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J Biol Chem. 2014 Jan 31;289(5):2888-98. doi: 10.1074/jbc.M113.530980. Epub 2013 Dec 9.

Role of the LEXE motif of protein-primed DNA polymerases in the interaction with the incoming nucleotide.

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From the Instituto de Biología Molecular "Eladio Viñuela" (Consejo Superior de Investigaciones Científicas), Centro de Biología Molecular "Severo Ochoa" (Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid), C/Nicolás Cabrera 1, Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain.


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 ϕ29 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 Mg(2+) ions, a reaction highly improved when Mn(2+) was used as metal activator. These results, together with previous crystallographic resolution of ϕ29 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 Mg(2+). Recovery of both reactions with Mn(2+) supports a role for Glu-486 in the interaction with the pyrophosphate moiety of the dNTP.


Bacteriophage; DNA Polymerase; DNA Replication; Nucleic Acid Synthesis; Viral Polymerase

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