Mutants of Tn3 resolvase which do not require accessory binding sites for recombination activity
1999; Springer Nature; Volume: 18; Issue: 5 Linguagem: Inglês
10.1093/emboj/18.5.1407
ISSN1460-2075
Autores Tópico(s)DNA Repair Mechanisms
ResumoArticle1 March 1999free access Mutants of Tn3 resolvase which do not require accessory binding sites for recombination activity Patricia H. Arnold Patricia H. Arnold Institute of Biomedical and Life Sciences, University of Glasgow, 56 Dumbarton Road, Glasgow, G11 6NU UK Search for more papers by this author David G. Blake David G. Blake Cyclacel Ltd, Dundee Technology Park, 5 Whitehall Crescent, Dundee, DD1 4AR Scotland, UK Search for more papers by this author Nigel D.F. Grindley Nigel D.F. Grindley Department of Molecular Biophysics and Biochemistry, Yale University, PO Box 208114, 266 Whitney Avenue, New Haven, CT, 06520-8114 USA Search for more papers by this author Martin R. Boocock Martin R. Boocock Institute of Biomedical and Life Sciences, University of Glasgow, 56 Dumbarton Road, Glasgow, G11 6NU UK Search for more papers by this author W.Marshall Stark Corresponding Author W.Marshall Stark Institute of Biomedical and Life Sciences, University of Glasgow, 56 Dumbarton Road, Glasgow, G11 6NU UK Search for more papers by this author Patricia H. Arnold Patricia H. Arnold Institute of Biomedical and Life Sciences, University of Glasgow, 56 Dumbarton Road, Glasgow, G11 6NU UK Search for more papers by this author David G. Blake David G. Blake Cyclacel Ltd, Dundee Technology Park, 5 Whitehall Crescent, Dundee, DD1 4AR Scotland, UK Search for more papers by this author Nigel D.F. Grindley Nigel D.F. Grindley Department of Molecular Biophysics and Biochemistry, Yale University, PO Box 208114, 266 Whitney Avenue, New Haven, CT, 06520-8114 USA Search for more papers by this author Martin R. Boocock Martin R. Boocock Institute of Biomedical and Life Sciences, University of Glasgow, 56 Dumbarton Road, Glasgow, G11 6NU UK Search for more papers by this author W.Marshall Stark Corresponding Author W.Marshall Stark Institute of Biomedical and Life Sciences, University of Glasgow, 56 Dumbarton Road, Glasgow, G11 6NU UK Search for more papers by this author Author Information Patricia H. Arnold1,2, David G. Blake3, Nigel D.F. Grindley4, Martin R. Boocock1 and W.Marshall Stark 1 1Institute of Biomedical and Life Sciences, University of Glasgow, 56 Dumbarton Road, Glasgow, G11 6NU UK 2Gynecology and Breast Research Laboratory, Sloan-Kettering Memorial Cancer Center, 1275 York Avenue, New York, NY, 10021 USA 3Cyclacel Ltd, Dundee Technology Park, 5 Whitehall Crescent, Dundee, DD1 4AR Scotland, UK 4Department of Molecular Biophysics and Biochemistry, Yale University, PO Box 208114, 266 Whitney Avenue, New Haven, CT, 06520-8114 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (1999)18:1407-1414https://doi.org/10.1093/emboj/18.5.1407 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Tn3 resolvase promotes site-specific recombination between two res sites, each of which has three resolvase dimer-binding sites. Catalysis of DNA-strand cleavage and rejoining occurs at binding site I, but binding sites II and III are required for recombination. We used an in vivo screen to detect resolvase mutants that were active on res sites with binding sites II and III deleted (that is, only site I remaining). Mutations of amino acids Asp102 (D102) or Met103 (M103) were sufficient to permit catalysis of recombination between site I and a full res, but not between two copies of site I. A double mutant resolvase, with a D102Y mutation and an additional activating mutation at Glu124 (E124Q), recombined substrates containing only two copies of site I, in vivo and in vitro. In these novel site I×site I reactions, product topology is no longer restricted to the normal simple catenane, indicating synapsis by random collision. Furthermore, the mutants have lost the normal specificity for directly repeated sites and supercoiled substrates; that is, they promote recombination between pairs of res sites in linear molecules, or in inverted repeat in a supercoiled molecule, or in separate molecules. Introduction Tn3 resolvase is a member of a large family of related site-specific recombinases which includes the γδ and Tn21 resolvases, and the DNA invertases Gin and Hin. The structure of γδ resolvase has been solved both alone and in complex with res binding site I (Sanderson et al., 1990; Yang and Steitz, 1995). Tn3 resolvase differs from γδ resolvase in only 35 residues, and its structure is thought to be very similar (reviewed in Grindley, 1994). The typical reaction promoted by resolvase in vitro is depicted in Figure 1. A supercoiled plasmid containing directly repeated 114 bp res sites (analogous to the cointegrate intermediate of Tn3 replicative transposition) is resolved into two smaller circular molecules, each with one res site. The two circles are linked as a simple catenane (Figure 1). Figure 1.