Crystal structure of the specific DNA-binding domain of Tc3 transposase of C.elegans in complex with transposon DNA
1997; Springer Nature; Volume: 16; Issue: 19 Linguagem: Inglês
10.1093/emboj/16.19.6044
ISSN1460-2075
AutoresGertie van Pouderoyen, René F. Ketting, Anastassis Perrakis, Ronald H.A. Plasterk, Titia K. Sixma,
Tópico(s)Fungal and yeast genetics research
ResumoArticle1 October 1997free access Crystal structure of the specific DNA-binding domain of Tc3 transposase of C.elegans in complex with transposon DNA Gertie van Pouderoyen Gertie van Pouderoyen Divisions of Molecular Carcinogenesis, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands Search for more papers by this author René F. Ketting René F. Ketting Divisions of Molecular Biology, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands Search for more papers by this author Anastassis Perrakis Anastassis Perrakis Divisions of Molecular Carcinogenesis, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands Search for more papers by this author Ronald H.A. Plasterk Ronald H.A. Plasterk Divisions of Molecular Biology, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands Search for more papers by this author Titia K. Sixma Corresponding Author Titia K. Sixma Divisions of Molecular Carcinogenesis, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands Search for more papers by this author Gertie van Pouderoyen Gertie van Pouderoyen Divisions of Molecular Carcinogenesis, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands Search for more papers by this author René F. Ketting René F. Ketting Divisions of Molecular Biology, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands Search for more papers by this author Anastassis Perrakis Anastassis Perrakis Divisions of Molecular Carcinogenesis, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands Search for more papers by this author Ronald H.A. Plasterk Ronald H.A. Plasterk Divisions of Molecular Biology, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands Search for more papers by this author Titia K. Sixma Corresponding Author Titia K. Sixma Divisions of Molecular Carcinogenesis, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands Search for more papers by this author Author Information Gertie van Pouderoyen1, René F. Ketting2, Anastassis Perrakis1, Ronald H.A. Plasterk2 and Titia K. Sixma 1 1Divisions of Molecular Carcinogenesis, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands 2Divisions of Molecular Biology, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands *Corresponding author. E-mail: [email protected] The EMBO Journal (1997)16:6044-6054https://doi.org/10.1093/emboj/16.19.6044 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The crystal structure of the complex between the N-terminal DNA-binding domain of Tc3 transposase and an oligomer of transposon DNA has been determined. The specific DNA-binding domain contains three α-helices, of which two form a helix–turn–helix (HTH) motif. The recognition of transposon DNA by the transposase is mediated through base-specific contacts and complementarity between protein and sequence-dependent deformations of the DNA. The HTH motif makes four base-specific contacts with the major groove, and the N-terminus makes three base-specific contacts with the minor groove. The DNA oligomer adopts a non-linear B-DNA conformation, made possible by a stretch of seven G:C base pairs at one end and a TATA sequence towards the other end. Extensive contacts (seven salt bridges and 16 hydrogen bonds) of the protein with the DNA backbone allow the protein to probe and recognize the sequence-dependent DNA deformation. The DNA-binding domain forms a dimer in the crystals. Each monomer binds a separate transposon end, implying that the dimer plays a role in synapsis, necessary for the simultaneous cleavage of both transposon termini. Introduction Tc3 of Caenorhabditis elegans is a member of the Tc1/mariner family of transposable elements. Members of that family are found in a wide variety of organisms, ranging from fungi to humans (Doak et al., 1994). Transposable elements are small stretches of DNA that can move from one position in the genome to another. The proteins responsible for the excision and insertion of the transposon into the genome are generally encoded by the transposon sequences. In the Tc1/mariner family, the transposons encode only a single protein, the transposase, that is capable of performing the entire transposition reaction in vitro (Lampe et al., 1996; Vos et al., 1996). The Tc1/mariner transposase genes are flanked by terminal inverted repeats. The sequences of the terminal inverted repeats are not conserved between different elements, apart from the four most terminal nucleotides. Another shared property of this family is that the transposon DNA is inserted into TA sequences of the host genome (for a review, see Plasterk, 1996). The first step in