Single-strand DNA processing: phylogenomics and sequence diversity of a superfamily of potential prokaryotic HuH endonucleases.

BACKGROUND: Some mobile genetic elements target the lagging strand template during DNA replication. Bacterial examples are insertion sequences IS608 and ISDra2 (IS200/IS605 family members). They use obligatory single-stranded circular DNA intermediates for excision and insertion and encode a transposase, TnpAIS200, which recognizes subterminal secondary structures at the insertion sequence ends. Similar secondary structures, Repeated Extragenic Palindromes (REP), are present in many bacterial genomes. TnpAIS200-related proteins, TnpAREP, have been identified and could be responsible for REP sequence proliferation. These proteins share a conserved HuH/Tyrosine core domain responsible for catalysis and are involved in processes of ssDNA cleavage and ligation. Our goal is to characterize the diversity of these proteins collectively referred as the TnpAY1 family. RESULTS: A genome-wide analysis of sequences similar to TnpAIS200 and TnpAREP in prokaryotes revealed a large number of family members with a wide taxonomic distribution. These can be arranged into three distinct classes and 12 subclasses based on sequence similarity. One subclass includes sequences similar to TnpAIS200. Proteins from other subclasses are not associated with typical insertion sequence features. These are characterized by specific additional domains possibly involved in protein/DNA or protein/protein interactions. Their genes are found in more than 25% of species analyzed. They exhibit a patchy taxonomic distribution consistent with dissemination by horizontal gene transfers followed by loss. The tnpAREP genes of five subclasses are flanked by typical REP sequences in a REPtron-like arrangement. Four distinct REP types were characterized with a subclass specific distribution. Other subclasses are not associated with REP sequences but have a large conserved domain located in C-terminal end of their sequence. This unexpected diversity suggests that, while most likely involved in processing single-strand DNA, proteins from different subfamilies may play a number of different roles. CONCLUSIONS: We established a detailed classification of TnpAY1 proteins, consolidated by the analysis of the conserved core domains and the characterization of additional domains. The data obtained illustrate the unexpected diversity of the TnpAY1 family and provide a strong framework for future evolutionary and functional studies. By their potential function in ssDNA editing, they may confer adaptive responses to host cell physiology and metabolism.

Identification and Characterization of Domesticated Bacterial Transposases.

Selfish genetic elements, such as insertion sequences and transposons are found in most genomes. Transposons are usually identifiable by their high copy number within genomes. In contrast, REP-associated tyrosine transposases (RAYTs), a recently described class of bacterial transposase, are typically present at just one copy per genome. This suggests that RAYTs no longer copy themselves and thus they no longer function as a typical transposase. Motivated by this possibility we interrogated thousands of fully sequenced bacterial genomes in order to determine patterns of RAYT diversity, their distribution across chromosomes and accessory elements, and rate of duplication. RAYTs encompass exceptional diversity and are divisible into at least five distinct groups. They possess features more similar to housekeeping genes than insertion sequences, are predominantly vertically transmitted and have persisted through evolutionary time to the point where they are now found in 24% of all species for which at least one fully sequenced genome is available. Overall, the genomic distribution of RAYTs suggests that they have been coopted by host genomes to perform a function that benefits the host cell.

Structuring the bacterial genome: Y1-transposases associated with REP-BIME sequences.

REPs are highly repeated intergenic palindromic sequences often clustered into structures called BIMEs including two individual REPs separated by short linker of variable length. They play a variety of key roles in the cell. REPs also resemble the sub-terminal hairpins of the atypical IS200/605 family of insertion sequences which encode Y1 transposases (TnpA(IS200/IS605)). These belong to the HUH endonuclease family, carry a single catalytic tyrosine (Y) and promote single strand transposition. Recently, a new clade of Y1 transposases (TnpA(REP)) was found associated with REP/BIME in structures called REPtrons. It has been suggested that TnpA(REP) is responsible for REP/BIME proliferation over genomes. We analysed and compared REP distribution and REPtron structure in numerous available E. coli and Shigella strains. Phylogenetic analysis clearly indicated that tnpA(REP) was acquired early in the species radiation and was lost later in some strains. To understand REP/BIME behaviour within the host genome, we also studied E. coli K12 TnpA(REP) activity in vitro and demonstrated that it catalyses cleavage and recombination of BIMEs. While TnpA(REP) shared the same general organization and similar catalytic characteristics with TnpA(IS200/IS605) transposases, it exhibited distinct properties potentially important in the creation of BIME variability and in their amplification. TnpA(REP) may therefore be one of the first examples of transposase domestication in prokaryotes.

