Site-specific DNA-binding Proteins Research Articles

Prolonged Combinatorial Pharmacological Therapy Targeting the Epigenome Increases Fetal Hemoglobin to Clinically Relevant Levels in Normal, Nonanemic Baboons

Elevated levels of Fetal Hemoglobin (HbF; α2γ2) reduce the symptoms associated with Sickle Cell Disease (SCD) and increase the lifespan of patients. An effective pharmacological therapy that increases HbF would advance the treatment of SCD. HbF levels are developmentally regulated by transcriptional repression of the γ-globin gene in adult erythroid cells. This is achieved by a co-repressor complex containing multiple enzymes that catalyze repressive epigenetic modifications of chromatin targeted to the γ-globin promoter by site-specific DNA binding proteins. Pharmacological inhibitors of repressive epigenetic enzymes within the complex including DNMT1, LSD1 (KDM1A), G9A, and HDACs increase HbF in cell line and animal models and SCD patients. Many of these drugs have been studied in Phase 1 trials, but only the DNMT inhibitor decitabine (DAC) has advanced to Phase 3 trials. The successful development of a pharmacological therapy to increase HbF for SCD has thus far been hindered by dose-limiting side effects including neutropenia, thrombocytopenia, and/or thrombophilia. The goal of this current work is to develop a pharmacological therapy employing a combination of drugs that target different repressive epigenetic enzymes within the co-repressor complex based on the hypothesis that administration of these drugs in combination will reduce the dose and/or time of exposure to any single agent to minimize adverse effects while still stimulating clinically meaningful HbF elevations. To test this hypothesis, baboons, long considered the best animal model to test the effect of HbF-inducing agents due to conservation of the structure and developmental regulation of the β-globin gene locus between baboons and humans, were treated with a combination of two drugs, DAC and RN-1, that inhibit DNMT1 and LSD1 respectively. Analysis of seven baboons used in our studies showed that they comprised two phenotypic groups differentiated by baseline pre-treatment HbF expression (Figure 1). Two baboons of each phenotypic group were chosen to study the effect of DAC and RN-1, alone and in combination, on HbF. Either RN-1 (0.25mg/kg/d) or DAC (0.5mg/kg/d), were administered subcutaneously individually or in combination to normal, non-anemic animals for 2 days. The effect on γ-globin expression was measured by flow cytometry analysis of F-retics and RT-PCR of γ-globin mRNA in peripheral blood at least 3X per week post-drug treatments. Each drug induced a significant increase in F-retics and peripheral blood γ-globin mRNA in both phenotypic groups that peaked on d8 (Figure 1) while differences in F-retics and γ-globin mRNA between the two groups were maintained. Combined administration (RN-1 (0.25mg/kg/d; DAC (0.5mg/kg/d); 2d) increased levels of F-retics and γ-globin mRNA to greater than additive levels (Figure 1) while the difference in γ-globin mRNA (p<0.02), but not F-retics, between the two groups was still observed. To assess the effect of sustained administration of the two drug combination on HbF expression and evaluate possible hematological toxicity, two normal baboons, one of the low HbF group (PA 9254) and one of the high HbF group (PA 9194), were treated weekly with 12 total courses of the DAC-RN-1 combination (DAC, 0.5mg/kg/d; RN-1, 0.25mg/kg/d; 2d/wk). F cells increased approximately 6 fold to 60.5% in the high HbF animal and >10 fold to 42% in the low HbF baboon (Table 1). HPLC analysis of globin chain expression showed that HbF (γ/γ+β) increased tenfold in each animal (Table 1). The calculated % HbF/ F cell values were 33% for PA 9194 (High HbF group) and 23.5% for PA 9254 (Low HbF group). No instances of moderate or severe neutropenia, thrombocytopenia, or thrombophilia were observed during the treatment period (Table 1). Decreased Hct, Hb, and RBCs were apparent in the high HbF group baboon (PA 9194) but not the low HbF group baboon (PA 9254). In conclusion, combinatorial administration of DAC and RN-1 increased HbF and F cell numbers in a greater than additive manner. Prolonged treatment of two normal, nonanemic baboons increased F cells and HbF to levels predicted to be clinically significant in the absence of neutropenia, thrombocytopenia and/or thrombophilia throughout the treatment period although a reduction in Hct, RBCs, and Hb was observed in one animal. These results strongly suggest additional drug combinations, dosing, and schedules to increase HbF for the treatment of SCD should be pursued. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Chromosome segregation in Archaea: SegA-and SegB-DNA complex structures provide insights into segrosome assembly.

