Targeting IS608 transposon integration to highly specific sequences by structure-based transposon engineering.

Transposable elements are efficient DNA carriers and thus important tools for transgenesis and insertional mutagenesis. However, their poor target sequence specificity constitutes an important limitation for site-directed applications. The insertion sequence IS608 from Helicobacter pylori recognizes a specific tetranucleotide sequence by base pairing, and its target choice can be re-programmed by changes in the transposon DNA.

Insertion Sequence IS26 Reorganizes Plasmids in Clinically Isolated Multidrug-Resistant Bacteria by Replicative Transposition.

Unlabelled: Carbapenemase-producing Enterobacteriaceae (CPE), which are resistant to most or all known antibiotics, constitute a global threat to public health. Transposable elements are often associated with antibiotic resistance determinants, suggesting a role in the emergence of resistance. One insertion sequence, IS26, is frequently associated with resistance determinants, but its role remains unclear.

The emerging diversity of transpososome architectures.

DNA transposases are enzymes that catalyze the movement of discrete pieces of DNA from one location in the genome to another. Transposition occurs through a series of controlled DNA strand cleavage and subsequent integration reactions that are carried out by nucleoprotein complexes known as transpososomes. Transpososomes are dynamic assemblies which must undergo conformational changes that control DNA breaks and ensure that, once started, the transposition reaction goes to completion.

The amino acid linker between the endonuclease and helicase domains of adeno-associated virus type 5 Rep plays a critical role in DNA-dependent oligomerization.

The adeno-associated virus (AAV) genome encodes four Rep proteins, all of which contain an SF3 helicase domain. The larger Rep proteins, Rep78 and Rep68, are required for viral replication, whereas Rep40 and Rep52 are needed to package AAV genomes into preformed capsids; these smaller proteins are missing the site-specific DNA-binding and endonuclease domain found in Rep68/78. Other viral SF3 helicases, such as the simian virus 40 large T antigen and the papillomavirus E1 protein, are active as hexameric assemblies.

DNA recognition and the precleavage state during single-stranded DNA transposition in D. radiodurans.

Bacterial insertion sequences (ISs) from the IS200/IS605 family encode the smallest known DNA transposases and mobilize through single-stranded DNA transposition. Transposition by one particular family member, ISDra2 from Deinococcus radiodurans, is dramatically stimulated upon massive γ irradiation. We have determined the crystal structures of four ISDra2 transposase/IS end complexes; combined with in vivo activity assays and fluorescence anisotropy binding measurements, these have revealed the molecular basis of strand discrimination and transposase action.

Single-stranded DNA transposition is coupled to host replication.

DNA transposition has contributed significantly to evolution of eukaryotes and prokaryotes. Insertion sequences (ISs) are the simplest prokaryotic transposons and are divided into families on the basis of their organization and transposition mechanism. Here, we describe a link between transposition of IS608 and ISDra2, both members of the IS200/IS605 family, which uses obligatory single-stranded DNA intermediates, and the host replication fork.

Integrating prokaryotes and eukaryotes: DNA transposases in light of structure.

DNA rearrangements are important in genome function and evolution. Genetic material can be rearranged inadvertently during processes such as DNA repair, or can be moved in a controlled manner by enzymes specifically dedicated to the task. DNA transposases comprise one class of such enzymes.

Resetting the site: redirecting integration of an insertion sequence in a predictable way.

Target site choice is a complex and poorly understood aspect of DNA transposition despite its importance in rational transposon-mediated gene delivery. Though most transposons choose target sites essentially randomly or with some slight sequence or structural preferences, insertion sequence IS608 from Helicobacter pylori, which transposes using single-stranded DNA, always inserts just 3' of a TTAC tetranucleotide. Our results from studies on the IS608 transposition mechanism demonstrated that the transposase recognizes its target site by co-opting an internal segment of transposon DNA and utilizes it for specific recognition of the target sites through base-pairing.

In vitro reconstitution of a single-stranded transposition mechanism of IS608.

Bacterial insertion sequences (IS) play an important role in restructuring their host genomes. IS608, from Helicobacter pylori, belongs to a newly recognized and widespread IS group with a unique transposition mechanism. We have reconstituted the entire set of transposition cleavage and strand transfer reactions in vitro and find that, unlike any other known transposition system, they strictly require single-strand DNA.

