title: Total analysis and purification of cellular proteins binding to cisplatin-damaged DNA using submicron beads.
abstract: A high-performance affinity purification technique has been developed for cisplatin (CDDP)-damaged DNA binding proteins directly from crude nuclear extracts of HeLaS3 cell using novel submicron beads synthesized by copolymerization of styrene and glycidyl methacrylate (GMA). The beads dramatically decreased both nonspecific protein adsorption on solid surfaces and elution volume and simplified the handling procedure. Preparation of the beads for purification was carried out by immobilization of telomeric repeats, (TTAGGG)(n), on the surface after the reaction with CDDP. At least nine proteins clearly showed higher affinity to CDDP-DNA and were identified by amino acid sequence analysis including HMGB (high mobility group), hUBF (human upstream binding factor), and Ku autoantigen, which were previously reported to be components of CDDP-damaged DNA binding proteins.
authors.affiliation: Institute of Molecular and Cell Biology, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki 305-8566, Japan.t.tomohiro@aist.co.jp
authors.forename: Takenori
authors.initials: T
authors.lastname: Tomohiro
authors.forename: Jun-ichi
authors.initials: J
authors.lastname: Sawada Ji
authors.forename: Chika
authors.initials: C
authors.lastname: Sawa
authors.forename: Hironori
authors.initials: H
authors.lastname: Nakura
authors.forename: Shuhei
authors.initials: S
authors.lastname: Yoshida
authors.forename: Masato
authors.initials: M
authors.lastname: Kodaka
authors.forename: Mamoru
authors.initials: M
authors.lastname: Hatakeyama
authors.forename: Haruma
authors.initials: H
authors.lastname: Kawaguchi
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Handa
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Okuno
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity; D002945:Cisplatin / Q000737:chemistry / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000737:chemistry; D018736:DNA Adducts / Q000737:chemistry / Q000378:metabolism*; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D004852:Epoxy Compounds / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D008689:Methacrylates / Q000737:chemistry; D008863:Microspheres; D011485:Protein Binding; D020058:Styrene / Q000737:chemistry
chemical_list: D003432:Cross-Linking Reagents; D018736:DNA Adducts; D004268:DNA-Binding Proteins; D004852:Epoxy Compounds; D008689:Methacrylates; C057270:cisplatin-DNA adduct; D020058:Styrene; D002945:Cisplatin; C007870:glycidyl methacrylate