Journal of Molecular Biology
Volume 365, Issue 4, 26 January 2007, Pages 1005-1016
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Structure-based Analysis of HU–DNA Binding

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Abstract

HU and IHF are prokaryotic proteins that induce very large bends in DNA. They are present in high concentrations in the bacterial nucleoid and aid in chromosomal compaction. They also function as regulatory cofactors in many processes, such as site-specific recombination and the initiation of replication and transcription. HU and IHF have become paradigms for understanding DNA bending and indirect readout of sequence. While IHF shows significant sequence specificity, HU binds preferentially to certain damaged or distorted DNAs. However, none of the structurally diverse HU substrates previously studied in vitro is identical with the distorted substrates in the recently published Anabaena HU(AHU)–DNA cocrystal structures. Here, we report binding affinities for AHU and the DNA in the cocrystal structures. The binding free energies for formation of these AHU–DNA complexes range from ∼10–14.5 kcal/mol, representing Kd values in the nanomolar to low picomolar range, and a maximum stabilization of at least ∼6.3 kcal/mol relative to complexes with undistorted, non-specific DNA. We investigated IHF binding and found that appropriate structural distortions can greatly enhance its affinity. On the basis of the coupling of structural and relevant binding data, we estimate the amount of conformational strain in an IHF-mediated DNA kink that is relieved by a nick (at least 0.76 kcal/mol) and pinpoint the location of the strain. We show that AHU has a sequence preference for an A+T-rich region in the center of its DNA-binding site, correlating with an unusually narrow minor groove. This is similar to sequence preferences shown by the eukaryotic nucleosome.

Introduction

HU and IHF are closely related eubacterial, DNA-bending proteins characterized by two overlapping and functionally relevant modes of DNA binding: a non-specific mode and a specific mode. In their capacity as histone-like architectural factors, HU and IHF bind to DNA non-specifically and are found in high concentrations in the bacterial nucleoid,1., 2. but their exact role in nucleoid compaction is still debated.3 Sequence-specific binding, largely by IHF,4 plays a major role in facilitating processes such as replication initiation, site-specific recombination and transcription, when sharp bends or DNA loops are required for assembly of multi-component nucleoprotein complexes.5 HU is able to regulate such processes by binding DNA in a topology-dependent or structure-specific manner.6., 7.

These proteins form dimers with a compact, positively charged body and two long β ribbon arms that track along the DNA minor groove. They induce flexible bends in the DNA of ∼105° to >180° (Figure 1(a)).8., 9., 10., 11. Most of the bending is concentrated in two large kinks, spaced 9 bp apart, where the base-stacking is disrupted by a large roll angle and a highly conserved proline residue at the tip of each arm is intercalated between base pairs. IHF recognizes specific DNA sequences through indirect readout of the sequence-dependent conformational parameters of its binding site. Although HU binding is generally described as non-sequence specific, DNA modifications that stabilize the particular unusual conformation seen in the crystal would be expected to enhance binding.

Structure-specific binding of HU to DNA in vitro is well documented,12., 13., 14., 15., 16., 17., 18. and, given genetic data showing that HU-deficient cells are quite sensitive to UV damage,19., 20. it suggests that HU may be involved in DNA repair. However, in cases where high-affinity binding by HU to non-canonical (e.g nicked or mispaired) duplexes has been described there is no accompanying structural data, and models have been based on the IHF–DNA cocrystal structure.6., 7., 8., 14. Here, we present affinity data for a set of distorted duplexes based on that found in our recent cocrystal structures of Anabaena HU (AHU)–DNA complexes.10 These data can thus be used to estimate the strain in known DNA conformations.

Section snippets

Results

The DNA duplexes in the IHF–DNA and AHU–DNA cocrystal structures are shown in Figure 2(a) and (b). The AHU–DNA duplex was one of four oligonucleotides (TR3 for top right 3) that were annealed to create the doubly nicked IHF site for crystallization. The fact that AHU crystallized with small amounts of TR3 in the presence of higher concentrations of a less-distorted DNA duplex led us to predict that the TR3 duplex would bind very tightly to AHU in vitro. In fact, the affinity of AHU for a

Discussion

Among the substrates we tested, the free energy of binding, ΔG°, ranged from a rough estimate of ∼−6.3 kcal/mol (or weaker) for non-specific binding to −14.5 kcal/mol for the tightest specific complex (summarized in Table 1). The largest release of free energy upon binding occurs when the unbound duplex itself contains helix-destabilizing distortions and specifically, when those distortions are positioned so that both intercalating side-chains on the protein can contact them. Duplexes 1 and 2,

Protein and DNA purification

AHU was cloned and purified as described.10 E. coli strain RJ1878 (a gift from R. Johnson), a derivative of BL21(DE3) that lacks functional chromosomal HU genes was used for protein expression. After a two-step ammonium sulfate precipitation, AHU was further purified on a heparin column (Amersham Pharmacia) followed by ion-exchange on a Mono-S column (Amersham Pharmacia). The possibility of endonuclease contamination was excluded by incubating the protein with supercoiled plasmid and 10 mM MgCl2

Acknowledgements

We thank Reid Johnson for E. coli strain RJ1878, Ying Zhang Pigli for expert help with subcloning and protein purification, Adam Conway and Arabela Grigorescu for helpful comments on the manuscript, Thomas Lynch for helpful technical advice, and John Marko for interesting discussions of kinking. We thank Shu-wei Yang, Howard Nash, and Kiyoshi Mizuuchi for their contributions to the doubly nicked IHF crystal structure, and Craig Ogata for help with data collection. This study was supported by

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    Present address: K. K. Swinger, Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, IL 60208, USA.

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