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.     

Twelve species of the nucleoid-associated protein from Escherichia coli. Sequence recognition specificity and DNA binding affinity.

The genome of Escherichia coli is composed of a single molecule of circular DNA with the length of about 47,000 kilobase pairs, which is associated with about 10 major DNA-binding proteins, altogether forming the nucleoid. We expressed and purified 12 species of the DNA-binding protein, i.e. CbpA (curved DNA-binding protein A), CbpB or Rob (curved DNA-binding protein B or right arm of the replication origin binding protein), DnaA (DNA-binding protein A), Dps (DNA-binding protein from starved cells), Fis (factor for inversion stimulation), Hfq (host factor for phage Q(beta)), H-NS (histone-like nucleoid structuring protein), HU (heat-unstable nucleoid protein), IciA (inhibitor of chromosome initiation A), IHF (integration host factor), Lrp (leucine-responsive regulatory protein), and StpA (suppressor of td(-) phenotype A). The sequence specificity of DNA binding was determined for all the purified nucleoid proteins using gel-mobility shift assays. Five proteins (CbpB, DnaA, Fis, IHF, and Lrp) were found to bind to specific DNA sequences, while the remaining seven proteins (CbpA, Dps, Hfq, H-NS, HU, IciA, and StpA) showed apparently sequence-nonspecific DNA binding activities. Four proteins, CbpA, Hfq, H-NS, and IciA, showed the binding preference for the curved DNA. From the apparent dissociation constant (K(d)) determined using the sequence-specific or nonspecific DNA probes, the order of DNA binding affinity were determined to be: HU > IHF > Lrp > CbpB(Rob) > Fis > H-NS > StpA > CbpA > IciA > Hfq/Dps, ranging from 25 nM (HU binding to the non-curved DNA) to 250 nM (Hfq binding to the non-curved DNA), under the assay conditions employed.     

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.     

Replication fork passage drives asymmetric dynamics of a critical nucleoid‐associated protein in Caulobacter

In bacteria, chromosome dynamics and gene expression are modulated by nucleoid‐associated proteins (NAPs), but little is known about how NAP activity is coupled to cell cycle progression. Using genomic techniques, quantitative cell imaging, and mathematical modeling, our study in Caulobacter crescentus identifies a novel NAP (GapR) whose activity over the cell cycle is shaped by DNA replication. GapR activity is critical for cellular function, as loss of GapR causes severe, pleiotropic defects in growth, cell division, DNA replication, and chromosome segregation. GapR also affects global gene expression with a chromosomal bias from origin to terminus, which is associated with a similar general bias in GapR binding activity along the chromosome. Strikingly, this asymmetric localization cannot be explained by the distribution of GapR binding sites on the chromosome. Instead, we present a mechanistic model in which the spatiotemporal dynamics of GapR are primarily driven by the progression of the replication forks. This model represents a simple mechanism of cell cycle regulation, in which DNA‐binding activity is intimately linked to the action of DNA replication.     

Real‐time detection of condensin‐driven DNA compaction reveals a multistep binding mechanism

Condensin, a conserved member of the SMC protein family of ring‐shaped multi‐subunit protein complexes, is essential for structuring and compacting chromosomes. Despite its key role, its molecular mechanism has remained largely unknown. Here, we employ single‐molecule magnetic tweezers to measure, in real time, the compaction of individual DNA molecules by the budding yeast condensin complex. We show that compaction can proceed in large steps, driving DNA molecules into a fully condensed state against forces of up to 2 pN. Compaction can be reversed by applying high forces or adding buffer of high ionic strength. While condensin can stably bind DNA in the absence of ATP, ATP hydrolysis by the SMC subunits is required for rendering the association salt insensitive and for the subsequent compaction process. Our results indicate that the condensin reaction cycle involves two distinct steps, where condensin first binds DNA through electrostatic interactions before using ATP hydrolysis to encircle the DNA topologically within its ring structure, which initiates DNA compaction. The finding that both binding modes are essential for its DNA compaction activity has important implications for understanding the mechanism of chromosome compaction.     

