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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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Structure of ternary complex (PolIVdA:dTMPnPP). (A) The structure of PolIV is displayed in ribbon representation with the palm domain coloured cyan, fingers domain coloured yellow, thumb domain coloured orange, PAD domain coloured green and linker between Thumb and PAD domain coloured wheat. DNA is displayed in stick representation, and atoms are coloured according to element. The template and incoming nucleotide—dA and dTMPnPP—are coloured according to element. The Mg2+ ions are shown in the form of blue spheres. (B) Active site of PolIV. A close up of the active site of PolIVdA:dTMPnPP is shown with the residues that form interactions with the template dA (G33, R38, G39, V40, S42, A56, T248, K291, F295 and R330) and incoming dTMPnPP (D8, M9, D10, C11, F12, F13, S42, T43, R49, S55, D103 and K157) displayed in stick representation. DNA residues and incoming nucleotide are also displayed in stick representation, and all the residues are coloured according to constituent elements. The Mg2+ ions are shown in the form of a blue spheres.
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gkt146-F1: Structure of ternary complex (PolIVdA:dTMPnPP). (A) The structure of PolIV is displayed in ribbon representation with the palm domain coloured cyan, fingers domain coloured yellow, thumb domain coloured orange, PAD domain coloured green and linker between Thumb and PAD domain coloured wheat. DNA is displayed in stick representation, and atoms are coloured according to element. The template and incoming nucleotide—dA and dTMPnPP—are coloured according to element. The Mg2+ ions are shown in the form of blue spheres. (B) Active site of PolIV. A close up of the active site of PolIVdA:dTMPnPP is shown with the residues that form interactions with the template dA (G33, R38, G39, V40, S42, A56, T248, K291, F295 and R330) and incoming dTMPnPP (D8, M9, D10, C11, F12, F13, S42, T43, R49, S55, D103 and K157) displayed in stick representation. DNA residues and incoming nucleotide are also displayed in stick representation, and all the residues are coloured according to constituent elements. The Mg2+ ions are shown in the form of a blue spheres.

Mentions: We have determined the structure of four functional complexes of PolIV corresponding to the four possible Watson–Crick pairs. To our knowledge, these are the first structures of a prokaryotic Y-family DNA polymerase captured in the functional state. The four complexes PolIVdG:dCMPnPP, PolIVdC:dGMPnPP, PolIVdA:dTMPnPP and PolIVdT:dAMPnPP crystallized in the space group P21 with roughly similar cell constants of a = 86.7 Å, b = 56.9 Å, c = 111.3 Å, α = γ = 90° and β = 94.3° (Table 1). X-ray diffraction data could be collected to a maximal resolution of 2.67, 2.33, 2.38 and 2.48 Å for the PolIVdG:dCMPnPP, PolIVdC:dGMPnPP, PolIVdA:dTMPnPP and PolIVdT:dAMPnPP complexes, respectively. In all the structures, the asymmetric unit is composed of two complexes. The final refined structure for each complex includes PolIV residues 1–342, nucleotides 1–18 for the template strand, nucleotides 1–14 to 17 for the primer strand, incoming dNTP, two Mg2+ ions and water molecules (Figure 1A). The structures show the presence of the four domains characteristic of Y-family dPols, namely, the palm (residues 1–10, 74–165), fingers (11–73), thumb (166–230) and the PAD or little finger (241–341) (Figure 1A). The palm domain houses the catalytic residues—Asp8, Asp103 and Glu104.Figure 1.


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)

Structure of ternary complex (PolIVdA:dTMPnPP). (A) The structure of PolIV is displayed in ribbon representation with the palm domain coloured cyan, fingers domain coloured yellow, thumb domain coloured orange, PAD domain coloured green and linker between Thumb and PAD domain coloured wheat. DNA is displayed in stick representation, and atoms are coloured according to element. The template and incoming nucleotide—dA and dTMPnPP—are coloured according to element. The Mg2+ ions are shown in the form of blue spheres. (B) Active site of PolIV. A close up of the active site of PolIVdA:dTMPnPP is shown with the residues that form interactions with the template dA (G33, R38, G39, V40, S42, A56, T248, K291, F295 and R330) and incoming dTMPnPP (D8, M9, D10, C11, F12, F13, S42, T43, R49, S55, D103 and K157) displayed in stick representation. DNA residues and incoming nucleotide are also displayed in stick representation, and all the residues are coloured according to constituent elements. The Mg2+ ions are shown in the form of a blue spheres.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt146-F1: Structure of ternary complex (PolIVdA:dTMPnPP). (A) The structure of PolIV is displayed in ribbon representation with the palm domain coloured cyan, fingers domain coloured yellow, thumb domain coloured orange, PAD domain coloured green and linker between Thumb and PAD domain coloured wheat. DNA is displayed in stick representation, and atoms are coloured according to element. The template and incoming nucleotide—dA and dTMPnPP—are coloured according to element. The Mg2+ ions are shown in the form of blue spheres. (B) Active site of PolIV. A close up of the active site of PolIVdA:dTMPnPP is shown with the residues that form interactions with the template dA (G33, R38, G39, V40, S42, A56, T248, K291, F295 and R330) and incoming dTMPnPP (D8, M9, D10, C11, F12, F13, S42, T43, R49, S55, D103 and K157) displayed in stick representation. DNA residues and incoming nucleotide are also displayed in stick representation, and all the residues are coloured according to constituent elements. The Mg2+ ions are shown in the form of a blue spheres.
Mentions: We have determined the structure of four functional complexes of PolIV corresponding to the four possible Watson–Crick pairs. To our knowledge, these are the first structures of a prokaryotic Y-family DNA polymerase captured in the functional state. The four complexes PolIVdG:dCMPnPP, PolIVdC:dGMPnPP, PolIVdA:dTMPnPP and PolIVdT:dAMPnPP crystallized in the space group P21 with roughly similar cell constants of a = 86.7 Å, b = 56.9 Å, c = 111.3 Å, α = γ = 90° and β = 94.3° (Table 1). X-ray diffraction data could be collected to a maximal resolution of 2.67, 2.33, 2.38 and 2.48 Å for the PolIVdG:dCMPnPP, PolIVdC:dGMPnPP, PolIVdA:dTMPnPP and PolIVdT:dAMPnPP complexes, respectively. In all the structures, the asymmetric unit is composed of two complexes. The final refined structure for each complex includes PolIV residues 1–342, nucleotides 1–18 for the template strand, nucleotides 1–14 to 17 for the primer strand, incoming dNTP, two Mg2+ ions and water molecules (Figure 1A). The structures show the presence of the four domains characteristic of Y-family dPols, namely, the palm (residues 1–10, 74–165), fingers (11–73), thumb (166–230) and the PAD or little finger (241–341) (Figure 1A). The palm domain houses the catalytic residues—Asp8, Asp103 and Glu104.Figure 1.

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