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An Improved Method for Identifying Specific DNA-Protein-Binding Sites In Vitro

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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.

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Abbreviations

ChIP:

Chromatin immunoprecipitation

SSDP:

Specific sites of DNA-protein-binding

DdrO:

A DNA damage response protein from Deinococcus radiodurans

EMSA:

Electrophoretic mobility shift assay

SPR:

Surface plasmon resonance analyses

PBM:

Protein-binding microarray

ChIA-PET:

Chromatin interaction analysis by paired-end tag sequencing

DHS:

DNase I hypersensitive sites

ENCODE:

Encyclopedia of DNA elements

DDR:

DNA damage response

recA :

Recombinant A

MBP:

Maltose-binding protein

TEV:

Tobacco etch virus

IPTG:

Isopropyl β-d-1-thiogalactopyranoside

BPB:

Bromophenol blue

RDRM:

Radiation and desiccation response motif

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Acknowledgement

This work was supported by grants from the National Natural Science Foundation of China (31210103904, 31370102, 31570058), the Natural Science Foundation of Zhejiang Province (LY13C010001) and the Public Project of Zhejiang Province (2014C33024) and the Project for Experimental Technology of Zhejiang University.

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    Authors and Affiliations

    1. 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

      Liangyan Wang, Huizhi Lu, Yunguang Wang, Su Yang, Hong Xu, Kaiying Cheng, Ye Zhao, Bing Tian & Yuejin Hua

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    1. Liangyan Wang
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    2. Huizhi Lu
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    3. Yunguang Wang
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    4. Su Yang
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    Correspondence to Bing Tian or Yuejin Hua.

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    The authors declare no financial or commercial conflict of interest.

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    Liangyan Wang and Huizhi Lu have contributed equally to the paper as first authors.

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    Wang, L., Lu, H., Wang, Y. et al. An Improved Method for Identifying Specific DNA-Protein-Binding Sites In Vitro. Mol Biotechnol 59, 59–65 (2017). https://doi.org/10.1007/s12033-017-9993-y

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