Mu Transposition Research Articles

Clustering of rRNA operons in E. coli is disrupted by σH.

Chromosomal organization in E. coli as examined by Hi-C methodology indicates that long-range interactions are sparse. Yet, spatial co-localization or 'clustering' of 6/7 ribosomal RNA (rrn) operons distributed over half the 4.6 Mbp genome has been captured by two other methodologies - fluorescence microscopy and Mu transposition. Our current understanding of the mechanism of clustering is limited to mapping essential cis elements. To identify trans elements, we resorted to perturbing the system by chemical and physical means and observed that heat shock disrupts clustering. Levels of σH are known to rise as a cellular response to the shock. We show that elevated expression of σH alone is sufficient to disrupt clustering, independent of heat stress. The anti-clustering activity of σH does not depend on its transcriptional activity but requires core-RNAP interaction and DNA-binding activities. This activity of σH is suppressed by ectopic expression of σD suggesting a competition for core-RNAP. A query of the other five known σ factors of E. coli found that elevated expression of FecI, the ECF σ factor that controls iron citrate transport, also perturbs clustering and is also suppressed by σD. We discuss a possible scenario for how these membrane-associated σ factors participate in clustering of distant rrn loci.

High-Throughput Mapping of Chromosomal Conformations in E. coli Under Physiological Conditions Using Massively Multiplexed Mu Transposition.

Many approaches for measuring three-dimensional chromosomal conformations rely upon formaldehyde crosslinking followed by subsequent proximity ligation, a family of methods exemplified by 3C, Hi-C, etc. Here we provide an alternative crosslinking-free procedure for high-throughput identification of long-range contacts in the chromosomes of enterobacteria, making use of contact-dependent transposition of phage Mu to identify distant loci in close contact. The procedure described here will suffice to provide a comprehensive map of transposition frequencies between tens of thousands of loci in a bacterial genome, with the resolution limited by the diversity of the insertion site library used and the sequencing depth applied.

Targeted DNA integration in human cells without double-strand breaks using CRISPR-associated transposases.

Conventional genome engineering with CRISPR-Cas9 creates double-strand breaks (DSBs) that lead to undesirable byproducts and reduce product purity. Here we report an approach for programmable integration of large DNA sequences in human cells that avoids the generation of DSBs by using Type I-F CRISPR-associated transposases (CASTs). We optimized DNA targeting by the QCascade complex through protein design and developed potent transcriptional activators by exploiting the multi-valent recruitment of the AAA+ ATPase TnsC to genomic sites targeted by QCascade. After initial detection of plasmid-based integration, we screened 15 additional CAST systems from a wide range of bacterial hosts, identified a homolog from Pseudoalteromonas that exhibits improved activity and further increased integration efficiencies. Finally, we discovered that bacterial ClpX enhances genomic integration by multiple orders of magnitude, likely by promoting active disassembly of the post-integration CAST complex, akin to its known role in Mu transposition. Our work highlights the ability to reconstitute complex, multi-component machineries in human cells and establishes a strong foundation to exploit CRISPR-associated transposases for eukaryotic genome engineering.

Deep sequencing reveals new roles for MuB in transposition immunity and target-capture, and redefines the insular Ter region of E. coli

BackgroundThe target capture protein MuB is responsible for the high efficiency of phage Mu transposition within the E. coli genome. However, some targets are off-limits, such as regions immediately outside the Mu ends (cis-immunity) as well as the entire ~ 37 kb genome of Mu (Mu genome immunity). Paradoxically, MuB is responsible for cis-immunity and is also implicated in Mu genome immunity, but via different mechanisms. This study was undertaken to dissect the role of MuB in target choice in vivo.ResultsWe tracked Mu transposition from six different starting locations on the E. coli genome, in the presence and absence of MuB. The data reveal that Mu’s ability to sample the entire genome during a single hop in a clonal population is independent of MuB, and that MuB is responsible for cis-immunity, plays a minor role in Mu genome immunity, and facilitates insertions into transcriptionally active regions. Unexpectedly, transposition patterns in the absence of MuB have helped extend the boundaries of the insular Ter segment of the E. coli genome.ConclusionsThe results in this study demonstrate unambiguously the operation of two distinct mechanisms of Mu target immunity, only one of which is wholly dependent on MuB. The study also reveals several interesting and hitherto unknown aspects of Mu target choice in vivo, particularly the role of MuB in facilitating the capture of promoter and translation start site targets, likely by displacing macromolecular complexes engaged in gene expression. So also, MuB facilitates transposition into the restricted Ter region of the genome.

