Nonequilibrium dynamics and ultraslow relaxation of confined DNA during viral packaging

Many viruses use molecular motors that generate large forces to package DNA to near-crystalline densities inside preformed viral proheads. Besides being a key step in viral assembly, this process is of interest as a model for understanding the physics of charged polymers under tight 3D confinement. A large number of theoretical studies have modeled DNA packaging, and the nature of the molecular dynamics and the forces resisting the tight confinement is a subject of wide debate. Here, we directly measure the packaging of single DNA molecules in bacteriophage phi29 with optical tweezers. Using a new technique in which we stall the motor and restart it after increasing waiting periods, we show that the DNA undergoes nonequilibrium conformational dynamics during packaging. We show that the relaxation time of the confined DNA is >10 min, which is longer than the time to package the viral genome and 60,000 times longer than that of the unconfined DNA in solution. Thus, the confined DNA molecule becomes kinetically constrained on the timescale of packaging, exhibiting glassy dynamics, which slows the motor, causes significant heterogeneity in packaging rates of individual viruses, and explains the frequent pausing observed in DNA translocation. These results support several recent hypotheses proposed based on polymer dynamics simulations and show that packaging cannot be fully understood by quasistatic thermodynamic models.DNA packaging is both a critical step in viral assembly and a unique model for understanding the physics of polymers under strong confinement. Before packaging, the DNA (∼6–60 µm long) forms a loose random coil of diameter ∼1–3 µm. After translocation into the viral prohead (∼50–100 nm in diameter), a ∼10,000-fold volume compaction is achieved. Packaging is driven by a powerful molecular motor that must work against the large forces resisting confinement arising from DNA bending, repulsion between DNA segments, and entropy loss (1–8).DNA packaging in bacteriophages phi29, lambda, and T4 has been directly measured via single-molecule manipulation with optical tweezers and the packaging motors have been shown to generate forces of >60 pN, among the highest known for biomotors, while translocating DNA at rates ranging from ∼100 bp (for phage phi29, which packages a 19.3-kbp genome into a 42 × 54-nm prohead shell) up to as high as ∼2,000 bp/s (for phage T4, which packages a 171-kbp genome into a 120 × 86-nm prohead) (9–15). The force resisting packaging rises steeply with prohead filling and has been proposed to play an important role in driving viral DNA ejection (16).Recently, a variety of theoretical models for viral DNA packaging have been proposed (3–5, 17–21). The simplest treat DNA as an elastic rod with repulsive self-interactions and assume that packaging is a quasistatic thermodynamic process, i.e., that the DNA is able to continuously relax to a free-energy minimum state (3–5, 19–21). The DNA arrangement is generally assumed to be an inverse spool with local hexagonal close packing between DNA segments, as suggested by electron microscopy and X-ray scattering studies (22, 23). Such models yield exact analytical predictions that reproduce many of the experimental trends, including the sharp rise in resistance during the latter stages of packaging (3–5, 20).Dynamic simulations, however, predict differing results. Depending on model and simulation protocol, some predict rapid equilibration into ordered spool or folded toroid conformations, whereas others predict nonequilibrium dynamics and disordered conformations (3, 6, 24–31). The packaged DNA conformation also depends on ionic conditions, capsid size and shape, and shape of the internal core structure found in some phages (6, 30). Notably, some electron microscopy studies have also been interpreted as suggesting ordered spooled conformations (22), whereas others have been interpreted as suggesting partly disordered conformations (29). Although some simulations predict nonequilibrium dynamics, several potential caveats are that (i) the DNA has been represented by coarse-grained polymer models with various approximations for physical interactions (6), (ii) the packaging rate used in the simulations is >10⁵ times higher than the measured packaging rate due to computational constraints (3, 26–28), and (iii) it has been pointed out by some authors that simulation timescale cannot be directly related to experimental timescale because of the use of coarse-grained models for DNA (25, 28). As noted in early modeling studies, the calculations based on quasistatic models may represent a lower bound on the required packaging forces due to dissipative dynamic losses (4). Whether nonequilibrium dynamics play a significant role in real systems has thus remained an important open question.     

Structure-based cleavage mechanism of Thermus thermophilus Argonaute DNA guide strand-mediated DNA target cleavage

