title: Expression and purification of human FANCI and FANCD2 using Escherichia coli cells.
abstract: The DNA interstrand crosslink (ICL) is an extremely deleterious DNA lesion that covalently crosslinks complementary strands and prevents the strand-separation reaction. In higher eukaryotes, the Fanconi anemia proteins, FANCI and FANCD2, form a heterodimer and play essential roles in ICL repair. Human FANCI and FANCD2 are large proteins with molecular masses of 149kDa and 164kDa, respectively, and were reportedly purified using a baculovirus expression system with insect cells. We have established a novel expression and purification procedure for human FANCD2 and FANCI, using Escherichia coli cells. The human FANCD2 and FANCI proteins purified by this bacterial expression method formed a stable heterodimer, and exhibited DNA binding and histone chaperone activities, as previously reported for the proteins purified by the baculovirus system. Therefore, these purification methods for human FANCI and FANCD2 provide novel procedures to facilitate structural and biochemical studies of human FANCI and FANCD2.
authors.affiliation: Laboratory of Structural Biology, Graduate School of Advanced Science & Engineering, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Takahashi
authors.affiliation: Laboratory of Structural Biology, Graduate School of Advanced Science & Engineering, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan.
authors.forename: Koichi
authors.initials: K
authors.lastname: Sato
authors.affiliation: Laboratory of Structural Biology, Graduate School of Advanced Science & Engineering, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan.
authors.forename: Mayo
authors.initials: M
authors.lastname: Shimomuki
authors.affiliation: Laboratory of DNA Damage Signaling, Department of Late Effects Studies, Radiation Biology Center, Kyoto University, Kyoto 606-8501, Japan.
authors.forename: Minoru
authors.initials: M
authors.lastname: Takata
authors.affiliation: Laboratory of Structural Biology, Graduate School of Advanced Science & Engineering, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan. Electronic address: kurumizaka@waseda.jp.
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Kurumizaka
mesh_terms: D002843:Chromatin / Q000235:genetics; D004249:DNA Damage / Q000235:genetics; D004260:DNA Repair / Q000235:genetics; D004926:Escherichia coli; D052236:Fanconi Anemia Complementation Group D2 Protein / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D051856:Fanconi Anemia Complementation Group Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D005786:Gene Expression Regulation; D006801:Humans; D009154:Mutation
chemical_list: D002843:Chromatin; C499905:FANCD2 protein, human; C519921:FANCI protein, human; D052236:Fanconi Anemia Complementation Group D2 Protein; D051856:Fanconi Anemia Complementation Group Proteins
keywords: Bacterial expression; DNA interstrand crosslink; FANCI–FANCD2 complex; Fanconi anemia