title: The novel function of a short region K253xRxxxD259 conserved in the exonuclease domain of hyperthermostable DNA polymerase I from Pyrococcus horikoshii.
abstract: The DNA polymerase gene of the hyperthermophile Pyrococcus horikoshii was successfully overexpressed after removing an intein. The importance of an amino acid sequence around a highly conserved Asp was studied by site-directed mutagenesis. The results indicated that Lys253, Arg255, and Asp259 form a novel functional motif, K253xRxxxD259 (outside known motifs Exo I, II, and III), that is important not only for exonuclease activity but also for polymerizing activity, confirming functional interdependence between the polymerase and exonuclease domains. The short loop region, K253G254R255, probably contributes to binding to DNA substrates. Moreover, the negative charge and the side-chain length of D259 might play a supporting role in coordinating the conserved Mg2+ to the correct position at the active center in the exonuclease domain.
authors.affiliation: National Institute of Bioscience and Human-Technology, Tsukuba, Ibaraki, Japan.
authors.forename: R
authors.initials: R
authors.lastname: Zheng
authors.forename: E
authors.initials: E
authors.lastname: Matsui
authors.forename: Y
authors.initials: Y
authors.lastname: Shen
authors.forename: K V
authors.initials: KV
authors.lastname: Musti
authors.forename: Y
authors.initials: Y
authors.lastname: Feng
authors.forename: S
authors.initials: S
authors.lastname: Darnis
authors.forename: Y
authors.initials: Y
authors.lastname: Kawarabayasi
authors.forename: H
authors.initials: H
authors.lastname: Kikuchi
authors.forename: K
authors.initials: K
authors.lastname: Harata
authors.forename: I
authors.initials: I
authors.lastname: Matsui
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D017124:Conserved Sequence; D004256:DNA Polymerase I / Q000737:chemistry / Q000235:genetics*; D017931:DNA Primers; D004795:Enzyme Stability; D005092:Exonucleases / Q000737:chemistry / Q000235:genetics*; D006358:Hot Temperature; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D019711:Pyrococcus / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid
chemical_list: D017931:DNA Primers; D004256:DNA Polymerase I; D005092:Exonucleases