Mechanism of IS200/IS605 family DNA transposases: activation and transposon-directed target site selection.

The smallest known DNA transposases are those from the IS200/IS605 family. Here we show how the interplay of protein and DNA activates TnpA, the Helicobacter pylori IS608 transposase, for catalysis. First, transposon end binding causes a conformational change that aligns catalytically important protein residues within the active site.

Structural basis for dimerization of LAP2alpha, a component of the nuclear lamina.

Lamina-associated polypeptides (LAPs) are important components of the nuclear lamina, the dense network of filaments that supports the nuclear envelope and also extends into the nucleoplasm. The main protein constituents of the nuclear lamina are the constitutively expressed B-type lamins and the developmentally regulated A- and C-type lamins. LAP2alpha is the only non-membrane-associated member of the LAP family.

Purification, crystallization and preliminary crystallographic analysis of the Hermes transposase.

DNA transposition is the movement of a defined segment of DNA from one location to another. Although the enzymes that catalyze transposition in bacterial systems have been well characterized, much less is known about the families of transposase enzymes that function in higher organisms. Active transposons have been identified in many insect species, providing tools for gene identification and offering the possibility of altering the genotypes of natural insect populations.

Binding and unwinding: SF3 viral helicases.

The SF3 helicases, distinct from the more prevalent SF1 and SF2 helicases, were originally identified in the genomes of small DNA and RNA viruses. The first crystal structures of SF3 helicases have been determined, revealing a closer structural relationship to AAA+ proteins than to RecA, consistent with their participation in replication initiation. In conjunction with origin-binding domains, SF3 helicases are responsible for distorting DNA before replication forks can be assembled.

Transposition of hAT elements links transposable elements and V(D)J recombination.

Transposons are DNA sequences that encode functions that promote their movement to new locations in the genome. If unregulated, such movement could potentially insert additional DNA into genes, thereby disrupting gene expression and compromising an organism's viability. Transposable elements are classified by their transposition mechanisms and by the transposases that mediate their movement.

The carboxy-terminal portion of TnsC activates the Tn7 transposase through a specific interaction with TnsA.

Tn7 transposition requires the assembly of a nucleoprotein complex containing four self-encoded proteins, transposon ends, and target DNA. Within this complex, TnsC, the molecular switch that regulates transposition, and TnsA, one part of the transposase, interact directly. Here, we demonstrate that residues 504-555 of TnsC are responsible for TnsA/TnsC interaction.

The nuclease domain of adeno-associated virus rep coordinates replication initiation using two distinct DNA recognition interfaces.

Integration into a particular location in human chromosomes is a unique property of the adeno-associated virus (AAV). This reaction requires the viral Rep protein and AAV origin sequences. To understand how Rep recognizes DNA, we have determined the structures of the Rep endonuclease domain separately complexed with two DNA substrates: the Rep binding site within the viral inverted terminal repeat and one of the terminal hairpin arms.

Two 14-3-3 binding motifs are required for stable association of Forkhead transcription factor FOXO4 with 14-3-3 proteins and inhibition of DNA binding.

The 14-3-3 proteins, a family of dimeric regulatory proteins, are involved in many biologically important processes. The common feature of 14-3-3 proteins is their ability to bind to other proteins in a phosphorylation-dependent manner. Through these binding interactions, 14-3-3 proteins work as molecular scaffolds, modulating the biological functions of their partners.

A mob of reps.

Emerging structural results confirm that the large Rolling Circle Replication initiator superfamily is composed of two classes of proteins that are circularly permutated with respect to each other, as initially suggested by sequence analysis. The two classes are united by the same endonucleolytic mechanism and a conserved Mg(2+) binding site containing multiple histidine ligands unique to this superfamily.

Structural unity among viral origin binding proteins: crystal structure of the nuclease domain of adeno-associated virus Rep.

Adeno-associated virus (AAV), unique among animal viruses in its ability to integrate into a specific chromosomal location, is a promising vector for human gene therapy. AAV Replication (Rep) protein is essential for viral replication and integration, and its amino terminal domain possesses site-specific DNA binding and endonuclease activities required for replication initiation and integration. This domain displays a novel endonuclease fold and demonstrates an unexpected structural relationship to other viral origin binding proteins such as the papillomavirus E1 protein and the SV40 T antigen.