Advertisement

Target Capture during Mos1 Transposition*

  • Aude Pflieger
    Footnotes
    Affiliations
    EA 6306 Innovation Moléculaire et Thérapeutique, Université François Rabelais, UFR des Sciences et Techniques, UFR de Pharmacie, 37200 Tours, France

    Groupe de Recherche en Hypertension Pulmonaire, CRIUCPQ, Québec, Québec G1V 4G5, Canada
    Search for articles by this author
  • Jerôme Jaillet
    Affiliations
    EA 6306 Innovation Moléculaire et Thérapeutique, Université François Rabelais, UFR des Sciences et Techniques, UFR de Pharmacie, 37200 Tours, France
    Search for articles by this author
  • Agnès Petit
    Affiliations
    Centre d'Etudes des Pathologies Respiratoires INSERM UMR 1100, Université François Rabelais, UFR de Médecine, 37032 Tours Cedex 1, France,
    Search for articles by this author
  • Corinne Augé-Gouillou
    Correspondence
    To whom correspondence may be addressed: EA 6306 Innovation Moléculaire et Thérapeutique, Université François Rabelais, UFR des Sciences et Techniques, UFR de Pharmacie, 31 Avenue Monge, 37200 Tours, France. Tel.: 33-2-47-36-74-72
    Affiliations
    EA 6306 Innovation Moléculaire et Thérapeutique, Université François Rabelais, UFR des Sciences et Techniques, UFR de Pharmacie, 37200 Tours, France
    Search for articles by this author
  • Sylvaine Renault
    Correspondence
    To whom correspondence may be addressed: EA 6306 Innovation Moléculaire et Thérapeutique, Université François Rabelais, UFR des Sciences et Techniques, UFR de Pharmacie, 31 Avenue Monge, 37200 Tours, France. Tel.: 33-2-47-36-74-72
    Affiliations
    EA 6306 Innovation Moléculaire et Thérapeutique, Université François Rabelais, UFR des Sciences et Techniques, UFR de Pharmacie, 37200 Tours, France
    Search for articles by this author
  • Author Footnotes
    * This work was supported by the Région Centre (InhDDE Project), the University of Tours, and the Centre National de la Recherche Scientifique.
    This article contains supplemental Table S1 and Figs. S1–S3.
    1 Postdoctoral fellow of the InhDDE Project (Region Centre, France).
Open AccessPublished:November 22, 2013DOI:https://doi.org/10.1074/jbc.M113.523894
      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.

