Assembly Of Synaptic Complexes Research Articles

A di-acetyl-decorated chromatin signature couples liquid condensation to suppress DNA end synapsis

Appropriate DNA end synapsis, regulated by core components of the synaptic complex including KU70-KU80, LIG4, XRCC4, and XLF, is central to non-homologous end joining (NHEJ) repair of chromatinized DNA double-strand breaks (DSBs). However, it remains enigmatic whether chromatin modifications can influence the formation of NHEJ synaptic complex at DNA ends, and if so, how this is achieved. Here, we report that the mitotic deacetylase complex (MiDAC) serves as a key regulator of DNA end synapsis during NHEJ repair in mammalian cells. Mechanistically, MiDAC removes combinatorial acetyl marks on histone H2A (H2AK5acK9ac) around DSB-proximal chromatin, suppressing hyperaccumulation of bromodomain-containing protein BRD4 that would otherwise undergo liquid-liquid phase separation with KU80 and prevent the proper installation of LIG4-XRCC4-XLF onto DSB ends. This study provides mechanistic insight into the control of NHEJ synaptic complex assembly by a specific chromatin signature and highlights the critical role of H2A hypoacetylation in restraining unscheduled compartmentalization of DNA repair machinery.

Correction to ‘Mechanisms of Cre recombinase synaptic complex assembly and activation illuminated by Cryo-EM’

Correction to ‘Mechanisms of Cre recombinase synaptic complex assembly and activation illuminated by Cryo-EM’

Mechanisms of Cre recombinase synaptic complex assembly and activation illuminated by Cryo-EM.

Cre recombinase selectively recognizes DNA and prevents non-specific DNA cleavage through an orchestrated series of assembly intermediates. Cre recombines two loxP DNA sequences featuring a pair of palindromic recombinase binding elements and an asymmetric spacer region, by assembly of a tetrameric synaptic complex, cleavage of an opposing pair of strands, and formation of a Holliday junction intermediate. We used Cre and loxP variants to isolate the monomeric Cre-loxP (54 kDa), dimeric Cre2-loxP (110 kDa), and tetrameric Cre4-loxP2 assembly intermediates, and determined their structures using cryo-EM to resolutions of 3.9, 4.5 and 3.2 Å, respectively. Progressive and asymmetric bending of the spacer region along the assembly pathway enables formation of increasingly intimate interfaces between Cre protomers and illuminates the structural bases of biased loxP strand cleavage order and half-the-sites activity. Application of 3D variability analysis to the tetramer data reveals constrained conformational sampling along the pathway between protomer activation and Holliday junction isomerization. These findings underscore the importance of protein and DNA flexibility in Cre-mediated site selection, controlled activation of alternating protomers, the basis for biased strand cleavage order, and recombination efficiency. Such considerations may advance development of site-specific recombinases for use in gene editing applications.

In Vitro Reconstitution of BRCA1-BARD1/RAD51-Mediated Homologous DNA Pairing.

RAD51-mediated homologous recombination (HR) is a conserved mechanism for the repair of DNA double-strand breaks and the maintenance of DNA replication forks. Several breast and ovarian tumor suppressors, including BRCA1 and BARD1, have been implicated in HR since their discovery in the 1990s. However, a holistic understanding of how they participate in HR has been hampered by the immense challenge of expressing and purifying these large and unstable protein complexes for mechanistic analysis. Recently, we have overcome such a challenge for the BRCA1-BARD1 complex, allowing us to demonstrate its pivotal role in HR via the promotion of RAD51-mediated DNA strand invasion. In this chapter, we describe detailed procedures for the expression and purification of the BRCA1-BARD1 complex and in vitro assays using this tumor suppressor complex to examine its ability to promote RAD51-mediated homologous DNA pairing. This includes two distinct biochemical assays, namely, D-loop formation and synaptic complex assembly. These methods are invaluable for studying the BRCA1-BARD1 complex and its functional interplay with other factors in the HR process.