(A) The reaction catalysed by Tn3 resolvase. A supercoiled plasmid (represented as a circle) containing directly repeated res sites (represented as arrowheads) is recombined to yield two product circles, which are linked as a simple (2-noded) catenane. (B) The recombination site, res. The three resolvase-binding sites are shown as boxes (site I, pale grey; sites II and III, dark grey). The arabic numbers indicate the lengths of the various segments in base pairs. The staggered line in site I shows where resolvase breaks and rejoins the DNA during recombination. (C) A model for the productive synaptic complex (greatly simplified). Double-stranded DNA is represented by thick black lines; res accessory sites by dark grey boxes; binding site Is by pale grey boxes; and resolvase subunits by shaded balls. The dashed arrows indicate possible contacts between resolvase/DNA at the accessory sites and at the site Is. Download figure Download PowerPoint Studies on several resolvase/res systems in vitro have provided some insight into the intermediates of the reaction, and have led to models for how selectivity for recombination between directly repeated res sites, and unique product topology are achieved (reviewed in Grindley, 1994; Stark and Boocock, 1995). Resolution is proposed to require formation of a synaptic intermediate in which the 'accessory' binding sites II and III of the two res sites are intertwined, allowing juxtaposition and subsequent catalysis of strand exchange at the two site Is (Figure 1). The functions of the accessory binding sites (and resolvase bound there) are therefore proposed to be 'architectural' and regulatory. They may be crucial in holding the two copies of res together prior to recombination, the specific intertwining of the accessory sites required to form the synaptic complex is implicated in topological selectivity, and the structure which they create is somehow necessary for activation of the catalytic function of resolvase bound at site I (Benjamin and Cozzarelli, 1988; Grindley, 1993; Stark et al., 1994; Watson et al., 1996). It is not clear whether this activation is purely architectural (that is, by holding the two site Is together in a configuration suitable for catalysis), or if there are direct 'activating' contacts of resolvase subunits bound at sites II and III with those bound at site I. The DNA invertases Gin and Hin are clearly related to resolvase in sequence and mechanism, and also are topologically selective, but recombination at the gix and hix sites does not require accessory invertase-binding sites. However, recombination does require an enhancer element which binds the host protein FIS, within the same supercoiled substrate molecule. Models for invertase recombination, in which the FIS-bound enhancer forms a synaptic complex with the gix (or hix) sites, are strikingly similar to the model for resolvase recombination described above, except that the FIS–enhancer complex takes the place of the intertwined sites II and III of res (Kanaar et al., 1989; Heichman and Johnson, 1990). Recent mutational analyses suggest that activation involves direct contacts of specific residues of FIS with Gin/Hin subunits (Spaeny-Dekking et al., 1995; Deufel et al., 1997; Safo et al., 1997). It proved possible to isolate mutants of the invertases which do not require the FIS–enhancer complex to promote inversion (Haffter and Bickle, 1988; Klippel et al., 1988). FIS-independent Gin mutants have been studied in detail. These mutants have lost selectivity for inverted sites; that is, they recombine sites in direct repeat or on different molecules. Also, whilst the natural reaction gives exclusively unknotted circular inversion products, the mutants give products with many different topologies, consistent with recombination after synapsis by random collision (Klippel et al., 1993; Crisona et al., 1994). It has been shown that the requirement of resolvase for two full res sites could be reduced by careful choice of in vitro reaction conditions, so that some recombination could be observed between a full res and a site I in a supercoiled plasmid (Bednarz et al., 1990). However, the efficiency of reaction was very low; also, wild-type resolvase does not promote this reaction to a detectable level in vivo (Wells and Grindley, 1984; see below). Here, we report the isolation of resolvase mutants which have reduced or zero requirement for accessory sites to promote recombination. We describe the unusual properties of the mutants in vitro, and discuss implications for the mechanisms of activation and catalysis. Results Isolation of mutants Our first attempts at isolation of activating resolvase mutants by chemical mutagenesis of the whole reading frame were unsuccessful (D.G.Blake, unpublished). We decided, therefore, to focus mutagenesis on a specific part of the reading frame (94–120, corresponding to amino acid residues of the E-helix and the preceding β-strand and loop; Figure 2B) which we thought most likely to yield activating mutants (see Discussion). Our strategy was