      Introduction

      Transposable elements are prominent in the make up of a major fraction of many eukaryotic genomes. They contribute to genome plasticity in many ways. They act as insertional mutagens, alter the regulation of gene expression, and provide coding information for the emergence of new functions
      • Babatz T.D.
      • Burns K.H.
      Functional impact of the human mobilome.
      • Kunarso G.
      • Chia N.-Y.
      • Jeyakani J.
      • Hwang C.
      • Lu X.
      • Chan Y.-S.
      • Ng H.-H.
      • Bourque G.
      Transposable elements have rewired the core regulatory network of human embryonic stem cells.
      • Cordaux R.
      • Udit S.
      • Batzer M.A.
      • Feschotte C.
      Birth of a chimeric primate gene by capture of the transposase gene from a mobile element.
      • Chalopin D.
      • Galiana D.
      • Volff J.-N.
      Genetic innovation in vertebrates. Gypsy integrase genes and other genes derived from transposable elements.
      . The study of their transposition pathways may shed light onto their impact on the dynamics of eukaryotic genome evolution. Among these transposons, Itm elements (to which belong mariner elements) are one of the most widespread groups of transposons. They transpose via a DNA intermediate, using a cut and paste mechanism (
      • Plasterk R.H.
      • van Luenen H.G.
      ). Elements of mariner are discrete DNA segments containing an ORF coding a single protein, the transposase, surrounded by inverted terminal repeats (ITRs).
      The abbreviations used are:
      ITR
      inverted terminal repeat
      M
      mismatched
      MBP
      maltose-binding protein
      N
      nicked
      PC-ITR
      precleaved ITR
      PN-ITR
      prenicked ITR
      PIC
      preintegration complex
      SEC
      single end complex
      TCC
      target capture complex
      UC-ITR
      uncleaved ITR.
      The active transposase is a homodimer that binds to one ITR. The second ITR is recruited to form a paired end complex, which is the catalytic complex where the strand transfer reactions promoting excision take place. The excision product containing the two cleaved ITR with a transposase dimer is known as a preintegration complex (PIC), in reference to the equivalent complex described for HIV integrase (
      • Maertens G.N.
      • Hare S.
      • Cherepanov P.
      The mechanism of retroviral integration from x-ray structures of its key intermediates.
      ). Paired end complex assembly and excision of mariner are now well understood
      • Jaillet J.
      • Genty M.
      • Cambefort J.
      • Rouault J.-D.
      • Augé-Gouillou C.
      Regulation of mariner transposition. The peculiar case of Mos1.
      • Carpentier G.
      • Jaillet J.
      • Pflieger A.
      • Adet J.
      • Renault S.
      • Augé-Gouillou C.
      Transposase-transposase interactions in MOS1 complexes. A biochemical approach.
      • Richardson J.M.
      • Colloms S.D.
      • Finnegan D.J.
      • Walkinshaw M.D.
      Molecular architecture of the Mos1 paired-end complex. The structural basis of DNA transposition in a eukaryote.
      • Claeys Bouuaert C.
      • Chalmers R.
      Transposition of the human Hsmar1 transposon. Rate-limiting steps and the importance of the flanking TA dinucleotide in second strand cleavage.
      . In contrast, little is known about the subsequent step, namely the target capture. The target DNA is thought to be associated with the PIC in the so-called target capture complex (TCC) that drives the transposon to integrate at its new site in the genome.
      Target capture has been extensively studied for prokaryotic model elements (Tn5, Tn7, and Tn10), the Mu phage, and two eukaryotic transposons (RAG1/2 and Himar1). All the related transposases display an RNase H-like catalytic domain that contains a DD(E/D) catalytic triad (
      • Nowotny M.
      Retroviral integrase superfamily. The structural perspective.
      ). However, these transposases are of different origins: Tn5 and Tn10 belong to the IS10 superfamily, and Tn7 is the prototype of a single family. Concerning the eukaryotic transposases, RAG1/2 belongs to the Transib family, whereas Himar1 belongs to the mariner family. These different origins result in differences in the transposition mechanisms. For instance, hairpins are produced during the excision at the ends of Tn5 and Tn10 but on the flanking DNA for RAG1/2. In contrast, mariner transposition does not require any hairpin (
      • Claeys Bouuaert C.
      • Chalmers R.M.
      Gene therapy vectors. The prospects and potentials of the cut-and-paste transposons.
      ). This suggests that the paired end and preintegration complexes are organized differently depending on the transposase involved. This is not the only difference existing between these well studied transposons. The target structure appears crucial in the recognition and/or integration of various elements (Tn7, Tn10, RAG1/2 transposases, and HIV-1 integrase)
      • Kuduvalli P.N.
      • Rao J.E.
      • Craig N.L.
      Target DNA structure plays a critical role in Tn7 transposition.
      • Pribil P.A.
      • Haniford D.B.
      Target DNA bending is an important specificity determinant in target site selection in Tn10 transposition.
      • Pribil P.A.
      • Wardle S.J.
      • Haniford D.B.
      Enhancement and rescue of target capture in Tn10 transposition by site-specific modifications in target DNA.
      • Tsai C.-L.
      • Chatterji M.
      • Schatz D.G.
      DNA mismatches and GC-rich motifs target transposition by the RAG1/RAG2 transposase.
      • Müller H.P.
      • Varmus H.E.
      DNA bending creates favored sites for retroviral integration. An explanation for preferred insertion sites in nucleosomes.
      , whereas the only determinant for mariner target recognition and strand transfer is the presence of a TA dinucleotide, which is duplicated upon integration (
      • Lohe A.R.
      • De Aguiar D.
      • Hartl D.L.
      Mutations in the mariner transposase. The D,D(35)E consensus sequence is nonfunctional.
      ,
      • Claeys Bouuaert C.
      • Chalmers R.
      Hsmar1 transposition is sensitive to the topology of the transposon donor and the target.
      ). Moreover, some elements have specific (attTn7 for Tn7) (
      • Kuduvalli P.N.
      • Rao J.E.
      • Craig N.L.