We report on crystal structures of ternary Thermus thermophilus Argonaute (TtAgo) complexes with 5′-phosphorylated guide DNA and a series of DNA targets. These ternary complex structures of cleavage-incompatible, cleavage-compatible, and postcleavage states solved at improved resolution up to 2.2 Å have provided molecular insights into the orchestrated positioning of catalytic residues, a pair of Mg²⁺ cations, and the putative water nucleophile positioned for in-line attack on the cleavable phosphate for TtAgo-mediated target cleavage by a RNase H-type mechanism. In addition, these ternary complex structures have provided insights into protein and DNA conformational changes that facilitate transition between cleavage-incompatible and cleavage-compatible states, including the role of a Glu finger in generating a cleavage-competent catalytic Asp-Glu-Asp-Asp tetrad. Following cleavage, the seed segment forms a stable duplex with the complementary segment of the target strand.Argonaute (Ago) proteins, critical components of the RNA-induced silencing complex, play a key role in guide strand-mediated target RNA recognition, cleavage, and product release (reviewed in refs. 1–3). Ago proteins adopt a bilobal scaffold composed of an amino terminal PAZ-containing lobe (N and PAZ domains), a carboxyl-terminal PIWI-containing lobe (Mid and PIWI domains), and connecting linkers L1 and L2. Ago proteins bind guide strands whose 5′-phosphorylated and 3′-hydroxyl ends are anchored within Mid and PAZ pockets, respectively (4–7), with the anchored guide strand then serving as a template for pairing with the target strand (8, 9). The cleavage activity of Ago resides in the RNase H fold adopted by the PIWI domain (10, 11), whereby the enzyme’s Asp-Asp-Asp/His catalytic triad (12–15) initially processes loaded double-stranded siRNAs by cleaving the passenger strand and subsequently processes guide-target RNA duplexes by cleaving the target strand (reviewed in refs. 16–18). Such Mg²⁺ cation-mediated endonucleolytic cleavage of the target RNA strand (19, 20) resulting in 3′-OH and 5′-phosphate ends (21) requires Watson–Crick pairing of the guide and target strands spanning the seed segment (positions 2–2′ to 8–8′) and the cleavage site (10′–11′ step on the target strand) (9). Insights into target RNA recognition and cleavage have emerged from structural (9), chemical (22), and biophysical (23) experiments.Notably, bacterial and archaeal Ago proteins have recently been shown to preferentially bind 5′-phosphoryated guide DNA (14, 15) and use an activated water molecule as the nucleophile (reviewed in ref. 24) to cleave both RNA and DNA target strands (9). Structural studies have been undertaken on bacterial and archaeal Ago proteins in the free state (10, 15) and bound to a 5′-phosphorylated guide DNA strand (4) and added target RNA strand (8, 9). The structural studies of Thermus thermophilus Ago (TtAgo) ternary complexes have provided insights into the nucleation, propagation, and cleavage steps of target RNA silencing in a bacterial system (9). These studies have highlighted the conformational transitions on proceeding from Ago in the free state to the binary complex (4) to the ternary complexes (8, 9) and have emphasized the requirement for a precisely aligned Asp-Asp-Asp triad and a pair of Mg²⁺ cations for cleavage chemistry (9), typical of RNase H fold-mediated enzymes (24, 25). Structural studies have also been extended to binary complexes of both human (5, 6) and yeast (7) Agos bound to 5′-phosphorylated guide RNA strands.Despite these singular advances in the structural biology of RNA silencing, further progress was hampered by the modest resolution (2.8- to 3.0-Å resolution) of TtAgo ternary complexes with guide DNA (4) and added target RNAs (8, 9). This precluded identification of water molecules coordinated with the pair of Mg²⁺ cations, including the key water that acts as a nucleophile and targets the cleavable phosphate between positions 10′-11′ on the target strand. We have now extended our research to TtAgo ternary complexes with guide DNA and target DNA strands, which has permitted us to grow crystals of ternary complexes that diffract to higher (2.2–2.3 Å) resolution in the cleavage-incompatible, cleavage-compatible, and postcleavage steps. These high-resolution structures of TtAgo ternary complexes provide snapshots of distinct key steps in the catalytic cleavage pathway, opening opportunities for experimental probing into DNA target cleavage as a defense mechanism against plasmids and possibly other mobile elements (26, 27).     

WWOX,the common fragile site FRA16D gene product,regulates ATM activation and the DNA damage response