to create random mutations in this region, by ligating a double-stranded oligonucleotide containing random changes from the canonical sequence into a gap made by cutting the resolvase-encoding DNA at two appropriate restriction enzyme sites. In order to allow mutagenesis of this and other selected regions of resolvase, a large part of the reading frame DNA was reassembled from double-stranded synthetic oligonucleotides, thereby introducing unique restriction sites at appropriate intervals by basepair substitutions which did not affect the amino acid translation (Figure 2B). Mutant DNA libraries were made from pAT5, a plasmid which gave a low level of resolvase expression found to be suitable for in vivo screening. The libraries were used to transform the Escherichia coli strains DS941/pDB34, DS941/pDB35 and DS941/pDB37. DS941 is a galK− strain; pDB34, pDB35, and pDB37 are low copy number plasmids with a galK gene bounded by potential recombination sites (pDB34, res×res; pDB35, site I× site I; pDB37, res×site I; Figure 2A). Resolution of the test plasmid separates the galK gene from the origin of replication and antibiotic resistance gene, and progeny cells become galK−. galK− colonies are pale yellow ('white') on MacConkey agar plates containing galactose, and galK+ colonies are red. Figure 2.(A) Left, the plasmids used for in vivo screening of resolvase mutants, pDB34 (res×res), pDB35 (site I×site I), and pDB37 (res×site I). See Materials and methods for further details. The res elements are indicated by arrowheads. The gene marked repA encodes an initiator protein required at the pSC101 origin of replication (Manen and Caro, 1991). Right, pAT5, the resolvase-expressing plasmid used for the in vivo screening experiments. (B) Schematic representation of the modified resolvase coding sequence in pAT5, as used for the mutagenesis. The restriction sites marked are not in the natural resolvase coding sequence, except for the BamHI site, and are unique in pAT5, except for PstI. The secondary structure of resolvase indicated is as in Yang and Steitz (1995). The α-helices are represented as boxes, and the β-sheet components with vertical dashes. Download figure Download PowerPoint No mutants were found which could give sufficient resolution of a test plasmid with two copies of site I to give white colonies (∼5000 mutants were screened). However, a few white colonies were observed when the test plasmid had one res and one site I (pDB37). Visualization of the cell DNA after agarose gel electrophoresis showed that the test plasmid was resolved in these colonies (data not shown). The resolvase-expressing plasmids were isolated and re-tested for their ability to give white colonies on retransformation into the test strain (Figure 3). The resolvase reading frames of candidate mutant pAT5 plasmids were then sequenced. Figure 3.In vivo screening of the activity of resolvases and mutants. The MacConkey agar plates are arranged so that in each panel, the upper left sector contains colonies of DS941/pDB34 (res×res) transformed with the pAT5-derived resolvase-expressing plasmid, the upper right sector is from DS941/pDB37 (res×site I), and the bottom sector is from DS941/pDB35 (site I×site I). The pDB test plasmid has been resolved in 'white' (pale yellow) colonies, but not in red colonies. See Materials and methods for further details. Download figure Download PowerPoint All of the pAT5 variants isolated by virtue of their ability to resolve the res×site I test plasmid were found to carry mutations leading to substitutions at residue D102 and/or residue M103. The single substitutions D102Y and D102V were sufficient for a 'white' phenotype, as was M103I. D102Y had the strongest phenotype. Several mutants were isolated which had multiple changes, but all were mutant at D102 and/or M103. These were: D102A/M103I; D102Y/A113T; D102Y/Q116H; D102I/M103W; D102T/M103T; and I97V/G101S/D102Y/A113T. Further screens using PCR-based mutagenesis of the entire resolvase catalytic domain produced several 'white' mutants, which were also altered at D102 (S.Wenwieser and M.R.Boocock, data not shown). A γδ resolvase mutant E124Q has recently been reported to be hyperactive in vitro; that is, it promotes recombination between res sites on linear molecules, unlike wild-type γδ resolvase, and also gives enhanced amounts of products in which the DNA has been cleaved in both strands at the centre of site I (M.R.Boocock, X.Zhu and N.D.F.Grindley, manuscript in preparation). We created the equivalent Tn3 resolvase mutant (also E124Q) by cloning synthetic oligonucleotides between the BamHI and EagI sites of the 'cassetted' reading frame (Figure 2B). Expression of Tn3 E124Q resolvase in vivo did not promote resolution of the res×site I or site I×site I test plasmids. Surprisingly, however, an equivalent plasmid expressing the γδ resolvase E124Q mutant did give white colonies, indicative of resolution, with the res×site I substrate plasmid (Figure 3). A Tn3 resolvase D102Y/E124Q double mutant