COMPACTION AND SUPERCOILING OF SINGLE, LONG DNA MOLECULES BY HU PROTEIN

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Abstract
The bacterial cell contains the highly conserved protein HU in abundance. To characterize its architectural role, we studied the elastic behavior of single, supercoilable DNA molecules (tens of kilobases long) in solution with HU from B. stearothermophilus (BstHU) by a micromanipulation assay. We point out quantitative yet notable differences to the behavior of HU from E. coli (EcoHU) observed by others. Our main contribution here, however, is to characterize the interaction of BstHU with single molecules of DNA in arbitrary states of supercoiling. BstHU clearly distinguishes under- and overwound substrates, breaking the characteristic symmetry in the elastic response of bare DNA. We demonstrate that BstHU shifts the preferred linking number of the complex, consistent with a model in which bound proteins untwist the double helix. The model qualitatively explains various features, such as overall compaction and weaker dependence on supercoiling, by a softening of the DNA to twist and bending. Previously reported reversal of binding effects at protein concentrations above a threshold also extends to supercoiling. All observed effects are highly sensitive to salt concentrations. Their range and magnitude lend HU great versatility in dynamically altering the physical properties and organization of the nucleoid.
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