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. 2012;7(11):e49885.
doi: 10.1371/journal.pone.0049885. Epub 2012 Nov 14.

Physical Organization of DNA by Multiple Non-Specific DNA-binding Modes of Integration Host Factor (IHF)

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Free PMC article

Physical Organization of DNA by Multiple Non-Specific DNA-binding Modes of Integration Host Factor (IHF)

Jie Lin et al. PLoS One. .
Free PMC article

Abstract

The integration host factor (IHF) is an abundant nucleoid-associated protein and an essential co-factor for phage λ site-specific recombination and gene regulation in E. coli. Introduction of a sharp DNA kink at specific cognate sites is critical for these functions. Interestingly, the intracellular concentration of IHF is much higher than the concentration needed for site-specific interactions, suggesting that non-specific binding of IHF to DNA plays a role in the physical organization of bacterial chromatin. However, it is unclear how non-specific DNA association contributes to DNA organization. By using a combination of single DNA manipulation and atomic force microscopy imaging methods, we show here that distinct modes of non-specific DNA binding of IHF result in complex global DNA conformations. Changes in KCl and IHF concentrations, as well as tension applied to DNA, dramatically influence the degree of DNA-bending. In addition, IHF can crosslink DNA into a highly compact DNA meshwork that is observed in the presence of magnesium at low concentration of monovalent ions and high IHF-DNA stoichiometries. Our findings provide important insights into how IHF contributes to bacterial chromatin organization, gene regulation, and biofilm formation.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Influences of IHF on DNA force response in the absence of magnesium.
(A) Top-panel: Schematic diagram of the transverse magnetic tweezers setup used in this paper. Bottom panel: force-extension curves of λ-DNA according to the Marko-Siggia formula for the protein-free DNA persistence length of 50 nm (black) and a reduced persistence length of 25 nm (red). (B–D) Effects of IHF on the force response of λ-DNA at varying concentrations of KCl and pH 7.4. Force-extension curves of DNA in the force-decreasing (filled triangles) and force-increasing (open triangles) scans at the indicated concentrations of IHF in 200 mM KCl (B), 100 mM KCl (C), and 50 mM KCl (D), respectively. (E) Force-extension curves measured in 50 mM KCl by force jumping. (F) DNA extension as a function of the IHF concentration at 0.1 pN at different KCl concentrations. Data at 0.1 pN were obtained from the force-extension curves at corresponding KCl concentrations in Figure 1B–C and 1E by linear interpolation using two nearest neighbouring data points adjacent to 0.1 pN. (G) Decreasing KCl concentration from 200 mM to 50 mM drives a switch from smaller to higher degrees of DNA bending. Filled triangles represent force-extension curves of DNA incubated in 200 mM KCl at the indicated concentration of IHF. Open triangles represent force-extension curves of DNA after lowering the KCl concentration to 50 mM and removing IHF. (H) The DNA bending angle as a function of the spacing of IHF bound to DNA that causes 50% reduction in DNA extension at 0.1 pN.
Figure 2
Figure 2. AFM analysis of linearized double-stranded Φx174 DNA incubated with varying concentrations of IHF.
IHF heterodimer to DNA base pair ratio is indicated in each image panel. (A) Naked DNA that was not incubated with IHF in 50 mM KCl. Similar DNA conformation was found in 200 mM KCl with 1250 nM IHF, which is the highest protein concentration (Inset figure). (B–C) DNA molecules incubated in 100 mM KCl with 250 nM IHF (B) and 1250 nM IHF (C) respectively. (D-F) DNA molecules incubated in 50 mM KCl with 50 nM IHF (D), 250 nM IHF (E) and 1250 nM IHF (F), respectively.
Figure 3
Figure 3. Effects of magnesium on DNA condensation in the presence of IHF.
(A) Force-extension curves obtained by force jumping. Triangles and circles represent data obtained in the absence and presence of 2 mM MgCl2, respectively. For 250 nM and 1250 nM IHF, data are not shown for force <0.6 pN because DNA extension was below the minimal extension (∼2 µm) that could be measured by our instrument. (B) DNA folding time course at various values of lower force and unfolding time course at the high force of ∼12 pN in 1250 nM IHF. (C–F) Atomic force microscopy analysis of DNA molecules incubated in 50 mM KCl and 2 mM MgCl2 with 1250 nM IHF (C), 310 nM IHF (D), 78 nM IHF (E) and 31 nM IHF (F). IHF heterodimer to DNA base pair ratio is indicated in each image panel.
Figure 4
Figure 4. Schematic model of IHF-DNA interaction.
The conformational states of the DNA-IHF complex and their dependence on force, [IHF], [KCl] and [MgCl2] are summarized here. Yellow represents an IHF dimer, and blue represents dsDNA. Dark red right trangles indicate increasing values of force and [KCl].

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Grant support

This work was supported by grants by the Ministry of Education of Singapore [MOE2008-T2-1-096 to JY]; the Mechanobiology Institute Singapore [Internal Funding to JY]; and the Academic Research Council Singapore [Tier 1 to PD]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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