transposition is the recognition of the transposon DNA by a specific DNA-binding domain of the transposase protein. For Tc3A, the Tc3 transposase, it has been shown that the N-terminal domain is responsible for this specific DNA binding. This domain binds a region ∼12 bases away from the DNA cleavage site (van Luenen et al., 1993; Colloms et al., 1994) (Figure 1). Between the N-terminal specific DNA-binding domain and the catalytic domain, another DNA recognition domain is present. This second, more C-terminal domain recognizes DNA sequences located more towards the cleavage site (R.F.Ketting and R.H.A.Plasterk, unpublished results). Such a bipartite DNA binding has also been shown for the related Tc1 transposase (Tc1A) of C.elegans (Vos and Plasterk, 1994). In general, there is little sequence conservation in the N-terminal regions of Tc1/mariner transposases and no apparent conservation of the inverted repeat sequence. Thus, the specific DNA-binding domain of each transposase of this family recognizes the termini of its own transposon specifically, and the transposase protein of a given element will act only upon its own transposon ends. Figure 1.Schematic representation of Tc3 transposase protein and Tc3 transposon DNA. Shaded boxes indicate which part of the protein and DNA were co-crystallized in this study. The numbering of the DNA oligomer used in this study is indicated. The arrows under the inverted repeats of the DNA indicate the two almost identical binding sites of Tc3A separated by ∼180 bp at each transposon end (Colloms et al., 1994). The binding site region used in this study (indicated by a gray box) is identical in these two sites. The function of the internal binding site is not very clear, because it does not seem essential for efficient transposition (H.G.A.M.van Luenen and R.H.A.Plasterk, personal communication). The sequence of the 65 amino acid N-terminal fragment of Tc3A differs at position 41 (Val instead of Glu) from the sequence in the GenBank database. Download figure Download PowerPoint Protein sequence alignments have revealed a weak similarity between the N-terminal region of the Tc1-like Minos element and a DNA-binding domain of the Pax/paired family, the paired domain, found in mammalian and Drosophila genes (Franz et al., 1994). The paired domain is a conserved DNA-binding domain found in a set of transcription factors (Pax proteins) that play important roles in development. The significance of the similarity is enhanced by the identification of the bipartite nature of the paired DNA-binding domain (Czerny et al., 1993; Xu et al., 1995). The difference from the Tc3A bipartite DNA binding is that the C-terminal part of the paired domain could not be shown to bind to DNA (Xu et al., 1995). However, in other members of the paired domain family (Pax proteins), a C-terminal part plays a role in site-specific recognition (Czerny et al., 1993; Epstein et al., 1994a,b; Xu et al., 1995). Furthermore, secondary structure elements are similar for the Tc1A and Tc3A DNA-binding domains (as predicted with PHDsec, Rost and Sander, 1993). Part of the catalytic domain is also involved in DNA binding. In a South-Western assay, it has been shown that a region between amino acids 98 and 159 shows non-specific DNA-binding activity (Colloms et al., 1994). The catalytic domain of Tc3A shares sequence homology with the IS630-Tc1 family and contains a catalytic triad, DDE motif, of carboxyl groups (Figure 1) (Doak et al., 1994), that is important for the Tc3 transposition activity (van Luenen et al., 1994). Other polynucleotidyl transferases also use three (sometimes four) carboxyl groups to position one or two catalytic divalent metal ions (Yang and Steitz, 1995). This family includes ribonuclease H (Doolittle et al., 1989), retroviral/retrotransposases (Fayet et al., 1990; Kulkosky et al., 1992), Mu transposase (Baker and Luo, 1994) and RuvC resolvase (Ariyoshi et al., 1994). The crystal structures of the catalytic domains of several of these proteins (Katayanagi et al., 1990, 1992; Yang et al., 1990; Ariyoshi et al., 1994; Dyda et al., 1994; Rice and Mizuuchi, 1995) revealed that they share a structurally related core in which the catalytic carboxyl groups are oriented in a similar way. It is likely that the catalytic domain of the Tc3 transposase has a similar three-dimensional fold. The detailed mechanism of transposition has many variations, for example in invoking single or double strand breaks of the host DNA, in the target site specificity or in the number of proteins involved (for a review, see Plasterk, 1995). Synapsis (assembly of multiple proteins, the transposon DNA ends and the target DNA) is thought to be required for proper transposition. This has been studied extensively for phage Mu, in which tetramerization of the transposase on the transposon ends is essential for transposition (for a review, see Chaconas et al., 1996). Another example