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Topological Probes of a Cooperative, Nonspecific Protein–DNA Complex

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Biophysics of DNA-Protein Interactions

Part of the book series: Biological and Medical Physics, Biomedical Engineering ((BIOMEDICAL))

Abstract

Many proteins bind DNA with moderate cooperativity and low sequence discrimination. Important among these are bacterial and eukaryotic chromosome-structuring proteins [2, 22] and single-stranded DNA-binding proteins [14]. In addition, many sequence-specific DNA-binding proteins interact with nontarget sequences cooperatively and with low sequence discrimination as part of their target-search mechanisms. Examples can be found among the transcription-regulatory proteins (human glucocorticoid receptor [10] and NFkB [23]; E. coli CAP [27, 30] and lambda repressor [24]), among the bacterial restriction endonucleases [31], and among the DNA-repair enzymes [18]. The ubiquity of such complexes calls for a better understanding of the interactions that stabilize them and the structures that result. Here we describe DNA-directed cross-linking and topoisomer analyses that are useful for characterizing topologies of protein–DNA complexes. Each is a variation on a classical approach, adapting it to features that are present in cooperative, nonspecific assemblies.

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Notes

  1. 1.

    E. coli topoisomerase I acts preferentially on negatively supercoiled DNAs, while the vaccinia enzyme relaxes both negatively and positively supercoiled molecules [8, 13, 29]. Topoisomer distributions obtained with the vaccinia enzyme are offset in the direction of positive supercoiling when compared to those obtained with the E. coli enzyme; this is an expected consequence of the preferential binding of vaccinia topoisomerase with positively supercoiled DNA. When this offset is taken into account, the linking differences observed with vaccinia and E. coli topoisomerases were mutually consistent.

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Acknowledgments

Mass spectrometric analyses were performed at the University of Kentucky Center for Structural Biology Protein Core Facility. This facility is supported, in part, by funds from NIH National Center for Research Resources (NCRR) grant P20 RR020171. We gratefully acknowledge the help of Dr. Carol Beach in acquiring these data. This research was supported by NIH grant GM-070662 (to M.G.F.).

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Authors and Affiliations

  1. Center for Structural Biology, Department of Molecular and Cellular Biochemistry, University of Kentucky, 741 South Limestone, Lexington, KY, 40536-0509, USA

    Michael G. Fried

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  1. Claire A. Adams
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  2. Manana Melikishvili
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  3. Michael G. Fried
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Correspondence to Michael G. Fried .

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Editors and Affiliations

  1. Dept. Physics, Northeastern University, Forsyth Street 110, Boston, 02115, Massachusetts, USA

    Mark C. Williams

  2. College of Medicine, Dept. Biochemistry & Molecular Biology, Mayo Clinic, First Street SW., 200, Rochester, 55905, Minnesota, USA

    L. James Maher III

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Adams, C.A., Melikishvili, M., Fried, M.G. (2010). Topological Probes of a Cooperative, Nonspecific Protein–DNA Complex. In: Williams, M., Maher, L. (eds) Biophysics of DNA-Protein Interactions. Biological and Medical Physics, Biomedical Engineering. Springer, New York, NY. https://doi.org/10.1007/978-0-387-92808-1_12

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  • DOI: https://doi.org/10.1007/978-0-387-92808-1_12

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