Acinetobacter insertion sequence ISAba11 belongs to a novel family that encodes transposases with a signature HHEK motif.

Experimental and in silico PCR analysis targeting ISAba11 and TnAbaR islands in 196 epidemiologically unrelated Acinetobacter strains representative of ≥19 species were performed. The first two Acinetobacter baumannii ISAba11 elements identified had been found to map to the same site on TnAbaR transposons. However, no further evidence of physical linkage between the two elements was demonstrated. Indeed, examination of 25 definite or putative insertion sites suggested limited sequence specificity. Importantly, an aacC1-tagged version of ISAba11 was shown to actively transpose in A. baumannii. Similarity searches identified nine iso-ISAba11 elements in Acinetobacter and one in Enhydrobacter and single representatives of four distant homologs in bacteria belonging to the phyla "Cyanobacteria" and Proteobacteria. Phylogenetic, sequence, and structural analyses of ISAba11 and/or its associated transposase (Tnp(ISAba11)) suggested that these elements be assigned to a new family. All five homologs encode transposases with a shared extended signature comprising 16 invariant residues within the N2, N3, and C1 regions, four of which constituted the cardinal ISAba11 family HHEK motif that is substituted for the YREK DNA binding motif conserved in the IS4 family. Additionally, ISAba11 family members were associated with either no flanking direct repeat (DR) or an ISAba11-typical 5-bp DR and possessed variable-length terminal inverted repeats that exhibited extensive intrafamily sequence identity. Given the limited pairwise identity among Tnp(ISAba11) homologs and the observed restricted distribution of ISAba11, we propose that substantial gaps persist in the evolutionary record of ISAba11 and that this element represents a recent though potentially highly significant entrant into the A. baumannii gene pool.

The catalytic domain of all eukaryotic cut-and-paste transposase superfamilies.

Cut-and-paste DNA transposable elements are major components of eukaryotic genomes and are grouped into superfamilies (e.g., hAT, P) based on sequence similarity of the element-encoded transposase. The transposases from several superfamilies possess a protein domain containing an acidic amino acid triad (DDE or DDD) that catalyzes the "cut and paste" transposition reaction. However, it was unclear whether this domain was shared by the transposases from all superfamilies. Through multiple-alignment of transposase sequences from a diverse collection of previously identified and recently annotated elements from a wide range of organisms, we identified the putative DDE/D triad for all superfamilies. Furthermore, we identified additional highly conserved amino acid residues or motifs within the DDE/D domain that together form a "signature string" that is specific to each superfamily. These conserved residues or motifs were exploited as phylogenetic characters to infer evolutionary relationships among all superfamilies. The phylogenetic analysis revealed three major groups that were not previously discerned and led us to revise the classification of several currently recognized superfamilies. Taking the data together, this study suggests that all eukaryotic cut-and-paste transposable element superfamilies have a common evolutionary origin and establishes a phylogenetic framework for all future cut-and-paste transposase comparisons.

Structural characterization of ISCR8, ISCR22, and ISCR23, subgroups of IS91-like insertion elements.