RAD51AP1 mediates RAD51 activity through nucleosome interaction

RAD51-associated protein 1 (RAD51AP1) is a key protein in the homologous recombination (HR) DNA repair pathway. Loss of RAD51AP1 leads to defective HR, genome instability, and telomere erosion. RAD51AP1 physically interacts with the RAD51 recombinase and promotes RAD51-mediated capture of donor DNA, synaptic complex assembly, and displacement-loop formation when tested with nucleosome-free DNA substrates. In cells, however, DNA is packaged into chromatin, posing an additional barrier to the complexities of the HR reaction. In this study, we show that RAD51AP1 binds to nucleosome core particles (NCPs), the minimum basic unit of chromatin in which approximately two superhelical turns of 147 bp double-stranded DNA are wrapped around one histone octamer with no free DNA ends remaining. We identified a C-terminal region in RAD51AP1, including its previously mapped DNA-binding domain, as critical for mediating the association between RAD51AP1 and both the NCP and the histone octamer. Using in vitro surrogate assays of HR activity, we show that RAD51AP1 is capable of promoting duplex DNA capture and initiating joint-molecule formation with the NCP and chromatinized template DNA, respectively. Together, our results suggest that RAD51AP1 directly assists in the RAD51-mediated search for donor DNA in chromatin. We present a model, in which RAD51AP1 anchors the DNA template through affinity for its nucleosomes to the RAD51-ssDNA nucleoprotein filament.

DNA binding induces a cis-to-trans switch in Cre recombinase to enable intasome assembly

Mechanistic understanding of DNA recombination in the Cre-loxP system has largely been guided by crystallographic structures of tetrameric synaptic complexes. Those studies have suggested a role for protein conformational dynamics that has not been well characterized at the atomic level. We used solution nuclear magnetic resonance (NMR) spectroscopy to discover the link between intrinsic flexibility and function in Cre recombinase. Transverse relaxation-optimized spectroscopy (TROSY) NMR spectra show the N-terminal and C-terminal catalytic domains (CreNTD and CreCat) to be structurally independent. Amide 15N relaxation measurements of the CreCat domain reveal fast-timescale dynamics in most regions that exhibit conformational differences in active and inactive Cre protomers in crystallographic tetramers. However, the C-terminal helix αN, implicated in assembly of synaptic complexes and regulation of DNA cleavage activity via trans protein-protein interactions, is unexpectedly rigid in free Cre. Chemical shift perturbations and intra- and intermolecular paramagnetic relaxation enhancement (PRE) NMR data reveal an alternative autoinhibitory conformation for the αN region of free Cre, wherein it packs in cis over the protein DNA binding surface and active site. Moreover, binding to loxP DNA induces a conformational change that dislodges the C terminus, resulting in a cis-to-trans switch that is likely to enable protein-protein interactions required for assembly of recombinogenic Cre intasomes. These findings necessitate a reexamination of the mechanisms by which this widely utilized gene-editing tool selects target sites, avoids spurious DNA cleavage activity, and controls DNA recombination efficiency.

UAF1 DNA Binding Activity Is Critical for RAD51-Mediated Homologous DNA Pairing

Background: In the Fanconi anemia (FA) DNA repair pathway, DNA damage induces the mono-ubiquitination of the FANCI-FANCD2 (ID2) heterodimer by the FA core complex through its inherent E3 ligase activity. The timely deubiquitination of ID2 by USP1-UAF1 deubiquitinase complex is also critically important for the FA DNA repair. UAF1 has a DNA binding activity, which is required for FANCD2 deubiquitination. UAF1 also enhances RAD51-mediated homologous DNA pairing in a manner that is dependent on complex formation with RAD51AP1. UAF1 deficient cells are impaired for DNA repair by homologous recombination (HR).The biochemical and cellular functions of UAF1 DNA binding activity in HR remain elusive. Methods:UAF1 wild type and DNA binding mutant proteins were purified and used to define its biochemical properties in HR. In vitroD-loop formation and synaptic complex assembly assay were performed to discover the DNA binding of UAF1 in RAD51 recombinase enhancement. U2OS-DR-GFP cell lines with impaired UAF1 or RAD51AP1DNA binding were generated to examine HR efficiency and DNA damage resistance. Results:UAF1 preferentially binds an HR-intermediate-like DNA substrate (D-loop, Fig.1). The DNA binding deficient mutant of UAF1 is unable to stimulate RAD51AP1 promotion of RAD51-mediated D-loop (Fig. 2) and the ability to recruit homologous DNA to form the presynaptic complex formation in HR (Fig. 3). In cells, the UAF1 DNA-binding mutant is compromised for the ability to repair DNA damage and to implement HR (Fig. 4). Such activity correlates with the ability to confer resistance to DNA cross linking agents such as mitomycin C (Fig. 4). The DNA binding of UAF1 and RAD51AP1 have a coordinated role in HR-directed DNA damage repair (Fig. 5). Conclusions: UAF1 DNA binding activity is indispensable for its function in enhancing RAD51-mediated homologous DNA pairing within the context of the UAF1-RAD51AP1 complex. UAF1 DNA binding deficiency causes DNA damage sensitivity and impairs HR efficiency in cells. Translational Applicability:Our findings reveal a critical role of UAF1 DNA binding in DNA repair and genome maintenance. The identification of UAF1's role in repair will enable targeted efforts to improve molecular approaches for FA therapy. Disclosures No relevant conflicts of interest to declare.