Genomic instability is a hallmark of cancer. The WW domain-containing oxidoreductase (WWOX) is a tumor suppressor spanning the common chromosomal fragile site FRA16D. Here, we report a direct role of WWOX in DNA damage response (DDR) and DNA repair. We show that Wwox deficiency results in reduced activation of the ataxia telangiectasia-mutated (ATM) checkpoint kinase, inefficient induction and maintenance of γ-H2AX foci, and impaired DNA repair. Mechanistically, we show that, upon DNA damage, WWOX accumulates in the cell nucleus, where it interacts with ATM and enhances its activation. Nuclear accumulation of WWOX is regulated by its K63-linked ubiquitination at lysine residue 274, which is mediated by the E3 ubiquitin ligase ITCH. These findings identify a novel role for the tumor suppressor WWOX and show that loss of WWOX expression may drive genomic instability and provide an advantage for clonal expansion of neoplastic cells.Genomic instability is a common characteristic of human cancers. The DNA damage response (DDR) maintains the integrity of the genome in response to DNA damage. DDR is a complex signaling process that results in cell cycle arrest followed by either DNA repair or apoptosis if the DNA damage is too extensive to be repaired (1–3). Key mammalian damage response sensors are ataxia telangiectasia-mutated (ATM), ATM and Rad3-related, and DNA-dependent PKs (4, 5). Disruption of the DDR machinery in human cells leads to genomic instability and an increased risk of cancer progression (6, 7).The WW domain-containing oxidoreductase (WWOX) gene spans the common fragile site (CFS) FRA16D (8, 9). Genomic alterations affecting the WWOX locus have been reported in several types of cancer and include homozygous and hemizygous deletions (10–13). Ectopic expression of WWOX in WWOX-negative cancer cells attenuates cell growth and suppresses tumor growth in immunocompromised mice (10, 11, 14). Importantly, targeted ablation of Wwox in mice results in higher incidence of spontaneous lesions resembling osteosarcomas and lung and mammary tumors (14–16). These findings suggest WWOX as a tumor suppressor. The WWOX protein contains two N-terminal WW domains mediating WWOX interaction with PP(proline)x(amino acid)Y(tyrosine)-containing proteins (11, 17) and a central short-chain deyhdrogenase/reductase domain that has been proposed to function in steroidogenesis (18). Recent characterization of WWOX domains revealed that they interact, mainly through the WW1 domain, with multiprotein networks (3). The mechanism by which WWOX suppresses tumorigenicity is, however, not well-known.In vitro, CFSs are defined as gaps or breaks on metaphase chromosomes that occur in cells treated with inhibitors of DNA replication (19, 20). In vivo, CFSs are preferential targets of replication stress in preneoplastic lesions (21), and emerging evidence suggests that they represent early warning sensors for DNA damage (22–24). Both genetic and epigenetic factors are thought to regulate the fragility of CFS (25, 26). Recent profiling studies of CFS provide evidence that the functional fragility of CFS is tissue-specific (27–29). High-throughput genomic analyses of 3,131 cancer specimens (12) and 746 cancer cell lines (13) have recently identified large deletions in CFSs, including the FRA16D/WWOX locus. Although these deletions have been linked to the presence of DNA replication stress (30), the molecular function of gene products of CFSs, including the WWOX protein, is poorly understood. Here, we identify a direct role of WWOX in the DDR and show that the WWOX gene product functions as a modulator of the DNA damage checkpoint kinase ATM.     

Reconstitution of long and short patch mismatch repair reactions using Saccharomyces cerevisiae proteins

A problem in understanding eukaryotic DNA mismatch repair (MMR) mechanisms is linking insights into MMR mechanisms from genetics and cell-biology studies with those from biochemical studies of MMR proteins and reconstituted MMR reactions. This type of analysis has proven difficult because reconstitution approaches have been most successful for human MMR whereas analysis of MMR in vivo has been most advanced in the yeast Saccharomyces cerevisiae. Here, we describe the reconstitution of MMR reactions using purified S. cerevisiae proteins and mispair-containing DNA substrates. A mixture of MutS homolog 2 (Msh2)–MutS homolog 6, Exonuclease 1, replication protein A, replication factor C-Δ1N, proliferating cell nuclear antigen and DNA polymerase δ was found to repair substrates containing TG, CC, +1 (+T), +2 (+GC), and +4 (+ACGA) mispairs and either a 5′ or 3′ strand interruption with different efficiencies. The Msh2–MutS homolog 3 mispair recognition protein could substitute for the Msh2–Msh6 mispair recognition protein and showed a different specificity of repair of the different mispairs whereas addition of MutL homolog 1–postmeiotic segregation 1 had no affect on MMR. Repair was catalytic, with as many as 11 substrates repaired per molecule of Exo1. Repair of the substrates containing either a 5′ or 3′ strand interruption occurred by mispair binding-dependent 5′ excision and subsequent resynthesis with excision tracts of up to ∼2.9 kb occurring during the repair of the substrate with a 3′ strand interruption. The availability of this reconstituted MMR reaction now makes possible detailed biochemical studies of the wealth of mutations identified that affect S. cerevisiae MMR.DNA mismatch repair (MMR) is a critical DNA repair pathway that is coupled to DNA replication in eukaryotes where it corrects misincorporation errors made during DNA replication (1–9). This pathway prevents mutations and acts to prevent the development of cancer (10, 11). MMR also contributes to gene conversion by repairing mispaired bases that occur during the formation of recombination intermediates (3, 4, 12). Finally, MMR acts to suppress recombination between divergent but homologous DNA sequences, thereby preventing the formation of genome rearrangements that can result from nonallelic homologous recombination (4, 13–15).Our knowledge of the mechanism of eukaryotic MMR comes from several general lines of investigation (3–9). Studies of bacterial MMR have provided a basic mechanistic framework for comparative studies (5). Genetic and cell-biology studies, primarily in Saccharomyces cerevisiae, have identified eukaryotic MMR genes, provided models for how their gene products define MMR pathways, and elucidated some of the details of how MMR pathways interact with replication (1–4). Reconstitution studies, primarily in human systems, have identified some of the catalytic features of eukaryotic MMR (7–9, 16, 17). Biochemical and structural studies of S. cerevisiae and human MMR proteins have provided information about the function of individual MMR proteins (6–9).In eukaryotic MMR, mispairs are bound by MutS homolog 2 (Msh2)–MutS homolog 6 (Msh6) and Msh2–MutS homolog 3 (Msh3), two partially redundant complexes of MutS-related proteins (3, 4, 18, 19). These complexes recruit a MutL-related complex, called MutL homoloh 1 (Mlh1)–postmeiotic segregation 1 (Pms1) in S. cerevisiae and Mlh1–postmeiotic segregation 2 (Pms2) in human and mouse (3, 4, 20–23). The Mlh1–Pms1/Pms2 complex has an endonuclease activity suggested to play a role in the initiation of the excision step of MMR (24, 25). Downstream of mismatch recognition is a mispair excision step that can be catalyzed by Exonuclease 1 (Exo1) (26–28); however, defects in both S. cerevisiae and mouse Exo1 result in only a partial MMR deficiency, suggesting the existence of additional excision mechanisms (26, 27, 29). DNA polymerase δ, the single-strand DNA binding protein replication protein A (RPA), the sliding clamp proliferating cell nuclear antigen (PCNA), and the clamp loader replication factor C (RFC) are also required for MMR at different steps, including activation of Mlh1–Pms1/Pms2, stimulation of Exo1, potentially in Exo1-independent mispair excision, and in the gap-filling resynthesis steps of MMR (3, 16, 17, 24, 27, 30–36). Although much is known about these core MMR proteins, it is not well understood how eukaryotic MMR is coupled to DNA replication (1, 2), how excision is targeted to the newly replicated strand (1, 25, 37–39), or how different MMR mechanisms such as Exo1-dependent and -independent subpathways are selected or how many such subpathways exist (1, 24, 27, 29).S. cerevisiae has provided a number of tools for studying MMR, including forward genetic screens for mutations affecting MMR, including dominant and separation-of-function mutations, the ability to evaluate structure-based mutations in vivo, cell biological tools for visualizing and analyzing MMR proteins in vivo, and overproduction of individual MMR proteins for biochemical analysis. However, linking these tools with biochemical systems that catalyze MMR reactions in vitro for mechanistic studies has not yet been possible. Here, we describe the development of MMR reactions reconstituted using purified proteins for the analysis of MMR mechanisms.     

