Many DNA polymerases have conserved sequences required for 3'-->5' exonuclease activity, which contributes to the accuracy of DNA replication by removing misincorporated nucleotides prior to chain elongation. Using amino acid sequence alignments, we predicted the putative active site of the 3'-->5' exonuclease of Escherichia coli DNA polymerase II. Site-directed mutagenesis at D155A, E157A, D155A/E157A, D228A, Y330F, and D334A, which are in the predicted exonuclease active regions, specifically inactivated 3'-->5' exonucleolytic activity but not DNA-polymerizing activity of E. coli DNA polymerase II. Furthermore, all of the mutants were diminished in the in vitro proofreading ability, as judged by their increased insertion and extension of wrong nucleotides. These findings indicate that the 3'-->5' exonuclease region of the E. coli DNA polymerase II is in the amino-terminal part of the protein, as it is in other DNA polymerases, and are consistent with the proposal of an evolutionary conserved 3'-->5' exonuclease active site in most DNA-dependent DNA polymerases of both prokaryotic and eukaryotic origin by Bernad et al. (Bernad, A., Blanco, L., Lazaro, J. M., Martin, G., and Salas, M. (1989) Cell 59, 219-228).
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