DNA Affinity Purification: A Pulldown Assay for Identifying and Analyzing Proteins Binding to Nucleic Acids.

ABSTRACT

The interaction of proteins with DNA plays a central role in gene regulation. We describe a DNA affinity purification method that allows for identification and analysis of protein complex components. For example, a DNA probe carrying a transcription factor binding site is used to purify proteins from a nuclear extract. The proteins binding to the probe are then identified by mass spectrometry. In similar experiments, proteins purified by this pulldown method can be analyzed by Western blot. Employing this method, we found that the DREAM transcriptional repressor complex binds to CHR transcriptional elements in promoters of cell cycle genes. This complex is important for cell cycle-dependent repression and as part of the p53-DREAM pathway serves as a link for indirect transcriptional repression of target genes by the tumor suppressor p53. In general, the methods described can be applied for the identification and analysis of proteins binding to DNA.