HU, the major histone-like protein of E. coli, modulates the binding of IHF to oriC.

HU is one of the most abundant DNA binding proteins of bacteria. Unlike IHF, integration host factor of Escherichia coli, with which HU shares many properties, including a strong sequence homology and similar predicted structure, HU seems to bind non-specifically to DNA whereas IHF binds to specific sites. In this work we compare the binding characteristics of HU and IHF to a DNA fragment containing the minimal origin of replication of E. coli (oriC) and we analyse the effect of HU on the binding capacity of IHF to this oriC fragment. We show that HU interacts randomly and non-specifically with oriC as opposed to the specific binding of IHF to this same DNA sequence. In addition, we show that HU can modulate the binding of IHF to its specific oriC site. Depending on the relative concentrations of HU and IHF, HU is able either to activate or to inhibit the binding of IHF to oriC.     

E. coli DNA binding protein HU forms nucleosomelike structure with circular double-stranded DNA.

The incubation of the E coli DNA binding protein HU with relaxed circular SV40 DNA in the presence of pure nicking-closing enzyme introduces up to 18 negative superhelical turns in the DNA molecules as measured by agarose gel electrophoresis. The maximal density of supercoiling is obtained at a HU-DNA mass ratio of 1. Reconstituted DNA-HU complexes prefixed with glutaraldehyde appear as condensed circular structures having an average of 14 "beads" per circular SV40 DNA molecule, with a "bead" diameter of 180 +/- 23 A. The circular SV40 DNA is condensed by a ratio of 2.0-2.5 relative to naked DNA. This is similar to the ratio (2.4) measured for chromatin formed by reassociation of relaxed SV40 DNA with the four core histones.     

Preferential binding of tumor suppressor p53 to positively or negatively supercoiled DNA involves the C-terminal domain.

The C-terminal domain of the tumor suppressor protein p53 is the site of non-specific DNA binding. Purified p53 produced from baculovirus-infected insect cells binds preferentially to supercoiled DNA, forming bands with lower electrophoretic mobility. This non-covalent binding does not change the linking number of the DNA. An anti-p53 antibody targeting the C-terminal domain partially competes with supercoiled DNA in binding to p53, while antibodies targeted to the N terminus of p53 supershift the complex bands. A synthetic peptide corresponding to amino acid residues 319-393 of human p53 also displays preferential binding to supercoiled DNA, while a mutant peptide, which is unable to form tetramers, is inactive. The center of the equilibrium distribution of topoisomers formed by relaxation with topoisomerase I is not shifted in the presence of p53 although the distribution is broadened. The preferential binding of p53 is exhibited toward both positively and negatively supercoiled DNA. These observations are consistent with a model in which p53 binds to right-handed or left-handed strand crossings.     

Preferential binding of bacteriophage Mu repressor to supercoiled Mu DNA

It was shown, using a relatively simple assay, that Mu repressor, cI, binds specifically to a region which spans the leftmost HindIII cleavage site on the phage genome. This extends the observations of Kwoh and Zipser [Nature (London) 277, 489-491 (1979)], who were able to define a binding region to the left of this site. These results provide support for the idea that the eight blocks of repeated DNA sequences, which also span the HindIII cleavage site, are involved in repressor binding. These results also indicate that cI repressor has a marked preference for supercoiled DNA.     

The histone-like protein HU binds specifically to DNA recombination and repair intermediates

The heterodimeric HU protein associated with the Escherichia coli nucleoid shares some properties with histones and HMG proteins. HU binds DNA junctions and DNA containing a nick much more avidly than double-stranded (ds-) DNA. Cells lacking HU are extremely sensitive to gamma irradiation and we wondered how HU could play a role in maintaining the integrity of the bacterial chromosome. We show that HU binds with high affinity to DNA repair and recombination intermediates, including DNA invasions, DNA overhangs and DNA forks. The DNA structural motif that HU specifically recognizes in all these structures consists of a ds-DNA module joined to a second module containing either ds- or single-stranded (ss-) DNA. The two modules rotate freely relative to one another. Binding specificity results from the simultaneous interaction of HU with these two modules: HU arms bind the ds-DNA module whereas the HU body contacts the 'variable' module containing either ds- or ss-DNA. Both structural motifs are recognized by HU at least 1000-fold more avidly than duplex DNA.     

Immotile phenotype of an Escherichia coli mutant lacking the histone-like protein HU

The histone-like protein HU in Escherichia coli are encoded by the hupA and hupB genes. A hupA-hupB double deletion mutant has now been shown to express an immotile phenotype. The motility of hupA or hupB single mutants was similar to that of wild-type cells. SDS-polyacrylamide gel electrophoresis revealed that the amount of flagellin in the hupA-hupB double deletion mutant was markedly reduced compared with the wild-type strain, suggesting that the immotile phenotype of the double deletion mutant is caused by a loss of flagella.     

