In vitro footprinting of promoter regions within supercoiled plasmid DNA.
Polypurine/polypyrimidine (pPu/pPy) tracts, which exist in the promoter regions of many growth-related genes, have been proposed to be very dynamic in their conformation. In this chapter, we describe a detailed protocol for DNase I and S1 nuclease footprinting experiments with supercoiled plasmid DNA containing the promoter regions to probe whether there are conformational transitions to B-type DNA, melted DNA, and G-quadruplex structures within this tract. This is demonstrated with the proximal promoter region of the human vascular endothelial growth factor (VEGF) gene, which also contains multiple binding sites for Sp1 and Egr-1 transcription factors.
Sun
Daekyu
D
Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, BIO5 Institute, Tucson, AZ, USA. sun@pharmacy.arizona.edu
D001483:Base Sequence; D018983:DNA Footprinting / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D004278:DNA, Superhelical / Q000235:genetics* / Q000378:metabolism; D003851:Deoxyribonucleases / Q000378:metabolism; D004586:Electrophoresis; D006801:Humans; D009691:Nucleic Acid Denaturation; D010957:Plasmids / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics*; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics
D017931:DNA Primers; D004278:DNA, Superhelical; D042461:Vascular Endothelial Growth Factor A; D003851:Deoxyribonucleases