Compensating for over-production inhibition of the Hsmar1 transposon in using a series of constitutive promoters.

Background: Transposable elements (TEs) are a diverse group of self-mobilizing DNA elements. Transposition has been exploited as a powerful tool for molecular biology and genomics. However, transposition is sometimes limited because of auto-regulatory mechanisms that presumably allow them to cohabit within their hosts without causing excessive genomic damage.

Targeted DNA transposition in vitro using a dCas9-transposase fusion protein.

Homology-directed genome engineering is limited by transgene size. Although DNA transposons are more efficient with large transgenes, random integrations are potentially mutagenic. Here we present an in vitro mechanistic study that demonstrates efficient Cas9 targeting of the mariner transposon Hsmar1.

The roles of the human SETMAR (Metnase) protein in illegitimate DNA recombination and non-homologous end joining repair.

SETMAR is a fusion between a SET-domain methyltransferase gene and a mariner-family transposase gene, which is specific to anthropoid primates. However, the ancestral SET gene is present in all other mammals and birds. SETMAR is reported to be involved in transcriptional regulation and a diverse set of reactions related to DNA repair.

Human SETMAR is a DNA sequence-specific histone-methylase with a broad effect on the transcriptome.

Transposons impart dynamism to the genomes they inhabit and their movements frequently rewire the control of nearby genes. Occasionally, their proteins are domesticated when they evolve a new function. SETMAR is a protein methylase with a sequence-specific DNA binding domain.

Transposase subunit architecture and its relationship to genome size and the rate of transposition in prokaryotes and eukaryotes.

Cut-and-paste transposons are important tools for mutagenesis, gene-delivery and DNA sequencing applications. At the molecular level, the most thoroughly understood are Tn5 and Tn10 in bacteria, and mariner and hAT elements in eukaryotes. All bacterial cut-and-paste transposases characterized to date are monomeric prior to interacting with the transposon end, while all eukaryotic transposases are multimers.

A single active site in the mariner transposase cleaves DNA strands of opposite polarity.

The RNase H structural fold defines a large family of nucleic acid metabolizing enzymes that catalyze phosphoryl transfer reactions using two divalent metal ions in the active site. Almost all of these reactions involve only one strand of the nucleic acid substrates. In contrast, cut-and-paste transposases cleave two DNA strands of opposite polarity, which is usually achieved via an elegant hairpin mechanism.

The Invertebrate Lysozyme Effector ILYS-3 Is Systemically Activated in Response to Danger Signals and Confers Antimicrobial Protection in C. elegans.

Little is known about the relative contributions and importance of antibacterial effectors in the nematode C. elegans, despite extensive work on the innate immune responses in this organism. We report an investigation of the expression, function and regulation of the six ilys (invertebrate-type lysozyme) genes of C.

Mariner and the ITm Superfamily of Transposons.

The IS630-Tc1-mariner (ITm) family of transposons is one of the most widespread in nature. The phylogenetic distribution of its members shows that they do not persist for long in a given lineage, but rely on frequent horizontal transfer to new hosts. Although they are primarily selfish genomic-parasites, ITm transposons contribute to the evolution of their hosts because they generate variation and contribute protein domains and regulatory regions.

One to rule them all: A highly conserved motif in mariner transposase controls multiple steps of transposition.

The development of transposon-based genome manipulation tools can benefit greatly from understanding transposons' inherent regulatory mechanisms. The Tc1-mariner transposons, which are being widely used in biotechnological applications, are subject to a self-inhibitory mechanism whereby increasing transposase expression beyond a certain point decreases the rate of transposition. In a recent paper, Liu and Chalmers performed saturating mutagenesis on the highly conserved WVPHEL motif in the mariner-family transposase from the Hsmar1 element.

Crosstalk between transposase subunits during cleavage of the mariner transposon.

Mariner transposition is a complex reaction that involves three recombination sites and six strand breaking and joining reactions. This requires precise spatial and temporal coordination between the different components to ensure a productive outcome and minimize genomic instability. We have investigated how the cleavage events are orchestrated within the mariner transpososome.

Hyperactive mariner transposons are created by mutations that disrupt allosterism and increase the rate of transposon end synapsis.

New applications for transposons in vertebrate genetics have spurred efforts to develop hyperactive variants. Typically, a genetic screen is used to identify several hyperactive point mutations, which are then incorporated in a single transposase gene. However, the mechanisms responsible for the increased activity are unknown.

Accuracy and efficiency define Bxb1 integrase as the best of fifteen candidate serine recombinases for the integration of DNA into the human genome.

Background: Phage-encoded serine integrases, such as φC31 integrase, are widely used for genome engineering. Fifteen such integrases have been described but their utility for genome engineering has not been compared in uniform assays.

Results: We have compared fifteen serine integrases for their utility for DNA manipulations in mammalian cells after first demonstrating that all were functional in E.

Temperature triggers immune evasion by Neisseria meningitidis.

