Role of the LExE Motif of Protein-primed DNA Polymerases in the Interaction with the Incoming Nucleotide [Enzymology]

December 9th, 2013 by Santos, E., L&aacutezaro, J. M., P&eacuterez-Arnaiz, P., Salas, M., de Vega, M.

The LExE motif, conserved in eukaryotic type DNA polymerases, is placed close to the polymerization active site. Previous studies suggested that the second glutamate was involved in binding a third non-catalytic ion in bacteriophage RB69 DNA polymerase. In the protein-primed DNA polymerases 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 φ29 DNA polymerase residues Gly481, Trp483, Ala484 and Glu486. We show that mutations at Gly481 and Trp483 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 φ29 DNA polymerase ternary complex allow us to infer that Gly481 and Trp483 could form a pocket that orients Val250 to interact with the dNTP. Mutants at Glu486 are also defective in polymerization and, as mutants at Gly481 and Trp483, in the pyrophosphorolytic activity with Mg2+. Recovery of both reactions with Mn2+ supports a role for Glu486 in the interaction with the pyrophosphate moiety of the dNTP.