HU binding to bent DNA: a fluorescence resonance energy transfer and anisotropy study

HU, an architectural DNA-binding protein, either stabilizes DNA in a bent conformation or induces a bend upon binding to give other proteins access to the DNA. In this study, HU binding affinity for a bent DNA sequence relative to a linear sequence was investigated using fluorescence anisotropy measurements. A static bend was achieved by the introduction of two phased A4T4 tracts in a 20 bp duplex. Binding affinity for 20 bp duplexes containing two phased A-tracts in either a 5'-3' or 3'-5' orientation was found to be almost 10-fold higher than HU binding to a random sequence 20 bp duplex (6.1 vs 0.68 microM(-1)). The fluorescence technique of resonance energy transfer was used to quantitatively determine the static bend of the DNA duplexes and the HU-induced bend. DNA molecules were 5'-end labeled with fluorescein as the donor or rhodamine as the acceptor. From the efficiency of energy transfer, the end-to-end distance of the DNA duplexes was calculated. The end-to-end distance relative to DNA contour length (R/R(C)) yields a bend angle for the A-tract duplex of 45 +/- 7 degrees in the absence of HU and 70 +/- 3 degrees in the presence of HU. The bend angle calculated for the T4A4 tract duplex was 62 +/- 4 degrees after binding two HU dimers. Fluorescence anisotropy measurements reveal that HU binds in a 1:1 stoichiometry to the A4T4 tract duplex but a 2:1 stoichiometry to the T4A4 tract and random sequence duplex. These findings suggest that HU binding and recognition of DNA may be governed by a structural mechanism.     

Effects of HU binding on the equilibrium cyclization of mismatched, curved, and normal DNA

The effects of HU, the histone-like protein from Escherichia coli, on the equilibrium cyclization of duplex DNAs have been observed as a function of protein concentration and DNA sequence. The results indicate that the presence of HU significantly enhances the extent of cyclization and increases the melting temperature, T(m), of the cyclized form of the DNA by >10 K. The stabilization of equilibrium cyclization by HU binding is at least -1.2 kcal/mol. The results are consistent with two HU homotypic dimers binding to each of the three 29-mer duplexes studied. One of the 29-mer duplexes contains a central dA tract, one contains mismatched sites, and one a conventional sequence. Stepwise or microscopic association constants, determined from the fluorescence data, range from 1.5 to 0.6 micro M(-1). The binding affinity of the HU dimer is strongest for the mismatched duplex and lowest for the dA tract, consistent with HU dimers having a preference for flexible DNA substrates. These results demonstrate the utility of the equilibrium cyclization approach to monitor DNA-protein interactions. These results have been considered along with those previously obtained to refine a model for the interaction of HU with duplex DNA.     

DNA binding mode transitions of Escherichia coli HU(alphabeta): evidence for formation of a bent DNA--protein complex on intact, linear duplex DNA

Escherichia coli HU_αβ, a major nucleoid-associated protein, organizes chromosomal DNA and facilitates numerous DNA transactions. Using isothermal titration calorimetry, fluorescence resonance energy transfer and a series of DNA lengths (8 bp, 15 bp, 34 bp, 38 bp and 160 bp) we established that HU_αβ interacts with duplex DNA using three different nonspecific binding modes. Both the HU to DNA molar ratio ([HU]/[DNA]) and DNA length dictate the dominant HU binding mode. On sufficiently long DNA (≥ 34 bp), at low [HU]/[DNA], HU populates a noncooperative 34 bp binding mode with a binding constant of 2.1 ± 0.4 × 10⁶ M^− 1, and a binding enthalpy of + 7.7 ± 0.6 kcal/mol at 15 °C and 0.15 M Na⁺. With increasing [HU]/[DNA], HU bound in the noncooperative 34 bp mode progressively converts to two cooperative (ω∼20) modes with site sizes of 10 bp and 6 bp. These latter modes exhibit smaller binding constants (1.1 ± 0.2 × 10⁵ M^− 1 for the 10 bp mode, 3.5 ± 1.4 × 10⁴ M^− 1 for the 6 bp mode) and binding enthalpies (4.2 ± 0.3 kcal/mol for the 10 bp mode, − 1.6 ± 0.3 kcal/mol for the 6 bp mode). As DNA length increases to 34 bp or more at low [HU]/[DNA], the small modes are replaced by the 34 bp binding mode. Fluorescence resonance energy transfer data demonstrate that the 34 bp mode bends DNA by 143 ± 6° whereas the 6 bp and 10 bp modes do not. The model proposed in this study provides a novel quantitative and comprehensive framework for reconciling previous structural and solution studies of HU, including single molecule (force extension measurement), fluorescence, and electrophoretic gel mobility-shift assays. In particular, it explains how HU condenses or extends DNA depending on the relative concentrations of HU and DNA.     