      Target DNA structure plays a critical role in Tn7 transposition.
      ) or preferred (HisG1 for Tn10, G(CT)(CT)(CT)(AT)(AG)(AG)(AG)C for Tn5) (
      • Benjamin H.W.
      • Kleckner N.
      Intramolecular transposition by Tn10.
      ,
      • Shevchenko Y.
      • Bouffard G.G.
      • Butterfield Y.S.
      • Blakesley R.W.
      • Hartley J.L.
      • Young A.C.
      • Marra M.A.
      • Jones S.J.
      • Touchman J.W.
      • Green E.D.
      Systematic sequencing of cDNA clones using the transposon Tn5.
      ) targets, whereas mariner elements display an essentially random integration pattern. However, it has been shown that Mos1 may have preferred integration spots such as the one in the chloramphenicol resistance gene of Tn9 (
      • Crénès G.
      • Ivo D.
      • Hérisson J.
      • Dion S.
      • Renault S.
      • Bigot Y.
      • Petit A.
      The bacterial Tn9 chloramphenicol resistance gene. An attractive DNA segment for Mos1 mariner insertions.
      ). No structural features explaining the preferential integration of Mos1 have yet been identified (
      • Crénès G.
      • Moundras C.
      • Demattei M.-V.
      • Bigot Y.
      • Petit A.
      • Renault S.
      Target site selection by the mariner-like element, Mos1.
      ). In addition, contrarily to RAG1/2 that creates a 5-bp duplication upon insertion (
      • Matthews A.G.
      • Elkin S.K.
      • Oettinger M.A.
      Ordered DNA release and target capture in RAG transposition.
      ), mariner elements generate a 2-bp duplication.
      All these remarks suggest that target capture complexes are organized differently depending on the transposase involved and result in different integration pathways. An example is the timing of the target capture during various transposition cycles. Tn7 binds its specific target attTn7 before excising (
      • Kuduvalli P.N.
      • Rao J.E.
      • Craig N.L.
      Target DNA structure plays a critical role in Tn7 transposition.
      ), whereas Mu captures its target DNA at different points of the reaction pathway (
      • Matthews A.G.
      • Elkin S.K.
      • Oettinger M.A.
      Ordered DNA release and target capture in RAG transposition.
      ,
      • Naigamwalla D.Z.
      • Chaconas G.
      A new set of Mu DNA transposition intermediates. Alternate pathways of target capture preceding strand transfer.
      ). In contrast, Tn5, Tn10, and RAG1/2 bind their target after excision (
      • Matthews A.G.
      • Elkin S.K.
      • Oettinger M.A.
      Ordered DNA release and target capture in RAG transposition.
      ,
      • Sakai J.
      • Kleckner N.
      The Tn10 synaptic complex can capture a target DNA only after transposon excision.
      • Gradman R.J.
      • Ptacin J.L.
      • Bhasin A.
      • Reznikoff W.S.
      • Goryshin I.Y.
      A bifunctional DNA binding region in Tn5 transposase.
      • Neiditch M.B.
      • Lee G.S.
      • Landree M.A.
      • Roth D.B.
      RAG transposase can capture and commit to target DNA before or after donor cleavage.
      • Lipkow K.
      • Buisine N.
      • Chalmers R.
      Promiscuous target interactions in the mariner transposon Himar1.
      ), and Himar1 could perform target capture both before and after excision (
      • Lipkow K.
      • Buisine N.
      • Chalmers R.
      Promiscuous target interactions in the mariner transposon Himar1.
      ). It has been demonstrated that Tn10, Himar1, and RAG1/2 capture their target in a two-step procedure. First, a labile interaction occurs between the target and the excised element. Second, the complex is stabilized, so that the target cannot be replaced by another DNA, resulting in what is known as “target commitment” (
      • Sakai J.
      • Kleckner N.
      The Tn10 synaptic complex can capture a target DNA only after transposon excision.
      ,
      • Neiditch M.B.
      • Lee G.S.
      • Landree M.A.
      • Roth D.B.
      RAG transposase can capture and commit to target DNA before or after donor cleavage.
      ,
      • Lipkow K.
      • Buisine N.
      • Chalmers R.
      Promiscuous target interactions in the mariner transposon Himar1.
      ).
      The variety of the target capture mechanisms makes it necessary to describe them for each transposon family. In particular, within the mariner family, the fact that the first model to be studied, namely Himar1 (
      • Lipkow K.
      • Buisine N.
      • Chalmers R.
      Promiscuous target interactions in the mariner transposon Himar1.
      ), displayed a target capture contrasting with other transposases (Tn5, Tn10, and RAG1/2), leaves open the question of the chronology of events (excision/target capture/integration or target capture/excision/integration) for the other mariner elements. In this perspective, the Mos1 element is of particular interest, because it is the only eukaryotic transposon for which the complete transposition cycle can be reconstituted step by step in vitro and for which crystallographic data of the excised element (precleaved ITRs with two transposases) are available (
      • Richardson J.M.
      • Colloms S.D.
      • Finnegan D.J.
      • Walkinshaw M.D.
      Molecular architecture of the Mos1 paired-end complex. The structural basis of DNA transposition in a eukaryote.
      ). Based on the crystal structure, a target capture complex has been recently proposed to account for the fact that a channel exists between the two transposases that might provide a niche to the target DNA (
      • Richardson J.M.
      • Colloms S.D.
      • Finnegan D.J.
      • Walkinshaw M.D.
      Molecular architecture of the Mos1 paired-end complex. The structural basis of DNA transposition in a eukaryote.
      ). However, there is no biochemical evidence to support this model.
      Here, we confirm that the organization of the Mos1 target capture complex is consistent with that predicted by cut and paste models of transposition. We show that target capture takes place only after Mos1 element has been fully excised. The TA dinucleotide present in the target is of particular importance not only for the strand transfer, but also for the target capture itself. DNA bending of the target significantly improves the efficiency of capture, highlighting the role of the +2 position relative to the TA dinucleotide. The various models previously published for Mos1 are discussed in the light of our findings.

