SETMAR, a case of primate co-opted genes: towards new perspectives.

BACKGROUND: We carry out a review of the history and biological activities of one domesticated gene in higher primates, SETMAR, by discussing current controversies. Our purpose is to open a new outlook that will serve as a framework for future work about SETMAR, possibly in the field of cognition development. MAIN BODY: What is newly important about SETMAR can be summarized as follows: (1) the whole protein sequence is under strong purifying pressure; (2) its role is to strengthen existing biological functions rather than to provide new ones; (3) it displays a tissue-specific pattern of expression, at least for the alternative-splicing it undergoes. Studies reported here demonstrate that SETMAR protein(s) may be involved in essential networks regulating replication, transcription and translation. Moreover, during embryogenesis, SETMAR appears to contribute to brain development. SHORT CONCLUSION: Our review underlines for the first time that SETMAR directly interacts with genes involved in brain functions related to vocalization and vocal learning. These findings pave the way for future works regarding SETMAR and the development of cognitive abilities in higher primates.

SETMAR Shorter Isoform: A New Prognostic Factor in Glioblastoma.

Recent evidence suggests that the chimeric protein SETMAR is a factor of interest in cancer, especially in glioblastoma. However, little is known about the expression of this protein in glioblastoma tissues, and no study has been done to assess if SETMAR could be a prognostic and/or diagnostic marker of glioblastoma. We analyzed protein extracts of 47 glioblastoma samples coming from a local and a national cohort of patients. From the local cohort, we obtained localized biopsies from the central necrosis area, the tumor, and the perilesional brain. From the French Glioblastoma Biobank (FGB), we obtained three types of samples: from the same tumors before and after treatment, from long survivors, and from very short survivors. We studied the correlations between SETMAR amounts, clinical profiles of patients and other associated proteins (PTN, snRNP70 and OLIG2). In glioblastoma tissues, the shorter isoform of SETMAR (S-SETMAR) was predominant over the full-length isoform (FL-SETMAR), and the expression of both SETMAR variants was higher in the tumor compared to the perilesional tissues. Data from the FGB showed that SETMAR amounts were not different between the initial tumors and tumor relapses after treatment. These data also showed a trend toward higher amounts of S-SETMAR in long survivors. In localized biopsies, we found a positive correlation between good prognosis and large amounts of S-SETMAR in the perilesional area. This is the main result presented here: survival in Glioblastoma is correlated with amounts of S-SETMAR in perilesional brain, which should be considered as a new relevant prognosis marker.

The epigenetic regulation of HsMar1, a human DNA transposon.

BACKGROUND: Both classes of transposable elements (DNA and RNA) are tightly regulated at the transcriptional level leading to the inactivation of transposition via epigenetic mechanisms. Due to the high copies number of these elements, the hypothesis has emerged that their regulation can coordinate a regulatory network of genes. Herein, we investigated whether transposition regulation of HsMar1, a human DNA transposon, differs in presence or absence of endogenous HsMar1 copies. In the case where HsMar1 transposition is regulated, the number of repetitive DNA sequences issued by HsMar1 and distributed in the human genome makes HsMar1 a good candidate to regulate neighboring gene expression by epigenetic mechanisms. RESULTS: A recombinant active HsMar1 copy was inserted in HeLa (human) and CHO (hamster) cells and its genomic excision monitored. We show that HsMar1 excision is blocked in HeLa cells, whereas CHO cells are competent to promote HsMar1 excision. We demonstrate that de novo HsMar1 insertions in HeLa cells (human) undergo rapid silencing by cytosine methylation and apposition of H3K9me3 marks, whereas de novo HsMar1 insertions in CHO cells (hamster) are not repressed and enriched in H3K4me3 modifications. The overall analysis of HsMar1 endogenous copies in HeLa cells indicates that neither full-length endogenous inactive copies nor their Inverted Terminal Repeats seem to be specifically silenced, and are, in contrast, devoid of epigenetic marks. Finally, the setmar gene, derived from HsMar1, presents H3K4me3 modifications as expected for a human housekeeping gene. CONCLUSIONS: Our work highlights that de novo and old HsMar1 are not similarly regulated by epigenetic mechanisms. Old HsMar1 are generally detected as lacking epigenetic marks, irrespective their localisation relative to the genes. Considering the putative existence of a network associating HsMar1 old copies and SETMAR, two non-mutually exclusive hypotheses are proposed: active and inactive HsMar1 copies are not similarly regulated or/and regulations concern only few loci (and few genes) that cannot be detected at the whole genome level.