The simultaneous binding of two double-stranded DNA molecules by Escherichia coli RecA protein

We have characterized the double-stranded DNA (dsDNA) binding properties of RecA protein, using an assay based on changes in the fluorescence of 4',6-diamidino-2-phenylindole (DAPI)-dsDNA complexes. Here we use fluorescence, nitrocellulose filter-binding, and DNase I-sensitivity assays to demonstrate the binding of two duplex DNA molecules by the RecA protein filament. We previously established that the binding stoichiometry for the RecA protein-dsDNA complex is three base-pairs per RecA protein monomer, in the presence of ATP. In the presence of ATPgammaS, however, the binding stoichiometry depends on the MgCl2 concentration. The stoichiometry is 3 bp per monomer at low MgCl2 concentrations, but changes to 6 bp per monomer at higher MgCl2 concentrations, with the transition occurring at approximately 5 mM MgCl2. Above this MgCl2 concentration, the dsDNA within the RecA nucleoprotein complex becomes uncharacteristically sensitive to DNase I digestion. For these reasons we suggest that, at the elevated MgCl2 conditions, the RecA-dsDNA nucleoprotein filament can bind a second equivalent of dsDNA. These results demonstrate that RecA protein has the capacity to bind two dsDNA molecules, and they suggest that RecA or RecA-like proteins may effect homologous recognition between intact DNA duplexes.     

The bacterial histone-like protein HU specifically recognizes similar structures in all nucleic acids. DNA,RNA, and their hybrids

HU, a major component of the bacterial nucleoid, shares properties with histones, high mobility group proteins (HMGs), and other eukaryotic proteins. HU, which participates in many major pathways of the bacterial cell, binds without sequence specificity to duplex DNA but recognizes with high affinity DNA repair intermediates. Here we demonstrate that HU binds to double-stranded DNA, double-stranded RNA, and linear DNA-RNA duplexes with a similar low affinity. In contrast to this nonspecific binding to total cellular RNA and to supercoiled DNA, HU specifically recognizes defined structures common to both DNA and RNA. In particular HU binds specifically to nicked or gapped DNA-RNA hybrids and to composite RNA molecules such as DsrA, a small non-coding RNA. HU, which modulates DNA architecture, may play additional key functions in the bacterial machinery via its RNA binding capacity. The simple, straightforward structure of its binding domain with two highly flexible beta-ribbon arms and an alpha-helical platform is an alternative model for the elaborate binding domains of the eukaryotic proteins that display dual DNA- and RNA-specific binding capacities.     

Effects of the nucleoid protein HU on the structure, flexibility, and ring-closure properties of DNA deduced from Monte Carlo simulations

The histone-like HU (heat unstable) protein plays a key role in the organization and regulation of the Escherichia coli genome. The nonspecific nature of HU binding to DNA complicates analysis of the mechanism by which the protein contributes to the looping of DNA. Conventional models of the looping of HU-bound duplexes attribute the changes in biophysical properties of DNA brought about by the random binding of protein to changes in the effective parameters of an ideal helical wormlike chain. Here, we introduce a novel Monte Carlo approach to study the effects of nonspecific HU binding on the configurational properties of DNA directly. We randomly decorated segments of an ideal double-helical DNA with HU molecules that induce the bends and other structural distortions of the double helix find in currently available X-ray structures. We find that the presence of HU at levels approximating those found in the cell reduces the persistence length by roughly threefold compared with that of naked DNA. The binding of protein has particularly striking effects on the cyclization properties of short duplexes, altering the dependence of ring closure on chain length in a way that cannot be mimicked by a simple wormlike model and accumulating at higher-than-expected levels on successfully closed chains. Moreover, the uptake of protein on small minicircles depends on chain length, taking advantage of the HU-induced deformations of DNA structure to facilitate ligation. Circular duplexes with bound HU show much greater propensity than protein-free DNA to exist as negatively supercoiled topoisomers, suggesting a potential role of HU in organizing the bacterial nucleoid. The local bending and undertwisting of DNA by HU, in combination with the number of bound proteins, provide a structural rationale for the condensation of DNA and the observed expression levels of reporter genes in vivo.     

