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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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Ser42 interacts with bases of the nascent base pair. The figure shows the interactions between Ser42 and (A) template dG and incoming dCMPnPP in the case of PolIVdG:dCMPnPP, (B) incoming dGMPnPP in the case of PolIVdC:dGMPnPP, (C) template dA and incoming dTMPnPP in the case of PolIVdA:dTMPnPP and (D) incoming dAMPnPP in the case of PolIVdT:dAMPnPP. The amino acid and nucleotide residues are shown in stick representations and coloured according to element, and water molecules are shown in the form of red spheres. The direct and water-mediated hydrogen bonds formed between Ser42 and the bases are highlighted by dotted lines.
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gkt146-F2: Ser42 interacts with bases of the nascent base pair. The figure shows the interactions between Ser42 and (A) template dG and incoming dCMPnPP in the case of PolIVdG:dCMPnPP, (B) incoming dGMPnPP in the case of PolIVdC:dGMPnPP, (C) template dA and incoming dTMPnPP in the case of PolIVdA:dTMPnPP and (D) incoming dAMPnPP in the case of PolIVdT:dAMPnPP. The amino acid and nucleotide residues are shown in stick representations and coloured according to element, and water molecules are shown in the form of red spheres. The direct and water-mediated hydrogen bonds formed between Ser42 and the bases are highlighted by dotted lines.

Mentions: The side chains of majority of the residues that line the active site form van der Waal interactions with the atoms of the base moieties of the incipient base pair. However, unlike the other residues, Ser42 forms direct and water-mediated polar interactions with the atoms of the bases in the nascent base pair in all four complexes (Figure 2). This residue is located close to the midline of the Watson–Crick base pair towards the minor groove and is, therefore, positioned correctly to interact with the base atoms. In the case of PolIVdG:dCMPnPP complex, the –OH group of the side chain of this residue forms a hydrogen bond with the N2 atom (2.7 Å) of the template guanine and the O2 atom (3.3 Å) of the incoming nucleotide dCMPnPP (Figure 2A). In the PolIVdC:dGMPnPP complex, this residue forms both direct (3.0 Å) and water-mediated hydrogen bonds with the N2 atom of dGMPnPP (Figure 2B). For the PolIVdA:dTMPnPP complex, Ser42 forms direct (2.9 Å) and water-mediated interactions with the O2 atom of dTMPnPP (Figure 2C). In addition, Ser42 interacts with the N3 atom of dA through two bridging water molecules. Finally, in the case of the PolIVdT:dAMPnPP complex, Ser42 forms an interaction with the N3 atom (3.2 Å) of dAMPnPP (Figure 2D). Overall, it is clear that the Ser42 residue interacts with the bases of the nascent base pair and could influence fidelity of replication.Figure 2.


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)

Ser42 interacts with bases of the nascent base pair. The figure shows the interactions between Ser42 and (A) template dG and incoming dCMPnPP in the case of PolIVdG:dCMPnPP, (B) incoming dGMPnPP in the case of PolIVdC:dGMPnPP, (C) template dA and incoming dTMPnPP in the case of PolIVdA:dTMPnPP and (D) incoming dAMPnPP in the case of PolIVdT:dAMPnPP. The amino acid and nucleotide residues are shown in stick representations and coloured according to element, and water molecules are shown in the form of red spheres. The direct and water-mediated hydrogen bonds formed between Ser42 and the bases are highlighted by dotted lines.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt146-F2: Ser42 interacts with bases of the nascent base pair. The figure shows the interactions between Ser42 and (A) template dG and incoming dCMPnPP in the case of PolIVdG:dCMPnPP, (B) incoming dGMPnPP in the case of PolIVdC:dGMPnPP, (C) template dA and incoming dTMPnPP in the case of PolIVdA:dTMPnPP and (D) incoming dAMPnPP in the case of PolIVdT:dAMPnPP. The amino acid and nucleotide residues are shown in stick representations and coloured according to element, and water molecules are shown in the form of red spheres. The direct and water-mediated hydrogen bonds formed between Ser42 and the bases are highlighted by dotted lines.
Mentions: The side chains of majority of the residues that line the active site form van der Waal interactions with the atoms of the base moieties of the incipient base pair. However, unlike the other residues, Ser42 forms direct and water-mediated polar interactions with the atoms of the bases in the nascent base pair in all four complexes (Figure 2). This residue is located close to the midline of the Watson–Crick base pair towards the minor groove and is, therefore, positioned correctly to interact with the base atoms. In the case of PolIVdG:dCMPnPP complex, the –OH group of the side chain of this residue forms a hydrogen bond with the N2 atom (2.7 Å) of the template guanine and the O2 atom (3.3 Å) of the incoming nucleotide dCMPnPP (Figure 2A). In the PolIVdC:dGMPnPP complex, this residue forms both direct (3.0 Å) and water-mediated hydrogen bonds with the N2 atom of dGMPnPP (Figure 2B). For the PolIVdA:dTMPnPP complex, Ser42 forms direct (2.9 Å) and water-mediated interactions with the O2 atom of dTMPnPP (Figure 2C). In addition, Ser42 interacts with the N3 atom of dA through two bridging water molecules. Finally, in the case of the PolIVdT:dAMPnPP complex, Ser42 forms an interaction with the N3 atom (3.2 Å) of dAMPnPP (Figure 2D). Overall, it is clear that the Ser42 residue interacts with the bases of the nascent base pair and could influence fidelity of replication.Figure 2.

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