A single XLF dimer bridges DNA ends during nonhomologous end joining.

Non-homologous end joining (NHEJ) is the primary pathway of DNA double-strand break repair in vertebrate cells, yet it remains unclear how NHEJ factors assemble a synaptic complex that bridges DNA ends. To address the role of XRCC4-like factor (XLF) in synaptic complex assembly, we employed single-molecule fluorescence imaging in Xenopus laevis egg extract, a system that efficiently joins DNA ends. We find that a single XLF dimer binds to DNA substrates just prior to formation of a ligation-competent synaptic complex between DNA ends. The interaction of both globular head domains of the XLF dimer with XRCC4 is required for efficient formation of this synaptic complex. In contrast to a model in which filaments of XLF and XRCC4 bridge DNA ends, our results indicate that binding of a single XLF dimer facilitates the assembly of a stoichiometrically well-defined synaptic complex.

The Role of UAF1 in the Fanconi Anemia Pathway Regulation of Homologous Recombination-Mediated Genome Maintenance

Background: Fanconi anemia (FA), a cancer-prone genetic disease, is caused by defects in the FA-DNA repair pathway. In response to DNA interstrand crosslink (ICL)-induced DNA damage, FANCI-FANCD2 mono-ubiquitination licenses the execution of downstream DNA damage signaling and repair steps, including repair by homologous recombination (HR) that utilizes the recombinase RAD51 and its cohort of accessory factors. Timely deubiquitination of FANCD2 by the UAF1-USP1 deubiquitinating enzyme complex is also critically important for the FA pathway. As such, UAF1 depletion results in persistent FANCD2 ubiquitination and DNA damage hypersensitivity. UAF1 deficient cells are also impaired for DNA repair by homologous recombination. UAF1 physically associates with RAD51AP1, a protein that enhances the activity of the RAD51 recombinase. It remains to be defined how UAF1 regulates homologous recombination and genome stability.Methods: Highly purified proteins were used to define the DNA binding activity and protein interaction of UAF1. In vitroD-loop formation reaction and synaptic complex assembly assay were used to discover the function of UAF1 in RAD51 recombinase enhancement. HeLa and U2OS-DR-GFP cell lines with impaired UAF1-RAD51AP1 interaction or UAF1 DNA binding were generated to examine DNA-damage agent sensitivity and HR efficiency.Results: (1) UAF1 possesses a DNA binding activity capable of engaging ssDNA, dsDNA and has a preference for the D-loop DNA substrate. We further identified that the N-terminus but not C-terminal SLD domain of UAF1 binds DNA. (2) UAF1 forms a dimeric complex with RAD51AP1. Our results also revealed a trimeric complex of RAD51-RAD51AP1-UAF1, with RAD51AP1 providing a tethering function between the other two proteins. (3) The RAD51AP1-UAF1 interaction interface was defined showing a novel SIM motif in the middle portion of RAD51AP1and the SLD1-SLD2 domain of UAF1 mediate protein complex formation. Based on the domain mapping results, point mutants of RAD51AP1 and UAF1 that are specifically compromised for the formation of the RAD51AP1-UAF1 complex were generated. (4) UAF1 synergizes with RAD51AP1 in the RAD51-mediated D-loop reaction and that this functional synergy requires the RAD51AP1-UAF1 complex and also the DNA and RAD51 binding attributes of RAD51AP1. (5) RAD51AP1-UAF1 works in conjunction with the RAD51 presynaptic filament in the capture of the duplex DNA partner and in the assembly of the synaptic complex. (6) Human cell lines impaired for RAD51AP1-UAF1 complex formation are compromised for the ability to repair DNA damage and to execute HR. (7) DNA repair function of the RAD51AP1-UAF1 complex is likely USP1-independent.Conclusions: The physical interaction between UAF1 and RAD51AP1 is indispensable for functional synergy in vitro and, accordingly, for the biological function of UAF1 in HR and DNA damage repair. Our findings provide insights into a novel USP1-independent regulatory mechanism of UAF1 on homologous recombination-mediated genome maintenance. DisclosuresNo relevant conflicts of interest to declare.