derivative of pAT5 was made by splicing the D102Y and E124Q coding sequences. Expression of the D102Y/E124Q resolvase promoted resolution of both res×site I and site I×site I substrates in vivo (Figure 3). In vitro properties of resolvase mutants The three Tn3 resolvase mutants, D102Y, E124Q and D102Y/E124Q, were overexpressed and purified. All three mutants were unimpaired in their ability to resolve a standard substrate with directly repeated res sites (pMA21; Figure 4B). Tn3 E124Q resolvase had some tendency to give enhanced levels of products with double-strand breaks at site I, but this property was much less marked than in the case of the γδ resolvase E124Q mutant (M.R.Boocock, X.Zhu and N.D.F.Grindley, manuscript in preparation). However, only D102Y and D102Y/E124Q were active in vitro on a res×site I substrate (pAL265) under standard conditions (Figure 4C), and only the double mutant showed any recombination activity under any conditions on a site I×site I substrate (pAL225; Figure 4D; data not shown). Recombination by the D102Y and D102Y/E124Q mutant resolvases is most efficient with the 'standard' substrate pMA21, and least efficient with pAL225 which has no accessory sites (Figure 4), showing that although the accessory sites are no longer required, they still stimulate recombination. Figure 4.Recombination activities of mutant resolvases in vitro. (A) The plasmids used in the in vitro experiments. res is represented as a black arrowhead, and site I as an unfilled arrowhead. The numbers indicate the positions of cleavage by restriction enzymes and by resolvase (i.e. centre of binding site I of res). (B) Reactions of pMA21 with Tn3 resolvase and mutants. Following incubation with resolvase for 1 h, each sample was divided into three equal aliquots and treated as indicated (see Materials and methods). nc, nicked circular substrate (pMA21); sc, supercoiled plasmid substrate; cat, supercoiled catenane resolution product; non-r, non-recombinant PstI + HindIII digestion products; rec, recombinant PstI + HindIII digestion products; hcat, catenane resolution product, with one supercoiled and one nicked DNA circle; rc, free circular resolution product; 2, 4, products with two topological nodes (i.e. fully nicked catenane) or four nodes (probably knot iteration product). (C) Reactions of pAL265 with Tn3 resolvase and mutants. Following incubation with resolvase for 5 h, samples were treated as in the legend to (B). The annotation of the gels is as in (B); inv, bands from PstI + HindIII digestion of inversion recombination products. (D) Reactions of pAL225 with D102Y/E124Q resolvase. Following incubation for 5 h, samples were treated as in (B). The annotation is as in (B and C); inter, products of intermolecular reactions. (E) Reactions of pMA21, linearized prior to reaction with PstI + HindIII, with Tn3 resolvase and mutants. After reaction with resolvase for 16 h, samples were loaded directly onto the gel. The annotation is as in (D). Download figure Download PowerPoint The topologies of the reaction products were analysed by nicking the products with DNase I, followed by agarose gel electrophoresis (Figure 4). The major or exclusive product from treatment of the res×res substrate pMA21 with each of the four proteins was a simple (2-noded) catenane (Figure 4B); restriction digests confirmed that resolution products were predominant. Both D102Y and D102Y/E124Q recombined the res×site I substrate pAL265. Again, the predominant restriction pattern was that of resolution, and the products were mainly 2-noded catenanes, although there was more evidence of products with alternative topologies (3- and 4-noded; Figure 4C). The behaviour of D102Y/E124Q with the site I×site I substrate pAL225 was quite different; approximately equal amounts of resolution and inversion products were observed in restriction digests, and the products were of varying topologies, as evidenced by the 'ladder' of topologically complex monomeric products, and free circular resolution products, observed by gel electrophoresis after nicking (Figure 4D). Both intra- and intermolecular recombination had occurred. The monomeric products with greater mobility than nicked substrate circle in Figure 4D are the result of intramolecular reactions, whereas the species running more slowly than nicked circle are products of intermolecular reactions. D102Y and D102Y/E124Q had the ability to recombine res sites on linear DNA (Figure 4E), indicating loss of the normal requirement for supercoiling in the substrate. These two mutants also promoted recombination between res sites in inverted repeat in a supercoiled plasmid (data not shown); this reaction is not observed with wild-type resolvase. We carried out assays for binding of resolvase to res and binding site I of res, and for synapsis of full or partial res sites, to determine how, if at all, the mutants were affected in these properties. Binding of D102Y to res (Figure 5) was essentially identical to binding of the wild-type protein; a