Mycoplasma gallisepticum produces a histone-like protein that recognizes base mismatches in DNA

Mycoplasmas are the smallest known microorganisms, with drastically reduced genome sizes. One of the essential biochemical pathways lost in mycoplasmas is methylation-mediated DNA repair (MMR), which is responsible for correction of base substitutions, insertions, and deletions in both bacteria and higher organisms. We found that the histone-like protein encoded by the himA/hup_2 gene of Mycoplasma gallisepticum (mgHU) recognizes typical MMR substrates, in contrast to homologues from other species. The recognition of substitution mismatches is sequence-dependent, with affinities decreasing in the following order: CC > CT = TT > AA = AC. Insertions or deletions of one nucleotide are also specifically recognized with the following sequence-dependent preference: A = T > C. One-nucleotide lesions involving guanine are bound only weakly, and this binding is indistinguishable from binding to intact DNA. Although mgHU is dissimilar to Escherichia coli HU, expression in a slow-growing hupAB E. coli strain restores wild-type growth. The results indicate that mgHU executes all essential functions of bacterial architectural proteins. The origin and the possible role of enhanced specificity for typical MMR substrates are discussed.     

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.     

The essential histone-like protein HU plays a major role in Deinococcus radiodurans nucleoid compaction

The nucleoid of radioresistant bacteria, including D .  radiodurans , adopts a highly condensed structure that remains unaltered after exposure to high doses of irradiation. This structure may contribute to radioresistance by preventing the dispersion of DNA fragments generated by irradiation. In this report, we focused our study on the role of HU protein, a nucleoid-associated protein referred to as a histone-like protein, in the nucleoid compaction of D. radiodurans. We demonstrate, using a new system allowing conditional gene expression, that HU is essential for viability in D. radiodurans . Using a tagged HU protein and immunofluorescence microscopy, we show that HU protein localizes all over the nucleoid and that when HU is expressed from a thermosensitive plasmid, its progressive depletion at the non-permissive temperature generates decondensation of DNA before fractionation of the nucleoid into several entities and subsequent cell lysis. We also tested the effect of the absence of Dps, a protein also involved in nucleoid structure. In contrast to the drastic effect of HU depletion, no change in nucleoid morphology and cell viability was observed in dps mutants compared with the wild-type, reinforcing the major role of HU in nucleoid organization and DNA compaction in D. radiodurans .     

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.     

Level of Hu protein binding with DNA and the Hu/DNA ratio in Escherichia coli cells with various generation periods

The possibility of quantitative determination of protein HU in E. coli cell lysates was demonstrated, using enzyme immunoassay with monospecific polyclonal antibodies against HU and homogeneous protein HU. The protein HU/DNA ratio in two cultures of E. coli with different generation periods was found to be constant despite the differences in the levels of proteins HU and DNA. Protein HU was found to be represented by approximately 420.10(3) copies per fast growing. E. coli cell and by 320.10(3) copies per slowly growing cell. These amounts of the HU protein correspond to a ratio of one protein HU molecule per 44 +/- 8 base pairs of DNA and can provide for the nucleosome-like organization of approximately 30% of E. coli DNA length. The constant HU/DNA ratio which is similar to constant histones/DNA ratio provides additional evidence in favour of functional similarity of the HU E. coli DNA-binding protein to histones.     

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.     

Genomic analysis of DNA binding and gene regulation by homologous nucleoid-associated proteins IHF and HU in Escherichia coli K12

IHF and HU are two heterodimeric nucleoid-associated proteins (NAP) that belong to the same protein family but interact differently with the DNA. IHF is a sequence-specific DNA-binding protein that bends the DNA by over 160°. HU is the most conserved NAP, which binds non-specifically to duplex DNA with a particular preference for targeting nicked and bent DNA. Despite their importance, the in vivo interactions of the two proteins to the DNA remain to be described at a high resolution and on a genome-wide scale. Further, the effects of these proteins on gene expression on a global scale remain contentious. Finally, the contrast between the functions of the homo- and heterodimeric forms of proteins deserves the attention of further study. Here we present a genome-scale study of HU- and IHF binding to the Escherichia coli K12 chromosome using ChIP-seq. We also perform microarray analysis of gene expression in single- and double-deletion mutants of each protein to identify their regulons. The sequence-specific binding profile of IHF encompasses ～30% of all operons, though the expression of <10% of these is affected by its deletion suggesting combinatorial control or a molecular backup. The binding profile for HU is reflective of relatively non-specific binding to the chromosome, however, with a preference for A/T-rich DNA. The HU regulon comprises highly conserved genes including those that are essential and possibly supercoiling sensitive. Finally, by performing ChIP-seq experiments, where possible, of each subunit of IHF and HU in the absence of the other subunit, we define genome-wide maps of DNA binding of the proteins in their hetero- and homodimeric forms.     