Genetic differentiation of Mutator insertion polymorphisms and association with agronomic traits in waxy and common maize.

As waxy maize is considered a key economic crop in Korea, an understanding of its genetic variation and differentiation is fundamental for the selective plant breeding. The maize genome is primarily composed of transposable elements, for which large and stable insertions generate variations that reflect selection during evolution. This study was to elucidate the genetic diversity based on the contribution of TEs and to investigate the effect of Mu transposition on the genetic divergence of waxy and common maize. We also performed an association analysis on these inbred lines to determine the Mu insertions associated with agronomic traits. In this study, we utilized a Mutator-based transposon display method to study the genetic diversity and population structure of 40 waxy and 40 common inbred lines of maize in the Gangwon Agricultural Research and Extension Services collection at the Maize Research Institute. We detected polymorphisms in 86.33% of 278 Mutator (Mu) anchored loci, reflecting the activity of the Mu element and its contribution to genetic variation. Common maize showed a substantial amount of genetic diversity, which was greater than that observed in waxy maize. Principal-coordinate and neighbor-joining cluster analyzes consistently supported the presence of two genetically distinct groups. However, the distribution of genetic variation within the populations was much higher than the genetic differentiation among the populations. To explore the contribution of the Mu element to phenotypic variation, we analyzed the associations with ten important agronomical traits. On the basis of the combined results from two models (QGLM and Q + KLM), we found significant associations between seven Mu loci and four different traits. These results will assist waxy maize breeders in choosing parental lines and be useful for marker-assisted selection.

A Well-Mixed E. coli Genome: Widespread Contacts Revealed by Tracking Mu Transposition

The three-dimensional structures of chromosomes are increasingly being recognized as playing a major role in cellular regulatory states. The efficiency and promiscuity of phage Mu transposition was exploited to directly measure invivo interactions between genomic loci in E.coli. Two global organizing principles have emerged: first, the chromosome is well-mixed and uncompartmentalized, with transpositions occurring freely between all measured loci; second, several gene families/regions show "clustering": strong three-dimensional co-localization regardless of linear genomic distance. The activities of the SMC/condensin protein MukB and nucleoid-compacting protein subunit HU-α are essential for the well-mixed state; HU-α is also needed for clustering of 6/7 ribosomal RNA-encoding loci. The data are explained by a model in which the chromosomal structure is driven by dynamic competition between DNA replication and chromosomal relaxation, providing a foundation for determining how region-specific properties contribute to both chromosomal structure and gene regulation.

An N-terminal motif in NLR immune receptors is functionally conserved across distantly related plant species.

The molecular codes underpinning the functions of plant NLR immune receptors are poorly understood. We used in vitro Mu transposition to generate a random truncation library and identify the minimal functional region of NLRs. We applied this method to NRC4-a helper NLR that functions with multiple sensor NLRs within a Solanaceae receptor network. This revealed that the NRC4 N-terminal 29 amino acids are sufficient to induce hypersensitive cell death. This region is defined by the consensus MADAxVSFxVxKLxxLLxxEx (MADA motif) that is conserved at the N-termini of NRC family proteins and ~20% of coiled-coil (CC)-type plant NLRs. The MADA motif matches the N-terminal α1 helix of Arabidopsis NLR protein ZAR1, which undergoes a conformational switch during resistosome activation. Immunoassays revealed that the MADA motif is functionally conserved across NLRs from distantly related plant species. NRC-dependent sensor NLRs lack MADA sequences indicating that this motif has degenerated in sensor NLRs over evolutionary time.

