Fidelity of Dpo4: effect of metal ions, nucleotide selection and pyrophosphorolysis

We report the crystal structures of a translesion DNA polymerase, Dpo4, complexed with a matched or mis-matched 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 Mg2þ with Mn2þ, which has a relaxed coordination requirement and tolerates misalignment, the catalytic efficiency of misincorporation increases dramatically. Mn2þ also enhances translesion synthesis by Dpo4. Subtle conforma-tional changes that lead to the proper metal ion coordina-tion 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 promot-ing 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