Analysis of ISCR8 (ISPps1) revealed that this group of insertion elements has to be subdivided into three subgroups: ISCR8, ISCR22, and ISCR23. The distinction of three subgroups is supported by phylogenetic analysis of the transposase open reading frames (ORFs). Comparison of over 20 complete and partial ISCR8/22/23 elements identified oriIS candidate sequences for all groups and a terIS candidate sequence for ISCR8. The oriIS sequences, their distance to the transposase ORFs, and the sequence of this intervening region are group specific, further supporting the definition of two new ISCR elements. ISCR8/22/23 have a very broad host range, including Gram-positive and Gram-negative bacteria, among which are several (opportunistic) pathogens. The IS often resides on plasmids or in the vicinity of other mobile elements and is mostly associated with genes for the degradation of halo- or nitro-aromatics. However, in one case ISCR8 was found in the neighborhood of an antibiotic resistance determinant in Klebsiella pneumoniae. ISCR8 resembles other IS91 family elements in mediating genetic rearrangements by homologous recombination between two copies. In Delftia acidovorans this led to the loss of the genes encoding dichlorprop cleavage. In conclusion, this study shows that ISCR8 could be a fully functional and active member of the IS91 family of insertion elements.

Phantom, a new subclass of Mutator DNA transposons found in insect viruses and widely distributed in animals.

Transposons of the Mutator (Mu) superfamily have been shown to play a critical role in the evolution of plant genomes. However, the identification of Mutator transposons in other eukaryotes has been quite limited. Here we describe a previously uncharacterized group of DNA transposons designated Phantom identified in the genomes of a wide range of eukaryotic taxa, including many animals, and provide evidence for its inclusion within the Mutator superfamily. Interestingly three Phantom proteins were also identified in two insect viruses and phylogenetic analysis suggests horizontal movement from insect to virus, providing a new line of evidence for the role of viruses in the horizontal transfer of DNA transposons in animals. Many of the Phantom transposases are predicted to harbor a FLYWCH domain in the amino terminus, which displays a WRKY-GCM1 fold characteristic of the DNA binding domain (DBD) of Mutator transposases and of several transcription factors. While some Phantom elements have terminal inverted repeats similar in length and structure to Mutator elements, some display subterminal inverted repeats (sub-TIRs) and others have more complex termini reminiscent of so-called Foldback (FB) transposons. The structural plasticity of Phantom and the distant relationship of its encoded protein to known transposases may have impeded the discovery of this group of transposons and it suggests that structure in itself is not a reliable character for transposon classification.

Automatic classification within families of transposable elements: application to the mariner Family.

The higher levels of the classification of transposable elements (TEs) from Classes to Superfamilies or Families, is regularly updated, but the lower levels (below the Family) have received little investigation. In particular, this applies to the Families that include a large number of copies. In this article we propose an automatic classification of DNA sequences. This procedure is based on an aggregation process using a pairwise matrix of distances, allowing us to define several groups characterized by a sphere with a central sequence and a radius. This method was tested on the mariner Family, because this is probably one of the most extensively studied Families. Several Subfamilies had already been defined from phylogenetic analyses based on multiple alignments of complete or partial amino-acid sequences of the transposase. The classification obtained here from DNA sequences of 935 items matches the phylogenies of the transposase. The rate of error from a posteriori re-assignment is relatively low.

Passport, a native Tc1 transposon from flatfish, is functionally active in vertebrate cells.

The Tc1/mariner family of DNA transposons is widespread across fungal, plant and animal kingdoms, and thought to contribute to the evolution of their host genomes. To date, an active Tc1 transposon has not been identified within the native genome of a vertebrate. We demonstrate that Passport, a native transposon isolated from a fish (Pleuronectes platessa), is active in a variety of vertebrate cells. In transposition assays, we found that the Passport transposon system improved stable cellular transgenesis by 40-fold, has an apparent preference for insertion into genes, and is subject to overproduction inhibition like other Tc1 elements. Passport represents the first vertebrate Tc1 element described as both natively intact and functionally active, and given its restricted phylogenetic distribution, may be contemporaneously active. The Passport transposon system thus complements the available genetic tools for the manipulation of vertebrate genomes, and may provide a unique system for studying the infiltration of vertebrate genomes by Tc1 elements.

Analysis of the DDE motif in the Mutator superfamily.

