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. 2005 Sep 7;24(17):2957-67.
doi: 10.1038/sj.emboj.7600786. Epub 2005 Aug 18.

Fidelity of Dpo4: Effect of Metal Ions, Nucleotide Selection and Pyrophosphorolysis

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Free PMC article

Fidelity of Dpo4: Effect of Metal Ions, Nucleotide Selection and Pyrophosphorolysis

Alexandra Vaisman et al. EMBO J. .
Free PMC article

Abstract

We report the crystal structures of a translesion DNA polymerase, Dpo4, complexed with a matched or mismatched incoming nucleotide and with a pyrophosphate product after misincorporation. These structures suggest two mechanisms by which Dpo4 may reject a wrong incoming nucleotide with its preformed and open active site. First, a mismatched replicating base pair leads to poor base stacking and alignment of the metal ions and as a consequence, inhibits incorporation. By replacing Mg2+ with Mn2+, which has a relaxed coordination requirement and tolerates misalignment, the catalytic efficiency of misincorporation increases dramatically. Mn2+ also enhances translesion synthesis by Dpo4. Subtle conformational changes that lead to the proper metal ion coordination may, therefore, be a key step in catalysis. Second, the slow release of pyrophosphate may increase the fidelity of Dpo4 by stalling mispaired primer extension and promoting pyrophosphorolysis that reverses the polymerization reaction. Indeed, Dpo4 has robust pyrophosphorolysis activity and degrades the primer strand in the presence of pyrophosphate. The correct incoming nucleotide allows DNA synthesis to overcome pyrophosphorolysis, but an incorrect incoming nucleotide does not.

Figures

Figure 1
Figure 1
Ribbon diagrams of the T/dATP (A), T/dGTP-1 (B) and T/dGTP-2 (C) and T/G (D) structures around the active site. Dpo4 is shown as ribbons. The three conserved carboxylates in the active site, the last two base pairs of the primer/template and the replicating base pair are shown as ball-and-stick models. The template strand is shown in blue and the primer strand in purple. The incoming nucleotide is shown in different colors for each crystal structure. The metal ions are shown as green spheres. The 2FoFc electron density maps are contoured at 1σ level and superimposed onto the nucleic acid portion.
Figure 2
Figure 2
Structural comparison of Dpo4 and T7 DNA polymerase. (A) The replicating base pairs in three Dpo4 structures (T/dGTP, T/dATP and Ab-2A) are shown as ball-and-stick models. The two metal ions (A and B) are shown as green spheres. The A-metal ion position differs in each structure. The conformation of the triphosphate is denoted as ‘chair-like' and ‘goat tail-like'. (B) Superposition of T/dATP, T/dGTP and Ab-2A structures. The Cα traces, DNA and nucleotide substrate are shown in stick models. A zoom-in stereo view of the finger domain with the replicating base pair and metal ions (outlined in gray) is shown on the right. The colors representing each structure are indicated. (C) Superposition of the metal ion coordination in Dpo4 (Ab-2A, yellow and brown colors) and T7 DNA polymerase (PDB: 1T7P, blue and green colors) in a stereo view. The oxygen atoms of the three conserved carboxylates and those involved in metal ion coordination are highlighted in red. The metal ion coordination is schematically drawn on the right. Red indicates ligands conserved in both polymerases, light green in Dpo4 only and blue in T7 only. The hypothesized 3′-OH of the primer strand is shown in gray.
Figure 3
Figure 3
Effect of divalent metal ion on the fidelity and lesion bypass ability of Dpo4. (A) Specificity of nucleotide incorporation. (B) Mispaired primer extension. Bypass of (C) a synthetic abasic site and (D) a cis-syn CPD. (E) Terminal nucleotidyl transferase activity of Dpo4. Reactions were performed in the presence of 5 mM Mg2+ or Mn2+. The local template sequence is shown to the left of each gel. Dpo4 concentration in each reaction was 2 nM, or as indicated at the bottom of the gels. Primer elongations were calculated as the percent of total primer termini, and are indicated at the bottom of the gels.
Figure 4
Figure 4
Pyrophosphorolytic activity of Dpo4. Comparison of polymerization and pyrophosphorolytic activities of Dpo4 with correctly paired (A) or mispaired (B) primer–template termini. Effect of increasing PPi concentrations on primer extension and degradation was studied in the absence of added dNTP (panel I), in the presence of the next correct dATP (panel II) or in the presence of incorrect dGTP nucleotide (panel III). The local template sequence is shown to the left of the gel. PPi-dependent inhibition of primer elongation (panel IV) or primer degradation (panel V) in reactions shown in panels I, II and III was calculated as percent of total primer termini. Open triangles (▵) represent the presence of dATP, open diamonds (◊) the presence of dGTP and open circles (○) the absence of dNTP.
Figure 5
Figure 5
Comparison of extension of a preformed primer/template terminal mismatch and mismatch formed as a result of nucleotide misincorporation. (A) PPi-dependent inhibition of primer extension in the presence of 100 μM incorrect dGTP and 10 μM of the next correct dCTP. (B) PPi-dependent inhibition of G:T mismatch extension in the presence of 10 μM correct dCTP and dGTP. Reactions were performed in the presence of 8 nM Dpo4. The local template sequence is shown to the left of the gels. Mismatch extension was calculated as 100 × (N1+N2+⋯+Nn)/(Nm+N1+N2+⋯+Nn). Primer elongation was calculated as 100 × (Nm+N1+N2+⋯+Nn)/(N0+Nm+N1+N2+⋯+Nn).
Figure 6
Figure 6
Comparison of pyrophosphorolytic activity of Dpo4, Dbh, Dpo4-LF-Dbh and Dbh-LF-Dpo4. (A) PPi-dependent inhibition of dATP (100 μM) incorporation and (B) pyrophosphorolysis catalyzed by 2.5 nM Dpo4 (panel I), 5 nM Dbh-LF-Dpo4 (panel II), 10 nM Dbh (panel III) or 25 nM Dpo4-LF-Dbh (panel IV). The local template sequence is shown to the left of each set of reactions.

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