title: UV laser cross-linking: a real-time assay to study dynamic protein/DNA interactions during chromatin remodeling.
abstract: We describe the use of laser ultraviolet (UV) cross-linking to study the interaction of transcription factors with in vitro assembled chromatinized DNA templates in real time. Because the laser source delivers a high density of photons in a single ns pulse, the cross-linking reaction is completed in less than 1 microseconds, allowing the investigator to freeze rapid dynamic changes in protein-DNA interactions. Using this approach, we have sampled the dynamic equilibrium of the glucocorticoid receptor (GR) and the chromatin remodeling complex (SWI/SNF) during adenosine triphosphate (ATP)-dependent chromatin remodeling on a chromatinized mouse mammary tumor virus promoter in vitro. UV laser cross-linking shows that the GR and SWI/SNF complex undergoes a periodic binding and displacement event during the process of chromatin remodeling. The assay provides unique information regarding the equilibrium of protein-DNA interactions in real time and can be easily adapted to study the dynamic events in the assembly and disassembly of other multiprotein complexes on chromatin or DNA templates.
authors.affiliation: Laboratory of Receptor Biology and Gene Expression, Bldg 41 Room B602, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892-5055, USA.
authors.forename: Akhilesh K
authors.initials: AK
authors.lastname: Nagaich
authors.forename: Gordon L
authors.initials: GL
authors.lastname: Hager
mesh_terms: D000818:Animals; D000906:Antibodies / Q000378:metabolism; D001681:Biological Assay / Q000379:methods*; D001756:Blastoderm / Q000737:chemistry; D016466:CHO Cells / Q000737:chemistry / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D002843:Chromatin / Q000235:genetics; D042002:Chromatin Assembly and Disassembly / Q000235:genetics*; D003199:Computer Systems; D006224:Cricetinae; D003432:Cross-Linking Reagents; D004247:DNA / Q000378:metabolism*; D004265:DNA Helicases; D004273:DNA, Neoplasm / Q000378:metabolism; D004330:Drosophila / Q000737:chemistry / Q000196:embryology; D006367:HeLa Cells / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007834:Lasers; D008280:Magnetics; D051379:Mice; D008863:Microspheres; D009687:Nuclear Proteins / Q000276:immunology / Q000378:metabolism*; D014020:Tissue Extracts / Q000737:chemistry; D014157:Transcription Factors / Q000276:immunology / Q000378:metabolism*; D014466:Ultraviolet Rays
chemical_list: D000906:Antibodies; D002843:Chromatin; D003432:Cross-Linking Reagents; D004273:DNA, Neoplasm; D009687:Nuclear Proteins; C084011:SMARCA2 protein, human; C499503:Smarca2 protein, mouse; D014020:Tissue Extracts; D014157:Transcription Factors; D004247:DNA; C084108:SMARCA4 protein, human; C513194:Smarca4 protein, mouse; D004265:DNA Helicases