Genome segregation is a vital process in all organisms. Chromosome partitioning remains obscure in Archaea, the third domain of life. Here, we investigated the SegAB system from Sulfolobus solfataricus. SegA is a ParA Walker-type ATPase and SegB is a site-specific DNA-binding protein. We determined the structures of both proteins and those of SegA–DNA and SegB–DNA complexes. The SegA structure revealed an atypical, novel non-sandwich dimer that binds DNA either in the presence or in the absence of ATP. The SegB structure disclosed a ribbon–helix–helix motif through which the protein binds DNA site specifically. The association of multiple interacting SegB dimers with the DNA results in a higher order chromatin-like structure. The unstructured SegB N-terminus plays an essential catalytic role in stimulating SegA ATPase activity and an architectural regulatory role in segrosome (SegA–SegB–DNA) formation. Electron microscopy results also provide a compact ring-like segrosome structure related to chromosome organization. These findings contribute a novel mechanistic perspective on archaeal chromosome segregation.

Physical Modeling of a Sliding Clamp Mechanism for the Spreading of ParB at Short Genomic Distance from Bacterial Centromere Sites.

SummaryBacterial ParB partitioning proteins involved in chromosomes and low-copy-number plasmid segregation are cytosine triphosphate (CTP)-dependent molecular switches. CTP-binding converts ParB dimers to DNA clamps, allowing unidimensional diffusion along the DNA. This sliding property has been proposed to explain the ParB spreading over large distances from parS centromere sites where ParB is specifically loaded. We modeled such a “clamping and sliding” mechanism as a typical reaction-diffusion system, compared it to the F plasmid ParB DNA binding pattern, and found that it can account neither for the long range of ParB binding to DNA nor for the rapid assembly kinetics observed in vivo after parS duplication. Also, it predicts a strong effect on the F plasmid ParB binding pattern from the presence of a roadblock that is not observed in ChIP-sequencing (ChIP-seq). We conclude that although “clamping and sliding” can occur at short distances from parS, another mechanism must apply for ParB recruitment at larger genomic distances.

Variants in nuclear factor I genes influence growth and development.

The nuclear factor one (NFI) site-specific DNA-binding proteins represent a family of transcription factors that are important for the development of multiple organ systems, including the brain. During brain development in mice, the expression patterns of Nfia, Nfib, and Nfix overlap, and knockout mice for each of these exhibit overlapping brain defects, including megalencephaly, dysgenesis of the corpus callosum, and enlarged ventricles, which implies a common but not redundant function in brain development. In line with these models, human phenotypes caused by haploinsufficiency of NFIA, NFIB, and NFIX display significant overlap, sharing neurodevelopmental deficits, macrocephaly, brain anomalies, and variable somatic overgrowth. Other anomalies may be present depending on the NFI gene involved. The possibility of variants in NFI genes should therefore be considered in individuals with intellectual disability and brain overgrowth, with individual NFI-related conditions being differentiated from one another by additional signs and symptoms. The exception is provided by specific NFIX variants that act in a dominant negative manner, as these cause a recognizable entity with more severe cognitive impairment and marked bone dysplasia, Marshall-Smith syndrome. NFIX duplications are associated with a phenotype opposite to that of haploinsufficiency, characterized by short stature, small head circumference, and delayed bone age. The spectrum of NFI-related disorders will likely be further expanded, as larger cohorts are assessed.

The meiotic-specific Mek1 kinase in budding yeast regulates interhomolog recombination and coordinates meiotic progression with double-strand break repair.

Recombination, along with sister chromatid cohesion, is used during meiosis to physically connect homologous chromosomes so that they can be segregated properly at the first meiotic division. Recombination is initiated by the introduction of programmed double strand breaks (DSBs) into the genome, a subset of which is processed into crossovers. In budding yeast, the regulation of meiotic DSB repair is controlled by a meiosis-specific kinase called Mek1. Mek1 kinase activity promotes recombination between homologs, rather than sister chromatids, as well as the processing of recombination intermediates along a pathway that results in synapsis of homologous chromosomes and the distribution of crossovers throughout the genome. In addition, Mek1 kinase activity provides a readout for the number of DSBs in the cell as part of the meiotic recombination checkpoint. This checkpoint delays entry into the first meiotic division until DSBs have been repaired by inhibiting the activity of the meiosis-specific transcription factor Ndt80, a site-specific DNA binding protein that activates transcription of over 300 target genes. Recent work has shown that Mek1 binds to Ndt80 and phosphorylates it on multiple sites, including the DNA binding domain, thereby preventing Ndt80 from activating transcription. As DSBs are repaired, Mek1 is removed from chromosomes and its activity decreases. Loss of the inhibitory Mek1 phosphates and phosphorylation of Ndt80 by the meiosis-specific kinase, Ime2, promote Ndt80 activity such that Ndt80 transcribes its own gene in a positive feedback loop, as well as genes required for the completion of recombination and entry into the meiotic divisions. Mek1 is therefore the key regulator of meiotic recombination in yeast.