of the importance of synapsis has been shown for the Moloney murine leukemia virus. When one of the two ends of this viral DNA is mutated to block cleavage, cleavage is inhibited at the other (wild-type) end as well. This indicates that cleavage at either end (the first step in the integration process) only takes place when both ends contribute to the synapsis and are recognized by the integrase protein (Murphy and Goff, 1992). To understand how the Tc3 transposase recognizes its own transposon DNA ends, we studied the structure of the N-terminal specific DNA-binding domain (Tc3A-N) in complex with transposon DNA. This is the first crystal structure of a DNA-binding region of a transposase in complex with DNA. With this, we can begin to understand the mode of recognition by transposases of their DNA substrates, and eventually the mode of binding and regulation of simultaneous cleavage at the two ends of the transposon. Results and discussion The overall structure of the protein–DNA complex We have determined the X-ray structure of the 65 amino acid DNA-binding domain of Tc3 transposase (Tc3A-N) in complex with transposon DNA (Figure 2A) at 2.45 Å resolution. The parts of the transposase and transposon used in co-crystallization are indicated by shaded boxes in Figure 1. The structure shows that Tc3A-N contains three α-helices (residues 9–20, 25–32 and 36–44) typical of proteins sharing the helix–turn–helix (HTH) motif, such as homeodomains. The first helix is involved in dimerization of the protein domains in the crystal. Each monomer binds a DNA molecule. The second and third helix form the HTH motif and are involved in DNA recognition in the major groove. The N-terminus of the protein interacts with DNA in the locally narrowed minor groove. The C-terminus (residues 53–65 and the 6-histidine tag) is not visible in the final electron density, probably due to flexibility. The 20/21 DNA oligomer is in a non-linear B-DNA conformation and both the major and minor groove interact with the protein. Figure 2.Protein–DNA contacts. (A) A schematic view with ribbons drawn through the Cαs of the Tc3A DNA-binding domain (yellow) and through the phosphate backbone of the DNA strands (blue and magenta). (B) Sketch summarizing the hydrogen bonding (indicated by green dotted lines, base-specific H-bonds by green solid lines) and salt bridging contacts (blue lines) between the Tc3A domain and the DNA. Gray boxes indicate residues involved in base-specific contacts. Hydrogen bonds are at a maximum distance of 3.5 Å and salt bridges at a maximum of 4.0 Å (Barlow and Thorton, 1983). (C) and (D) Stereo views (Kraulis, 1991) of the HTH DNA contacts in the major groove, and the N-terminus of Tc3A bound in the minor groove of DNA, respectively. Hydrogen bonds are indicated with green dotted lines. Download figure Download PowerPoint The HTH motif makes base-specific contacts in the major groove Part of the recognition process of Tc3A for its own transposon DNA is mediated by direct readout of four bases by the HTH motif. The HTH motif, comprising helices 2 and 3, interacts in the major groove of the DNA. There are also extensive contacts with mainly one DNA backbone strand: 10 H-bonds and seven salt bridges (Figure 2B and C). Within the HTH motif, the N-terminus of helix 2 is involved in extensive contacts with the phosphate groups in one DNA backbone strand. However, one side chain (His26) is making a purine-specific hydrogen bond to G7 (for base numbering see Figure 1). Backbone amides of Leu25 and His26 make hydrogen bonds to phosphate group 7. The dipole moment (Hol et al., 1978) of helix 2 interacts favorably with phosphate group 7 as well. The contacts with one of the DNA backbones are consolidated by Ser24 (in the loop connecting helices 1 and 2) and Arg30, making hydrogen bonds to phosphate group 6. More salt bridges are made by His26 with phosphates 6 and 7 and by Arg30 with phosphates 5 and 6. Two amino acids in the turn of the HTH motif make contacts with the other DNA backbone: Arg34 is hydrogen bonded to sugar 109 and salt bridged to phosphate 110. Ser35 donates a hydrogen to phosphate 110. The N-terminal part of helix 3 (the recognition helix in the HTH motif) is involved in base-specific contacts with three bases (including one mediated via a water molecule) and several DNA backbone contacts. Arg36 makes one guanine- and one purine-specific hydrogen bond to the guanine base at position 8. Arg40 makes a thymine-specific hydrogen bond, via a water molecule, to T9. His37 recognizes G110 via a hydrogen bond to the purine-specific N7 or to the guanine-specific O6 of G110. We are not able to distinguish this at the current resolution. Helix 3 makes additional hydrogen bonds through Arg36 via a water molecule to phosphates 7 and 8 and through Cys38 to phosphate 110. Salt bridges are made by both Arg36 and Arg40 to phosphate group 8. Minor groove interactions Additional