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Sci. STKE, 26 October 2004
Vol. 2004, Issue 256, p. pl13
[DOI: 10.1126/stke.2562004pl13]

PROTOCOLS

UV Laser Cross-linking: A Real-Time Assay to Study Dynamic Protein/DNA Interactions During Chromatin Remodeling

Akhilesh K. Nagaich and Gordon L. Hager*

Laboratory of Receptor Biology and Gene Expression, Bldg 41 Room B602, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892–5055, USA.

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.

*Corresponding author: Telephone, 301-496-9867; fax, 301-496-4951

Citation: A. K. Nagaich, G. L. Hager, UV Laser Cross-linking: A Real-Time Assay to Study Dynamic Protein/DNA Interactions During Chromatin Remodeling. Sci. STKE 2004, pl13 (2004).

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