A Dynamic <i>E. coli</i> Genome: Widespread DNA Contacts Revealed by Monitoring Mu Transposition

The problem of compacting genomes while still performing cellular processes is common to all life forms. The current view for E. coli is a compacted genome with well-organized domains where replication initiates and terminates. Some studies have suggested that these and other regions are sequestered or compartmentalized such that there is little to no interaction between them. In this work, we have exploited the high efficiency and promiscuity of phage Mu transposition to directly measure the in vivo rates of interactions between genomic loci, and have developed new tools for analyzing the proximity of loci across the genome. We observe widespread contacts between all regions of the E. coli chromosome, revealing a dynamic, effectively un-compartmentalized genome. We detect long-range interactions between several genes in different gene families such as dna and rrna, implicating spatial proximity of many distantly co-regulated genes for the first time in a prokaryote. We also see a higher interaction between the two halves of the chromosome during replication, consistent with the deduced proximity and higher mobility of the chromosomal arms as they segregate during replication. Our work advances a new view of genome organization in E. coli.

Mu transpososome activity-profiling yields hyperactive MuA variants for highly efficient genetic and genome engineering.

The phage Mu DNA transposition system provides a versatile species non-specific tool for molecular biology, genetic engineering and genome modification applications. Mu transposition is catalyzed by MuA transposase, with DNA cleavage and integration reactions ultimately attaching the transposon DNA to target DNA. To improve the activity of the Mu DNA transposition machinery, we mutagenized MuA protein and screened for hyperactivity-causing substitutions using an in vivo assay. The individual activity-enhancing substitutions were mapped onto the MuA–DNA complex structure, containing a tetramer of MuA transposase, two Mu end segments and a target DNA. This analysis, combined with the varying effect of the mutations in different assays, implied that the mutations exert their effects in several ways, including optimizing protein–protein and protein–DNA contacts. Based on these insights, we engineered highly hyperactive versions of MuA, by combining several synergistically acting substitutions located in different subdomains of the protein. Purified hyperactive MuA variants are now ready for use as second-generation tools in a variety of Mu-based DNA transposition applications. These variants will also widen the scope of Mu-based gene transfer technologies toward medical applications such as human gene therapy. Moreover, the work provides a platform for further design of custom transposases.

Target DNA bending by the Mu transpososome promotes careful transposition and prevents its reversal

The transposition of bacteriophage Mu serves as a model system for understanding DDE transposases and integrases. All available structures of these enzymes at the end of the transposition reaction, including Mu, exhibit significant bends in the transposition target site DNA. Here we use Mu to investigate the ramifications of target DNA bending on the transposition reaction. Enhancing the flexibility of the target DNA or prebending it increases its affinity for transpososomes by over an order of magnitude and increases the overall reaction rate. This and FRET confirm that flexibility is interrogated early during the interaction between the transposase and a potential target site, which may be how other DNA binding proteins can steer selection of advantageous target sites. We also find that the conformation of the target DNA after strand transfer is involved in preventing accidental catalysis of the reverse reaction, as conditions that destabilize this conformation also trigger reversal.

A set of mini-Mu transposons for versatile cloning of circular DNA and novel dual-transposon strategy for increased efficiency

Mu transposition-based cloning of DNA circles employs in vitro transposition reaction to deliver both the plasmid origin of replication and a selectable marker into the target DNA of interest. We report here the construction of a platform for the purpose that contains ten mini-Mu transposons with five different replication origins, enabling a variety of research approaches for the discovery and study of circular DNA. We also demonstrate that the simultaneous use of two transposons, one with the origin of replication and the other with selectable marker, is beneficial as it improves the cloning efficiency by reducing the fraction of autointegration-derived plasmid clones. The constructed transposons now provide a set of new tools for the studies on DNA circles and widen the applicability of Mu transposition based approaches to clone circular DNA from various sources.

The N-terminal domain of MuB protein has striking structural similarity to DNA-binding domains and mediates MuB filament-filament interactions.