base-specific recognition (of three bases closer to the DNA cleavage site) occurs by binding of the N-terminus in the minor groove. Six more hydrogen bonds are made with the DNA backbone by both the N- and C-termini of the domain (Figure 2). The side chains of residues Pro2 and Arg3 are inserted in the minor groove. Since Met1 is missing (as shown by sequencing of the purified protein), Pro2 is at the N-terminus and thus positively charged. The two positively charged N-terminal residues bind like a thumb and finger between the negatively charged phosphate backbones of the locally relatively narrow minor groove (Figure 2D). Pro2 makes a pyrimidine-specific hydrogen bond to T14 and a hydrogen bond to sugar 15. The rest of Pro2 is in van der Waals contact with sugars 15 and 108. Arg3 makes one pyrimidine-specific hydrogen bond to T12 and one to sugar 12. The backbone amide of Arg3 most likely makes a purine-specific H-bond to A109, but a H-bond to sugar 109 cannot be excluded. The backbone amide of Gly4 is hydrogen bonded to sugar 109 and the backbone amide of Ala6 to phosphate 16. The last ordered amino acids of the C-terminus (49–52) are close to the minor groove as well. Tyr49 OH and Ser52 Oγ are donating hydrogens to phosphate groups 109 and 108 respectively. There is some electron density visible beyond Ser52 in the minor groove, although it is not continuous and cannot be interpreted, indicating that the flexible C-terminus is continuing along the minor groove towards the DNA cleavage site. Protein dimer The Tc3A domain behaves like a 25 kDa protein on a gel filtration column, while the mol. wt is only 8.3 kDa. The 19 flexible C-terminal amino acids are likely to cause a shift to higher molecular weight; however, the difference between the expected 8.3 kDa for a monomeric domain and the observed 25 kDa peak cannot be attributed to that only. It is more likely that Tc3A-N is forming a dimer in solution. In the crystal, we observe protein dimers as well, in which each protein monomer binds one DNA molecule. Helix 1 is involved in the dimerization of two protein domains by a 2-fold crystallographic rotation. Each protein domain makes, at one side, contact with DNA and, at the opposite side, contact with a protein of a symmetry-related complex (Figure 3). The accessible surface of a monomer of the Tc3A domain is 3999 Å2 (calculated with GRASP, Nicholls et al., 1991). Upon dimerization with another Tc3A domain, 12% (475 Å2) of this surface is buried on each monomer. The contact, which involves mainly helix 1 of each domain, is predominantly hydrophobic (24 van der Waals contacts), and only two hydrogen bonds occur (between the carbonyl oxygen of Ala13 of both molecules and the Nϵ of Gln14 of the symmetry-related molecules). The dimer found in the crystals, and presumably in solution, may also be present in active transposition complexes. Figure 3.Ribbon diagram displaying the dimer of the Tc3A DNA-binding domains with the two DNA oligomers of the transposon ends bound. Download figure Download PowerPoint DNA conformation and recognition through sequence-dependent conformation The DNA in the crystallized complex is not in a linear conformation (Figure 2A), but bent at both ends in different directions and planes. The bending of the DNA is stabilized (Strauss and Maher, 1994) by the large amount of positive charge on the protein, which is located mainly at the interface with DNA (Figure 4). Figure 4.The Tc3A domain bound to DNA. The protein is shown in an electrostatic surface representation with positively and negatively charged regions in blue and red respectively (GRASP-scale −10 to +10). DNA is shown in stick representation, with carbons in white, nitrogens in blue, oxygens in red and phosphors in yellow. Download figure Download PowerPoint The conformation of the DNA was analyzed with the program CURVES (Lavery and Sklenar, 1989) using the global parameters. The average helical twist of 33.5° (10.7 residues per turn) and the average rise per base pair of 3.41 Å are typical for B-DNA. The bends of the DNA are reflected in increases in the roll and tilt angles of the base pairs and in deviations of the major and minor groove widths and depths, compared with average B-DNA (Stofer and Lavery, 1994). To define the sugar puckers, higher resolution data will be needed. DNA sequences highly enriched in G:C base pairs have a tendency to form low twist angles (underwinding) and positive roll angles of the base pairs, resulting in a widening of the minor groove and narrowing of the major groove (Travers, 1993). The seven consecutive G:C base pairs (2:119–8:113) in this structure indeed have a relatively low twist angle (30.5° on average) and a positive roll angle (∼12° per base pair). The roll angles of this stretch add up to an 82° bend of the DNA. In the crystal, one end of each DNA molecule bends into the minor groove of a symmetry-related DNA molecule, with the base pairs