Modulation of DNA conformations through the formation of alternative high-order HU-DNA complexes

HU is an abundant, highly conserved protein associated with the bacterial chromosome. It belongs to a small class of proteins that includes the eukaryotic proteins TBP, SRY, HMG-I and LEF-I, which bind to DNA non-specifically at the minor groove. HU plays important roles as an accessory architectural factor in a variety of bacterial cellular processes such as DNA compaction, replication, transposition, recombination and gene regulation. In an attempt to unravel the role this protein plays in shaping nucleoid structure, we have carried out fluorescence resonance energy transfer measurements of HU-DNA oligonucleotide complexes, both at the ensemble and single-pair levels. Our results provide direct experimental evidence for concerted DNA bending by HU, and the abrogation of this effect at HU to DNA ratios above about one HU dimer per 10-12 bp. These findings support a model in which a number of HU molecules form an ordered helical scaffold with DNA lying in the periphery. The abrogation of these nucleosome-like structures for high HU to DNA ratios suggests a unique role for HU in the dynamic modulation of bacterial nucleoid structure.     

Mechanisms of specific and nonspecific binding of architectural proteins in prokaryotic gene regulation

IHF and HU are small basic proteins of eubacteria that bind as homodimers to double-stranded DNA and bend the duplex to promote architectures required for gene regulation. These architectural proteins share a common alpha/beta fold but exhibit different nucleic acid binding surfaces and distinct functional roles. With respect to DNA-binding specificity, for example, IHF is sequence specific, while HU is not. We have employed Raman difference spectroscopy and gel mobility assays to characterize the molecular mechanisms underlying such differences in DNA recognition. Parallel studies of solution complexes of IHF and HU with the same DNA nonadecamer (5' --> 3' sequence: TC TAAGTAGTTGATTCATA, where the phage lambda H1 consensus sequence of IHF is underlined) show the following. (i) The structure of the targeted DNA site is altered much more dramatically by IHF than by HU binding. (ii) In the IHF complex, the structural perturbations encompass both the sugar-phosphate backbone and the bases of the consensus sequence, whereas only the DNA backbone is altered by HU binding. (iii) In the presence of excess protein, complexes of order higher than 1 dimer per duplex are detected for HU:DNA, though not for IHF:DNA. The results differentiate structural motifs of IHF:DNA and HU:DNA solution complexes, provide Raman signatures of prokaryotic sequence-specific and nonspecific recognition, and suggest that the architectural role of HU may involve the capability to recruit additional binding partners to even relatively short DNA sequences.     

Specific binding of cruciform DNA structures by a protein from human extracts.

A gel electrophoresis binding assay has been used to probe extracts from cultured human lymphoblasts for proteins that bind cruciform structures in duplex DNA. Proteins have been detected that form complexes with synthetic X- and Y-junctions. Several lines of evidence suggest that binding is specific for DNA structure rather than sequence: (1) X- and Y-structures were bound whereas linear duplexes containing identical DNA sequences were not, (2) Binding occurred with equal efficiency to two X-junctions that were constructed from DNA strands of different sequence, (3) One X-junction successfully competed with another for binding whereas linear duplex DNA did not; and (4) protein-DNA complexes were observed at probe:non-specific competitor DNA ratios of 1:10,000.     

Complexation of the quinolone antibiotic norfloxacin with DNA

¹H-NMR spectroscopy (500 MHz) was used to study the complexation of the antibacterial agent norfloxacin (NOR) with DNA tetramers 5′-d(TpGpCpA) and 5′-d(CpGpCpG) in aqueous solution. For the first time, the equilibrium parameters (equilibrium constants, enthalpy, and entropy) were obtained for NOR binding with single-stranded and duplex DNA tetramers. By analyzing the complexation parameters and the induced proton chemical shifts in NOR in various complexes, the character of NOR binding was identified as intercalation in the case of the duplex tetramers and as intercalation with external binding in the case of single-stranded tetramers. NOR proved to preferentially bind to GC sites in DNA duplexes.     