SETMAR isoforms in glioblastoma: A matter of protein stability.

Glioblastomas (GBMs) are the most frequent and the most aggressive brain tumors, known for their chemo- and radio-resistance, making them often incurable. We also know that SETMAR is a protein involved in chromatin dynamics and genome plasticity, of which overexpression confers chemo- and radio-resistance to some tumors. The relationships between SETMAR and GBM have never been explored. To fill this gap, we define the SETMAR status of 44 resected tumors and of GBM derived cells, at both the mRNA and the protein levels. We identify a new, small SETMAR protein (so called SETMAR-1200), enriched in GBMs and GBM stem cells as compared to the regular enzyme (SETMAR-2100). We show that SETMAR-1200 is able to increase DNA repair by non-homologous end-joining, albeit with a lower efficiency than the regular SETMAR protein. Interestingly, the regular/small ratio of SETMAR in GBM cells changes depending on cell type, providing evidence that SETMAR expression is regulated by alternative splicing. We also demonstrate that SETMAR expression can be regulated by the use of an alternative ATG. In conclusion, various SETMAR proteins can be synthesized in human GBM that may each have specific biophysical and/or biochemical properties and characteristics. Among them, the small SETMAR may play a role in GBMs biogenesis. On this basis, we would like to consider SETMAR-1200 as a new potential therapeutic target to investigate, in addition to the regular SETMAR protein already considered by others.

Genetic Inactivation of ATRX Leads to a Decrease in the Amount of Telomeric Cohesin and Level of Telomere Transcription in Human Glioma Cells.

Mutations in ATRX (alpha thalassemia/mental retardation syndrome X-linked), a chromatin-remodeling protein, are associated with the telomerase-independent ALT (alternative lengthening of telomeres) pathway of telomere maintenance in several types of cancer, including human gliomas. In telomerase-positive glioma cells, we found by immunofluorescence that ATRX localized not far from the chromosome ends but not exactly at the telomere termini. Chromatin immunoprecipitation (ChIP) experiments confirmed a subtelomeric localization for ATRX, yet short hairpin RNA (shRNA)-mediated genetic inactivation of ATRX failed to trigger the ALT pathway. Cohesin has been recently shown to be part of telomeric chromatin. Here, using ChIP, we showed that genetic inactivation of ATRX provoked diminution in the amount of cohesin in subtelomeric regions of telomerase-positive glioma cells. Inactivation of ATRX also led to diminution in the amount of TERRAs, noncoding RNAs resulting from transcription of telomeric DNA, as well as to a decrease in RNA polymerase II (RNAP II) levels at the telomeres. Our data suggest that ATRX might establish functional interactions with cohesin on telomeric chromatin in order to control TERRA levels and that one or the other or both of these events might be relevant to the triggering of the ALT pathway in cancer cells that exhibit genetic inactivation of ATRX.

Kinetic analysis of the interaction of Mos1 transposase with its inverted terminal repeats reveals new insight into the protein-DNA complex assembly.

Transposases are specific DNA-binding proteins that promote the mobility of discrete DNA segments. We used a combination of physicochemical approaches to describe the association of MOS1 (an eukaryotic transposase) with its specific target DNA, an event corresponding to the first steps of the transposition cycle. Because the kinetic constants of the reaction are still unknown, we aimed to determine them by using quartz crystal microbalance on two sources of recombinant MOS1: one produced in insect cells and the other produced in bacteria. The prokaryotic-expressed MOS1 showed no cooperativity and displayed a Kd of about 300 nM. In contrast, the eukaryotic-expressed MOS1 generated a cooperative system, with a lower Kd (∼ 2 nm). The origins of these differences were investigated by IR spectroscopy and AFM imaging. Both support the conclusion that prokaryotic- and eukaryotic-expressed MOS1 are not similarly folded, thereby resulting in differences in the early steps of transposition.

Target capture during Mos1 transposition.