      EXPERIMENTAL PROCEDURES

      Proteins

      The MBP-MOS1 protein was produced using the pVL1392 baculovirus transfer vector and the BaculoGoldTM baculovirus expression system (BD Biosciences) and then extracted from baculovirus-infected Sf21 cells. The fusion protein was purified on a amylose resin (New England Biolabs) as previously described (
      • Bouchet N.
      • Jaillet J.
      • Gabant G.
      • Brillet B.
      • Briseño-Roa L.
      • Cadene M.
      • Augé-Gouillou C.
      cAMP protein kinase phosphorylates the Mos1 transposase and regulates its activity. Evidences from mass spectrometry and biochemical analyses.
      ) (supplemental Fig. S1).

      ITR and Target DNA

      The sequences of the oligonucleotides used in the study are shown in supplemental Table S1. The precleaved ITR (PC-ITR) is 50 bp long (also used as a short ITR), and the long PC-ITR is 70 bp long. The uncleaved ITR (UC-ITR) and the prenicked ITR (PN-ITR) are 70 bp long. The TA targets are 30 bp long (also used as a short target) and 50 bp long (used as long target). GC target and targets containing mismatch and nick are 30 bp long. Oligonucleotides were provided by Eurofins MWG Biotech (Germany) or Eurogentec (Belgium) and purified by PAGE. Double-stranded DNA of ITRs and targets were obtained by annealing equimolar amounts of different complementary oligonucleotides. The wild type TA target was obtained by annealing T1 and T2. Targets exhibiting a nick at the integration site obtained by annealing T1, T3, and T3′ (N+1 target); T1, T4, and T4′ (N+2 target); T1, T5, and T5′ (N+3 target); and T3′, T4′, and T5′ were phosphorylated at the 5′ position using standard procedures. The targets exhibiting a mismatch were obtained by annealing T1 and T6 (M+1 target), T1 and T7 (M+2 target), and T1 and T8 (M+3 target). The GC target was obtained by annealing T9 and T10. Long targets were obtained by annealing T11 and T12 or T13 and T14. All targets were filled in with [α-32P]dATP by Klenow exo-minus in presence of dTTP. PC-ITR was obtained by annealing I4 and I5, UC-ITR was obtained by annealing I1 and I2, and PN-ITR was obtained by annealing I2, I3, and I4. The transferred strand (I2 or I5) was 5′ end-labeled with [γ-32P]ATP by T4 polynucleotide kinase prior to annealing.

      Preintegration Complex Assembly

      PICs were formed using 250 nm transposase (MBP-MOS1) and 250 nm PC-ITR in 10 mm Tris, pH 9, 50 mm NaCl, 0.5 mm DTT, 5% glycerol, and 5 mm MgCl2 in 20 μl. Reactions were carried out for 3 h at 30 °C. For PIC analyses, labeled ITRs were used instead of unlabeled ITRs, and the reaction products were analyzed by EMSA onto 6% native polyacrylamide gel in 0.25× TBE (Tris-borate-EDTA) buffer. The gel was dried and scanned. When specified, the PICs were assembled using UC-ITR or PN-ITR instead of PC-ITR.

      Target Capture Assays

      PICs were formed as described above using cold ITRs. After incubation, 250 nm of labeled targets were added with 5 mm EDTA. The addition of EDTA chelates MgCl2, blocking the strand transfer. The use of labeled target and EDTA allowed the detection of complexes if and only if the complexes contained a target and avoided strand transfer. For TCC assembly, reactions were carried out for another hour (30 °C). All the targets used contained a single dinucleotide TA, except for the GC target, which did not contain any TA. Complexes were analyzed by EMSA onto 6% native polyacrylamide gel in 0.25× TBE buffer. The gel was dried and scanned.

      Stoichiometry of Target Capture Complex

      Determination of the Number of Transposases in TCC

      Cold PICs were prepared as described above but with an overnight incubation before proceeding to TCC assembly with a labeled target. The TCCs were then digested for 0–5 h by factor Xa (1 μg) in the presence of 5 mm CaCl2 at 30 °C. Reaction products were analyzed by EMSA onto 6% native polyacrylamide gel. The gel was dried and scanned.

      Determination of the Number of Targets in TCC

      Cold PICs were prepared as described above. Then labeled short (30 bp) and/or long (50 bp) targets were added before performing target capture. Reaction products were analyzed by EMSA onto 6% native polyacrylamide gel. The gel was dried and scanned.

      Determination of the Number of ITRs in TCC

      Cold PICs were prepared as described above using a mixture of equimolar unlabeled short (50 bp) and long (70 bp) PC-ITRs at a final concentration of 250 nm. Standard target capture (with a labeled target) assays were performed as described above. Reaction products were analyzed by EMSA onto 6% native polyacrylamide gel. The gel was dried and scanned.