DNA protection by histone-like protein HU from the hyperthermophilic eubacterium Thermotoga maritima

In mesophilic prokaryotes, the DNA-binding protein HU participates in nucleoid organization as well as in regulation of DNA-dependent processes. Little is known about nucleoid organization in thermophilic eubacteria. We show here that HU from the hyperthermophilic eubacterium Thermotoga maritima HU bends DNA and constrains negative DNA supercoils in the presence of topoisomerase I. However, while binding to a single site occludes approximately 35 bp, association of T. maritima HU with DNA of sufficient length to accommodate multiple protomers results in an apparent shorter occluded site size. Such complexes consist of ordered arrays of protomers, as revealed by the periodicity of DNase I cleavage. Association of TmHU with plasmid DNA yields a complex that is remarkably resistant to DNase I-mediated degradation. TmHU is the only member of this protein family capable of occluding a 35 bp nonspecific site in duplex DNA; we propose that this property allows TmHU to form exceedingly stable associations in which DNA flanking the kinks is sandwiched between adjacent proteins. We suggest that T. maritima HU serves an architectural function when associating with a single 35 bp site, but generates a very stable and compact aggregate at higher protein concentrations that organizes and protects the genomic DNA.     

Nonspecific DNA binding and bending by HUαβ: interfaces of the three binding modes characterized by salt-dependent thermodynamics

Previous isothermal titration calorimetry (ITC) and Förster resonance energy transfer studies demonstrated that Escherichia coli HU_αβ binds nonspecifically to duplex DNA in three different binding modes: a tighter-binding 34-bp mode that interacts with DNA in large (> 34 bp) gaps between bound proteins, reversibly bending it by 140^o and thereby increasing its flexibility, and two weaker, modestly cooperative small site-size modes (10 bp and 6 bp) that are useful for filling gaps between bound proteins shorter than 34 bp. Here we use ITC to determine the thermodynamics of these binding modes as a function of salt concentration, and we deduce that DNA in the 34-bp mode is bent around—but not wrapped on—the body of HU, in contrast to specific binding of integration host factor. Analyses of binding isotherms (8-bp, 15-bp, and 34-bp DNA) and initial binding heats (34-bp, 38-bp, and 160-bp DNA) reveal that all three modes have similar log-log salt concentration derivatives of the binding constants (Sk_i) even though their binding site sizes differ greatly; the most probable values of Sk_i on 34-bp DNA or larger DNA are − 7.5 ± 0.5. From the similarity of Sk_i values, we conclude that the binding interfaces of all three modes involve the same region of the arms and saddle of HU. All modes are entropy-driven, as expected for nonspecific binding driven by the polyelectrolyte effect. The bent DNA 34-bp mode is most endothermic, presumably because of the cost of HU-induced DNA bending, while the 6-bp mode is modestly exothermic at all salt concentrations examined. Structural models consistent with the observed Sk_i values are proposed.     

Massive parallel analysis of the binding specificity of histone-like protein HU to single- and double-stranded DNA with generic oligodeoxyribonucleotide microchips

A generic hexadeoxyribonucleotide microchip has been applied to test the DNA-binding properties of HU histone-like bacterial protein, which is known to have a low sequence specificity. All 4096 hexamers flanked within 8mers by degenerate bases at both the 3′- and 5′-ends were immobilized within the 100 × 100 × 20 mm polyacrylamide gel pads of the microchip. Single-stranded immobilized oligonucleotides were converted in some experiments to the double-stranded form by hybridization with a specified mixture of 8mers. The DNA interaction with HU was characterized by three type of measurements: (i) binding of FITC-labeled HU to microchip oligonucleotides; (ii) melting curves of complexes of labeled HU with single-stranded microchip oligonucleotides; (iii) the effect of HU binding on melting curves of microchip double-stranded DNA labeled with another fluorescent dye, Texas Red. Large numbers of measurements of these parameters were carried out in parallel for all or many generic microchip elements in real time with a multi-wavelength fluorescence microscope. Statistical analysis of these data suggests some preference for HU binding to G/C-rich single-stranded oligonucleotides. HU complexes with double-stranded microchip 8mers can be divided into two groups in which HU binding either increased the melting temperature (T_m) of duplexes or decreased it. The stabilized duplexes showed some preference for presence of the sequence motifs AAG, AGA and AAGA. In the second type of complex, enriched with A/T base pairs, the destabilization effect was higher for longer stretches of A/T duplexes. Binding of HU to labeled duplexes in the second type of complex caused some decrease in fluorescence. This decrease also correlates with the higher A/T content and lower T_m. The results demonstrate that generic microchips could be an efficient approach in analysis of sequence specificity of proteins.     

