Invariant Asp-1122 and Asp-1124 are essential residues for polymerization catalysis of family D DNA polymerase from Pyrococcus horikoshii

Family D DNA polymerase has recently been found in the Euryarchaeota subdom ain of Archaea. Its genes are adjacent to several other genes related to DN A replication, repair, and recombination in the genome, suggesting that thi s enzyme may be the major DNA replicase in Euryarchaeota. Although it, poss esses strong polymerization and proofreading activities, the motifs common to other DNA polymerase families are absent in its sequences. Here we repor t the mapping of the catalytic residues in a family D DNA polymerase from P yrococcus horikoshii. Site-directed alanine mutants for 28 conserved aspart ic acid or glutamic acid residues mere screened for polymerization and 3'-5 ' exonuclease activities. We identified the invariant aspartates Asp-1122 a nd Asp-1124 within the most conserved motif as the catalytic residues invol ved in DNA polymerization. Alanine mutation at either site caused a loss of polymerization activity, whereas the conserved mutants, D1122E, D1124N, an d D1124E, had slightly reduced polymerization activity. We also found that the 3'-5' exonuclease activity remains in D1122A and D1124A, indicating tha t the catalytic residues of DNA polymerization are different from those of the 3'-5' exonuclease activity. Furthermore me determined the molecular mas s of the recombinant enzyme by gel filtration and proposed a heterotetramer ic structure for this enzyme.