      Target Commitment

      The TCCs were assembled as previously described using either short or long labeled targets as the first target. To find out whether the target could be exchanged, the second labeled target (short if the first was long and vice versa) was added at 250 nm. A control reaction was performed by adding long and short targets at the same time. Reactions were carried out for 1 h more at 30 °C. Complexes were analyzed by EMSA onto 6% native polyacrylamide gel. The gel was dried and scanned. Each point was repeated five times. The signal obtained for each target (long or short) was quantified using ImageQuant software. The percentage of each target was calculated as the signal obtained with the long (or short) target divided by the signal obtained with long and short targets. The percentage of target commitment was calculated as the percentage of the long or short target in a target commitment assay minus the percentage of the long or short target in the control reaction.

      Integration Assays

      Cold PICs were formed using PC-ITR at 30 °C but for 30 min. Labeled targets were then added with or without 5 mm EDTA for 3 h at 30 or 4 °C. Reactions were stopped by adding 0.1 mg/ml proteinase K and 0.1% SDS, incubated at 65 °C for 10 min, and incubated then at 37 °C for 30 min. DNA products were purified by phenol chloroform extraction and ethanol precipitation. Integration products were resuspended in a standard denaturing loading buffer, boiled for 5 min, and then loaded onto an 8% denaturing urea-acrylamide gel. The gel was dried and scanned. The integration rate is the ratio of the integration signal divided by the free target signal plus the integration signal. The integration efficiency found using the TA target was normalized to 100%. Each point was repeated five times. A labeled G+A ladder was obtained by annealing of GA1 (labeled at the 5′ end with [γ-32P]ATP by T4 polynucleotide kinase) to its complement GA2 (supplemental Table S1). This product was chemically cleaved at G and A using the Maxam and Gilbert standard procedure. G+A products were resuspended in denaturing loading buffer.
      Integration products were analyzed by PCR, using F1 and F2 primers (supplemental Table S1). Integration products (3 μl) were amplified with GoTaq DNA polymerase (Promega) in conditions recommended by the manufacturer. 35 PCR cycles were carried out with an annealing temperature of 44 °C. PCR products were resolved on a 2% agarose gel containing ethidium bromide and purified using the Nucleospin extract II kit (Macherey-Nagel). PCR products were then cloned in the pGEM-T Easy System (Promega) and sequenced by Eurofins MWG Biotech (Germany).

      Statistical Analyses

      Quantification was done using ImageQuant software. Statistical analyses were performed using GraphPad Prism version 4.0c for Macintosh (GraphPad Software, La Jolla, CA). We used a Kruskal-Wallis one-way analysis of variance by ranks. This is a nonparametric method for testing whether samples originate from the same distribution or not. It is used to compare more than two samples that are not related. The null hypothesis is that the populations from which the samples originate all have the same median value. Analyses are done using an α level of 5% (α = 0.05). The calculated Kruskal-Wallis value is then compared with the critical value. If the Kruskal-Wallis test leads to significant results, then at least one of the samples is different from the other samples. The test does not identify where the differences lie, nor how many differences are actually present. To identify the differences, sample contrast determinations were carried out between individual sample pairs (or post hoc tests). Multiple comparisons were done using Dunn's test (pairwise comparisons) and the Bonferroni correction to find out whether the post hoc tests are significant.