Programmable motion of DNA origami mechanisms

DNA origami enables the precise fabrication of nanoscale geometries. We demonstrate an approach to engineer complex and reversible motion of nanoscale DNA origami machine elements. We first design, fabricate, and characterize the mechanical behavior of flexible DNA origami rotational and linear joints that integrate stiff double-stranded DNA components and flexible single-stranded DNA components to constrain motion along a single degree of freedom and demonstrate the ability to tune the flexibility and range of motion. Multiple joints with simple 1D motion were then integrated into higher order mechanisms. One mechanism is a crank–slider that couples rotational and linear motion, and the other is a Bennett linkage that moves between a compacted bundle and an expanded frame configuration with a constrained 3D motion path. Finally, we demonstrate distributed actuation of the linkage using DNA input strands to achieve reversible conformational changes of the entire structure on ∼minute timescales. Our results demonstrate programmable motion of 2D and 3D DNA origami mechanisms constructed following a macroscopic machine design approach.The ability to control, manipulate, and organize matter at the nanoscale has demonstrated immense potential for advancements in industrial technology, medicine, and materials (1–3). Bottom-up self-assembly has become a particularly promising area for nanofabrication (4, 5); however, to date designing complex motion at the nanoscale remains a challenge (6–9). Amino acid polymers exhibit well-defined and complex dynamics in natural systems and have been assembled into designed structures including nanotubes, sheets, and networks (10–12), although the complexity of interactions that govern amino acid folding make designing complex geometries extremely challenging. DNA nanotechnology, on the other hand, has exploited well-understood assembly properties of DNA to create a variety of increasingly complex designed nanostructures (13–15).Scaffolded DNA origami, the process of folding a long single-stranded DNA (ssDNA) strand into a custom structure (16–18), has enabled the fabrication of nanoscale objects with unprecedented geometric complexity that have recently been implemented in applications such as containers for drug delivery (19, 20), nanopores for single-molecule sensing (21–23), and templates for nanoparticles (24, 25) or proteins (26–28). The majority of these and other applications of DNA origami have largely focused on static structures. Natural biomolecular machines, in contrast, have a rich diversity of functionalities that rely on complex but well-defined and reversible conformational changes. Currently, the scope of biomolecular nanotechnology is limited by an inability to achieve similar motion in designed nanosystems.DNA nanotechnology has enabled critical steps toward that goal starting with the work of Mao et al. (29), who developed a DNA nanostructure that took advantage of the B–Z transition of DNA to switch states. Since then, efforts to fabricate dynamic DNA systems have primarily focused on strand displacement approaches (30) mainly on systems comprising a few strands or arrays of strands undergoing ∼nm-scale motions (31–37) in some cases guided by DNA origami templates (38–40). More recently, strand displacement has been used to reconfigure DNA origami nanostructures, for example opening DNA containers (19, 41, 42), controlling molecular binding (43, 44), or reconfiguring structures (45). The largest triggerable structural change was achieved by Han et al. in a DNA origami Möbius strip (one-sided ribbon structure) that could be opened to approximately double in size (45). Constrained motion has been achieved in systems with rotational motion (19, 20, 32, 41, 44, 46, 47) in some cases to open lid-like components (19, 20, 41) or detect molecular binding (44, 48, 49). A few of these systems achieved reversible conformational changes (32, 41, 44, 46), although the motion path and flexibility were not studied. Constrained linear motion has remained largely unexplored. Linear displacements on the scale of a few nanometers have been demonstrated via conformational changes of DNA structure motifs (50–55), strand invasion to open DNA hairpins (36, 55, 56), or the reversible sliding motion of a DNA tile actuator (56); these cases also did not investigate the motion path or flexibility of motion.Building on these prior studies, this work implements concepts from macroscopic machine design to build modular parts with constrained motion. We demonstrate an ability to tune the flexibility and range of motion and then integrate these parts into prototype mechanisms with designed 2D and 3D motion. We further demonstrate reversible actuation of a mechanism with complex conformational changes on minute timescales.     