HU binds and folds single-stranded DNA

The nucleoid-associated protein HU plays an important role in bacterial nucleoid organization and is involved in numerous processes including transposition, recombination and DNA repair. We show here that HU binds specifically DNA containing mismatched region longer than 3 bp as well as DNA bulges. HU binds single-stranded DNA (ssDNA) in a binding mode that is reminiscent but different from earlier reported specific HU interactions with double-helical DNA lesions. An HU dimer requires 24 nt of ssDNA for initial binding, and 12 nt of ssDNA for each additional dimer binding. In the presence of equimolar amounts of HU dimer and DNA, the ssDNA molecule forms an U-loop (hairpin-like) around the protein, providing contacts with both sides of the HU body. This mode differs from the binding of the single-strand-binding protein (SSB) to ssDNA: in sharp contrast to SSB, HU binds ssDNA non-cooperatively and does not destabilize double-helical DNA. Furthermore HU has a strong preference for poly(dG), while binding to poly(dA) is the weakest. HU binding to ssDNA is probably important for its capacity to cover and protect bacterial DNA both intact and carrying lesions.     

DNA looping and the helical repeat in vitro and in vivo: effect of HU protein and enhancer location on Hin invertasome assembly.

Site-specific DNA inversion by the Hin recombinase requires the formation of a multicomponent nucleo-protein structure called an invertasome. In this structure, the two recombination sites bound by Hin are assembled together at the Fis-bound recombinational enhancer with the requisite looping of the intervening DNA segments. We have analyzed the role of the HU protein in invertasome assembly when the enhancer is located at variable positions close to one of the recombination sites. In the absence of HU in vitro and in hupA hupB mutant cells in vivo, invertasome assembly is very inefficient when there is < 104 bp of DNA between the enhancer and recombination site. Invertasome assembly in the presence of HU in vitro or in vivo displayed a periodicity beginning with 60 bp of intervening DNA that reflected its helical repeat. The average helical repeat for this DNA region was calculated by autocorrelation and Fourier transformation to be 11.2 bp per turn for supercoiled DNA both in the presence of HU in vitro and in hup+ cells in vivo. HU is the only protein in Escherichia coli that can promote invertasome formation with short DNA lengths between the enhancer and recombination sites. However, the presence of certain polyamines and a protein activity present in HeLa nuclear extracts can efficiently substitute for HU in invertasome assembly. These data support a model in which HU binds non-specifically to the DNA between the enhancer and recombination site to facilitate DNA looping.     

Purification and functional analysis of recombinant Acholeplasma laidlawii histone-like HU protein

HU is a most abundant DNA-binding protein in bacteria. This protein is conserved either in its heterodimeric form or in one of its homodimeric forms in all bacteria, in plant chloroplasts, and in some viruses. HU protein non-specifically binds and bends DNA as a hetero- or homodimer and can participate in DNA supercoiling and DNA condensation. It also takes part in some DNA functions such as replication, recombination, and repair. HU does not recognize any specific sequences but shows some specificity to cruciform DNA and to repair intermediates, e.g., nick, gap, bulge, 3′-overhang, etc. To understand the features of HU binding to DNA and repair intermediates, a fast and easy HU proteins purification procedure is required. Here we report overproduction and purification of the HU homodimers. The method of HU purification allows obtaining a pure recombinant non-tagged protein cloned in Escherichia coli. We applied this method for purification of Acholeplasma laidlawii HU and demonstrated that this protein possesses a DNA - binding activity and is free of contaminating nuclease activity. Besides that we have shown that expression of A. laidlawii ihf_hu gene in a slow-growing hupAB E. coli strain restores the wild-type growth indicating that aclHU can perform the basic functions of E. coli HU in vivo.     

