Total analysis and purification of cellular proteins binding to cisplatin-damaged DNA using submicron beads.
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.
Tomohiro
Takenori
T
Institute of Molecular and Cell Biology, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki 305-8566, Japan.t.tomohiro@aist.co.jp
Sawada Ji
Jun-ichi
J
Sawa
Chika
C
Nakura
Hironori
H
Yoshida
Shuhei
S
Kodaka
Masato
M
Hatakeyama
Mamoru
M
Kawaguchi
Haruma
H
Handa
Hiroshi
H
Okuno
Hiroaki
H
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
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