PRDM14 Is a Unique Epigenetic Regulator Stabilizing Transcriptional Networks for Pluripotency.

PR-domain containing protein 14 (PRDM14) is a site-specific DNA-binding protein and is required for establishment of pluripotency in embryonic stem cells (ESCs) and primordial germ cells (PGCs) in mice. DNA methylation status is regulated by the balance between de novo methylation and passive/active demethylation, and global DNA hypomethylation is closely associated with cellular pluripotency and totipotency. PRDM14 ensures hypomethylation in mouse ESCs and PGCs through two distinct layers, transcriptional repression of the DNA methyltransferases Dnmt3a/b/l and active demethylation by recruitment of TET proteins. However, the function of PRDM14 remains unclear in other species including humans. Hence, here we focus on the unique characteristics of mouse PRDM14 in the epigenetic regulation of pluripotent cells and primordial germ cells. In addition, we discuss the expression regulation and function of PRDM14 in other species compared with those in mice.

Measuring Search Times in Site Specific DNA Binding

Facilitated diffusion is the accepted theory of how site-specific DNA binding proteins search for their targets. When DNA sliding is included in the search strategy, protein search times are predicted to exhibit a bi-model dependence on the sliding length, with a minimum occurring at an optimal sliding length. This peak in specific association rate as sliding length is varied has rarely been directly measured. We are studying facilitated diffusion using restriction endonucleases as a model system. Our multiplexing single molecule technique is based on DNA tethering of microbeads and can measure the time of cleavage of up to hundreds of DNAs in a single experiment. We use diffusion controlled conditions to directly measure the search time of proteins which cleave dsDNA. In the work reported here, we vary the siding length by varying the salt concentration from a few mM up to 150 mM NaCl. We have discovered a peak in specific association rates of both EcoRI and NdeI, consistent with the predictions of facilitated diffusion. These peaks both occur near 80 mM NaCl, significantly less than physiological salt concentrations. I will discuss how our technique works and how we are using the theory of facilitated diffusion to understand our results. I will further discuss our efforts to directly verify DNA sliding in these systems using single molecule fluorescent imaging.

ParB Partition Proteins: Complex Formation and Spreading at Bacterial and Plasmid Centromeres.

In bacteria, active partition systems contribute to the faithful segregation of both chromosomes and low-copy-number plasmids. Each system depends on a site-specific DNA binding protein to recognize and assemble a partition complex at a centromere-like site, commonly called parS. Many plasmid, and all chromosomal centromere-binding proteins are dimeric helix-turn-helix DNA binding proteins, which are commonly named ParB. Although the overall sequence conservation among ParBs is not high, the proteins share similar domain and functional organization, and they assemble into similar higher-order complexes. In vivo, ParBs “spread,” that is, DNA binding extends away from the parS site into the surrounding non-specific DNA, a feature that reflects higher-order complex assembly. ParBs bridge and pair DNA at parS and non-specific DNA sites. ParB dimers interact with each other via flexible conformations of an N-terminal region. This review will focus on the properties of the HTH centromere-binding protein, in light of recent experimental evidence and models that are adding to our understanding of how these proteins assemble into large and dynamic partition complexes at and around their specific DNA sites.

Methods for Investigating DNA Accessibility with Single Nucleosomes.

Nucleosomes are the fundamental organizing unit of all eukaryotic genomes. Understanding how proteins gain access to DNA-binding sites located within nucleosomes is important for understanding DNA processing including transcription, replication, and repair. Single-molecule total internal reflection fluorescence (smTIRF) microscopy measurements can provide key insight into how proteins gain and maintain access to DNA sites within nucleosomes. Here, we describe methods for smTIRF experiments including the preparation of fluorophore-labeled nucleosomes, the smTIRF system, data acquisition, analysis, and controls. These methods are presented for investigating transcription factor binding within nucleosomes. However, they are applicable for investigating the binding of any site-specific DNA-binding protein within nucleosomes.

