Role of the LEXE Motif of Protein-primed DNA Polymerases in the Interaction with the Incoming Nucleotide

Background: LEXE motif is conserved among the eukaryotic DNA polymerases. Results: Substitutions at LEXE residues decrease the replication and pyrophosphorolytic activities of 29 DNA polymerase and compromise nucleotide insertion fidelity. Conclusion: The results suggest a role for LEXE residues in making interactions with the incoming nucleotide. Significance: Results support the proposal of a third metal ion as a general feature of the two-metal ion mechanism. The LEXE motif, conserved in eukaryotic type DNA polymerases, is placed close to the polymerization active site. Previous studies suggested that the second Glu was involved in binding a third noncatalytic ion in bacteriophage RB69 DNA polymerase. In the protein-primed DNA polymerase subgroup, the LEXE motif lacks the first Glu in most cases, but it has a conserved Phe/Trp and a Gly preceding that position. To ascertain the role of those residues, we have analyzed the behavior of mutants at the corresponding phi 29 DNA polymerase residues Gly-481, Trp-483, Ala-484, and Glu-486. We show that mutations at Gly-481 and Trp-483 hamper insertion of the incoming dNTP in the presence of Mg2+ ions, a reaction highly improved when Mn2+ was used as metal activator. These results, together with previous crystallographic resolution of phi 29 DNA polymerase ternary complex, allow us to infer that Gly-481 and Trp-483 could form a pocket that orients Val-250 to interact with the dNTP. Mutants at Glu-486 are also defective in polymerization and, as mutants at Gly-481 and Trp-483, in the pyrophosphorolytic activity with Mg2+. Recovery of both reactions with Mn2+ supports a role for Glu-486 in the interaction with the pyrophosphate moiety of the dNTP.