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Methods Mol Biol. 2012;922:55-83. doi: 10.1007/978-1-62703-032-8_4.

SSB-DNA binding monitored by fluorescence intensity and anisotropy.

Author information

1
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO, USA.

Abstract

Fluorescence methods have proven to be extremely useful tools for quantitative studies of the equilibria and kinetics of protein-DNA interactions. If the protein contains tryptophan (Trp), as is often the case, and there is a change in intrinsic Trp fluorescence of the protein, one can use this change in signal (quenching/enhancement) to monitor binding. One can also attach an extrinsic fluorophore to either the protein or the DNA and monitor binding due to a change in fluorescence intensity or a change in fluorescence anisotropy. Such equilibrium studies can provide important quantitative information on stoichiometries (occluded site size, number of binding sites) and energetics (affinities and cooperativities) of the interactions. This information is needed to understand the mechanisms of protein-DNA interactions. A critical aspect of such approaches for systems that have non-unity stoichiometries (e.g., a protein that binds multiple ligands) is knowledge of the relationship between the change in fluorescence signal (intensity or anisotropy) and the average extent of binding. Here we describe procedures for using fluorescence approaches to examine the stoichiometries and equilibrium binding affinities of Escherichia coli single-stranded DNA-binding protein (SSB) and Deinococcus radiodurans SSB with long polymeric ssDNA to determine an occluded site size. We also provide examples of studies of SSB binding to shorter oligonucleotides to demonstrate analysis and fitting of the data to an appropriate model (monitoring fluorescence intensity or anisotropy) to obtain quantitative estimates of equilibrium binding parameters. We emphasize that the solution conditions (especially salt concentration and type) can influence not only the binding affinity, but also the mode by which an SSB oligomer binds ssDNA.

PMID:
22976177
PMCID:
PMC4342854
DOI:
10.1007/978-1-62703-032-8_4
[Indexed for MEDLINE]
Free PMC Article

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