DNA transposition contributes to genomic plasticity. Target capture is a key step in the transposition process, because it contributes to the selection of new insertion sites. Nothing or little is known about how eukaryotic mariner DNA transposons trigger this step. In the case of Mos1, biochemistry and crystallography have deciphered several inverted terminal repeat-transposase complexes that are intermediates during transposition. However, the target capture complex is still unknown. Here, we show that the preintegration complex (i.e., the excised transposon) is the only complex able to capture a target DNA. Mos1 transposase does not support target commitment, which has been proposed to explain Mos1 random genomic integrations within host genomes. We demonstrate that the TA dinucleotide used as the target is crucial both to target recognition and in the chemistry of the strand transfer reaction. Bent DNA molecules are better targets for the capture when the target DNA is nicked two nucleotides apart from the TA. They improve strand transfer when the target DNA contains a mismatch near the TA dinucleotide.

cAMP protein kinase phosphorylates the Mos1 transposase and regulates its activity: evidences from mass spectrometry and biochemical analyses.

Genomic plasticity mediated by transposable elements can have a dramatic impact on genome integrity. To minimize its genotoxic effects, it is tightly regulated either by intrinsic mechanisms (linked to the element itself) or by host-mediated mechanisms. Using mass spectrometry, we show here for the first time that MOS1, the transposase driving the mobility of the mariner Mos1 element, is phosphorylated. We also show that the transposition activity of MOS1 is downregulated by protein kinase AMP cyclic-dependent phosphorylation at S170, which renders the transposase unable to promote Mos1 transposition. One step in the transposition cycle, the assembly of the paired-end complex, is specifically inhibited. At the cellular level, we provide evidence that phosphorylation at S170 prevents the active transport of the transposase into the nucleus. Our data suggest that protein kinase AMP cyclic-dependent phosphorylation may play a double role in the early stages of genome invasion by mariner elements.

Natural stilbenoids isolated from grapevine exhibiting inhibitory effects against HIV-1 integrase and eukaryote MOS1 transposase in vitro activities.

Polynucleotidyl transferases are enzymes involved in several DNA mobility mechanisms in prokaryotes and eukaryotes. Some of them such as retroviral integrases are crucial for pathogenous processes and are therefore good candidates for therapeutic approaches. To identify new therapeutic compounds and new tools for investigating the common functional features of these proteins, we addressed the inhibition properties of natural stilbenoids deriving from resveratrol on two models: the HIV-1 integrase and the eukaryote MOS-1 transposase. Two resveratrol dimers, leachianol F and G, were isolated for the first time in Vitis along with fourteen known stilbenoids: E-resveratrol, E-piceid, E-pterostilbene, E-piceatannol, (+)-E-ε-viniferin, E-ε-viniferinglucoside, E-scirpusin A, quadragularin A, ampelopsin A, pallidol, E-miyabenol C, E-vitisin B, hopeaphenol, and isohopeaphenol and were purified from stalks of Vitis vinifera (Vitaceae), and moracin M from stem bark of Milliciaexelsa (Moraceae). These compounds were tested in in vitro and in vivo assays reproducing the activity of both enzymes. Several molecules presented significant inhibition on both systems. Some of the molecules were found to be active against both proteins while others were specific for one of the two models. Comparison of the differential effects of the molecules suggested that the compounds could target specific intermediate nucleocomplexes of the reactions. Additionally E-pterostilbene was found active on the early lentiviral replication steps in lentiviruses transduced cells. Consequently, in addition to representing new original lead compounds for further modelling of new active agents against HIV-1 integrase, these molecules could be good tools for identifying such reaction intermediates in DNA mobility processes.

Regulation of mariner transposition: the peculiar case of Mos1.

BACKGROUND: Mariner elements represent the most successful family of autonomous DNA transposons, being present in various plant and animal genomes, including humans. The introduction and co-evolution of mariners within host genomes imply a strict regulation of the transposon activity. Biochemical data accumulated during the past decade have led to a convergent picture of the transposition cycle of mariner elements, suggesting that mariner transposition does not rely on host-specific factors. This model does not account for differences of transposition efficiency in human cells between mariners. We thus wondered whether apparent similarities in transposition cycle could hide differences in the intrinsic parameters that control mariner transposition. PRINCIPAL FINDINGS: We find that Mos1 transposase concentrations in excess to the Mos1 ends prevent the paired-end complex assembly. However, we observe that Mos1 transposition is not impaired by transposase high concentration, dismissing the idea that transposase over production plays an obligatory role in the down-regulation of mariner transposition. Our main finding is that the paired-end complex is formed in a cooperative way, regardless of the transposase concentration. We also show that an element framed by two identical ITRs (Inverted Terminal Repeats) is more efficient in driving transposition than an element framed by two different ITRs (i.e. the natural Mos1 copy), the latter being more sensitive to transposase concentration variations. Finally, we show that the current Mos1 ITRs correspond to the ancestral ones. CONCLUSIONS: We provide new insights on intrinsic properties supporting the self-regulation of the Mos1 element. These properties (transposase specific activity, aggregation, ITR sequences, transposase concentration/transposon copy number ratio...) could have played a role in the dynamics of host-genomes invasion by Mos1, accounting (at least in part) for the current low copy number of Mos1 within host genomes.