P Transposable Elements in Drosophila and other Eukaryotic Organisms.

P transposable elements were discovered in Drosophila as the causative agents of a syndrome of genetic traits called hybrid dysgenesis. Hybrid dysgenesis exhibits a unique pattern of maternal inheritance linked to the germline-specific small RNA piwi-interacting (piRNA) pathway. The use of P transposable elements as vectors for gene transfer and as genetic tools revolutionized the field of Drosophila molecular genetics. P element transposons have served as a useful model to investigate mechanisms of cut-and-paste transposition in eukaryotes. Biochemical studies have revealed new and unexpected insights into how eukaryotic DNA-based transposons are mobilized. For example, the P element transposase makes unusual 17nt-3' extended double-strand DNA breaks at the transposon termini and uses guanosine triphosphate (GTP) as a cofactor to promote synapsis of the two transposon ends early in the transposition pathway. The N-terminal DNA binding domain of the P element transposase, called a THAP domain, contains a C2CH zinc-coordinating motif and is the founding member of a large family of animal-specific site-specific DNA binding proteins. Over the past decade genome sequencing efforts have revealed the presence of P element-like transposable elements or P element transposase-like genes (called THAP9) in many eukaryotic genomes, including vertebrates, such as primates including humans, zebrafish and Xenopus, as well as the human parasite Trichomonas vaginalis, the sea squirt Ciona, sea urchin and hydra. Surprisingly, the human and zebrafish P element transposase-related THAP9 genes promote transposition of the Drosophila P element transposon DNA in human and Drosophila cells, indicating that the THAP9 genes encode active P element "transposase" proteins.

Kinetic gating mechanism of DNA damage recognition by Rad4/XPC.

The xeroderma pigmentosum C (XPC) complex initiates nucleotide excision repair by recognizing DNA lesions before recruiting downstream factors. How XPC detects structurally diverse lesions embedded within normal DNA is unknown. Here we present a crystal structure that captures the yeast XPC orthologue (Rad4) on a single register of undamaged DNA. The structure shows that a disulphide-tethered Rad4 flips out normal nucleotides and adopts a conformation similar to that seen with damaged DNA. Contrary to many DNA repair enzymes that can directly reject non-target sites as structural misfits, our results suggest that Rad4/XPC uses a kinetic gating mechanism whereby lesion selectivity arises from the kinetic competition between DNA opening and the residence time of Rad4/XPC per site. This mechanism is further supported by measurements of Rad4-induced lesion-opening times using temperature-jump perturbation spectroscopy. Kinetic gating may be a general mechanism used by site-specific DNA-binding proteins to minimize time-consuming interrogations of non-target sites.

Thymidine Glycol: The Effect on DNA Structure and DNA Binding by Site-Specific Proteins

Thymidine glycol (5,6-dihydroxy-5,6-dihydrothymidine, Tg) is a major type of oxidative damage in DNA. During chemical oligonucleotide synthesis, Tg residue was incorporated in the different positions of 17 b.p. DNA duplexes, which differ in one base pair in the internal part. According to UV-melting curves, Tg destabilizes the double helix in a sequence independent manner. In contrast, the localized alterations in duplex structure were shown by CD spectroscopy to depend on the type of base pairs flanking the Tg lesion. Molecular dynamics simulations demonstrate that Tg is partially out of the double helix. For the first time, Tg impact on several site-specific DNA-binding proteins is studied, namely p50 and p65 subunits of nuclear factor kappa-B (NF-κB) and DNA methyltransferase SsoII (M.SsoII). Our results show that p50/p50 and p65/p65 homodimers of NF-κB can tolerate a single Tg residue in the binding site quite well. Nevertheless the homodimers have different affinities to the oxidized κB site depending on the Tg position. M.SsoII can act as a transcription repressor when bound to the regulatory site. M.SsoII demonstrates decreased affinity and lowered methylation efficiency when its methylation site contains Tg in the central position. Single Tg in one half of the regulatory site decreases M.SsoII affinity to the oxidized DNA, whereas Tg presence in both half-sites prevents M.SsoII binding to such ligand.

Nuclear factor one transcription factors: Divergent functions in developmental versus adult stem cell populations.