Detection of genomic variations and DNA polymorphisms and impact on analysis of meiotic recombination and genetic mapping

DNA polymorphisms are important markers in genetic analyses and are increasingly detected by using genome resequencing. However, the presence of repetitive sequences and structural variants can lead to false positives in the identification of polymorphic alleles. Here, we describe an analysis strategy that minimizes false positives in allelic detection and present analyses of recently published resequencing data from Arabidopsis meiotic products and individual humans. Our analysis enables the accurate detection of sequencing errors, small insertions and deletions (indels), and structural variants, including large reciprocal indels and copy number variants, from comparisons between the resequenced and reference genomes. We offer an alternative interpretation of the sequencing data of meiotic products, including the number and type of recombination events, to illustrate the potential for mistakes in single-nucleotide polymorphism calling. Using these examples, we propose that the detection of DNA polymorphisms using resequencing data needs to account for nonallelic homologous sequences.DNA polymorphisms are ubiquitous genetic variations among individuals and include single nucleotide polymorphisms (SNPs), insertions and deletions (indels), and other larger rearrangements (1–3) (Fig. 1 A and B). They can have phenotypic consequences and also serve as molecular markers for genetic analyses, facilitating linkage and association studies of genetic diseases, and other traits in humans (4–6), animals, plants, (7–10) and other organisms. Using DNA polymorphisms for modern genetic applications requires low-error, high-throughput analytical strategies. Here, we illustrate the use of short-read next-generation sequencing (NGS) data to detect DNA polymorphisms in the context of whole-genome analysis of meiotic products.Open in a separate windowFig. 1.(A) SNPs and small indels between two ecotype genomes. (B) Possible types of SVs. Col genotypes are marked in blue and Ler in red. Arrows indicate DNA segments involved in SVs between the two ecotypes. (C) Meiotic recombination events including a CO and a GC (NCO). Centromeres are denoted by yellow dots.There are many methods for detecting SNPs (11–14) and structural variants (SVs) (15–25), including NGS, which can capture nearly all DNA polymorphisms (26–28). This approach has been widely used to analyze markers in crop species such as rice (29), genes associated with diseases (6, 26), and meiotic recombination in yeast and plants (30, 31). However, accurate identification of DNA polymorphisms can be challenging, in part because short-read sequencing data have limited information for inferring chromosomal context.Genomes usually contain repetitive sequences that can differ in copy number between individuals (26–28, 31); therefore, resequencing analyses must account for chromosomal context to avoid mistaking highly similar paralogous sequences for polymorphisms. Here, we use recently published datasets to describe several DNA sequence features that can be mistaken as allelic (32, 33) and describe a strategy for differentiating between repetitive sequences and polymorphic alleles. We illustrate the effectiveness of these analyses by examining the reported polymorphisms from the published datasets.Meiotic recombination is initiated by DNA double-strand breaks (DSBs) catalyzed by the topoisomerase-like SPORULATION 11 (SPO11). DSBs are repaired as either crossovers (COs) between chromosomes (Fig. 1C), or noncrossovers (NCOs). Both COs and NCOs can be accompanied by gene conversion (GC) events, which are the nonreciprocal transfer of sequence information due to the repair of heteroduplex DNA during meiotic recombination. Understanding the control of frequency and distribution of CO and NCO (including GC) events has important implications for human health (including cancer and aneuploidy), crop breeding, and the potential for use in genome engineering. COs can be detected relatively easily by using polymorphic markers in the flanking sequences, but NCO products can only be detected if they are accompanied by a GC event. Because GCs associated with NCO result in allelic changes at polymorphic sites without exchange of flanking sequences, they are more difficult to detect. Recent advances in DNA sequencing have made the analysis of meiotic NCOs more feasible (30–32, 34); however, SVs present a challenge in these analyses. We recommend a set of guidelines for detection of DNA polymorphisms by using genomic resequencing short-read datasets. These measures improve the accuracy of a wide range of analyses by using genomic resequencing, including estimation of COs, NCOs, and GCs.     