The eukaryotic Mutator family of transposable elements is widespread in plants. Active or potentially active copies are also found in fungi and protozoans, and sequences related to this family have been detected in metazoans as well. Members of this family are called Mutator-like elements (MULEs). They encode transposases, which contain a region conserved with transposases of the IS256 prokaryotic family, known to harbor a DDE catalytic domain. Different DDE or D34E motifs have been proposed in some groups of eukaryotic MULEs based on primary sequence conservation. On a large number of protein sequences related to, and representative of, all MULE families, we analyzed global conservation, the close environment of different acidic residues and the secondary structure. This allowed us to identify a potential DDE motif that is likely to be homologous to the one in IS256-like transposases. The characteristics of this motif are depicted in each known family of MULEs. Different hypotheses about the evolution of this triad are discussed.

The natural history of the WRKY-GCM1 zinc fingers and the relationship between transcription factors and transposons.

WRKY and GCM1 are metal chelating DNA-binding domains (DBD) which share a four stranded fold. Using sensitive sequence searches, we show that this WRKY-GCM1 fold is also shared by the FLYWCH Zn-finger domain and the DBDs of two classes of Mutator-like element (MULE) transposases. We present evidence that they share a stabilizing core, which suggests a possible origin from a BED finger-like intermediate that was in turn ultimately derived from a C2H2 Zn-finger domain. Through a systematic study of the phyletic pattern, we show that this WRKY-GCM1 superfamily is a widespread eukaryote-specific group of transcription factors (TFs). We identified several new members across diverse eukaryotic lineages, including potential TFs in animals, fungi and Entamoeba. By integrating sequence, structure, gene expression and transcriptional network data, we present evidence that at least two major global regulators belonging to this superfamily in Saccharomyces cerevisiae (Rcs1p and Aft2p) have evolved from transposons, and attained the status of transcription regulatory hubs in recent course of ascomycete yeast evolution. In plants, we show that the lineage-specific expansion of WRKY-GCM1 domain proteins acquired functional diversity mainly through expression divergence rather than by protein sequence divergence. We also use the WRKY-GCM1 superfamily as an example to illustrate the importance of transposons in the emergence of new TFs in different lineages.

Vibrio cholerae CTXPhi phage repressor gene is borne on an insertion sequence-like element and encodes a putative transposase.

Bioinformatics analyses show the presence of a novel insertion sequence-like element in the CTXPhi phage of Vibrio cholerae. The solitary open reading frame encompassed by the element is known to encode a repressor (RstR) of phage DNA replication and is responsible for phage heteroimmunity. Analysis of the nucleotide and protein sequence of the repressor and its flanking non-coding regions indicates that it resembles distinctly simple bacterial insertion elements in numerous aspects. Based on the results of extensive sequence analysis, we propose that the rstR gene is borne on an insertion sequence-like element and that the RstR protein encodes a putative transposase. We put forward the possibility that a bi-directional promoter could regulate the divergent transcription of the neighbouring genes of rstR and rstA. The possibility of overlapping reading frames yielding distinct repressor and transposase proteins is also expanded based on the analyses.

An Arabidopsis hAT-like transposase is essential for plant development.

A significant proportion of the genomes of higher plants and vertebrates consists of transposable elements and their derivatives. Autonomous DNA type transposons encode a transposase that enables them to mobilize to a new chromosomal position in the host genome by a cut-and-paste mechanism. As this is potentially mutagenic, the host limits transposition through epigenetic gene silencing and heterochromatin formation. Here we show that a transposase from Arabidopsis thaliana that we named DAYSLEEPER is essential for normal plant growth; it shares several characteristics with the hAT (hobo, Activator, Tam3) family of transposases. DAYSLEEPER was isolated as a factor binding to a motif (Kubox1) present in the upstream region of the Arabidopsis DNA repair gene Ku70. This motif is also present in the upstream regions of many other plant genes. Plants lacking DAYSLEEPER or strongly overexpressing this gene do not develop in a normal manner. Furthermore, DAYSLEEPER overexpression results in the altered expression of many genes. Our data indicate that transposase-like genes can be essential for plant development and can also regulate global gene expression. Thus, transposases can become domesticated by the host to fulfil important cellular functions.

Mutator-like element in the yeast Yarrowia lipolytica displays multiple alternative splicings.

