Abstract
So-called architectural DNA-binding proteins such as those of the HMGB-box family induce DNA bending and kinking. However, these proteins often display only a weak sequence preference, making the analysis of their DNA-binding characteristics difficult if not impossible in a standard electrophoretic mobility shift assay (EMSA). In contrast, such proteins often bind prebent DNAs with high affinity and specificity. A synthetic cruciform DNA structure will often provide an ideal binding site for such proteins, allowing their affinities for both bent and linear DNAs to be directly and simply determined by a modified form of EMSA.
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References
Bianchi ME, Beltrame M, Paonessa G (1989) Specific recognition of cruciform DNA by nuclear protein HMG1. Science 243:1056–1059
Ferrari S, Harley VR, Pontiggia A, Goodfellow PN, Lovell-Badge R, Bianchi ME (1992) SRY, like HMG1, recognizes sharp angles in DNA. EMBO J 11:4497–4506
Lilley DM (1992) DNA–protein interactions. HMG has DNA wrapped up. Nature 357:282–283
Van de Wetering M, Clevers H (1992) Sequence-specific interaction of the HMG box proteins TCF-1 and SRY occurs within the minor groove of a Watson-Crick double helix. EMBO J 11:3039–3044
Kuhn A, Stefanovsky V, Grummt I (1993) The nucleolar transcription activator UBF relieves Ku antigen-mediated repression of mouse ribosomal gene transcription. Nucleic Acids Res 21:2057–2063
Bazett-Jones DP, Leblanc B, Herfort M, Moss T (1994) Short-range DNA looping by the Xenopus HMG-box transcription factor, xUBF. Science 264:1134–1137
Stefanovsky VY, Bazett-Jones DP, Pelletier G, Moss T (1996) The DNA supercoiling architecture induced by the transcription factor xUBF requires three of its five HMG-boxes. Nucleic Acids Res 24:3208–3215
Stefanovsky VY, Pelletier G, Bazett-Jones DP, Moss T (2006) ERK modulates DNA bending and enhancesome structure by phosphorylating HMG1-boxes 1 and 2 of the RNA polymerase I transcription factor UBF. Biochemistry 45:3626–3634
Zannis-Hadjopoulos M, Yahyaoui W, Callejo M (2008) 14-3-3 cruciform-binding proteins as regulators of eukaryotic DNA replication. Trends Biochem Sci 33:44–50
Acknowledgments
This work was supported by an operating grant from the Canadian Institutes of Health Research.
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Stefanovsky, V.Y., Moss, T. (2015). The Cruciform DNA Mobility Shift Assay: A Tool to Study Proteins That Recognize Bent DNA. In: Leblanc, B., Rodrigue, S. (eds) DNA-Protein Interactions. Methods in Molecular Biology, vol 1334. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2877-4_12
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DOI: https://doi.org/10.1007/978-1-4939-2877-4_12
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2876-7
Online ISBN: 978-1-4939-2877-4
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