Insights into ParB spreading from the complex structure of Spo0J and parS

Spo0J (stage 0 sporulation protein J, a member of the ParB superfamily) is an essential component of the ParABS (partition system of ParA, ParB, and parS)-related bacterial chromosome segregation system. ParB (partition protein B) and its regulatory protein, ParA, act cooperatively through parS (partition S) DNA to facilitate chromosome segregation. ParB binds to chromosomal DNA at specific parS sites as well as the neighboring nonspecific DNA sites. Various ParB molecules can associate together and spread along the chromosomal DNA. ParB oligomer and parS DNA interact together to form a high-order nucleoprotein that is required for the loading of the structural maintenance of chromosomes proteins onto the chromosome for chromosomal DNA condensation. In this report, we characterized the binding of parS and Spo0J from Helicobacter pylori (HpSpo0J) and solved the crystal structure of the C-terminal domain truncated protein (Ct-HpSpo0J)-parS complex. Ct-HpSpo0J folds into an elongated structure that includes a flexible N-terminal domain for protein–protein interaction and a conserved DNA-binding domain for parS binding. Two Ct-HpSpo0J molecules bind with one parS. Ct-HpSpo0J interacts vertically and horizontally with its neighbors through the N-terminal domain to form an oligomer. These adjacent and transverse interactions are accomplished via a highly conserved arginine patch: RRLR. These interactions might be needed for molecular assembly of a high-order nucleoprotein complex and for ParB spreading. A structural model for ParB spreading and chromosomal DNA condensation that lead to chromosome segregation is proposed.The integrity of chromosomes and plasmids relies on precise DNA replication and segregation (1, 2). The initiation of DNA replication has to synchronize with the cell cycle to ensure precise chromosome segregation (3). In bacteria, the chromosome-encoded plasmid-partitioning system (Par) (4) and the structural maintenance of chromosomes (SMC) condensation complex (5) are two highly conserved systems associate with chromosome segregation and organization. SMC contributes to the overall stability and organization of genome (6–8). The partition system denoted ParABS is comprised of two proteins (ParA and ParB) and a centromere-like DNA element (parS) (9). ParB binds specifically to parS to form a complex. After binding ATP, ParA can interact with the ParB–parS complex to form a nucleoid–adaptor complex. ParB promotes the ATP hydrolysis activity of the complex to separate the chromosomes (9–13).In the bacterial chromosomal ParABS system, ParB has two functions: one is to regulate chromosome replication and sporulation (8, 12, 14) and the other is to participate in chromosome segregation (5, 15–17). ParB spreads along the chromosomal DNA by binding at specific parS and nonspecific DNA sites to form a high-order partition complex (18–20). This partition complex is required for the loading of SMC onto the chromosomal DNA (5). In addition, the N-terminal domain of ParB can interact with ParA and stimulate its ATPase activity (21). This nucleoid–adaptor complex, ParA–ParB–parS is used to drive chromosome segregation (22, 23). However, the detailed mechanism for this process is still unclear.Members of the ParB superfamily share similar functional domains: an N-terminal domain for protein–protein interactions, a central DNA-binding domain for parS binding, and a C-terminal domain for ParB dimerization (24). Two conserved N-terminal domain residues, Lys3 and Lys7, in the ParB from Bacillus subtilis (BsSpo0J), have been shown to interact with its regulatory protein BsSoj, a member of the ParA superfamily (3). The loss-of-function BsSpo0J R80A mutant was originally discovered by Autret et al. (25) and reportedly has disrupted focus formation by fluorescence microscopy. More recently, Graham et al. (20) showed that BsSpo0J bridges chromosomal DNA using single-molecule experiments. However, its R79A, R80A, and R82A mutants could not spread in vivo and did not bridge DNA in vitro. These highly conserved arginine residues were defined as an arginine patch (20). Furthermore, Broedersz et al. (26) studied the condensation and localization of ParB by computational simulation.The crystal structures of ParB superfamily proteins have been reported for a DNA-free form of TtSpo0J (from Thermus thermophilus, containing the N-terminal and the DNA-binding domains) (10) and three complexes: the RP4–KorB-O_B complex (from plasmid RP4, containing the DNA-binding domain) (27), the P1 ParB–parS complex (from Enterobacteria phage P1, containing the DNA-binding and the C-terminal domains) (28), and the F-SopB–sopC complex (from plasmid F, containing the DNA-binding domain) (29).The Helicobacter pylori ParABS system consists of HpSoj (ParA), HpSpo0J (ParB), and parS DNA (30, 31). Herein, we report the crystal structure of a C-terminal domain truncated HpSpo0J (Ct-HpSpo0J)–parS complex. The N-terminal and the DNA-binding domains are present on Ct-HpSpo0J. The structural details of the complex in combination with results from EMSAs, fluorescence anisotropy assay, and small angle X-ray scattering (SAXS) allow us to propose a model for ParB spreading as it relates to chromosome segregation.     