Transposase-transposase interactions in MOS1 complexes: a biochemical approach.

Transposases are proteins that have assumed the mobility of class II transposable elements. In order to map the interfaces involved in transposase-transposase interactions, we have taken advantage of 12 transposase mutants that impair mariner transposase-transposase interactions taking place during transposition. Our data indicate that transposase-transposase interactions regulating Mos1 transposition are sophisticated and result from (i) active MOS1 dimerization through the first HTH of the N-terminal domain, which leads to inverted terminal repeat (ITR) binding; (ii) inactive dimerization carried by part of the C-terminal domain, which prevents ITR binding; and (iii) oligomerization. Inactive dimers are nonpermissive in organizing complexes that produce ITR binding, but the interfaces (or interactions) supplied in this state could play a role in the various rearrangements needed during transposition. Oligomerization is probably not due to a specific MOS1 domain, but rather the result of nonspecific interactions resulting from incorrect folding of the protein. Our data also suggest that the MOS1 catalytic domain is a main actor in the overall organization of MOS1, thus playing a role in MOS1 oligomerization. Finally, we propose that MOS1 behaves as predicted by the pre-equilibrium existing model, whereby proteins are found to exist simultaneously in populations with diverse conformations, monomers and active and inactive dimers for MOS1. We were able to identify several MOS1 mutants that modify this pre-existing equilibrium. According to their properties, some of these mutants will be useful tools to break down the remaining gaps in our understanding of mariner transposition.

In vitro recombination and inverted terminal repeat binding activities of the Mcmar1 transposase.

The Mcmar1 mariner element (MLE) presents some intriguing features with two large, perfectly conserved, 355 bp inverted terminal repeats (ITRs) containing two 28 bp direct repeats (DRs). The presence of a complete ORF in Mcmar1 makes it possible to explore the transposition of this unusual MLE. Mcmar1 transposase (MCMAR1) was purified, and in vitro transposition assays showed that it is able to promote ITR-dependent DNA cleavages and recombination events, which correspond to plasmid fusions and transpositions with imprecise ends. Further analyses indicated that MCMAR1 is able to interact with the 355 bp ITR through two DRs: the EDR (external DR) is a high-affinity binding site for MCMAR1, whereas the IDR (internal DR) is a low-affinity binding site. The main complex detected within the EDR contained a transposase dimer and only one DNA molecule. We hypothesize that the inability of MCMAR1 to promote precise in vitro transposition events could be due to mutations in its ORF sequence or to the specific features of transposase binding to the ITR. Indeed, the ITR region spanning from EDR to IDR resembles a MITE and could be bent by specific host factors. This suggests that the assembly of the transposition complex is more complex than that of those involved in the mobility of the Mos1 and Himar1 mariner elements.

Site-directed integration of transgenes: transposons revisited using DNA-binding-domain technologies.

In the last 20 years, tools derived from DNA transposons have made major contributions to genetic studies from gene delivery to gene discovery. Various complementary and fairly ubiquitous DNA vehicles have been developed. Although many transposons are efficient DNA vehicles, they appear to have limited ability to target specific sequences, since all that is required at the integration locus is the presence of a short 2- to 4-bp sequence. Consequently, insertions mediated by transposon-based vectors occur somewhat randomly. In the past 5 years, strategies have emerged to enhance the site-specificity of transposon-based vectors, and to avoid random integrations. The first proposes that new target site specificity could be grafted onto a transposase by adding a new DNA-binding domain. Alternative strategies consist of indirectly targeting either the transposase or the transposon to a chosen genomic locus. The most important information available about each strategy are presented, and limitations and future prospects are discussed.

