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|Title:||A single molecule analysis of H-NS uncouples DNA binding affinity from DNA specificity||Authors:||Gulvady, R.
|Issue Date:||2018||Publisher:||Oxford University Press||Citation:||Gulvady, R., Gao, Y., Kenney, L.J., Yan, J. (2018). A single molecule analysis of H-NS uncouples DNA binding affinity from DNA specificity. Nucleic Acids Research 46 (19) : 10216-10224. ScholarBank@NUS Repository. https://doi.org/10.1093/nar/gky826||Rights:||Attribution-NonCommercial 4.0 International||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. © The Author(s) 2018.||Source Title:||Nucleic Acids Research||URI:||https://scholarbank.nus.edu.sg/handle/10635/209648||ISSN:||0305-1048||DOI:||10.1093/nar/gky826||Rights:||Attribution-NonCommercial 4.0 International|
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