      DISCUSSION

      The insertion sites of transposons have a major impact on genome structure and gene expression regulation (
      • Babatz T.D.
      • Burns K.H.
      Functional impact of the human mobilome.
      ,
      • Kunarso G.
      • Chia N.-Y.
      • Jeyakani J.
      • Hwang C.
      • Lu X.
      • Chan Y.-S.
      • Ng H.-H.
      • Bourque G.
      Transposable elements have rewired the core regulatory network of human embryonic stem cells.
      ). One way to know how insertion sites are selected is to study target capture, a critical albeit poorly known step in the transposition cycle of eukaryotic DNA transposons. To obtain information about the nature and dynamics of TCC, we developed a target capture assay using Mos1 as a model. This model might also help to better understand the mechanism of transposition of transposons of biotechnological interest (Sleeping Beauty and PiggyBac) for which biochemical approaches are not available. Our assay is based on a preformed PIC that is converted into a TCC by the recruitment of a DNA molecule. This assay was used to demonstrate that Mos1 proceeds to target capture only once the transposon has been excised from its donor site. In this respect, the transposition cycle of Mos1 (an eukaryotic transposon) is similar to that described for Tn5 (
      • Sakai J.
      • Kleckner N.
      The Tn10 synaptic complex can capture a target DNA only after transposon excision.
      ), Tn10 (
      • Gradman R.J.
      • Ptacin J.L.
      • Bhasin A.
      • Reznikoff W.S.
      • Goryshin I.Y.
      A bifunctional DNA binding region in Tn5 transposase.
      ) (prokaryotic transposons), and RAG1/2 (
      • Matthews A.G.
      • Elkin S.K.
      • Oettinger M.A.
      Ordered DNA release and target capture in RAG transposition.
      ). HIV integrase proceeds similarly, because the 3′ processing of viral ends must be done before target capture (
      • Krishnan L.
      • Engelman A.
      Retroviral integrase proteins and HIV-1 DNA integration.
      ). In contrast, Himar1 transposases can capture target after or before end cleavage. This apparent discrepancy between HIMAR1 and MOS1 probably results from the assays used. Indeed, the timing of HIMAR1 target capture was tested in the presence of Mg2+, thus allowing end cleavage during the TCC assays (
      • Lipkow K.
      • Buisine N.
      • Chalmers R.
      Promiscuous target interactions in the mariner transposon Himar1.
      ). In contrast, we used EDTA (which prevents end cleavage) to control the nature (cleaved versus un-cleaved) of the ITR ends. We thus propose that all mariner elements proceed to target capture only once the transposon has been excised, in agreement with prokaryotic DNA transposons and RAG1/2.
      Tn10 and Tn5 have been useful models for understanding the molecular mechanisms of target capture. These elements perform integration by resolving a hairpin present at the extremities of the excised element. This step results in blunt DNA ends that need a profound rearrangement when target capture occurs, resulting in the 3′OH of the transferred strand being better exposed (
      • Klenchin V.A.
      • Czyz A.
      • Goryshin I.Y.
      • Gradman R.
      • Lovell S.
      • Rayment I.
      • Reznikoff W.S.
      Phosphate coordination and movement of DNA in the Tn5 synaptic complex. Role of the (R)YREK motif.
      ). In the case of mariner elements, this rearrangement is not necessary, because the transferred strand is directly produced with a 3-bp overhang by the excision (
      • Dawson A.
      • Finnegan D.J.
      Excision of the Drosophila mariner transposon Mos1. Comparison with bacterial transposition and V(D)J recombination.
      ,
      • Miskey C.
      • Papp B.
      • Mátés L.
      • Sinzelle L.
      • Keller H.
      • Izsvák Z.
      • Ivics Z.
      The ancient mariner sails again. Transposition of the human Hsmar1 element by a reconstructed transposase and activities of the SETMAR protein on transposon ends.
      ) that actually precedes target capture. However, conformational changes have to occur, between the first and second DNA strand cleavage, to allow the second DNA strand cleavage to occur, thus generating a target capture-competent complex. These conformational changes have been recently documented for Mos1 by solution scattering methods and PIC crystallography (
      • Richardson J.M.
      • Colloms S.D.
      • Finnegan D.J.
      • Walkinshaw M.D.
      Molecular architecture of the Mos1 paired-end complex. The structural basis of DNA transposition in a eukaryote.
      ,
      • Cuypers M.G.
      • Trubitsyna M.
      • Callow P.
      • Forsyth V.T.
      • Richardson J.M.
      Solution conformations of early intermediates in Mos1 transposition.
      ). SEC2 presented first an elongated form, the two monomers interacting by the first helix turn helix domain. One monomer had to rotate for the recruitment of the second ITR (giving a compact SEC2), conducting to the compact crossed architecture observed in the PIC. These conformational changes may account for the fact that uncleaved and prenicked ITRs are more efficient in promoting target capture than precleaved ITRs. We assume that both of these ITRs, which need to be cleaved to allow PIC assembly, support conformational changes that lead to a target capture-competent complex. In contrast, the precleaved ITRs lead to less efficient target capture complexes. This suggests that this complex does not have the same conformation as the complex obtained with ITR cleaved by the transposase.
      Elements of mariner do not display integration proximate to the excision site (“local hopping”) in vivo. This is consistent with the fact the complete excision of the transposon precedes its reintegration. However, autointegration (i.e., the integration of a single ITR into the sequence of the transposon itself) has been detected in vitro for both Hsmar1 (
      • Claeys Bouuaert C.
      • Chalmers R.
      Transposition of the human Hsmar1 transposon. Rate-limiting steps and the importance of the flanking TA dinucleotide in second strand cleavage.
      ) (
      • Dawson A.
      • Finnegan D.J.
      Excision of the Drosophila mariner transposon Mos1. Comparison with bacterial transposition and V(D)J recombination.
      ,
      • Claeys Bouuaert C.
      • Liu D.
      • Chalmers R.
      A simple topological filter in a eukaryotic transposon as a mechanism to suppress genome instability.
      ) and Mos1, suggesting that partial local hopping may occur (
      • Sinzelle L.
      • Jégot G.
      • Brillet B.
      • Rouleux-Bonnin F.
      • Bigot Y.
      • Augé-Gouillou C.
      Factors acting on Mos1 transposition efficiency.
      ). Taken together, these observations imply that a more sophisticated mechanism prevents autointegrations and/or proximate integrations in vivo. One such mechanism could rely on the fact that the transposable element is mostly or totally insensitive to target commitment, excluding neighboring sequences as targets. In this case, the target capture complex would remain labile until the strand transfer reaction is completed. The element could “explore” several targets within the whole genome before being integrated at any given location, particularly at a distant location from the excision site. The lack of target commitment that we have demonstrated for Mos1 is consistent with the biology of mariner elements. It may account for the random distribution of mariner elements seen in natural populations (
      • Bryan G.
      • Garza D.
      • Hartl D.
      Insertion and excision of the transposable element mariner in Drosophila.
      ,
      • Lohe A.R.
      • Timmons C.
      • Beerman I.
      • Lozovskaya E.R.
      • Hartl D.L.
      Self-inflicted wounds, template-directed gap repair and a recombination hotspot. Effects of the mariner transposase.
      ) and for the results obtained in transgenesis assays using Mos1 as a vector (
      • Wang W.
      • Swevers L.
      • Iatrou K.
      Mariner (Mos1) transposase and genomic integration of foreign gene sequences in Bombyx mori cells.
      ,
      • Vallin E.
      • Gallagher J.
      • Granger L.
      • Martin E.
      • Belougne J.
      • Maurizio J.
      • Duverger Y.
      • Scaglione S.
      • Borrel C.
      • Cortier E.
      • Abouzid K.
      • Carre-Pierrat M.
      • Gieseler K.
      • Ségalat L.
      • Kuwabara P.E.
      • Ewbank J.J.
      A genome-wide collection of Mos1 transposon insertion mutants for the C. elegans research community.
      ). One of the consequences of the excision/target capture pathway that we elucidated in the present study is a possible risk of losing the excised element before it is integrated. However, the biochemistry of transposition makes this unlikely, because excision is the limiting step of the reaction (at least in vitro). This risk still exists even if double-stranded breaks are efficiently repaired using the sister chromosome. It may contribute to the stochastic loss of mariner elements that has been commonly observed (
      • Lohe A.R.
      • Moriyama E.N.