A new type of DNA transposon, Mutyl, has been identified in the sequenced genome of the yeast Yarrowia lipolytica. This transposon is 7,413 bp long and carries two open reading frames (ORFs) which potentially encode proteins of 459 and 1,178 amino acids, respectively. Whereas the first ORF shows no significant homology to previously described proteins, the second ORF shows sequence similarities with various Mutator-like element (MULE)-encoded transposases, including the bacterial transposase signature sequence. Other MULE features shared by Mutyl include a zinc finger motif in the putative transposase, a 22-bp-long imperfect inverted repeat at each end, and a 9- to 10-bp duplication of its target site in the chromosome. Of the five copies of Mutyl present in the genome, one has a deletion of the first 8 bases, and the others are full length with a single base change in one element. The first potential gene of Mutyl, mutB, was shown to be expressed in exponentially growing cells. Its sequence contains a predicted intron with two 5' splice sites, a single branch point, and two 3' splice sites. Its mRNA is alternatively spliced, as judged by reverse transcription-PCR, and generates four mRNAs corresponding to protein-coding sequences of 128, 156, 161, and 190 amino acids. Of the three distinct lineages characterized in Y. lipolytica, strains from the German lineage and the French lineage do not carry Mutyl. A study of the distribution of Mutyl in strains of the French lineage evidenced a recent transposition event. Taken together, these results indicate that Mutyl is still active.

Transposition from a gutless adeno-transposon vector stabilizes transgene expression in vivo.

A major limitation of adenovirus-mediated gene therapy for inherited diseases is the instability of transgene expression in vivo, which originates at least in part from the loss of the linear, extrachromosomal vector genomes. Herein we describe the production of a gene-deleted adenovirus-transposon vector that stably maintains virus-encoded transgenes in vivo through integration into host cell chromosomes. This system utilizes a donor transposon vector that undergoes Flp-mediated recombination and excision of its therapeutic payload in the presence of the Flp and Sleeping Beauty recombinases. Systemic in vivo delivery of this system resulted in efficient generation of transposon circles and stable transposase-mediated integration in mouse liver. Somatic integration was sufficient to maintain therapeutic levels of human coagulation Factor IX for more than six months in mice undergoing extensive liver proliferation. These vectors combine the versatility of adenoviral vectors with the integration capabilities of a eukaryotic DNA transposon and should prove useful in the treatment of genetic diseases.

The resolvase encoded by Xanthomonas campestris transposable element ISXc5 constitutes a new subfamily closely related to DNA invertases.

BACKGROUND: Conservative site-specific recombination is responsible for the resolution of cointegrates which result during the transposition of class II transposable elements. Resolution is catalysed by a transposon-encoded recombinase, resolvase, that belongs to a large family of recombinases, including DNA invertases. Resolvases and the related invertases are likely to employ similar reaction mechanisms during recombination. There are important differences, however. Resolvases require two accessory DNA binding sites within each of the two directly repeated recombination sites. Invertases instead need a host factor, Fis, and an enhancer type DNA sequence, in addition to two inversely orientated recombination sites. RESULTS: The resolvase encoded by transposable element ISXc5 from the gram-negative phytopathogen Xanthomonas campestris shows two features which distinguish it from other known resolvases. First, it is more closely phylogenetically related to invertases than other resolvases. In particular, two functionally important regions seem highly conserved between this resolvase and members of the invertase subfamily. Second, the enzyme exhibits a large extension of its carboxy-terminal domain with unknown function. We purified ISXc5 resolvase and analysed its resolution reaction in vitro. Our biochemical and DNA topological analysis reveals that critical features of resolution are similar, if not identical, to that carried out by gammadelta resolvase. However, despite its apparent similarity to invertases, we were unable to detect recombination on standard substrates for DNA inversion, in either the presence or absence of Fis. CONCLUSIONS: ISXc5 resolvase employs a reaction mechanism which is common to members of the resolvase family. Its position near the evolutionary borderline to invertases and its high degree of identity within two functionally important regions with members of the DNA invertase subfamily suggest that only a few replacements of critical residues may suffice to convert this resolvase into a functional, possibly Fis-dependent invertase.