Specific and cooperative binding of E. coli single-stranded DNA binding protein to mRNA.

Fluorometric titration of E. coli single-stranded DNA binding protein with various RNAs showed that the protein specifically and cooperatively binds to its own mRNA. The binding inhibited in vitro expression of ssb and bla but not nusA. This inhibition takes place at a physiological concentration of SSB. The function of the protein in gene regulation is discussed.     

The effect of Escherichia coli Uvr protein binding on the topology of supercoiled DNA.

The effects of the binding of the E. coli UvrA and UvrB proteins on the linking number (delta L) of superhelical DNA has been measured. The effects of cofactor ATP structure on UvrAB-nucleoprotein complex formation revealed that nucleotide binding, not hydrolysis, is sufficient to locally unwind the DNA helix of both ultraviolet light-damaged as well as undamaged DNAs. The extent of this unwinding is of the same order of magnitude as the nucleotide distances of the double incision sites generated by the UvrABC endonucleolytic reaction.     

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.     

Nanomechanics of negatively supercoiled diaminopurine-substituted DNA

Single molecule experiments have demonstrated a progressive transition from a B- to an L-form helix as DNA is gently stretched and progressively unwound. The particular sequence of a DNA segment defines both base stacking and hydrogen bonding that affect the partitioning and conformations of the two phases. Naturally or artificially modified bases alter H-bonds and base stacking and DNA with diaminopurine (DAP) replacing adenine was synthesized to produce linear fragments with triply hydrogen-bonded DAP:T base pairs. Both unmodified and DAP-substituted DNA transitioned from a B- to an L-helix under physiological conditions of mild tension and unwinding. This transition avoids writhing and the ease of this transition may prevent cumbersome topological rearrangements in genomic DNA that would require topoisomerase activity to resolve. L-DNA displayed about tenfold lower persistence length than B-DNA. However, left-handed DAP-substituted DNA was twice as stiff as unmodified L-DNA. Unmodified DNA and DAP-substituted DNA have very distinct mechanical characteristics at physiological levels of negative supercoiling and tension.     

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.     

DNA dynamic flexibility and protein recognition: differential stimulation by bacterial histone-like protein HU

The abundant E. coli "histone-like" protein HU is shown to be a differential effector of DNA recognition by three diverse control proteins. DNA recognition by lac repressor and catabolite activator protein is greatly stimulated, while specific aroH DNA recognition by trp repressor is inhibited. BaCl2, an agent previously shown to promote DNA bending, mimics the HU effect to give the same qualitative differential stimulation spectrum. The HU activation involves cooperativity, further suggesting that the various DNA bends and distortions induced during assembly of higher order HU:DNA structures are important for the HU stimulation. Thus, E. coli chromosomal DNA regulation is likely strongly influenced by HU protein that may promote a variety of alternative DNA structures that either facilitate or inhibit specific recognition by diverse control proteins.     

Binding of anti-Z-DNA antibodies to negatively supercoiled SV40 DNA.

The binding of anti-Z-DNA antibody preparations to negatively supercoiled, protein-free SC40 DNA was analyzed. Covalent cross-linking with 0.1% glutaraldehyde followed by DNA restriction endonucleolytic fragmentation and nitrocellulose filtration allowed accurate mapping of antibody binding sites. The critical superhelical density necessary to allow antibody binding was -sigma = 0.056. The major region of antibody-DNA interaction was found within an SV40 segment spanning viral map positions 40 to 474. This region coincides with the nucleosome free region in SV40 minichromosomes and harbours the early and late promoter regions including the SV40 enhancer segment. Although it is unknown whether alternative, non-B-DNA conformations are generated in vivo within SV40 minichromosomes our results emphasize the high degree of DNA structural flexibility that can be realized under negative torsional stress.     

The Escherichia coli histone-like protein HU affects DNA initiation, chromosome partitioning via MukB, and cell division via MinCDE.

Escherichia coli hupA hupB double mutants, lacking both subunits (HU1 and HU2) of the histone-like protein HU, accumulate secondary mutations. In some genetic backgrounds, these include mutations in the minCDE operon, inactivating this system of septation control and resulting in the formation of minicells. In the course of the characterization of hupA hupB mutants, we observed that the simultaneous absence of the HU2 subunit and the MukB protein, implicated in chromosome partitioning, is lethal for the bacteria; the integrity of either HU or MukB thus seems to be essential for bacterial growth. The HU protein has been shown to be involved in DNA replication in vitro; we show here that its inactivation in the hupA hupB double mutant disturbs the synchrony of replication initiation in vivo, as evaluated by flow cytometry. Our results suggest that global nucleoid structure, determined in part by the histone-like protein HU, plays a role in DNA replication initiation, in proper chromosome partitioning directed by the MukFEB proteins, and in correct septum placement directed by the MinCDE proteins.