MuB is an ATP-dependent DNA-binding protein that regulates the activity of MuA transposase and delivers the target DNA for transposition of phage Mu. Mechanistic insight into MuB function is limited to its AAA+ ATPase module, which upon ATP binding assembles into helical filaments around the DNA. However, the structure and function of the flexible N-terminal domain (NTD) appended to the AAA+ module remains uncharacterized. Here we report the solution structure of MuB NTD determined by NMR spectroscopy. The structure reveals a compact domain formed by four α-helices connected by short loops, and confirms the presence of a helix-turn-helix motif. High structural similarity and sequence homology with λ repressor-like DNA-binding domains suggest a possible role of MuB NTD in DNA binding. We also demonstrate that the NTD directly mediates the ability of MuB to establish filament-filament interactions. These findings lead us to a model in which the NTD interacts with the AAA+ spirals and perhaps also with the DNA bound within the filament, favoring MuB polymerization and filament clustering. We propose that the MuB NTD-dependent filament interactions might be an effective mechanism to bridge distant DNA regions during Mu transposition.

Transposable Phage Mu.

Transposable phage Mu has played a major role in elucidating the mechanism of movement of mobile DNA elements. The high efficiency of Mu transposition has facilitated a detailed biochemical dissection of the reaction mechanism, as well as of protein and DNA elements that regulate transpososome assembly and function. The deduced phosphotransfer mechanism involves in-line orientation of metal ion-activated hydroxyl groups for nucleophilic attack on reactive diester bonds, a mechanism that appears to be used by all transposable elements examined to date. A crystal structure of the Mu transpososome is available. Mu differs from all other transposable elements in encoding unique adaptations that promote its viral lifestyle. These adaptations include multiple DNA (enhancer, SGS) and protein (MuB, HU, IHF) elements that enable efficient Mu end synapsis, efficient target capture, low target specificity, immunity to transposition near or into itself, and efficient mechanisms for recruiting host repair and replication machineries to resolve transposition intermediates. MuB has multiple functions, including target capture and immunity. The SGS element promotes gyrase-mediated Mu end synapsis, and the enhancer, aided by HU and IHF, participates in directing a unique topological architecture of the Mu synapse. The function of these DNA and protein elements is important during both lysogenic and lytic phases. Enhancer properties have been exploited in the design of mini-Mu vectors for genetic engineering. Mu ends assembled into active transpososomes have been delivered directly into bacterial, yeast, and human genomes, where they integrate efficiently, and may prove useful for gene therapy.

An assay to monitor the activity of DNA transposition complexes yields a general quality control measure for transpositional recombination reactions.

Transposon-based technologies have many applications in molecular biology and can be used for gene delivery into prokaryotic and eukaryotic cells. Common transpositional activity measurement assays suitable for many types of transposons would be beneficial, as diverse transposon systems could be compared for their performance attributes. Therefore, we developed a general-purpose assay to enable and standardize the activity measurement for DNA transposition complexes (transpososomes), using phage Mu transposition as a test platform. This assay quantifies transpositional recombination efficiency and is based on an in vitro transposition reaction with a target plasmid carrying a lethal ccdB gene. If transposition targets ccdB, this gene becomes inactivated, enabling plasmid-receiving Escherichia coli cells to survive and to be scored as colonies on selection plates. The assay was validated with 3 mini-Mu transposons varying in size and differing in their marker gene constitution. Tests with different amounts of transposon DNA provided a linear response and yielded a 10-fold operational range for the assay. The colony formation capacity was linearly correlated with the competence status of the E.coli cells, enabling normalization of experimental data obtained with different batches of recipient cells. The developed assay can now be used to directly compare transpososome activities with all types of mini-Mu transposons, regardless of their aimed use. Furthermore, the assay should be directly applicable to other transposition-based systems with a functional in vitro reaction, and it provides a dependable quality control measure that previously has been lacking but is highly important for the evaluation of current and emerging transposon-based applications.

