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 subdomain of Archaea. Its genes are adjacent to several other genes related to DNA replication, repair, and recombination in the genome, suggesting that this enzyme may be the major DNA replicase in Euryarchaeota. Although it possesses strong polymerization and proofreading activities, the motifs common to other DNA polymerase families are absent in its sequences. Here we report the mapping of the catalytic residues in a family D DNA polymerase from Pyrococcus horikoshii. Site-directed alanine mutants for 28 conserved aspartic acid or glutamic acid residues were screened for polymerization and 3'-5' exonuclease activities. We identified the invariant aspartates Asp-1122 and Asp-1124 within the most conserved motif as the catalytic residues involved in DNA polymerization. Alanine mutation at either site caused a loss of polymerization activity, whereas the conserved mutants, D1122E, D1124N, and D1124E, had slightly reduced polymerization activity. We also found that the 3'-5' exonuclease activity remains in D1122A and D1124A, indicating that the catalytic residues of DNA polymerization are different from those of the 3'-5' exonuclease activity. Furthermore we determined the molecular mass of the recombinant enzyme by gel filtration and proposed a heterotetrameric structure for this enzyme.
Shen
Y
Y
National Institute of Bioscience and Human Technology, Tsukuba, Ibaraki 305, Japan.
Musti
K
K
Hiramoto
M
M
Kikuchi
H
H
Kawarabayashi
Y
Y
Matsui
I
I
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000378:metabolism*; D001483:Base Sequence; D001704:Biopolymers; D002384:Catalysis; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D019711:Pyrococcus / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid
D001704:Biopolymers; D017931:DNA Primers; D001224:Aspartic Acid; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V; D008274:Magnesium