title: Analysis of p53 binding to DNA by fluorescence imaging microscopy.
abstract: Transcription factors play a central role in cell biology through binding to target DNA elements and regulating gene expression. In this study, we used the p53 tumour suppressor as a model transcription factor to develop an imaging based assay to measure DNA binding. The assay utilizes fluorescence imaging microscopy to detect labelled p53 bound to DNA coated on microbeads. We demonstrate the ability to multiplex the assay by interrogating simultaneous binding to variant DNA sequences present on tractable beads. Additionally, the assay measures activation of p53 for increased DNA binding by a known peptide in addition to reactivation of mutant p53 by a small molecule. It may therefore be adaptable to a high-content imaging screen for compounds capable of restoring the function of mutant p53 associated with cancer.
authors.affiliation: Experimental Therapeutics Centre, Agency for Science and Technology and Research, Singapore 138669, Singapore.
authors.forename: Hwee Jing
authors.initials: HJ
authors.lastname: Ong
authors.forename: Jia Wei
authors.initials: JW
authors.lastname: Siau
authors.forename: Jing Bo
authors.initials: JB
authors.lastname: Zhang
authors.forename: Minghui
authors.initials: M
authors.lastname: Hong
authors.forename: Horst
authors.initials: H
authors.lastname: Flotow
authors.forename: Farid
authors.initials: F
authors.lastname: Ghadessy
mesh_terms: D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods*; D008863:Microspheres; D011485:Protein Binding; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; D016159:Tumor Suppressor Protein p53; D004247:DNA