Quantitative Detection of Small Molecule/DNA Complexes Employing a Force-Based and Label-Free DNA-Microarray

Force-based ligand detection is a promising method to characterize molecular complexes label-free at physiological conditions. Because conventional implementations of this technique, e.g., based on atomic force microscopy or optical traps, are low-throughput and require extremely sensitive and sophisticated equipment, this approach has to date found only limited application. We present a low-cost, chip-based assay, which combines high-throughput force-based detection of dsDNA·ligand interactions with the ease of fluorescence detection. Within the comparative unbinding force assay, many duplicates of a target DNA duplex are probed against a defined reference DNA duplex each. The fractions of broken target and reference DNA duplexes are determined via fluorescence. With this assay, we investigated the DNA binding behavior of artificial pyrrole-imidazole polyamides. These small compounds can be programmed to target specific dsDNA sequences and distinguish between D- and L-DNA. We found that titration with polyamides specific for a binding motif, which is present in the target DNA duplex and not in the reference DNA duplex, reliably resulted in a shift toward larger fractions of broken reference bonds. From the concentration dependence nanomolar to picomolar dissociation constants of dsDNA·ligand complexes were determined, agreeing well with prior quantitative DNAase footprinting experiments. This finding corroborates that the forced unbinding of dsDNA in presence of a ligand is a nonequilibrium process that produces a snapshot of the equilibrium distribution between dsDNA and dsDNA·ligand complexes.     

Binding of unnatural alpha,beta-oligocytidylates with DNA duplexes

Binding of short fluorescently labeled AT-containing DNA duplexes with modified oligocytidylates is studied. The latter are modified to contain unnatural alpha-anomers along with natural beta-nucleotides; the nucleotide composition is selected according to putative pattern of unconventional triplex formation between duplex and oligomer bases. Nondenaturing gel electrophoresis is used to study complexation of fluorescent duplexes with cytidyl oligomers and oligocytidylate self-association at low temperatures. A DNA duplex of random AT composition is shown to bind with an excess of the corresponding oligocytidylate in 0.1 M Tris-HCl in the presence of Mg2+. Binding is observed at neutral pH values, while more basic pH (8.0) prevents complexation of the AT duplex and oligocytidylate. Contrary to oligonucleotides of irregular composition, a regular dA30:dT30 duplex does not bind with the dC strand. It is also shown that alternating self-complementary duplex d(AT)16 and oligocytidylate d(CbetaCalpha)15 do not form complexes, and poly-dC self-associates are formed instead. The effect of 2'-O-methylation of the third strand on complex formation and self-association is also analyzed. The results suggest that a modified oligocytidylate binds with a random-composition duplex, albeit with lower efficiency.     

Interaction of EcoRII endonuclease with DNA substrates containing single recognition sites.

EcoRII is unusual among type II restriction enzymes in that, while it cleaves substrates such as pBR322 and bacteriophage lambda that contain several recognition sites for the enzyme efficiently, substrates such as the genomes of bacteriophages T3 and T7 which contain a small number of recognition sites are cut poorly by it. Interestingly, pBR322, or a short DNA duplex containing a single site for the enzyme, can activate the enzyme to cleave resistant substrates. We show here that, at low concentrations, activator short duplexes are themselves cleaved poorly by the enzyme. Further, the reaction shows substrate cooperativity, and at high concentrations, the duplexes are both activators and good substrates for the enzyme. This supports the model that the activation of EcoRII involves binding of more than one DNA molecule and provides a simple system to study the mechanism of activation. Using a gel mobility shift assay, we show that the enzyme forms sequence-specific, methylation-sensitive complexes with the duplexes in the absence of activating DNA. Therefore, resistance of the short duplexes to the enzyme at low concentrations cannot be due to an inability of the enzyme to bind the duplexes. Interestingly, these complexes are stable in the presence of Mg2+, the cofactor for the enzyme, and the complexes obtained in the presence of Mg2+ do not contain DNA that is cleaved by the enzyme. The inefficient step in the action of EcoRII on resistant substrates must occur subsequent to initial substrate binding and it is this step that the activating DNA must regulate.     