Sleeping Beauty Transposition.

Sleeping Beauty (SB) is a synthetic transposon that was constructed based on sequences of transpositionally inactive elements isolated from fish genomes. SB is a Tc1/mariner superfamily transposon following a cut-and-paste transpositional reaction, during which the element-encoded transposase interacts with its binding sites in the terminal inverted repeats of the transposon, promotes the assembly of a synaptic complex, catalyzes excision of the element out of its donor site, and integrates the excised transposon into a new location in target DNA. SB transposition is dependent on cellular host factors. Transcriptional control of transposase expression is regulated by the HMG2L1 transcription factor. Synaptic complex assembly is promoted by the HMGB1 protein and regulated by chromatin structure. SB transposition is highly dependent on the nonhomologous end joining (NHEJ) pathway of double-strand DNA break repair that generates a transposon footprint at the excision site. Through its association with the Miz-1 transcription factor, the SB transposase downregulates cyclin D1 expression that results in a slowdown of the cell-cycle in the G1 phase, where NHEJ is preferentially active. Transposon integration occurs at TA dinucleotides in the target DNA, which are duplicated at the flanks of the integrated transposon. SB shows a random genome-wide insertion profile in mammalian cells when launched from episomal vectors and "local hopping" when launched from chromosomal donor sites. Some of the excised transposons undergo a self-destructive autointegration reaction, which can partially explain why longer elements transpose less efficiently. SB became an important molecular tool for transgenesis, insertional mutagenesis, and gene therapy.

Single molecule TPM analysis of the catalytic pentad mutants of Cre and Flp site-specific recombinases: contributions of the pentad residues to the pre-chemical steps of recombination.

Cre and Flp site-specific recombinase variants harboring point mutations at their conserved catalytic pentad positions were characterized using single molecule tethered particle motion (TPM) analysis. The findings reveal contributions of these amino acids to the pre-chemical steps of recombination. They suggest functional differences between positionally conserved residues in how they influence recombinase-target site association and formation of ‘non-productive’, ‘pre-synaptic’ and ‘synaptic’ complexes. The most striking difference between the two systems is noted for the single conserved lysine. The pentad residues in Cre enhance commitment to recombination by kinetically favoring the formation of pre-synaptic complexes. These residues in Flp serve a similar function by promoting Flp binding to target sites, reducing non-productive binding and/or enhancing the rate of assembly of synaptic complexes. Kinetic comparisons between Cre and Flp, and between their derivatives lacking the tyrosine nucleophile, are consistent with a stronger commitment to recombination in the Flp system. The effect of target site orientation (head-to-head or head-to-tail) on the TPM behavior of synapsed DNA molecules supports the selection of anti-parallel target site alignment prior to the chemical steps. The integrity of the synapse, whose establishment/stability is fostered by strand cleavage in the case of Flp but not Cre, appears to be compromised by the pentad mutations.

Mechanistic insights into the role of Hop2-Mnd1 in meiotic homologous DNA pairing

The Hop2–Mnd1 complex functions with the DMC1 recombinase in meiotic recombination. Hop2–Mnd1 stabilizes the DMC1-single-stranded DNA (ssDNA) filament and promotes the capture of the double-stranded DNA partner by the recombinase filament to assemble the synaptic complex. Herein, we define the action mechanism of Hop2–Mnd1 in DMC1-mediated recombination. Small angle X-ray scattering analysis and electron microscopy reveal that the heterodimeric Hop2–Mnd1 is a V-shaped molecule. We show that the protein complex harbors three distinct DNA binding sites, and determine their functional relevance. Specifically, the N-terminal double-stranded DNA binding functions of Hop2 and Mnd1 co-operate to mediate synaptic complex assembly, whereas ssDNA binding by the Hop2 C-terminus helps stabilize the DMC1-ssDNA filament. A model of the Hop2-Mnd1-DMC1-ssDNA ensemble is proposed to explain how it mediates homologous DNA pairing in meiotic recombination.

