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High-resolution footprinting of sequence-specific protein–DNA contacts

Nature Biotechnology volume 20, pages 183186 (2002) | Download Citation

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Abstract

Gene transcription is regulated by proteins that bind specific DNA sequences and control the initiation of RNA synthesis. A major challenge is to map all of the regulatory sites in the genome and to identify the proteins that bind them. Because members of transcription factor families often exhibit similar sequence preferences, methods for determining intermolecular contacts in protein–DNA interfaces must be sensitive to even subtle structural differences. The most detailed structural views of protein–DNA interfaces have been obtained through X-ray crystallography and NMR spectroscopy, and these methods have revolutionized the understanding of the structural determinants of sequence-specific recognition1. Neither crystallography nor NMR, however, is particularly well-suited to high-throughput applications such as pan-genomic elucidation of regulatory sequences; in addition, these methods yield no information on the energetic contribution of particular contacts. Here we report a straightforward, high-resolution biochemical method for mapping, at single-nucleotide resolution, DNA bases that are subject to sequence-specific contacts by regulatory proteins.

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Acknowledgements

This work was supported by a grant from Variagenics, Inc. M.J.S. is the recipient of fellowships from the Alfred and Isabel Bader Foundation and from Hoffman-La Roche and Eli Lilly. A.E. gratefully acknowledges support from the Swiss National Science Foundation (postdoctoral fellowship 1998–1999) and from Harvard University (1999–2000). We thank Jia Wolfe, Jeff Olson, and Tomohiko Kawate of Variagenics, Inc. for sharing unpublished results, and members of the Verdine group for valuable discussions.

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    • Alexander Ernst

    Current address: Schering AG, D-13342 Berlin, Germany.

Affiliations

  1. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138.

    • Michael J. Storek
    • , Alexander Ernst
    •  & Gregory L. Verdine

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Correspondence to Gregory L. Verdine.

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DOI

https://doi.org/10.1038/nbt0202-183

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