A DNA‐binding protein defines the precise region of chromosome capture during Bacillus sporulation

During sporulation, Bacillus subtilis divides around the nucleoid near one cell pole, initially capturing approximately one quarter of one chromosome in the newly formed forespore compartment. While it is known that a specific region of the nucleoid is reproducibly captured in the forespore, the mechanism underlying the precision of capture is unknown. Here we describe a role for RefZ, a DNA‐binding protein that regulates FtsZ, and its cognate binding motifs (RBMs) in defining the specific region of chromosome initially captured in the forespore. RefZ is conserved across the Bacillus genus and remains functional as an inhibitor of cell division in a species‐swapping experiment. The RBMs are also conserved in their positioning relative to oriC across Bacillus, suggesting that the function of the RBMs is both important and position‐dependent in the genus. In B. subtilis, the RBMs flank the region of the chromosome captured at the time of cell division, and we find that RefZ binds the five oriC‐proximal RBMs with similar apparent affinity in units of two and four. refZ and RBM mutants capture chromosomal regions normally excluded from the forespore, suggesting that RefZ‐RBM complexes play a role in regulating the position of cell division relative to the chromosome during sporulation.     

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 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.     

Testing mechanisms of DNA sliding by architectural DNA-binding proteins: dynamics of single wild-type and mutant protein molecules in vitro and in vivo

Architectural DNA-binding proteins (ADBPs) are abundant constituents of eukaryotic or bacterial chromosomes that bind DNA promiscuously and function in diverse DNA reactions. They generate large conformational changes in DNA upon binding yet can slide along DNA when searching for functional binding sites. Here we investigate the mechanism by which ADBPs diffuse on DNA by single-molecule analyses of mutant proteins rationally chosen to distinguish between rotation-coupled diffusion and DNA surface sliding after transient unbinding from the groove(s). The properties of yeast Nhp6A mutant proteins, combined with molecular dynamics simulations, suggest Nhp6A switches between two binding modes: a static state, in which the HMGB domain is bound within the minor groove with the DNA highly bent, and a mobile state, where the protein is traveling along the DNA surface by means of its flexible N-terminal basic arm. The behaviors of Fis mutants, a bacterial nucleoid-associated helix-turn-helix dimer, are best explained by mobile proteins unbinding from the major groove and diffusing along the DNA surface. Nhp6A, Fis, and bacterial HU are all near exclusively associated with the chromosome, as packaged within the bacterial nucleoid, and can be modeled by three diffusion modes where HU exhibits the fastest and Fis the slowest diffusion.     

Nucleoid occlusion protein Noc recruits DNA to the bacterial cell membrane

To proliferate efficiently, cells must co‐ordinate division with chromosome segregation. In Bacillus subtilis, the nucleoid occlusion protein Noc binds to specific DNA sequences (NBSs) scattered around the chromosome and helps to protect genomic integrity by coupling the initiation of division to the progression of chromosome replication and segregation. However, how it inhibits division has remained unclear. Here, we demonstrate that Noc associates with the cell membrane via an N‐terminal amphipathic helix, which is necessary for function. Importantly, the membrane‐binding affinity of this helix is weak and requires the assembly of nucleoprotein complexes, thus establishing a mechanism for DNA‐dependent activation of Noc. Furthermore, division inhibition by Noc requires recruitment of NBS DNA to the cell membrane and is dependent on its ability to bind DNA and membrane simultaneously. Indeed, Noc production in a heterologous system is sufficient for recruitment of chromosomal DNA to the membrane. Our results suggest a simple model in which the formation of large membrane‐associated nucleoprotein complexes physically occludes assembly of the division machinery.     

Reorganization of inter‐chromosomal interactions in the 2q37‐deletion syndrome

Chromosomes occupy distinct interphase territories in the three‐dimensional nucleus. However, how these chromosome territories are arranged relative to one another is poorly understood. Here, we investigated the inter‐chromosomal interactions between chromosomes 2q, 12, and 17 in human mesenchymal stem cells (MSCs) and MSC‐derived cell types by DNA‐FISH. We compared our findings in normal karyotypes with a three‐generation family harboring a 2q37‐deletion syndrome, featuring a heterozygous partial deletion of histone deacetylase 4 (HDAC4) on chr2q37. In normal karyotypes, we detected stable, recurring arrangements and interactions between the three chromosomal territories with a tissue‐specific interaction bias at certain loci. These inter‐chromosomal interactions were confirmed by Hi‐C. Interestingly, the disease‐related HDAC4 deletion resulted in displaced inter‐chromosomal arrangements and altered interactions between the deletion‐affected chromosome 2 and chromosome 12 and/or 17 in 2q37‐deletion syndrome patients. Our findings provide evidence for a direct link between a structural chromosomal aberration and altered interphase architecture that results in a nuclear configuration, supporting a possible molecular pathogenesis.