DNA synapsis through transient tetramerization triggers cleavage by Ecl18kI restriction enzyme

To cut DNA at their target sites, restriction enzymes assemble into different oligomeric structures. The Ecl18kI endonuclease in the crystal is arranged as a tetramer made of two dimers each bound to a DNA copy. However, free in solution Ecl18kI is a dimer. To find out whether the Ecl18kI dimer or tetramer represents the functionally important assembly, we generated mutants aimed at disrupting the putative dimer–dimer interface and analysed the functional properties of Ecl18kI and mutant variants. We show by atomic force microscopy that on two-site DNA, Ecl18kI loops out an intervening DNA fragment and forms a tetramer. Using the tethered particle motion technique, we demonstrate that in solution DNA looping is highly dynamic and involves a transient interaction between the two DNA-bound dimers. Furthermore, we show that Ecl18kI cleaves DNA in the synaptic complex much faster than when acting on a single recognition site. Contrary to Ecl18kI, the tetramerization interface mutant R174A binds DNA as a dimer, shows no DNA looping and is virtually inactive. We conclude that Ecl18kI follows the association model for the synaptic complex assembly in which it binds to the target site as a dimer and then associates into a transient tetrameric form to accomplish the cleavage reaction.

Molecular Mechanism Underlying RAG1/RAG2 Synaptic Complex Formation

Two lymphoid cell-specific proteins, RAG1 and RAG2 (RAG), initiate V(D)J recombination by assembling a synaptic complex with recombination signal sequences (RSSs) abutting two different antigen receptor gene coding segments, and then introducing a DNA double strand break at the end of each RSS. Despite the biological importance of this system, the structure of the synaptic complex, and the RAG protein stoichiometry and arrangement of DNA within the synaptosome, remains poorly understood. Here we applied atomic force microscopy to directly visualize and characterize RAG synaptic complexes. We report that the pre-cleavage RAG synaptic complex contains about twice the protein content as a RAG complex bound to a single RSS, with a calculated mass consistent with a pair of RAG heterotetramers. In the synaptic complex, the RSSs are predominantly oriented in a side-by-side configuration with no DNA strand crossover. The mass of the synaptic complex, and the conditions under which it is formed in vitro, favors an association model of assembly in which isolated RAG-RSS complexes undergo synapsis mediated by RAG protein-protein interactions. The replacement of Mg2+ cations with Ca2+ leads to a dramatic change in protein stoichiometry for all RAG-RSS complexes, suggesting that the cation composition profoundly influences the type of complex assembled.

HMG-box domain stimulation of RAG1/2 cleavage activity is metal ion dependent

BackgroundRAG1 and RAG2 initiate V(D)J recombination by assembling a synaptic complex with a pair of antigen receptor gene segments through interactions with their flanking recombination signal sequence (RSS), and then introducing a DNA double-strand break at each RSS, separating it from the adjacent coding segment. While the RAG proteins are sufficient to mediate RSS binding and cleavage in vitro, these activities are stimulated by the architectural DNA binding and bending factors HMGB1 and HMGB2. Two previous studies (Bergeron et al., 2005, and Dai et al., 2005) came to different conclusions regarding whether only one of the two DNA binding domains of HMGB1 is sufficient to stimulate RAG-mediated binding and cleavage of naked DNA in vitro. Here we test whether this apparent discrepancy is attributed to the choice of divalent metal ion and the concentration of HMGB1 used in the cleavage reaction.ResultsWe show here that single HMG-box domains of HMGB1 stimulate RAG-mediated RSS cleavage in a concentration-dependent manner in the presence of Mn2+, but not Mg2+. Interestingly, the inability of a single HMG-box domain to stimulate RAG-mediated RSS cleavage in Mg2+ is overcome by the addition of partner RSS to promote synapsis. Furthermore, we show that mutant forms of HMGB1 which otherwise fail to stimulate RAG-mediated RSS cleavage in Mg2+ can be substantially rescued when Mg2+ is replaced with Mn2+.ConclusionThe conflicting data published previously in two different laboratories can be substantially explained by the choice of divalent metal ion and abundance of HMGB1 in the cleavage reaction. The observation that single HMG-box domains can promote RAG-mediated 23-RSS cleavage in Mg2+ in the presence, but not absence, of partner RSS suggests that synaptic complex assembly in vitro is associated with conformational changes that alter how the RAG and/or HMGB1 proteins bind and bend DNA in a manner that functionally replaces the role of one of the HMG-box domains in RAG-HMGB1 complexes assembled on a single RSS.