      • Lidholm D.A.
      • Hartl D.L.
      Horizontal transmission, vertical inactivation, and stochastic loss of mariner-like transposable elements.
      ). This can be perceived as the price to be paid for random distribution.
      Two recent publications have proposed a TCC model derived from the Mos1 PIC. The first model deals with the fact that this PIC has a channel between the catalytic domains, in which the modeled target DNA is straight and approximately perpendicular to both ITRs. This model predicts that the Arg186 MOS1 residue will bind to the target through the target TA dinucleotide, supporting subsequent integration (
      • Richardson J.M.
      • Colloms S.D.
      • Finnegan D.J.
      • Walkinshaw M.D.
      Molecular architecture of the Mos1 paired-end complex. The structural basis of DNA transposition in a eukaryote.
      ). The assay that we have developed allows us to discriminate between Mos1 target capture and strand transfer reactions. Hence, we have demonstrated that Arg186 is not involved in target capture and that it only plays a role in strand transfer reactions. Montaño and Rice (
      • Montaño S.P.
      • Rice P.A.
      Moving DNA around. DNA transposition and retroviral integration.
      ) have proposed an alternative model for the Mos1 TCC, in which the target DNA is strongly bent. This has been shown to occur in the intasome of the prototype foamy virus and in the Mu transpososome (
      • Maertens G.N.
      • Hare S.
      • Cherepanov P.
      The mechanism of retroviral integration from x-ray structures of its key intermediates.
      ,
      • Montaño S.P.
      • Pigli Y.Z.
      • Rice P.A.
      The Mu transpososome structure sheds light on DDE recombinase evolution.
      ). The Mu transpososome displays enhanced capture efficiency when mismatched targets are provided (
      • Yanagihara K.
      • Mizuuchi K.
      Mismatch-targeted transposition of Mu. A new strategy to map genetic polymorphism.
      ). We checked modified targets for their efficiency in the capture by Mos1.
      We have shown that nicks and mismatches influence integration, but in two different ways. Nicks enhance target capture up to 5-fold, indicating that the capture of the target is more efficient on a flexible target DNA molecule. Similar findings of enhanced target capture with nicked and mismatched targets have been observed with Tn10 transposase and RAG1/2 (
      • Pribil P.A.
      • Wardle S.J.
      • Haniford D.B.
      Enhancement and rescue of target capture in Tn10 transposition by site-specific modifications in target DNA.
      ,
      • Tsai C.-L.
      • Chatterji M.
      • Schatz D.G.
      DNA mismatches and GC-rich motifs target transposition by the RAG1/RAG2 transposase.
      ). Our results are consistent with the Mos1 TCC model of Montaño and Rice (
      • Montaño S.P.
      • Rice P.A.
      Moving DNA around. DNA transposition and retroviral integration.
      ). This suggests that target bending could serve to position the scissile phosphate in the active site.
      The target bending could also induce a local DNA deformation that would confer a high energy conformation to the scissile phosphate that is necessary for a nucleophilic attack and subsequent strand transfers, as detected with Tn10 (
      • Pribil P.A.
      • Wardle S.J.
      • Haniford D.B.
      Enhancement and rescue of target capture in Tn10 transposition by site-specific modifications in target DNA.
      ). Such severe deformation of the target and expansion of the major groove that makes it possible to position the scissile phosphate at the active sites have been detected in the prototype foamy virus and Mu TCCs (
      • Maertens G.N.
      • Hare S.
      • Cherepanov P.
      The mechanism of retroviral integration from x-ray structures of its key intermediates.
      ,
      • Cherepanov P.
      • Maertens G.N.
      • Hare S.
      Structural insights into the retroviral DNA integration apparatus.
      ). This interpretation was confirmed after using mismatched targets for which strand transfer reactions are strongly enhanced (up to 3-fold). Our results indicate that target DNA bends need to be strictly localized. In fact, the position of the mismatch is a main feature determining strand transfer efficiency. Using both nicked and mismatched targets highlighted the significance of the position +2 relative to the TA dinucleotide.
      During the past decade, all studies have failed to identify determinants of mariner integration other than the TA dinucleotide. Used as the target and duplicated upon the integration of all mariner elements, the TA dinucleotide was only known for its involvement in the excision process (
      • Claeys Bouuaert C.
      • Chalmers R.
      Transposition of the human Hsmar1 transposon. Rate-limiting steps and the importance of the flanking TA dinucleotide in second strand cleavage.
      ). We demonstrated here for the first time that the TA dinucleotide plays a crucial role in target capture and DNA strand transfer. Previous studies revealed that the local DNA sequences immediately surrounding the TA dinucleotide confer little target specificity, although they show a slight preference for TA dinucleotides that are flanked by A-T base pairs (
      • Claeys Bouuaert C.
      • Chalmers R.
      Transposition of the human Hsmar1 transposon. Rate-limiting steps and the importance of the flanking TA dinucleotide in second strand cleavage.
      ,
      • Crénès G.
      • Ivo D.
      • Hérisson J.
      • Dion S.
      • Renault S.
      • Bigot Y.
      • Petit A.
      The bacterial Tn9 chloramphenicol resistance gene. An attractive DNA segment for Mos1 mariner insertions.
      ,
      • Crénès G.
      • Moundras C.
      • Demattei M.-V.
      • Bigot Y.
      • Petit A.
      • Renault S.
      Target site selection by the mariner-like element, Mos1.
      ). It is tempting to imagine that the bending of the TA-rich targets is energetically more favorable for setting up the strand transfer reactions. This hypothesis is sustained by the lack of efficient strand transfer with TA-free targets. Accordingly, the target binding to TA dinucleotide is poorly specific, because displacement is observed with any sequence, but all containing TA. In addition, the target length, at least in vitro, influences the target capture efficiency, but the length is probably not the major factor, which is rather the target bendability, with respect with its length. Recently, it has been shown that the topology of the target affects integration, because negatively supercoiled DNA is a better target. One hypothesis accounting for this result is that supercoiling could underwind DNA, thus increasing its bendability (
      • Claeys Bouuaert C.
      • Chalmers R.
      Hsmar1 transposition is sensitive to the topology of the transposon donor and the target.
      ). This hypothesis fits in with our results.
      Mos1 has proved to be a valuable model for studying critical steps in transposition, especially because it offers a unique possibility to decipher in vitro key features of target selection. Other actors of the dynamics of transposition have been discovered in the course of in vivo studies. This is the case of HMGB1 during Sleeping Beauty transposition (
      • Zayed H.
      • Izsvák Z.
      • Khare D.
      • Heinemann U.
      • Ivics Z.
      The DNA-bending protein HMGB1 is a cellular cofactor of Sleeping Beauty transposition.
      ) or of nucleosomes positioning along the HIV integrasome (
      • Lohe A.R.
      • De Aguiar D.
      • Hartl D.L.
      Mutations in the mariner transposase. The D,D(35)E consensus sequence is nonfunctional.
      ). This indicates that in vitro biochemical results are relevant to the more complex in vivo situations. In this context, it would be interesting to investigate the local deformation of the target DNA in vivo. In fact, alterations of the DNA/chromatin structure have been shown to be essential to initiate replication and gene transcription. Interestingly, both replication and gene transcription involve TA-rich regions, which, in turn, have been shown to be essential for mariner transposition. This raises the issue of whether promoters and replication origins could be good mariner targets. Elements of mariner do not appear to have an insertion bias for active genes, suggesting that transcription is not a major factor in target capture nor in the subsequent integration. A possible relationship between mariner transposition and replication, especially at the level of its initiation (replication origins), remains an open issue. Eukaryotic genomes contain tens of thousands of replication origins distributed along the chromosomes in a way that matches the distribution of mariner elements. Moreover, the amplification of cut and paste transposons implies that replication and transposition have to be coupled. This correlation is in accordance with our hypothesis of target choice in AT-rich regions. This could provide new insights into the mechanisms that govern the dynamics of the transposition of DNA transposons into host genomes.