MuB gives a new twist to target DNA selection

Transposition target immunity is a phenomenon observed in some DNA transposons that are able to distinguish the host chromosome from their own DNA sequence, thus avoiding self-destructive insertions. The first molecular insight into target selection and immunity mechanisms came from the study of phage Mu transposition, which uses the protein MuB as a barrier to self-insertion. MuB is an ATP-dependent non-specific DNA binding protein that regulates the activity of the MuA transposase and captures target DNA for transposition. However, a detailed mechanistic understanding of MuB functioning was hindered by the poor solubility of the MuB-ATP complexes. Here we comment on the recent discovery that MuB is an AAA+ ATPase that upon ATP binding assembles into helical filaments that coat the DNA. Remarkably, the helical parameters of the MuB filament do not match those of the bound DNA. This intriguing mismatch symmetry led us to propose a model on how MuB targets DNA for transposition, favoring DNA bending and recognition by the transposase at the filament edge. We also speculate on a different protective role of MuB during immunity, where filament stickiness could favor the condensation of the DNA into a compact state that occludes it from the transposase.

TOPOLOGICAL ANALYSIS OF MU-TRANSPOSITION

An n-string tangle is a three dimensional ball with n-strings which are properly embedded in the ball. In early 90's, C. Ernst and D. Sumners first used a tangle to describe a DNA-protein complex. In this model, DNA is represented by a string and protein is represented by a ball. Mu is a protein which binds to DNA at three sites and a DNA-Mu complex is called Mu-transpososome. Knowing the DNA topology within Mu-transpososome is very important to understand DNA transposition by Mu protein. In 2002, Pathania et al. determined that the DNA configuration within the Mu transpososome is three branched and five noded [12]. In 2007, Darcy et al. analyzed this by using mathematical tangle and concluded that the three branched and five noded DNA configuration is the only biologically reasonable solution [4]. In this paper, based on the result of Pathania et al. and Darcy et al., the author determines the DNA topology within the DNA-Mu complex after the whole Mu transposition process. Furthermore, a new experiment is designed which can support the Pathania et al.'s result. The result of this new experiment is predicted through mathematical knot thory.

HU Induced Perturbation to the Structure and Dynamics of Flexible DNA Substrate Construct

Many proteins modulate the structure and dynamics of the bacterial chromosome. One of the most abundant of such proteins is HU (Heat_Unstable) protein, a member of the DNABII family of DNA-binding proteins. An architectural protein, HU introduces sharp bends (120-160 degrees) into the DNA upon binding in a site-specific manner. DNA bending induced by these proteins is important for transcriptional regulation, initiation of replication, Mu transposition, base excision repair, recombination, and negative supercoiling in bacteria. HU exhibits a strong preference for various distortions in DNA, such as nicks, gaps, cruciforms, and sticky ends, all of which share a common motif of a flexible junction. There is little structural or dynamical information related to the perturbation of these DNA motifs upon HU binding. Characterization of HU binding to these motifs provides insight into HU's role in the regulation of DNA structure and also gives insight into the general mode of site-specific recognition and binding for non-sequence-specific DNA binding proteins found in both prokaryotes and eukaryotes. We have employed various fluorescence spectroscopic techniques to address this problem. Through the use of fluorescence anisotropy we have determined the dissociation constant in solution for HU binding to DNA overhang constructs to be 0.3 nM, which is a 1000-fold lower than binding to duplex DNA. The stoichiometry of HU binding specifically to these substrates is one-to-one, while non-specific binding generally leads to larger protein to DNA ratios in bound complexes. Forster resonance energy transfer (FRET) results are indicative of protein-induced bending of the DNA substrate at the duplex/single strand junction and in the single stand region upon HU binding. We further explore the mechanism of HU-induced bending, structural recognition and binding through the use of FRET mapping techniques and time-resolved fluorescence methods.

The μ transpososome structure sheds light on DDE recombinase evolution.

