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In vitro Footprinting of Promoter Regions Within Supercoiled Plasmid DNA

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Drug-DNA Interaction Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 613))

Abstract

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.

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Acknowledgments

This research was supported by grants from the National Institutes of Health (CA109069). We are grateful to Drs. Allison Hays and Keith Fox for proofreading and editing the final version of the manuscript and figures. We also thank Drs Keping Xie and Kazuo Shin-ya for providing pGL3-V789 and telomestatin, respectively, for this study.

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Authors and Affiliations

  1. Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, BIO5 Institute, Tucson, AZ, USA

    Daekyu Sun

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  1. Daekyu Sun
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Correspondence to Daekyu Sun .

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Editors and Affiliations

  1. School of Biological Sciences, University of Southampton, Southampton, SO17 1BJ, United Kingdom

    Keith R. Fox

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Sun, D. (2010). In vitro Footprinting of Promoter Regions Within Supercoiled Plasmid DNA. In: Fox, K. (eds) Drug-DNA Interaction Protocols. Methods in Molecular Biology, vol 613. Humana Press. https://doi.org/10.1007/978-1-60327-418-0_14

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  • DOI: https://doi.org/10.1007/978-1-60327-418-0_14

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-60327-417-3

  • Online ISBN: 978-1-60327-418-0

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