      Acknowledgments

      We thank G. Carpentier for the construction of baculovirus expressing the MBP-MOS. We also thank P. Gaudray for helpful discussions. The autoradiograms were recovered using the PPF genome facilities (UFR Sciences & Techniques, University of Tours). Dr. M. Ghosh revised the English text.

      References

        • Babatz T.D.
        • Burns K.H.
        Functional impact of the human mobilome.
        Curr. Opin. Genet. Dev. 2013; 23: 264-270
        • Kunarso G.
        • Chia N.-Y.
        • Jeyakani J.
        • Hwang C.
        • Lu X.
        • Chan Y.-S.
        • Ng H.-H.
        • Bourque G.
        Transposable elements have rewired the core regulatory network of human embryonic stem cells.
        Nat. Genet. 2010; 42: 631-634
        • Cordaux R.
        • Udit S.
        • Batzer M.A.
        • Feschotte C.
        Birth of a chimeric primate gene by capture of the transposase gene from a mobile element.
        Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 8101-8106
        • Chalopin D.
        • Galiana D.
        • Volff J.-N.
        Genetic innovation in vertebrates. Gypsy integrase genes and other genes derived from transposable elements.
        Int. J. Evol. Biol. 2012; 2012: 724519
        • Plasterk R.H.
        • van Luenen H.G.
        Craig N.L. Craigie R. Gellert M. Lambowitz A.L. Mobile DNA II. ASM Press, Washington, D. C2002: 519-532
        • Maertens G.N.
        • Hare S.
        • Cherepanov P.
        The mechanism of retroviral integration from x-ray structures of its key intermediates.
        Nature. 2010; 468: 326-329
        • Jaillet J.
        • Genty M.
        • Cambefort J.
        • Rouault J.-D.
        • Augé-Gouillou C.
        Regulation of mariner transposition. The peculiar case of Mos1.
        PLoS One. 2012; 7: e43365
        • Carpentier G.
        • Jaillet J.
        • Pflieger A.
        • Adet J.
        • Renault S.
        • Augé-Gouillou C.
        Transposase-transposase interactions in MOS1 complexes. A biochemical approach.
        J. Mol. Biol. 2011; 405: 892-908
        • Richardson J.M.
        • Colloms S.D.
        • Finnegan D.J.
        • Walkinshaw M.D.
        Molecular architecture of the Mos1 paired-end complex. The structural basis of DNA transposition in a eukaryote.
        Cell. 2009; 138: 1096-1108
        • Claeys Bouuaert C.
        • Chalmers R.
        Transposition of the human Hsmar1 transposon. Rate-limiting steps and the importance of the flanking TA dinucleotide in second strand cleavage.
        Nucleic Acids Res. 2010; 38: 190-202
        • Nowotny M.
        Retroviral integrase superfamily. The structural perspective.
        EMBO Rep. 2009; 10: 144-151
        • Claeys Bouuaert C.
        • Chalmers R.M.
        Gene therapy vectors. The prospects and potentials of the cut-and-paste transposons.
        Genetica. 2010; 138: 473-484
        • Kuduvalli P.N.
        • Rao J.E.
        • Craig N.L.
        Target DNA structure plays a critical role in Tn7 transposition.
        EMBO J. 2001; 20: 924-932
        • Pribil P.A.
        • Haniford D.B.
        Target DNA bending is an important specificity determinant in target site selection in Tn10 transposition.
        J. Mol. Biol. 2003; 330: 247-259
        • Pribil P.A.
        • Wardle S.J.
        • Haniford D.B.
        Enhancement and rescue of target capture in Tn10 transposition by site-specific modifications in target DNA.
        Mol. Microbiol. 2004; 52: 1173-1186
        • Tsai C.-L.
        • Chatterji M.
        • Schatz D.G.
        DNA mismatches and GC-rich motifs target transposition by the RAG1/RAG2 transposase.
        Nucleic Acids Res. 2003; 31: 6180-6190
        • Müller H.P.
        • Varmus H.E.
        DNA bending creates favored sites for retroviral integration. An explanation for preferred insertion sites in nucleosomes.
        EMBO J. 1994; 13: 4704-4714
        • Lohe A.R.
        • De Aguiar D.
        • Hartl D.L.
        Mutations in the mariner transposase. The D,D(35)E consensus sequence is nonfunctional.
        Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 1293-1297
        • Claeys Bouuaert C.
        • Chalmers R.
        Hsmar1 transposition is sensitive to the topology of the transposon donor and the target.
        PLoS One. 2013; 8: e53690
        • Benjamin H.W.
        • Kleckner N.
        Intramolecular transposition by Tn10.
        Cell. 1989; 59: 373-383
        • Shevchenko Y.
        • Bouffard G.G.
        • Butterfield Y.S.
        • Blakesley R.W.
        • Hartley J.L.
        • Young A.C.
        • Marra M.A.
        • Jones S.J.
        • Touchman J.W.
        • Green E.D.
        Systematic sequencing of cDNA clones using the transposon Tn5.
        Nucleic Acids Res. 2002; 30: 2469-2477
        • Crénès G.
        • Ivo D.
        • Hérisson J.
        • Dion S.
        • Renault S.
        • Bigot Y.
        • Petit A.
        The bacterial Tn9 chloramphenicol resistance gene. An attractive DNA segment for Mos1 mariner insertions.
        Mol. Genet. Genomics. 2009; 281: 315-328
        • Crénès G.
        • Moundras C.
        • Demattei M.-V.
        • Bigot Y.
        • Petit A.
        • Renault S.
        Target site selection by the mariner-like element, Mos1.
        Genetica. 2010; 138: 509-517
        • Matthews A.G.
        • Elkin S.K.
        • Oettinger M.A.
        Ordered DNA release and target capture in RAG transposition.
        EMBO J. 2004; 23: 1198-1206
        • Naigamwalla D.Z.
        • Chaconas G.
        A new set of Mu DNA transposition intermediates. Alternate pathways of target capture preceding strand transfer.
        EMBO J. 1997; 16: 5227-5234
        • Sakai J.
        • Kleckner N.
        The Tn10 synaptic complex can capture a target DNA only after transposon excision.
        Cell. 1997; 89: 205-214
        • Gradman R.J.
        • Ptacin J.L.
        • Bhasin A.
        • Reznikoff W.S.
        • Goryshin I.Y.
        A bifunctional DNA binding region in Tn5 transposase.
        Mol. Microbiol. 2008; 67: 528-540
        • Neiditch M.B.
        • Lee G.S.
        • Landree M.A.
        • Roth D.B.
        RAG transposase can capture and commit to target DNA before or after donor cleavage.
        Mol. Cell Biol. 2001; 21: 4302-4310
        • Lipkow K.
        • Buisine N.
        • Chalmers R.
        Promiscuous target interactions in the mariner transposon Himar1.
        J. Biol. Chem. 2004; 279: 48569-48575
        • Bouchet N.
        • Jaillet J.
        • Gabant G.
        • Brillet B.
        • Briseño-Roa L.
        • Cadene M.
        • Augé-Gouillou C.
        cAMP protein kinase phosphorylates the Mos1 transposase and regulates its activity. Evidences from mass spectrometry and biochemical analyses.
        Nucleic Acids Res. 2013; (10.1093/nar/gkt874)
        • Augé-Gouillou C.
        • Brillet B.
        • Hamelin M.-H.
        • Bigot Y.
        Assembly of the mariner Mos1 synaptic complex.
        Mol. Cell Biol. 2005; 25: 2861-2870
        • Dawson A.
        • Finnegan D.J.
        Excision of the Drosophila mariner transposon Mos1. Comparison with bacterial transposition and V(D)J recombination.
        Mol. Cell. 2003; 11: 225-235
        • Cuypers M.G.
        • Trubitsyna M.
        • Callow P.
        • Forsyth V.T.
        • Richardson J.M.
        Solution conformations of early intermediates in Mos1 transposition.
        Nucleic Acids Res. 2013; 41: 2020-2033
        • Krishnan L.
        • Engelman A.
        Retroviral integrase proteins and HIV-1 DNA integration.
        J. Biol. Chem. 2012; 287: 40858-40866
        • Klenchin V.A.
        • Czyz A.
        • Goryshin I.Y.
        • Gradman R.
        • Lovell S.
        • Rayment I.
        • Reznikoff W.S.
        Phosphate coordination and movement of DNA in the Tn5 synaptic complex. Role of the (R)YREK motif.
        Nucleic Acids Res. 2008; 36: 5855-5862
        • Miskey C.
        • Papp B.
        • Mátés L.
        • Sinzelle L.
        • Keller H.
        • Izsvák Z.
        • Ivics Z.
        The ancient mariner sails again. Transposition of the human Hsmar1 element by a reconstructed transposase and activities of the SETMAR protein on transposon ends.
        Mol. Cell Biol. 2007; 27: 4589-4600
        • Claeys Bouuaert C.
        • Liu D.
        • Chalmers R.
        A simple topological filter in a eukaryotic transposon as a mechanism to suppress genome instability.
        Mol. Cell Biol. 2011; 31: 317-327
        • Sinzelle L.
        • Jégot G.
        • Brillet B.
        • Rouleux-Bonnin F.
        • Bigot Y.
        • Augé-Gouillou C.
        Factors acting on Mos1 transposition efficiency.
        BMC Mol. Biol. 2008; 9: 106
        • Bryan G.
        • Garza D.
        • Hartl D.
        Insertion and excision of the transposable element mariner in Drosophila.
        Genetics. 1990; 125: 103-114
        • Lohe A.R.
        • Timmons C.
        • Beerman I.
        • Lozovskaya E.R.
        • Hartl D.L.
        Self-inflicted wounds, template-directed gap repair and a recombination hotspot. Effects of the mariner transposase.
        Genetics. 2000; 154: 647-656
        • Wang W.
        • Swevers L.
        • Iatrou K.
        Mariner (Mos1) transposase and genomic integration of foreign gene sequences in Bombyx mori cells.
        Insect Mol. Biol. 2000; 9: 145-155
        • Vallin E.
        • Gallagher J.
        • Granger L.
        • Martin E.
        • Belougne J.
        • Maurizio J.
        • Duverger Y.
        • Scaglione S.
        • Borrel C.
        • Cortier E.
        • Abouzid K.
        • Carre-Pierrat M.
        • Gieseler K.
        • Ségalat L.
        • Kuwabara P.E.
        • Ewbank J.J.
        A genome-wide collection of Mos1 transposon insertion mutants for the C. elegans research community.
        PLoS One. 2012; 7: e30482
        • Lohe A.R.
        • Moriyama E.N.
        • Lidholm D.A.
        • Hartl D.L.
        Horizontal transmission, vertical inactivation, and stochastic loss of mariner-like transposable elements.
        Mol. Biol. Evol. 1995; 12: 62-72
        • Montaño S.P.
        • Rice P.A.
        Moving DNA around. DNA transposition and retroviral integration.
        Curr. Opin. Struct. Biol. 2011; 21: 370-378
        • Montaño S.P.
        • Pigli Y.Z.
        • Rice P.A.
        The Mu transpososome structure sheds light on DDE recombinase evolution.
        Nature. 2012; 491: 413-417
        • Yanagihara K.
        • Mizuuchi K.
        Mismatch-targeted transposition of Mu. A new strategy to map genetic polymorphism.
        Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 11317-11321
        • Cherepanov P.
        • Maertens G.N.
        • Hare S.
        Structural insights into the retroviral DNA integration apparatus.
        Curr. Opin. Struct. Biol. 2011; 21: 249-256
        • Zayed H.
        • Izsvák Z.
        • Khare D.
        • Heinemann U.
        • Ivics Z.
        The DNA-bending protein HMGB1 is a cellular cofactor of Sleeping Beauty transposition.
        Nucleic Acids Res. 2003; 31: 2313-2322