Rationally designed coiled-coil DNA looping peptides control DNA topology

Artificial DNA looping peptides were engineered to study the roles of protein and DNA flexibility in controlling the geometry and stability of protein-mediated DNA loops. These LZD (leucine zipper dual-binding) peptides were derived by fusing a second, C-terminal, DNA-binding region onto the GCN4 bZ...

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Bibliographic Details
Published in:Nucleic acids research 2013-09, Vol.41 (17), p.8253-8265
Main Authors: Gowetski, Daniel B, Kodis, Erin J, Kahn, Jason D
Format: Article
Language:English
Subjects:
Binding Sites
Cyclization
DNA - chemistry
DNA - metabolism
Electrophoretic Mobility Shift Assay
Leucine Zippers
Models, Molecular
Molecular Biology
Nucleic Acid Conformation
Peptides - chemical synthesis
Peptides - chemistry
Peptides - metabolism
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Summary:Artificial DNA looping peptides were engineered to study the roles of protein and DNA flexibility in controlling the geometry and stability of protein-mediated DNA loops. These LZD (leucine zipper dual-binding) peptides were derived by fusing a second, C-terminal, DNA-binding region onto the GCN4 bZip peptide. Two variants with different coiled-coil lengths were designed to control the relative orientations of DNA bound at each end. Electrophoretic mobility shift assays verified formation of a sandwich complex containing two DNAs and one peptide. Ring closure experiments demonstrated that looping requires a DNA-binding site separation of 310 bp, much longer than the length needed for natural loops. Systematic variation of binding site separation over a series of 10 constructs that cyclize to form 862-bp minicircles yielded positive and negative topoisomers because of two possible writhed geometries. Periodic variation in topoisomer abundance could be modeled using canonical DNA persistence length and torsional modulus values. The results confirm that the LZD peptides are stiffer than natural DNA looping proteins, and they suggest that formation of short DNA loops requires protein flexibility, not unusual DNA bendability. Small, stable, tunable looping peptides may be useful as synthetic transcriptional regulators or components of protein-DNA nanostructures.
ISSN:0305-1048
1362-4962
DOI:10.1093/nar/gkt553