Neisseria meningitidis has several strategies to evade complement-mediated killing, and these contribute to its ability to cause septicaemic disease and meningitis. However, the meningococcus is primarily an obligate commensal of the human nasopharynx, and it is unclear why the bacterium has evolved exquisite mechanisms to avoid host immunity. Here we demonstrate that mechanisms of meningococcal immune evasion and resistance against complement increase in response to an increase in ambient temperature.

The autoregulation of a eukaryotic DNA transposon.

How do DNA transposons live in harmony with their hosts? Bacteria provide the only documented mechanisms for autoregulation, but these are incompatible with eukaryotic cell biology. Here we show that autoregulation of Hsmar1 operates during assembly of the transpososome and arises from the multimeric state of the transposase, mediated by a competition for binding sites. We explore the dynamics of a genomic invasion using a computer model, supported by in vitro and in vivo experiments, and show that amplification accelerates at first but then achieves a constant rate.

Identification of multiple binding sites for the THAP domain of the Galileo transposase in the long terminal inverted-repeats.

Galileo is a DNA transposon responsible for the generation of several chromosomal inversions in Drosophila. In contrast to other members of the P-element superfamily, it has unusually long terminal inverted-repeats (TIRs) that resemble those of Foldback elements. To investigate the function of the long TIRs we derived consensus and ancestral sequences for the Galileo transposase in three species of Drosophilids.

Hsmar1 transposition is sensitive to the topology of the transposon donor and the target.

Hsmar1 is a member of the Tc1-mariner superfamily of DNA transposons. These elements mobilize within the genome of their host by a cut-and-paste mechanism. We have exploited the in vitro reaction provided by Hsmar1 to investigate the effect of DNA supercoiling on transposon integration.

H-NS mediates the dissociation of a refractory protein-DNA complex during Tn10/IS10 transposition.

Tn10/IS10 transposition takes place in the context of a protein-DNA complex called a transpososome. During the reaction, the transpososome undergoes several conformational changes. The host proteins IHF and H-NS, which also are global regulators of gene expression, play important roles in directing these architectural changes.

The transposon-like Correia elements encode numerous strong promoters and provide a potential new mechanism for phase variation in the meningococcus.

Neisseria meningitidis is the primary causative agent of bacterial meningitis. The genome is rich in repetitive DNA and almost 2% is occupied by a diminutive transposon called the Correia element. Here we report a bioinformatic analysis defining eight subtypes of the element with four distinct types of ends.

A simple topological filter in a eukaryotic transposon as a mechanism to suppress genome instability.

DNA transposition takes place within a higher-order complex known as the transpososome. Almost everything known about its assembly has been gleaned from bacterial transposons. Here we present a detailed analysis of transpososome assembly in the human Hsmar1 element.

Delivering the goods: viral and non-viral gene therapy systems and the inherent limits on cargo DNA and internal sequences.

Viruses have long been considered to be the most promising tools for human gene therapy. However, the initial enthusiasm for the use of viruses has been tarnished in the light of potentially fatal side effects. Transposons have a long history of use with bacteria in the laboratory and are now routinely applied to eukaryotic model organisms.

Transposition of the human Hsmar1 transposon: rate-limiting steps and the importance of the flanking TA dinucleotide in second strand cleavage.

Hsmar1 is a member of the mariner family of DNA transposons. Although widespread in nature, their molecular mechanism remains obscure. Many other cut-and-paste elements use a hairpin intermediate to cleave the two strands of DNA at each transposon end.

Base flipping in V(D)J recombination: insights into the mechanism of hairpin formation, the 12/23 rule, and the coordination of double-strand breaks.

Tn5 transposase cleaves the transposon end using a hairpin intermediate on the transposon end. This involves a flipped base that is stacked against a tryptophan residue in the protein. However, many other members of the cut-and-paste transposase family, including the RAG1 protein, produce a hairpin on the flanking DNA.

Gene therapy vectors: the prospects and potentials of the cut-and-paste transposons.

Gene therapy applications require efficient tools for the stable delivery of genetic information into eukaryotic genomes. Most current gene delivery strategies are based on viral vectors. However, a number of drawbacks, such as the limited cargo capacity, host immune response and mutational risks, highlight the need for alternative gene delivery tools.

Base flipping in tn10 transposition: an active flip and capture mechanism.

The bacterial Tn5 and Tn10 transposases have a single active site that cuts both strands of DNA at their respective transposon ends. This is achieved using a hairpin intermediate that requires the DNA to change conformation during the reaction. In Tn5 these changes are controlled in part by a flipped nucleoside that is stacked on a tryptophan residue in a hydrophobic pocket of the transposase.

Transposition of Mboumar-9: identification of a new naturally active mariner-family transposon.

Although mariner transposons are widespread in animal genomes, the vast majority harbor multiple inactivating mutations and only two naturally occurring elements are known to be active. Previously, we discovered a mariner-family transposon, Mboumar, in the satellite DNA of the ant Messor bouvieri. Several copies of the transposon contain a full-length open reading frame, including Mboumar-9, which has 64% nucleotide identity to Mos1 of Drosophila mauritiana.