@article{b96706327a964613aabdbc11ad1d5df4,
title = "A single molecule analysis of H-NS uncouples DNA binding affinity from DNA specificity",
abstract = "Heat-stable nucleoid structuring protein (H-NS) plays a crucial role in gene silencing within prokaryotic cells and is important in pathogenesis. It was reported that H-NS silences nearly 5% of the genome, yet the molecular mechanism of silencing is not well understood. Here, we employed a highly-sensitive single-molecule counting approach, and measured the dissociation constant (KD) of H-NS binding to single DNA binding sites. Charged residues in the linker domain of H-NS provided the most significant contribution to DNA binding affinity. Although H-NS was reported to prefer A/T-rich DNA (a feature of pathogenicity islands) over G/C-rich DNA, the dissociation constants obtained from such sites were nearly identical. Using a hairpin unzipping assay, we were able to uncouple non-specific DNA binding steps from nucleation site binding and subsequent polymerization. We propose a model in which H-NS initially engages with non-specific DNA via reasonably high affinity (∼60 nM KD) electrostatic interactions with basic residues in the linker domain. This initial contact enables H-NS to search along the DNA for specific nucleation sites that drive subsequent polymerization and gene silencing.",
author = "Ranjit Gulvady and Yunfeng Gao and Kenney, {Linda J.} and Jie Yan",
note = "Funding Information: National Research Foundation, Prime Minister{\textquoteright}s Office, Singapore and the Ministry of Education under the Research Centres of Excellence programme [to J.Y. and L.J.K.]; VA [IOBX-000372 to L.J.K.]; NIH [AI123640]. Funding for open access charge: National Research Foundation, Prime Minister{\textquoteright}s Office, Singapore and the Ministry of Education under the Research Centres of Excellence programme [to J.Y.]. Conflict of interest statement. None declared. Funding Information: We thank the protein expression core facility of the Mechanobiology Institute for protein purification. J.Y. and L.J.K. conceived the research. J.Y. and R.G. designed the single-molecule assay. Y.G. and L.J.K. designed the protein constructs. R.G. performed the experiments. R.G., L.J.K. and J.Y. interpreted the data and wrote the paper. National Research Foundation, Prime Minister{\textquoteright}s Office, Singapore and the Ministry of Education under the Research Centres of Excellence programme [to J.Y. and L.J.K.]; VA [IOBX-000372 to L.J.K.]; NIH [AI123640]. Funding for open access charge: National Research Foundation, Prime Minister{\textquoteright}s Office, Singapore and the Ministry of Education under the Research Centres of Excellence programme [to J.Y.]. Conflict of interest statement. None declared. Publisher Copyright: {\textcopyright} The Author(s) 2018.",
year = "2018",
month = nov,
day = "2",
doi = "10.1093/nar/gky826",
language = "English (US)",
volume = "46",
pages = "10216--10224",
journal = "Nucleic Acids Research",
issn = "0305-1048",
publisher = "Oxford University Press",
number = "19",
}