Physical properties of DNA components affecting the transposition efficiency of the mariner Mos1 element.

Previous studies have shown that the transposase and the inverted terminal repeat (ITR) of the Mos1 mariner elements are suboptimal for transposition; and that hyperactive transposases and transposon with more efficient ITR configurations can be obtained by rational molecular engineering. In an attempt to determine the extent to which this element is suboptimal for transposition, we investigate here the impact of the three main DNA components on its transposition efficiency in bacteria and in vitro. We found that combinations of natural and synthetic ITRs obtained by systematic evolution of ligands by exponential enrichment did increase the transposition rate. We observed that when untranslated terminal regions were associated with their respective natural ITRs, they acted as transposition enhancers, probably via the early transposition steps. Finally, we demonstrated that the integrity of the Mos1 inner region was essential for transposition. These findings allowed us to propose prototypes of optimized Mos1 vectors, and to define the best sequence features of their associated marker cassettes. These vector prototypes were assayed in HeLa cells, in which Mos1 vectors had so far been found to be inactive. The results obtained revealed that using these prototypes does not circumvent this problem. However, such vectors can be expected to provide new tools for the use in genome engineering in systems such as Caenorhabditis elegans in which Mos1 is very active.

Mariner Mos1 transposase optimization by rational mutagenesis.

Mariner transposons are probably the most widespread transposable element family in animal genomes. To date, they are believed not to require species-specific host factors for transposition. Despite this, Mos1, one of the most-studied mariner elements (with Himar1), has been shown to be active in insects, but inactive in mammalian genomes. To circumvent this problem, one strategy consists of both enhancing the activity of the Mos1 transposase (MOS1), and making it insensitive to activity-altering post-translational modifications. Here, we report rational mutagenesis studies performed to obtain hyperactive and non-phosphorylable MOS1 variants. Transposition assays in bacteria have made it possible to isolate numerous hyperactive MOS1 variants. The best mutant combinations, named FETY and FET, are 60- and 800-fold more active than the wild-type MOS1 version, respectively. However, there are serious difficulties in using them, notably because they display severe cytotoxicity. On the other hand, three positions lying within the HTH motif, T88, S99, and S104 were found to be sensitive to phosphorylation. Our efforts to obtain active non-phosphorylable mutants at S99 and S104 positions were unsuccessful, as these residues, like the co-linear amino acids in their close vicinity, are critical for MOS1 activity. Even if host factors are not essential for transposition, our data demonstrate that the host machinery is essential in regulating MOS1 activity.

Factors acting on Mos1 transposition efficiency.

BACKGROUND: Mariner-like elements (MLEs) are widespread DNA transposons in animal genomes. Although in vitro transposition reactions require only the transposase, various factors depending on the host, the physico-chemical environment and the transposon sequence can interfere with the MLEs transposition in vivo. RESULTS: The transposition of Mos1, first isolated from drosophila mauritiana, depends of both the nucleic acid sequence of the DNA stuffer (in terms of GC content), and its length. We provide the first in vitro experimental demonstration that MITEs of MLE origin, as small as 80 to 120-bp, are able to transpose. Excessive temperature down-regulates Mos1 transposition, yielding excision products unable to re-integrate. Finally, the super-helicity of the DNA transposon donor has a dramatic impact on the transposition efficiency. CONCLUSION: The study highlights how experimental conditions can bias interpretation of mariner excision frequency and quality. In vitro, the auto-integration pathway markedly limits transposition efficiency to new target sites, and this phenomenon may also limit events in the natural host. We propose a model for small transposons transposition that bypasses DNA bending constraints.

Molecular evidence for the evolution of ichnoviruses from ascoviruses by symbiogenesis.

