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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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A:C base pair in the Pol IV active site. The Ser42 and other residues lining the active site are coloured according to element and shown in stick representation. Water molecules are shown in the form of red spheres. The direct and water-mediated interactions formed between Ser42 and the O2 atom of incoming dCTP and N3 atom of dA, respectively, are shown in the form of dotted lines.
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gkt146-F5: A:C base pair in the Pol IV active site. The Ser42 and other residues lining the active site are coloured according to element and shown in stick representation. Water molecules are shown in the form of red spheres. The direct and water-mediated interactions formed between Ser42 and the O2 atom of incoming dCTP and N3 atom of dA, respectively, are shown in the form of dotted lines.

Mentions: In the PolIVdA:dCTP complex, the Ser42 residue forms direct interactions with the O2 atom of dCTP nucleotide (3.0 Å). In addition, this residue also forms water-mediated hydrogen bonds with the incoming and template nucleotide (Figure 5). The enzyme stabilizes the mismatch in a configuration similar to that of the Watson–Crick base pair with a C1′–C1′ distance of 10.5 Å (Figure 5). In the observed configuration, two hydrogen bond acceptors (N1 from dA and N3 from dCTP:2.7 Å) and two hydrogen bond donors (N6 from dA and N4 from dCTP:2.8 Å) are placed close to each other. Unless the N3 of dCTP is protonated, there does not seem to be any hydrogen-bonding possible between the adenine and cytosine bases. The interaction of the Ser42 residue with the template dA and the incoming dCTP, therefore, ensures that the incoming nucleotide is stabilized in a conformation compatible with productive catalysis. This observation is consistent with the inference that the Ser42 residue plays a critical role in the ability of PolIV to create substitution mutations.Figure 5.


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)

A:C base pair in the Pol IV active site. The Ser42 and other residues lining the active site are coloured according to element and shown in stick representation. Water molecules are shown in the form of red spheres. The direct and water-mediated interactions formed between Ser42 and the O2 atom of incoming dCTP and N3 atom of dA, respectively, are shown in the form of dotted lines.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt146-F5: A:C base pair in the Pol IV active site. The Ser42 and other residues lining the active site are coloured according to element and shown in stick representation. Water molecules are shown in the form of red spheres. The direct and water-mediated interactions formed between Ser42 and the O2 atom of incoming dCTP and N3 atom of dA, respectively, are shown in the form of dotted lines.
Mentions: In the PolIVdA:dCTP complex, the Ser42 residue forms direct interactions with the O2 atom of dCTP nucleotide (3.0 Å). In addition, this residue also forms water-mediated hydrogen bonds with the incoming and template nucleotide (Figure 5). The enzyme stabilizes the mismatch in a configuration similar to that of the Watson–Crick base pair with a C1′–C1′ distance of 10.5 Å (Figure 5). In the observed configuration, two hydrogen bond acceptors (N1 from dA and N3 from dCTP:2.7 Å) and two hydrogen bond donors (N6 from dA and N4 from dCTP:2.8 Å) are placed close to each other. Unless the N3 of dCTP is protonated, there does not seem to be any hydrogen-bonding possible between the adenine and cytosine bases. The interaction of the Ser42 residue with the template dA and the incoming dCTP, therefore, ensures that the incoming nucleotide is stabilized in a conformation compatible with productive catalysis. This observation is consistent with the inference that the Ser42 residue plays a critical role in the ability of PolIV to create substitution mutations.Figure 5.

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