of the two DNA molecules almost perpendicular (Figure 5A). Part of this bending is visible in the top part of Figure 2A as a DNA bend to the right, but it is also partly away from the viewer. Figure 5.Stereo view (Kraulis, 1991) of the DNA stacking in the crystal at both ends of the DNA oligomer. (A) View of one end along the 2-fold screw axis in the c-direction (water molecules are indicated as dots) and (B) view of the other end along the 2-fold axis in the a-direction. Download figure Download PowerPoint At the position of the bent G:C stretch, Tc3A-N makes numerous hydrogen bonds and salt bridges to only one of the DNA backbones (phosphates 5–8, Figure 2B and C). These contacts would not have been possible if the DNA was more linear. It is not clear whether the bend is present in the DNA alone, to which the protein adapts itself, or whether the bend is induced by the interaction with the protein. A third theoretical possibility, that the bend is caused or stabilized by the crystal contacts, seems unlikely given the exquisite complementarity of the protein and the deformed DNA. The bending at the other end of the DNA oligomer (more towards the DNA cleavage site in the lower part of Figure 2A) is reflected in mainly negative roll and tilt angles (around base pairs 13:108 and 14:107) and a narrowing of the minor groove (4 Å compared with 6 Å for average B-DNA; Stofer and Lavery, 1994). The bending is ∼30° and the direction is to the right in Figure 2A, and partly towards the viewer. This part of the DNA sequence contains AT bases, which have a tendency to form a narrow minor groove (Travers, 1993). In this narrow minor groove, the N-terminus is inserted, making hydrogen bonds and van der Waals contacts with both DNA backbones (Figure 2D). Such intimate protein–DNA contacts would not be possible with a wider minor groove. Again, the Tc3A domain is complementary to the sequence-dependent DNA conformation, contributing to the recognition by Tc3A of its own transposon DNA. Unusual DNA–DNA crystal contacts The DNA helix axis is roughly in the crystallographic b-direction. This is visible in the diffraction pattern as a fiber diffraction pattern at 3.4 Å along the b-axis. Although the DNA is mainly parallel to the b-axis, its non-linearity causes unusual crystal contacts. The A1 and T120 bases at one end of the oligomer do not form the expected base pair, but are flipped outwards. These bases, together with the last base pair (G2–C119), are interacting in the minor groove and are almost perpendicular to the base pairs of a symmetry-related DNA molecule (Figure 5A). Fraying of a DNA molecule at one end has been observed before (Joshua-Tor et al., 1992), where the flipped-out bases are also interacting in the minor groove of a symmetry-related molecule. However, the base pairs of the symmetry-related molecule are not perpendicular, as in this case. The other end of the DNA forms a shifted semi-continuous helix with the same end of a symmetry-related DNA molecule (Figure 5B). These two molecules are related by a 2-fold rotation axis in the a-direction (perpendicular to the paper in Figure 5B). The overhanging base T21 makes a triple helix by forming a Hoogsteen base pair with the symmetry-related base A101 in base pair A101:T20. Comparison with other proteins The three-dimensional fold of the Tc3A domain is a HTH fold. Proteins sharing this motif are the eukaryotic homeodomains and transcription factors, prokaryotic repressors and Hin recombinase, amongst others (for a review, see Luisi, 1995; Wintjens and Rooman, 1996). The Tc3A domain is most similar to the N-terminal part of the paired domain (Figure 6, Xu et al., 1995). The r.m.s. difference of 51 Cαs is 2.3 Å, according to the 3D alignment of the DALI-server) http://www.embl-heidelberg.de/d…; Holm and Sander, 1993), and the r.m.s. difference of the Cαs in the three helices and connecting loops is only 0.66 Å. Other similar structures are, for example, the Oct-1 POU domain (Klemm et al., 1994), C-myb DNA-binding domain (Ogata et al., 1995), engrailed homeodomain (Kissinger et al., 1990) and the paired homeodomain (Wilson et al., 1995). The three helices and connecting loops have r.m.s. Cα differences with Tc3A-N of 1.3–1.5 Å. Figure 6.A ribbon representation of the superposition of the Tc3A domain (light gray) and the equivalent part of the paired domain (dark gray), with their respective DNA molecules bound. Note the bending of the Tc3 oligomer in contrast to that bound to paired. Download figure Download PowerPoint Surprisingly, the recently determined Zn-containing N-terminal region of the functionally similar HIV integrase also contains a comparable HTH motif (Cai et al., 1997; Eijkelenboom et al., 1997). Superposition with the three helices and connecting loops of Tc3A-N results in a Cα r.m.s. deviaton of 2.0 Å. The C-terminus, however, folds in a different direction, where it provides two of the ligands to