Nuclear factor one (NFI) transcription factors are a group of site-specific DNA-binding proteins that are emerging as critical regulators of stem cell biology. During development NFIs promote the production of differentiated progeny at the expense of stem cell fate, with Nfi null mice exhibiting defects such as severely delayed brain and lung maturation, skeletomuscular defects and renal abnormalities, phenotypes that are often consistent with patients with congenital Nfi mutations. Intriguingly, recent research suggests that in adult tissues NFI factors play a qualitatively different role than during development, with NFIs serving to promote the survival and maintenance of slow-cycling adult stem cell populations rather than their differentiation. Here we review the role of NFI factors in development, largely focusing on their role as promoters of stem cell differentiation, and attempt to reconcile this with the emerging role of NFIs in adult stem cell niches.

Unusual characteristics of the DNA binding domain of epigenetic regulatory protein MeCP2 determine its binding specificity.

The protein MeCP2 mediates epigenetic regulation by binding methyl-CpG (mCpG) sites on chromatin. MeCP2 consists of six domains of which one, the methyl binding domain (MBD), binds mCpG sites in duplex DNA. We show that solution conditions with physiological or greater salt concentrations or the presence of nonspecific competitor DNA is necessary for the MBD to discriminate mCpG from CpG with high specificity. The specificity for mCpG over CpG is >100-fold under these solution conditions. In contrast, the MBD does not discriminate hydroxymethyl-CpG from CpG. The MBD is unusual among site-specific DNA binding proteins in that (i) specificity is not conferred by the enhanced affinity for the specific site but rather by suppression of its affinity for generic DNA, (ii) its specific binding to mCpG is highly electrostatic, and (iii) it takes up as well as displaces monovalent cations upon DNA binding. The MBD displays an unusually high affinity for single-stranded DNA independent of modification or sequence. In addition, the MBD forms a discrete dimer on DNA via a noncooperative binding pathway. Because the affinity of the second monomer is 1 order of magnitude greater than that of nonspecific binding, the MBD dimer is a unique molecular complex. The significance of these results in the context of neuronal function and development and MeCP2-related developmental disorders such as Rett syndrome is discussed.

Visualizing protein movement on DNA at the single-molecule level using DNA curtains

A fundamental feature of many nucleic-acid binding proteins is their ability to move along DNA either by diffusion-based mechanisms or by ATP-hydrolysis driven translocation. For example, most site-specific DNA-binding proteins must diffuse to some extent along DNA to either find their target sites, or to otherwise fulfill their biological roles. Similarly, nucleic-acid translocases such as helicases and polymerases must move along DNA to fulfill their functions. In both instances, the proteins must also be capable of moving in crowded environments while navigating through DNA-bound obstacles. These types of behaviors can be challenging to analyze by bulk biochemical methods because of the transient nature of the interactions, and/or heterogeneity of the reaction intermediates. The advent of single-molecule methodologies has overcome some of these problems, and has led to many new insights into the mechanisms that contribute to protein motion along DNA. We have developed DNA curtains as a tool to facilitate single molecule observations of protein-nucleic acid interactions, and we have applied these new research tools to systems involving both diffusive-based motion as well as ATP directed translocation. Here we highlight these studies by first discussing how diffusion contributes to target searches by proteins involved in post-replicative mismatch repair. We then discuss DNA curtain assays of two different DNA translocases, RecBCD and FtsK, which participate in homologous DNA recombination and site-specific DNA recombination, respectively.

Aptamer-guided gene targeting in yeast and human cells

Gene targeting is a genetic technique to modify an endogenous DNA sequence in its genomic location via homologous recombination (HR) and is useful both for functional analysis and gene therapy applications. HR is inefficient in most organisms and cell types, including mammalian cells, often limiting the effectiveness of gene targeting. Therefore, increasing HR efficiency remains a major challenge to DNA editing. Here, we present a new concept for gene correction based on the development of DNA aptamers capable of binding to a site-specific DNA binding protein to facilitate the exchange of homologous genetic information between a donor molecule and the desired target locus (aptamer-guided gene targeting). We selected DNA aptamers to the I-SceI endonuclease. Bifunctional oligonucleotides containing an I-SceI aptamer sequence were designed as part of a longer single-stranded DNA molecule that contained a region with homology to repair an I-SceI generated double-strand break and correct a disrupted gene. The I-SceI aptamer-containing oligonucleotides stimulated gene targeting up to 32-fold in yeast Saccharomyces cerevisiae and up to 16-fold in human cells. This work provides a novel concept and research direction to increase gene targeting efficiency and lays the groundwork for future studies using aptamers for gene targeting.