BACKGROUND: Female endoparasitic ichneumonid wasps inject virus-like particles into their caterpillar hosts to suppress immunity. These particles are classified as ichnovirus virions and resemble ascovirus virions, which are also transmitted by parasitic wasps and attack caterpillars. Ascoviruses replicate DNA and produce virions. Polydnavirus DNA consists of wasp DNA replicated by the wasp from its genome, which also directs particle synthesis. Structural similarities between ascovirus and ichnovirus particles and the biology of their transmission suggest that ichnoviruses evolved from ascoviruses, although molecular evidence for this hypothesis is lacking. RESULTS: Here we show that a family of unique pox-D5 NTPase proteins in the Glypta fumiferanae ichnovirus are related to three Diadromus pulchellus ascovirus proteins encoded by ORFs 90, 91 and 93. A new alignment technique also shows that two proteins from a related ichnovirus are orthologs of other ascovirus virion proteins. CONCLUSION: Our results provide molecular evidence supporting the origin of ichnoviruses from ascoviruses by lateral transfer of ascoviral genes into ichneumonid wasp genomes, perhaps the first example of symbiogenesis between large DNA viruses and eukaryotic organisms. We also discuss the limits of this evidence through complementary studies, which revealed that passive lateral transfer of viral genes among polydnaviral, bacterial, and wasp genomes may have occurred repeatedly through an intimate coupling of both recombination and replication of viral genomes during evolution. The impact of passive lateral transfers on evolutionary relationships between polydnaviruses and viruses with large double-stranded genomes is considered in the context of the theory of symbiogenesis.

Assembly of the Tc1 and mariner transposition initiation complexes depends on the origins of their transposase DNA binding domains.

In this review, we focus on the assembly of DNA/protein complexes that trigger transposition in eukaryotic members of the IS630-Tc1-mariner (ITm) super-family, the Tc1- and mariner-like elements (TLEs and MLEs). Elements belonging to this super-family encode transposases with DNA binding domains of different origins, and recent data indicate that the chimerization of functional domains has been an important evolutionary aspect in the generation of new transposons within the ITm super-family. These data also reveal that the inverted terminal repeats (ITRs) at the ends of transposons contain three kinds of motif within their sequences. The first two are well known and correspond to the cleavage site on the outer ITR extremities, and the transposase DNA binding site. The organization of ITRs and of the transposase DNA binding domains implies that differing pathways are used by MLEs and TLEs to regulate transposition initiation. These differences imply that the ways ITRs are recognized also differ leading to the formation of differently organized synaptic complexes. The third kind of motif is the transposition enhancers, which have been found in almost all the functional MLEs and TLEs analyzed to date. Finally, in vitro and in vivo assays of various elements all suggest that the transposition initiation complex is not formed randomly, but involves a mechanism of oriented transposon scanning.

Mariner Mos1 transposase dimerizes prior to ITR binding.

The mariner Mos1 synaptic complex consists of a tetramer of transposase molecules that bring together the two ends of the element. Such an assembly requires at least two kinds of protein-protein interfaces. The first is involved in "cis" dimerization, and consists of transposase molecules bound side-by-side on a single DNA molecule. The second, which is involved in "trans" dimerization, consists of transposase molecules bound to two different DNA molecules. Here, we used biochemical and genetic methods to enhance the definition of the regions involved in cis and trans-dimerization in the mariner Mos1 transposase. The cis and trans-dimerization interfaces were both found within the first 143 amino acid residues of the protein. The cis-dimerization activity was mainly contained in amino acids 1-20. The region spanning from amino acid residues 116-143, and containing the WVPHEL motif, was involved in the cis- to trans-shift as well as in trans-dimerization stabilization. Although the transposase exists mainly as a monomer in solution, we provide evidence that the transposase cis-dimer is the active species in inverted terminal repeat (ITR) binding. We also observed that the catalytic domain of the mariner Mos1 transposase modulates efficient transposase-transposase interactions in the absence of the transposon ends.

Conservation of Palindromic and Mirror Motifs within Inverted Terminal Repeats of mariner-like Elements.

The transposase of the mariner-like elements (MLEs) specifically binds as a dimer to the inverted terminal repeat of the transposon that encodes it. Two binding-motifs located within the inverted terminal sequences (ITR) are therefore recognized, as previously indicated, by biochemical data obtained with the Mos1 and Himar1 transposases. Here, we define the motifs that are involved in the binding of a MLE transposase to its ITR by analyzing the nucleic acid properties of the 5' and 3' ITR sequences from 45 MLEs, taking into account the fact that the transposase binds to the ITR, using its CRO binding domains and the general characteristics of the cro binding sites so far investigated. Our findings show that in all the MLE ITRs, the outer half was better conserved than the inner half. More interestingly, they allowed us to characterize conserved palindromic and mirror motifs specific to each "MLE species". The presence of the palindromic motifs was correlated to the binding of the transposase dimer, whereas the properties of the mirror motifs were shown to be responsible for the bend in each ITR that helps to stabilize transposase-ITR interactions.