title: A longer finger-subdomain of family A DNA polymerases found by metagenomic analysis strengthens DNA binding and primer extension abilities.
abstract: The family A DNA polymerases from thermophilic bacteria are useful for PCR. The DNA polymerase from Thermus aquaticus (Taq polymerase) was the original enzyme used when practical PCR was developed, and it has remained the standard enzyme for PCR to date. Knowledge gained from structure-function relationship studies of Taq polymerase is applicable to create PCR enzymes with enhanced performance. We collected the deduced amino acid sequences of the regions from motif A to motif C in the family A DNA polymerases from metagenomic sequence data, obtained by sequencing DNAs from microorganisms isolated from various hot spring areas in Japan. The corresponding regions of the polA gene for Taq polymerase were substituted with the metagenomic DNA gene fragments, and various chimeric DNA polymerases were prepared. Based on the properties of these chimeric enzymes and their sequences, we found an insertion sequence that affects the primer extension ability of the family A DNA polymerases. The insertion sequence is located in the finger subdomain, and it may enhance the affinity of the enzyme to DNA. Mutant Taq polymerases with the corresponding 9 amino acid insertion displayed enhanced PCR performance.
authors.affiliation: Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka, Japan; JST, CREST, Tokyo, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Yamagami
authors.affiliation: Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka, Japan.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Matsukawa
authors.affiliation: Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka, Japan.
authors.forename: Sae
authors.initials: S
authors.lastname: Tsunekawa
authors.affiliation: Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka, Japan; National Institute of Advanced Industrial Science and Technology, Tsukuba, Japan.
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Kawarabayasi
authors.affiliation: Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka, Japan; JST, CREST, Tokyo, Japan. Electronic address: sonoko@agr.kyushu-u.ac.jp.
authors.forename: Sonoko
authors.initials: S
authors.lastname: Ishino
authors.affiliation: Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka, Japan; JST, CREST, Tokyo, Japan. Electronic address: ishino@agr.kyushu-u.ac.jp.
authors.forename: Yoshizumi
authors.initials: Y
authors.lastname: Ishino
mesh_terms: D000595:Amino Acid Sequence; D017931:DNA Primers; D056186:Metagenomics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D019914:Taq Polymerase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D017931:DNA Primers; D019914:Taq Polymerase