Exchange protein directly activated by cAMP plays a critical role in bacterial invasion during fatal rickettsioses

Rickettsiae are responsible for some of the most devastating human infections. A high infectivity and severe illness after inhalation make some rickettsiae bioterrorism threats. We report that deletion of the exchange protein directly activated by cAMP (Epac) gene, Epac1, in mice protects them from an ordinarily lethal dose of rickettsiae. Inhibition of Epac1 suppresses bacterial adhesion and invasion. Most importantly, pharmacological inhibition of Epac1 in vivo using an Epac-specific small-molecule inhibitor, ESI-09, completely recapitulates the Epac1 knockout phenotype. ESI-09 treatment dramatically decreases the morbidity and mortality associated with fatal spotted fever rickettsiosis. Our results demonstrate that Epac1-mediated signaling represents a mechanism for host–pathogen interactions and that Epac1 is a potential target for the prevention and treatment of fatal rickettsioses.Rickettsiae are responsible for some of the most devastating human infections (1–4). It has been forecasted that temperature increases attributable to global climate change will lead to more widespread distribution of rickettsioses (5). These tick-borne diseases are caused by obligately intracellular bacteria of the genus Rickettsia, including Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever (RMSF) in the United States and Latin America (2, 3), and Rickettsia conorii, the causative agent of Mediterranean spotted fever endemic to southern Europe, North Africa, and India (6). A high infectivity and severe illness after inhalation make some rickettsiae (including Rickettsia prowazekii, R. rickettsii, Rickettsia typhi, and R. conorii) bioterrorism threats (7). Although the majority of rickettsial infections can be controlled by appropriate broad-spectrum antibiotic therapy if diagnosed early, up to 20% of misdiagnosed or untreated (1, 3) and 5% of treated RMSF cases (8) result in a fatal outcome caused by acute disseminated vascular endothelial infection and damage (9). Fatality rates as high as 32% have been reported in hospitalized patients diagnosed with Mediterranean spotted fever (10). In addition, strains of R. prowazekii resistant to tetracycline and chloramphenicol have been developed in laboratories (11). Disseminated endothelial infection and endothelial barrier disruption with increased microvascular permeability are the central features of SFG rickettsioses (1, 2, 9). The molecular mechanisms involved in rickettsial infection remain incompletely elucidated (9, 12). A comprehensive understanding of rickettsial pathogenesis and the development of novel mechanism-based treatment are urgently needed.Living organisms use intricate signaling networks for sensing and responding to changes in the external environment. cAMP, a ubiquitous second messenger, is an important molecular switch that translates environmental signals into regulatory effects in cells (13). As such, a number of microbial pathogens have evolved a set of diverse virulence-enhancing strategies that exploit the cAMP-signaling pathways of their hosts (14). The intracellular functions of cAMP are predominantly mediated by the classic cAMP receptor, protein kinase A (PKA), and the more recently discovered exchange protein directly activated by cAMP (Epac) (15). Thus, far, two isoforms, Epac1 and Epac2, have been identified in humans (16, 17). Epac proteins function by responding to increased intracellular cAMP levels and activating the Ras superfamily small GTPases Ras-proximate 1 and 2 (Rap1 and Rap2). Accumulating evidence demonstrates that the cAMP/Epac1 signaling axis plays key regulatory roles in controlling various cellular functions in endothelial cells in vitro, including cell adhesion (18–21), exocytosis (22), tissue plasminogen activator expression (23), suppressor of cytokine signaling 3 (SOCS-3) induction (24–27), microtubule dynamics (28, 29), cell–cell junctions, and permeability and barrier functions (30–37). Considering the critical importance of endothelial cells in rickettsioses, we examined the functional roles of Epac1 in rickettsial pathogenesis in vivo, taking advantage of the recently generated Epac1 knockout mouse (38) and Epac-specific inhibitors (39, 40) generated from our laboratory. Our studies demonstrate that Epac1 plays a key role in rickettsial infection and represents a therapeutic target for fatal rickettsioses.     