Analysis of P Element Transposase Protein-DNA Interactions during the Early Stages of Transposition

P elements are a family of transposable elements found in Drosophila that move by using a cut-and-paste mechanism and that encode a transposase protein that uses GTP as a cofactor for transposition. Here we used atomic force microscopy to visualize the initial interaction of transposase protein with P element DNA. The transposase first binds to one of the two P element ends, in the presence or absence of GTP, prior to synapsis. In the absence of GTP, these complexes remain stable but do not proceed to synapsis. In the presence of GTP or nonhydrolyzable GTP analogs, synapsis happens rapidly, whereas DNA cleavage is slow. Both atomic force microscopy and standard biochemical methods have been used to show that the P element transposase exists as a pre-formed tetramer that initially binds to either one of the two P element ends in the absence of GTP prior to synapsis. This initial single end binding may explain some of the aberrant P element-induced rearrangements observed in vivo, such as hybrid end insertion. The allosteric effect of GTP in promoting synapsis by P element transposase may be to orient a second site-specific DNA binding domain in the tetramer allowing recognition of a second high affinity transposase-binding site at the other transposon end.

Tn5 transposase loops DNA in the absence of Tn5 transposon end sequences

Transposases mediate transposition first by binding specific DNA end sequences that define a transposable element and then by organizing protein and DNA into a highly structured and stable nucleoprotein 'synaptic' complex. Synaptic complex assembly is a central checkpoint in many transposition mechanisms. The Tn5 synaptic complex contains two Tn5 transposase subunits and two Tn5 transposon end sequences, exhibits extensive protein-end sequence DNA contacts and is the node of a DNA loop. Using single-molecule and bulk biochemical approaches, we found that Tn5 transposase assembles a stable nucleoprotein complex in the absence of Tn5 transposon end sequences. Surprisingly, this end sequence-independent complex has structural similarities to the synaptic complex. This complex is the node of a DNA loop; transposase dimerization and DNA specificity mutants affect its assembly; and it likely has the same number of proteins and DNA molecules as the synaptic complex. Furthermore, our results indicate that Tn5 transposase preferentially binds and loops a subset of non-Tn5 end sequences. Assembly of end sequence-independent nucleoprotein complexes likely plays a role in the in vivo downregulation of transposition and the cis-transposition bias of many bacterial transposases.

Ordered assembly of the V(D)J synaptic complex ensures accurate recombination.

Recombination of gene segments at the immunoglobulin and T-cell receptor loci requires that the RAG1 and RAG2 proteins bring together DNA signal sequences (RSSs) with 12- and 23-bp spacers into a synaptic complex and cleave the DNA. A RAG1/2 multimer that can cleave both signals is shown to assemble on an isolated RSS, and the complementary RSS enters this complex as naked DNA. When RAG1/2 is allowed to bind 12 and 23 RSSs separately prior to their mixing, synaptic complex assembly and cleavage activity are greatly reduced, indicating that only a complex initially assembled on a single RSS leads to productive cleavage. RAG1/2 complexes assembled on 12 RSSs will only incorporate 23 partners, while complexes assembled on 23 RSSs show a 5- to 6-fold preference for 12 partners. Thus, initial assembly on a 12 RSS most accurately reflects the strict 12/23 coupled cleavage observed in the cell. Additional cellular factors such as chromatin may ensure that RAG1/2 first assembles on a 12 RSS, and then a free 23 RSS enters to activate cleavage.

The terminal inverted repeats of IS 911: requirements for synaptic complex assembly and activity

The bacterial insertion sequence IS 911 transposes via a covalently closed circular intermediate. Circle formation involves transposase-mediated pairing of both insertion sequence ends. While full-length transposase, OrfAB, binds poorly in vitro to IS 911 DNA fragments carrying a copy of the IS 911 end, truncated protein derivatives carrying the first 135 (OrfAB[1–135]) or 149 (OrfAB[1–149]) amino acid residues bind efficiently. They generate a paired-end complex containing two such fragments which resembles that expected for the first synaptic complex. Shorter protein derivatives lacking a region involved in multimerisation do not form these complexes but modify the binding of OrfAB[1–135] and OrfAB[1–149]. DNaseI footprinting demonstrated that OrfAB[1–149] protects a sub-terminal (internal) region of the inverted repeats which includes two blocks of sequence (β and γ) conserved between the left (IRL) and right (IRR) ends. DNA binding assays in vitro and measurement of recombination activity in vivo of sequential deletion derivatives of the two inverted repeats suggested a model in which the N-terminal region of OrfAB binds the conserved boxes β and γ in a sequence-specific manner and anchors the two insertion sequence ends into a paired-end complex. The external region of the inverted repeat is proposed to contact the C-terminal transposase domain carrying the catalytic site.