Applying 6-Methylisoxanthopterin-Enhanced Fluorescence To Examine Protein-DNA Interactions in the Picomolar Range

Incorporation of fluorescent nucleoside analogues into duplex DNA usually leads to a reduction in quantum yield, which significantly limits their potential use and application. We have identified two pentamer DNA sequences containing 6-methylisoxanthopterin (6-MI) (ATFAA and AAFTA, where F is 6-MI) that exhibit significant enhancement of fluorescence upon formation of duplex DNA with quantum yields close to that of monomeric 6-MI. The enhanced fluorescence dramatically increases the utility and sensitivity of the probe and is used to study protein-DNA interactions of nanomolar specificity in this work. The increased sensitivity of 6-MI allows anisotropy binding measurements to be performed at DNA concentrations of 1 nM and fluorescence intensity measurements at 50 pM DNA. The ATFAA sequence was incorporated into DNA constructs to measure the binding affinity of four different protein-DNA interactions that exhibit sequence-specific and non-sequence-specific recognition. In all cases, the K(d) values obtained were consistent with previously reported values measured by other methods. Time-resolved and steady-state fluorescence measurements demonstrate that 6-MI fluorescence is very sensitive to local distortion and reports on different degrees of protein-induced perturbations with single-base resolution, where the largest changes occur at the site of protein binding.     

Selective recognition of pyrimidine-pyrimidine DNA mismatches by distance-constrained macrocyclic bis-intercalators

Binding of three macrocyclic bis-intercalators, derivatives of acridine and naphthalene, and two acyclic model compounds to mismatch-containing and matched duplex oligodeoxynucleotides was analyzed by thermal denaturation experiments, electrospray ionization mass spectrometry studies (ESI-MS) and fluorescent intercalator displacement (FID) titrations. The macrocyclic bis-intercalators bind to duplexes containing mismatched thymine bases with high selectivity over the fully matched ones, whereas the acyclic model compounds are much less selective and strongly bind to the matched DNA. Moreover, the results from thermal denaturation experiments are in very good agreement with the binding affinities obtained by ESI-MS and FID measurements. The FID results also demonstrate that the macrocyclic naphthalene derivative BisNP preferentially binds to pyrimidine–pyrimidine mismatches compared to all other possible base mismatches. This ligand also efficiently competes with a DNA enzyme (M.TaqI) for binding to a duplex with a TT-mismatch, as shown by competitive fluorescence titrations. Altogether, our results demonstrate that macrocyclic distance-constrained bis-intercalators are efficient and selective mismatch-binding ligands that can interfere with mismatch-binding enzymes.     

The interaction of lamda-Cro repressor and its mutant covalent S-S-dimer lamda-CroV55C with symmetric and asymmetric DNA

Binding of lamda-Cro protein and mutant CroV55C disulfide bonded dimer to synthetic olygonucleotide duplexes were studied using a competition with distamycin A. The equilibrium binding constants for lamda operator OR3 and duplexes contained symmetry left or right halves of OR3 with one base pair deletion or insertion in center of duplex were calculated. The higher binding constant for Cro was detected with 17 bp symmetry duplex consist two left halves of OR3, for the mutant CroV55C higher binding constant was detected with 16 bp derivate of this duplex with the central GC base pair deletion.     

Structural perturbations induced in linear and circular DNA by the architectural protein HU from Bacillus stearothermophilus

HU is a small DNA-binding protein of eubacteria that is believed to induce or stabilize bending of the double helix and mediate nucleoid compaction in vivo. Although HU does not bind preferentially to specific DNA sequences, it is known to have high affinity for DNA sites containing structural anomalies, such as unpaired or mismatched bases, nicks, and four-way junctions. We have employed Raman spectroscopy to further investigate the structural basis of HU-DNA recognition in solution. Experiments were carried out on the homodimeric HU protein of Bacillus stearothermophilus (HUBst) and a 222-bp DNA fragment, which was isolated in linear (DNA(L222)) and circular (DNA(C222)) forms. In the absence of bound HUBst the Raman signatures of DNA(L222) and DNA(C222) are nearly superimposable, indicating that circularization produces no substantial change in the local B-DNA conformation. Conversely, the Raman signatures of DNA(L222) and DNA(C222) are perturbed significantly and specifically by HUBst binding. The HUBst-induced perturbations are markedly greater for the circularized DNA target. These results support an opportunistic molecular mechanism, in which HU binding is facilitated by intrinsic nonlinearity or flexibility in the DNA target. We propose that DNA segments which are bent or predisposed toward bending provide the high-affinity sites for HU attachment and nucleoid condensation. This model is consistent with the wide range of DNA bending angles reported in crystal structures of HU-DNA complexes.