mutated dinB gene and empty vector were nearly equal. These observations show that the Ser42 residue plays a critical role in the ability of PolIV to create substitution mutations.Figure 4.

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