UV laser cross-linking: a real-time assay to study dynamic protein/DNA interactions during chromatin remodeling.
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.
Nagaich
Akhilesh K
AK
Laboratory of Receptor Biology and Gene Expression, Bldg 41 Room B602, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892-5055, USA.
Hager
Gordon L
GL
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
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