the Zn ion, which is coordinated further by a histidine in helix 1 and a histidine in the loop between helices 1 and 2. Data on the function of this domain are not clear, and it is unknown whether it is involved in DNA binding, although the presence of the HTH domain is suggestive. The main differences between the N-terminal part of the paired domain and Tc3A-N are located in their N- and C-termini. The few residues preceding the first helix in the Tc3A domain adopt a conformation different from that of the longer N-terminus of the paired domain, which forms a small β-sheet (Figure 6). Helix 3 is longer in the paired domain than in the Tc3A domain, but the direction of the C-termini is similar. In the paired domain, the C-terminus is connected to the second domain, but the linker is not clearly visible in the electron density (Xu et al., 1995). In our crystals, only the loop towards the second DNA recognition domain (residues 53–65) is present, and it is invisible in the electron density as well. Docking on DNA It has been described earlier that there are several ways of docking an HTH motif on DNA (for example, see Suzuki and Gerstein, 1995; Wintjens and Rooman, 1996). The Tc3A-N protein is very similar to the N-terminal part of the paired domain and homeodomains. These have, however, a different way of docking on DNA. The homeodomains have only helix 3 (the recognition helix) inserted in the major groove, and the residues in the center of this helix are interacting with DNA. This recognition helix is relatively long and there are common features in the amino acid sequence playing a role in DNA interaction (Suzuki, 1993). The paired domain belongs to another family, which also includes the prokaryotic Hin recombinase and λ repressor. In this family, both helices 2 and 3 interact with DNA, and this family is distinguished by a relative short helix 2 of which the N-terminus interacts with the DNA backbone. Helix 3 is also interacting in the major groove, but at a different angle compared with the homeodomain family (Kissinger et al., 1990; Xu et al., 1995). The HTH motif of the Tc3A domain docks in a variant of the paired/Hin/λ family, although the non-linear DNA in the Tc3A complex is exceptional. There are nine residues (18%) in Tc3A-N that are identical to the N-terminal part of the paired domain. None of these residues are present at equivalent positions in the Hin recombinase (Feng et al., 1994) or the λ repressor (Beamer and Pabo, 1992). Only three of these residues (Arg30, Ser35 and Cys38 of Tc3A) are involved in similar DNA contacts. None of these are sequence specific; all three interact with the phosphate groups in the DNA backbone. The absence of conserved residues that interact with DNA is in contrast to the homeodomain family, which has anchor residues. The locations within the HTH fold of the side chains that interact with the bases of the DNA are not very conserved either. Some side chains are located at the N-terminus of helix 3, but not at identical positions. Others are situated towards in the center of helix 3, but again not at a fixed position. Tc3A-N has one exceptional side chain in helix 2 (His26) that is involved in a base contact. Correlation with biochemical data The double-stranded DNA oligomer used in this study was based on methylation interference (12 bp from G7 to A18) and footprinting (∼20 bases on each strand) studies (Colloms et al., 1994). The adenine and guanine bases which showed strong methylation interference are indeed in contact with the protein. A few bases with weak interference (G16, A17 and A18) are located where the flexible C-terminus of Tc3A-N is pointing. The middle of the 20/21 oligomer in the crystal is in contact with the protein, both in the major and minor groove, consistent with the prediction based on the methylation and footprinting studies (Colloms et al., 1994). The same 65 amino acid N-terminal domain with a histidine tag at the N-terminus (instead of the C-terminus as used in this study) did not bind transposon DNA, as shown by gel retardation assays (data not shown). The N-terminal residues of the protein (Pro2 and Arg3) are bound in the minor groove, and a histidine tag at the N-terminus would probably interfere with these contacts, making a protein–DNA complex impossible. Whether the N-terminal Met1 (absent in this study) is also absent in vivo in C.elegans is unclear. The base-specific contacts of Pro2 could be disturbed if Met1 were present, suggesting that Met1 is also absent in vivo. We were not able to see the amino acids beyond Ser52, although some electron density in the minor groove suggests that the chain is continuing along the minor groove in analogy with the loop in the paired domain. A smaller N-terminal part of Tc3A (amino acids 1–54) has been shown, however, not to bind to Tc3 transposon DNA (Colloms et al., 1994). This indicates t
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