Cytosolic HMGB1 expression in human renal clear cell cancer indicates higher pathological T classifications and tumor grades.

High mobility group box (HMGB) proteins are nuclear nonhistone chromosomal proteins that bend DNA, bind preferentially to distorted DNA structures, and promote the assembly of site-specific DNA binding proteins. Recent reports indicate that HMGB1 functions as a proinflammatory cytokine. Here, we studied expressions of HMGB1 and HMGB2 in human renal cancer. Immunohistological expressions of HMGB1 and HMGB2 were assessed in 39 surgically resected human renal cancer specimens. HMGB1 was expressed in the nucleus in 37 out of 39 (94.9%) renal clear cell cancers,while its expression in the cytosol was noted in 19 cases (48.7%). Cytosolic HMGB1 is expressed more frequently in cancers beyond the pT1b classification than in those at the pT1a classification. Higher tumor grades (≥ G2) were also significantly linked with the cytosolic expression of HMGB1. HMGB2 was expressed in the nucleus in 35 of 39 (89.7%) renal clear cell cancers, while its expression in the cytosol was observed in only 7 case (17.9%). Linkage between cytosolic expression of HMGB2 and T classifications was weakly observed, while that between nuclear expression and T classifications was not. HMGB1 expressed in the cytoplasm may be an effective marker indicating higher T classifications and tumor grades.

Eubacterial SpoVG Homologs Constitute a New Family of Site-Specific DNA-Binding Proteins

A site-specific DNA-binding protein was purified from Borrelia burgdorferi cytoplasmic extracts, and determined to be a member of the highly conserved SpoVG family. This is the first time a function has been attributed to any of these ubiquitous bacterial proteins. Further investigations into SpoVG orthologues indicated that the Staphylococcus aureus protein also binds DNA, but interacts preferentially with a distinct nucleic acid sequence. Site-directed mutagenesis and domain swapping between the S. aureus and B. burgdorferi proteins identified that a 6-residue stretch of the SpoVG α-helix contributes to DNA sequence specificity. Two additional, highly conserved amino acid residues on an adjacent β-sheet are essential for DNA-binding, apparently by contacts with the DNA phosphate backbone. Results of these studies thus identified a novel family of bacterial DNA-binding proteins, developed a model of SpoVG-DNA interactions, and provide direction for future functional studies on these wide-spread proteins.

Dissection of the ATPase Active Site of P1 ParA Reveals Multiple Active Forms Essential for Plasmid Partition

The segregation, or partition, of bacterial plasmids is driven by the action of plasmid-encoded partition ATPases, which work to position plasmids inside the cell. The most common type of partition ATPase, generally called ParA, is represented by the P1 plasmid ParA protein. ParA interacts with P1 ParB (the site-specific DNA binding protein that recognizes the parS partition site), and interacts with the bacterial chromosome via an ATP-dependent nonspecific DNA binding activity. ParA also regulates expression of the par genes by acting as a transcriptional repressor. ParA requires ATP for multiple steps and in different ways during the partition process. Here, we analyze the properties of mutations in P1 ParA that are altered in a key lysine in the Walker A motif of the ATP binding site. Four different residues at this position (Lys, Glu, Gln, Arg) result in four different phenotypes in vivo. We focus particularly on the arginine substitution (K122R) because it results in a worse-than-null and dominant-negative phenotype called ParPD. We show that ParAK122R binds and hydrolyzes ATP, although the latter activity is reduced compared with wild-type. ParAK122R interacts with ParB, but the consequences of the interaction are damaged. The ability of ParB to stimulate the ATPase activity of ParA in vitro and its repressor activity in vivo is defective. The K122R mutation specifically damages the disassembly of ParA-ParB-DNA partition complexes, which we believe explains the ParPD phenotype in vivo.

Effect of DNA conformation on facilitated diffusion

Within a living cell, site-specific DNA-binding proteins need to search the whole genome to find a target of ~10-20bp. That they find the target, and do so quickly, is vital for the correct functioning of the DNA, and of the cell as a whole. The current understanding is that this search is performed via facilitated diffusion, i.e. by combining three-dimensional bulk diffusion within the cytoplasm or nucleoplasm, with one-dimensional diffusion along the DNA backbone, to which the protein binds non-specifically. After reviewing the standard theory of facilitated diffusion, we discuss in the present article the still rather rare direct computer simulations of this process, focusing on the three-dimensional part of the search, and the effect of DNA looping and the general DNA conformation on its efficiency. We close by highlighting some open questions in this field.