title: An Improved Method for Identifying Specific DNA-Protein-Binding Sites In Vitro.
abstract: Binding of proteins to specific DNA sequences is essential for a variety of cellular processes such as DNA replication, transcription and responses to external stimuli. Chromatin immunoprecipitation is widely used for determining intracellular DNA fragments bound by a specific protein. However, the subsequent specific or accurate DNA-protein-binding sequence is usually determined by DNA footprinting. Here, we report an alternative method for identifying specific sites of DNA-protein-binding (designated SSDP) in vitro. This technique is mainly dependent on antibody-antigen immunity, simple and convenient, while radioactive isotope labeling and optimization of partial degradation by deoxyribonuclease (DNase) are avoided. As an example, the specific binding sequence of a target promoter by DdrO (a DNA damage response protein from Deinococcus radiodurans) in vitro was determined by the developed method. The central sequence of the binding site could be easily located using this technique.
authors.affiliation: Key Laboratory of Chinese Ministry of Agriculture and Zhejiang Province for Nuclear Agricultural Sciences, Institute of Nuclear Agricultural Sciences, Zhejiang University, No. 268, Kaixuan Road, Hangzhou, 310029, Zhejiang, China.
authors.forename: Liangyan
authors.initials: L
authors.lastname: Wang
authors.affiliation: Key Laboratory of Chinese Ministry of Agriculture and Zhejiang Province for Nuclear Agricultural Sciences, Institute of Nuclear Agricultural Sciences, Zhejiang University, No. 268, Kaixuan Road, Hangzhou, 310029, Zhejiang, China.
authors.forename: Huizhi
authors.initials: H
authors.lastname: Lu
authors.affiliation: Key Laboratory of Chinese Ministry of Agriculture and Zhejiang Province for Nuclear Agricultural Sciences, Institute of Nuclear Agricultural Sciences, Zhejiang University, No. 268, Kaixuan Road, Hangzhou, 310029, Zhejiang, China.
authors.forename: Yunguang
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Key Laboratory of Chinese Ministry of Agriculture and Zhejiang Province for Nuclear Agricultural Sciences, Institute of Nuclear Agricultural Sciences, Zhejiang University, No. 268, Kaixuan Road, Hangzhou, 310029, Zhejiang, China.
authors.forename: Su
authors.initials: S
authors.lastname: Yang
authors.affiliation: Key Laboratory of Chinese Ministry of Agriculture and Zhejiang Province for Nuclear Agricultural Sciences, Institute of Nuclear Agricultural Sciences, Zhejiang University, No. 268, Kaixuan Road, Hangzhou, 310029, Zhejiang, China.
authors.forename: Hong
authors.initials: H
authors.lastname: Xu
authors.affiliation: Key Laboratory of Chinese Ministry of Agriculture and Zhejiang Province for Nuclear Agricultural Sciences, Institute of Nuclear Agricultural Sciences, Zhejiang University, No. 268, Kaixuan Road, Hangzhou, 310029, Zhejiang, China.
authors.forename: Kaiying
authors.initials: K
authors.lastname: Cheng
authors.affiliation: Key Laboratory of Chinese Ministry of Agriculture and Zhejiang Province for Nuclear Agricultural Sciences, Institute of Nuclear Agricultural Sciences, Zhejiang University, No. 268, Kaixuan Road, Hangzhou, 310029, Zhejiang, China.
authors.forename: Ye
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: Key Laboratory of Chinese Ministry of Agriculture and Zhejiang Province for Nuclear Agricultural Sciences, Institute of Nuclear Agricultural Sciences, Zhejiang University, No. 268, Kaixuan Road, Hangzhou, 310029, Zhejiang, China. tianbing@zju.edu.cn.
authors.forename: Bing
authors.initials: B
authors.lastname: Tian
authors.affiliation: Key Laboratory of Chinese Ministry of Agriculture and Zhejiang Province for Nuclear Agricultural Sciences, Institute of Nuclear Agricultural Sciences, Zhejiang University, No. 268, Kaixuan Road, Hangzhou, 310029, Zhejiang, China. yjhua@zju.edu.cn.
authors.forename: Yuejin
authors.initials: Y
authors.lastname: Hua
mesh_terms: D000906:Antibodies / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D034301:Deinococcus / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D066298:In Vitro Techniques; D011401:Promoter Regions, Genetic; D011485:Protein Binding
chemical_list: D000906:Antibodies; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D004247:DNA
keywords: Antibody-antigen immunity; Chromatin immunoprecipitation; DNA footprinting; DNA-protein binding; Specific DNA-binding site