Bacterial protein HU dictates the morphology of DNA condensates produced by crowding agents and polyamines

Controlling the size and shape of DNA condensates is important in vivo and for the improvement of nonviral gene delivery. Here, we demonstrate that the morphology of DNA condensates, formed under a variety of conditions, is shifted completely from toroids to rods if the bacterial protein HU is present during condensation. HU is a non-sequence-specific DNA binding protein that sharply bends DNA, but alone does not condense DNA into densely packed particles. Less than one HU dimer per 225 bp of DNA is sufficient to completely control condensate morphology when DNA is condensed by spermidine. We propose that rods are favored in the presence of HU because rods contain sharply bent DNA, whereas toroids contain only smoothly bent DNA. The results presented illustrate the utility of naturally derived proteins for controlling the shape of DNA condensates formed in vitro. HU is a highly conserved protein in bacteria that is implicated in the compaction and shaping of nucleoid structure. However, the exact role of HU in chromosome compaction is not well understood. Our demonstration that HU governs DNA condensation in vitro also suggests a mechanism by which HU could act as an architectural protein for bacterial chromosome compaction and organization in vivo.     

The Bacillus subtilis DnaD protein: a putative link between DNA remodeling and initiation of DNA replication

The Bacillus subtilis DnaD protein is an essential protein and a component of the oriC and PriA primosomal cascades, which are responsible for loading the main replicative ring helicase DnaC onto DNA. We present evidence that DnaD also has a global DNA architectural activity, assembling into large nucleoprotein complexes on a plasmid and counteracting plasmid compaction in a manner analogous to that recently seen for the histone-like Escherichia coli HU proteins. This DNA-remodeling role may be an essential function for initiation of DNA replication in the Gram +ve B. subtilis, thus highlighting DnaD as the link between bacterial nucleoid reorganization and initiation of DNA replication.     

Hydroxyurea synchronization of bovine fetal spleen cells

Hydroxyurea (HU) was shown to be an effective synchronization agent for bovine fetal spleen (BFS) cells. Following exposure of cells to 2 mM HU for 32 h, DNA synthesis above background levels was not observed. BFS cells released from the HU block by washing began to synthesize DNA immediately. Within 2 h, 80–85% of the cells were in S phase, as determined by autoradiography, and the maximum rate of DNA synthesis occurred 2–4 h following removal of HU. The rapid induction of DNA synthesis in BFS cells and the high percentage of cells synthesizing DNA immediately after removal of HU demonstrate that HU produces a highly synchronized population of S phase BFS cells. Although RNA and protein synthesis were maintained at near normal rates early after cells were exposed to HU, the rates decreased to 40–50% of those observed in cells seeded in medium without HU by the time of release. These reduced rates of synthesis of RNA and protein in the absence of DNA synthesis may account for the low toxicity of HU for BFS cells.     

Hydroxyurea synchoronization of bovine fetal spleen cells.

Hydroxyurea (HU) was shown to be an effective synchronization agent for bovine fetal spleen (BFS) cells. Following exposure of cells to 2 mM HU for 32 h, DNA synthesis above background levels was not observed. BFS cells released from the HU block by washing began to synthesize DNA immediately. Within 2 h, 80–85% of the cells were in S phase, as determined by autoradiography, and the maximum rate of DNA synthesis occurred 2–4 h following removal of HU. The rapid induction of DNA synthesis in BFS cells and the high percentage of cells synthesizing DNA immediately after removal of HU demonstrate that HU produces a highly synchronized population of S phase BFS cells. Although RNA and protein synthesis were maintained at near normal rates early after cells were exposed to HU, the rates decreased to 40–50% of those observed in cells seeded in medium without HU by the time of release. These reduced rates of synthesis of RNA and protein in the absence of DNA synthesis may account for the low toxicity of HU for BFS cells.     

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.     

Cloning and expression in Escherichia coli of a gene,hup, encoding the histone-like protein HU of Bifidobacterium longum

A genomic DNA library of Bifidobacterium longum ATCC15707 was transfected into an Escherichia coli strain deficient in both HU and IHF, the growth of which is cold-sensitive because of the deficiency in these proteins. Cold-resistant colonies were selected and the DNA was cloned and sequenced. A polypeptide consisted of 93 amino acids, a predicted molecular mass of 9983 Da with an isoelectric point of 10.35, was deduced from an orf in the middle of the DNA fragment. The amino acid sequence was highly similar to HU family proteins, and 26 aas of N terminal was identical to a histone-like protein, HBI, a HU family protein of B. longum. Incapabilities of Mu phage propagation in an E. coli mutant deficient in HU or IHF could be suppressed by DNA bearing this orf. These results showed that the orf is a gene hup encoding HBI, a histone-like protein HU of B. longum.