Bacterial scaffold directs pole-specific centromere segregation

Bacteria use partitioning systems based on the ParA ATPase to actively mobilize and spatially organize molecular cargoes throughout the cytoplasm. The bacterium Caulobacter crescentus uses a ParA-based partitioning system to segregate newly replicated chromosomal centromeres to opposite cell poles. Here we demonstrate that the Caulobacter PopZ scaffold creates an organizing center at the cell pole that actively regulates polar centromere transport by the ParA partition system. As segregation proceeds, the ParB-bound centromere complex is moved by progressively disassembling ParA from a nucleoid-bound structure. Using superresolution microscopy, we show that released ParA is recruited directly to binding sites within a 3D ultrastructure composed of PopZ at the cell pole, whereas the ParB-centromere complex remains at the periphery of the PopZ structure. PopZ recruitment of ParA stimulates ParA to assemble on the nucleoid near the PopZ-proximal cell pole. We identify mutations in PopZ that allow scaffold assembly but specifically abrogate interactions with ParA and demonstrate that PopZ/ParA interactions are required for proper chromosome segregation in vivo. We propose that during segregation PopZ sequesters free ParA and induces target-proximal regeneration of ParA DNA binding activity to enforce processive and pole-directed centromere segregation, preventing segregation reversals. PopZ therefore functions as a polar hub complex at the cell pole to directly regulate the directionality and destination of transfer of the mitotic segregation machine.The bacterial cytoplasm is a complex mixture of dynamic macromolecules densely packed into a tiny compartment. Recent studies have revealed unexpected levels of organization of bacterial cytoplasmic components, including hundreds of proteins, specific lipids, mRNA molecules, and even the nucleoid itself (1). One strategy used by bacteria to generate subcellular organization of specific macromolecular complexes is active segregation by ParA-mediated molecular partitioning machines. ParA-based partitioning systems are found throughout bacteria and have been shown to spatially organize diverse macromolecular complexes to facilitate their equal distribution to progeny during cell division (2). An important question is how directionality is provided to ParA partitioning machines.One family of highly conserved ParA-based partitioning systems segregates plasmid or chromosomal centromeres to daughter cells during cell division. ParA-mediated DNA partitioning systems (Par systems) are composed of three core components: a centromeric DNA sequence parS, a site-specific DNA binding protein ParB that binds to the centromere parS sequence, and the ATPase ParA. Structural studies demonstrate that the activity of ParA is regulated by a molecular switch in which ATP-bound ParA forms dimers that bind tightly to DNA, and ParB stimulates ATP hydrolysis and release of ADP-bound ParA as monomers (3). During centromere partitioning in vivo, ATP-bound ParA assembles into a multimeric nucleoid-bound structure (4). At the centromere, ParB binds to the parS locus and nearby DNA to create a compact nucleoprotein complex (5). This ParB/parS complex binds to ParA subunits within the ParA/nucleoid structure, stimulating ATP hydrolysis and release of ParA-ADP (6–8). The multivalent ParB/parS complex has thus been proposed to bind to and shorten the ParA superstructure on the nucleoid, moving along a receding track via a dynamic disassembly mechanism (6, 8–10). The result of this process is the movement of the chromosomal centromere (parS) relative to the nucleoid bulk, and therefore to the cell itself.Whereas the fundamental operating principles of ParA-mediated movement seem conserved, how these machines target transfer to specific subcellular destinations is unknown. Many chromosomal Par systems maintain a single origin-proximal ParB/parS complex at the old cell pole and, after replication, move one newly replicated parS locus to the opposite pole (9, 11, 12). Polar protein complexes that interact with chromosome segregation factors have been identified in various bacteria, but the mechanistic consequences of these interactions have not been established (13–15). In Caulobacter, two distinct polar protein factors affect ParA-mediated centromere segregation: the new pole-specific protein TipN (16, 17) and the polar organizing protein PopZ (18, 19). TipN is a large, membrane-anchored, coiled-coil rich protein that localizes to the new pole throughout the cell cycle and, in addition to roles in localization of flagellar synthesis (16, 17), affects processive parS segregation via an unknown mechanism (6, 20).In contrast, PopZ is a small, acidic protein that forms a polymeric network at the cell pole (18, 19). In the prereplicative cell, PopZ localizes exclusively at the old cell pole, where it anchors the ParB-bound parS locus via direct interactions with ParB (18, 19). During chromosome replication initiation, PopZ releases ParB from the old pole and adopts a bipolar PopZ distribution that seems to capture ParB/parS complexes during the segregation process (18, 19). Whereas cells lacking tipN are only mildly elongated, popZ deletion causes severe filamentation (16–19), suggesting that PopZ plays a more important role in the regulation of segregation. However, the molecular mechanism by which PopZ affects segregation has remained elusive.Here we demonstrate that the multifunctional PopZ complex plays a crucial role in pole-directed movement of ParA-mediated chromosome segregation by interacting directly with ParA. We show that PopZ, but not TipN, is required for robust polar recruitment of ParA and demonstrate that a polar PopZ scaffold recruits and concentrates free ParA released during segregation. Recruitment of ParA within the PopZ matrix sequesters free ParA and locally regenerates ParA DNA binding activity. Active ParA complexes are released for recycling into nucleoid-bound structures near the cell pole, which we propose drives centromere segregation toward pole-localized PopZ. Thus, PopZ orchestrates a positive feedback mechanism that forces ParA-mediated centromere transfer to the cell pole. The polar PopZ scaffold complex creates a unique 3D microenvironment at the pole that spatially separates distinct centromere tethering and ParA-modulation activities, enabling coupling between chromosome segregation with the initiation of cell division.