Studies of bacteriophage Mu transposition paved the way for understanding retroviral integration and V(D)J recombination as well as many other DNA transposition reactions. Here we report the structure of Mu transposase (MuA) in complex with bacteriophage DNA ends and target DNA, determined from data that extend anisotropically to 5.2/5.2/3.7Å resolution, in conjunction with previously-determined structures of individual domains. The highly intertwined structure illustrates why chemical activity depends on formation of the synaptic complex, and reveals that individual domains play different roles when bound to different sites. It also suggests explanations for the increased stability of the final product complex and for its preferential recognition by the ATP-dependent unfoldase ClpX. Although MuA and many other recombinases share a structurally conserved “DDE” catalytic domain, comparisons among the limited set of available complex structures suggest that some conserved features, such as catalysis in trans and target DNA bending, arose through convergent evolution because they are important for function.

A New Role for Translation Initiation Factor 2 in Maintaining Genome Integrity

Escherichia coli translation initiation factor 2 (IF2) performs the unexpected function of promoting transition from recombination to replication during bacteriophage Mu transposition in vitro, leading to initiation by replication restart proteins. This function has suggested a role of IF2 in engaging cellular restart mechanisms and regulating the maintenance of genome integrity. To examine the potential effect of IF2 on restart mechanisms, we characterized its influence on cellular recovery following DNA damage by methyl methanesulfonate (MMS) and UV damage. Mutations that prevent expression of full-length IF2-1 or truncated IF2-2 and IF2-3 isoforms affected cellular growth or recovery following DNA damage differently, influencing different restart mechanisms. A deletion mutant (del1) expressing only IF2-2/3 was severely sensitive to growth in the presence of DNA-damaging agent MMS. Proficient as wild type in repairing DNA lesions and promoting replication restart upon removal of MMS, this mutant was nevertheless unable to sustain cell growth in the presence of MMS; however, growth in MMS could be partly restored by disruption of sulA, which encodes a cell division inhibitor induced during replication fork arrest. Moreover, such characteristics of del1 MMS sensitivity were shared by restart mutant priA300, which encodes a helicase-deficient restart protein. Epistasis analysis indicated that del1 in combination with priA300 had no further effects on cellular recovery from MMS and UV treatment; however, the del2/3 mutation, which allows expression of only IF2-1, synergistically increased UV sensitivity in combination with priA300. The results indicate that full-length IF2, in a function distinct from truncated forms, influences the engagement or activity of restart functions dependent on PriA helicase, allowing cellular growth when a DNA–damaging agent is present.

Insight into the Mechanism of Hu-Induced DNA Bending

The structure and dynamics of the bacterial chromosome are maintained by many proteins. One of the most abundant proteins, a member of the DNABII family of DNA-binding proteins, is HU (Heat_Unstable). An architectural protein is a term used to describe proteins that introduce sharp bends (120-160 degrees) into DNA upon binding. DNA bending induced by these proteins is important for transcriptional regulation, initiation of replication, mu transposition, base excision repair, recombination, and negative supercoiling in bacteria. HU exhibits a strong preference for various distortions in DNA; nicks, gaps, cruciform, and sticky ends. The distorted DNA structures seem to share a similar binding motif of a flexible junction. There is little structural or dynamical information related to perturbation of these DNA motifs upon HU binding. A better understanding of HU binding to these motifs would provide insight into HU’s role in the regulation of DNA structure. This information could also give insight into the general mode of recognition and binding for all non-sequence-specific DNA binding proteins found in both prokaryotes and eukaryotes. We have employed various fluorescence spectroscopy techniques to help answer questions related to this problem. Through the use of fluorescence anisotropy we have determined the dissociation constant in solution for HU binding to 3’overhang DNA substrate to be 3.41E-09+/- 1.2E-09 M, which is in good agreement with literature results. The stoichiometry for the specific binding of HU to these substrates is one to one, while non-specific binding may lead to higher order complexes. Fluorescence resonance energy transfer (FRET) results suggest bending of the DNA substrate upon HU binding. We hope to gain further information about the mechanism of HU-induced bending, structural recognition and binding through the use of such techniques as FRET mapping, Time-resolved fluorescence, and stop flow kinetics.