title: Structural basis for substrate recognition and processive cleavage mechanisms of the trimeric exonuclease PhoExo I.
abstract: Nucleases play important roles in nucleic acid processes, such as replication, repair and recombination. Recently, we identified a novel single-strand specific 3'-5' exonuclease, PfuExo I, from the hyperthermophilic archaeon Pyrococcus furiosus, which may be involved in the Thermococcales-specific DNA repair system. PfuExo I forms a trimer and cleaves single-stranded DNA at every two nucleotides. Here, we report the structural basis for the cleavage mechanism of this novel exonuclease family. A structural analysis of PhoExo I, the homologous enzyme from P. horikoshii OT3, showed that PhoExo I utilizes an RNase H-like active site and possesses a 3'-OH recognition site ∼9 Å away from the active site, which enables cleavage at every two nucleotides. Analyses of the heterotrimeric and monomeric PhoExo I activities showed that trimerization is indispensable for its processive cleavage mechanism, but only one active site of the trimer is required.
authors.affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.
authors.forename: Ken-Ichi
authors.initials: K
authors.lastname: Miyazono
authors.affiliation: Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, and Faculty of Agriculture, Kyushu University, Fukuoka, Japan.
authors.forename: Sonoko
authors.initials: S
authors.lastname: Ishino
authors.affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.
authors.forename: Kanae
authors.initials: K
authors.lastname: Tsutsumi
authors.affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.
authors.forename: Tomoko
authors.initials: T
authors.lastname: Ito
authors.affiliation: Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, and Faculty of Agriculture, Kyushu University, Fukuoka, Japan.
authors.forename: Yoshizumi
authors.initials: Y
authors.lastname: Ishino
authors.affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan amtanok@mail.ecc.u-tokyo.ac.jp.
authors.forename: Masaru
authors.initials: M
authors.lastname: Tanokura
mesh_terms: D019843:Archaeal Proteins / Q000737:chemistry* / Q000378:metabolism; D020134:Catalytic Domain; D053837:DNA Cleavage; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D055503:Protein Multimerization; D043902:Pyrococcus horikoshii / Q000201:enzymology; D016914:Ribonuclease H / Q000737:chemistry; D012260:Ribonucleases / Q000737:chemistry / Q000378:metabolism
chemical_list: D019843:Archaeal Proteins; D004277:DNA, Single-Stranded; D005090:Exodeoxyribonucleases; D012260:Ribonucleases; C028450:exodeoxyribonuclease I; D016914:Ribonuclease H