pattern of six bands corresponds to binding of six resolvase monomers, and the three alternate more intense bands correspond to cooperative formation of one, two or three on-site dimers (Blake et al., 1995; D.G.Blake, unpublished results). Similarly, binding to site I gave the characteristic weak monomer and strong dimer complexes. E124Q showed an aberrant binding pattern, consistent with less stable complex formation, and increased representation of the complexes of resolvase monomers. The double mutant D102Y/E124Q resembled E124Q in its binding properties. Synapsis of a pair of res sites can be detected by gel electrophoresis after protein–protein crosslinking (Watson et al., 1996); the three mutant resolvases and wild-type resolvase gave similar yields of synaptic complex in this assay (data not shown), despite the differences in binding noted above. No synapsis by any of the proteins was detectable in a plasmid containing two isolated site Is. Figure 5.Binding of Tn3 resolvase and mutants to res and site I. U, unbound DNA fragment. Numbers indicate the predicted numbers of resolvase monomers in the complexes. Download figure Download PowerPoint Discussion We have shown that mutation of just two amino acid residues confers on Tn3 resolvase the ability to promote recombination in the complete absence of the accessory binding sites II and III. Below, we consider what aspects of resolvase enzymology might have been changed by these mutations. Recombination between a complete res and site I, catalysed by wild-type resolvase, has been observed previously (Bednarz et al., 1990). The reaction was much slower than the equivalent res×res reaction, but surprisingly, the specificity of the natural reaction was maintained; only resolution products, and not inversion products, were seen, and the resolution product had the normal 2-noded catenane topology. It was suggested that non-specific DNA adjacent to site I was recruited into a synaptic complex with architecture similar to the normal one. The properties of the D102Y mutant suggest a similar scenario, since the reaction of the res×site I substrate gives a predominantly 2-noded catenane resolution product. The enhanced activity of the mutant on this substrate could be explained by enhanced ability to form a 'normal' synaptic complex using surrogate accessory site DNA, or the catalytic activity of the mutant could be less demanding of a specific synaptic complex structure. We favour the latter alternative, given the properties of the D102Y/E124Q double mutant, and the apparent lack of enhancement of synapsis by D102Y (see Results). The behaviour of the double mutant on res×res and res×site I substrates (predominance of resolution and 2-noded catenane products) also indicates a preference for reaction in a 'normal' synaptic complex, although the larger amounts of non-standard product topologies and evidence of inversion products in the restriction digests suggest reduced selectivity. In the site I×site I reactions catalysed by the double mutant resolvase, equal amounts of resolution and inversion products were observed, and product topology was variable with no clear bias towards 2-noded catenane. The 'ladder' of topologically complex monomer-sized products strongly suggests recombination following synapsis by random collision (for a discussion of the interpretation of product topology see Stark and Boocock, 1995). These observations clearly indicate that the standard synaptic complex has been dispensed with. Furthermore, as expected from the results of Bednarz et al. (1990), the left–right asymmetry of site I is apparently not detected by the recombination machinery; the inversion products are the result of joining of two left half-sites and two right half-sites, whereas only left-right junctions are observed in normal res×res recombination. A simple hypothesis is that site I×site I recombination follows synapsis by interaction of dimers bound at each site I. However, it is possible that the reaction is still stimulated by further resolvase subunits (perhaps bound at DNA adjacent to the site Is). We selected the sequence segment from amino acid residues 94–120 for saturation mutagenesis for the following reasons. First, several activating mutations in the invertases Gin and Cin, which confer FIS/enhancer-independent strand exchange activity, were in the homologous region of these proteins (Haffter and Bickle, 1988; Klippel et al., 1988). Secondly, it was predicted that this region of resolvase might have important functions during strand exchange. In the crystal structure of a γδ resolvase dimer bound to site I of res, an important component of the dimer interface is contributed by residues of the E-helix, the sidechains of which interact with the E-helix and other surfaces of the partner subunit (Figure 6). The invertase-activating mutations are in residues corresponding in resolvase to residues of the E-helix which make contacts with the other subunit. Also, a chemical modification of a γδ resolvase cysteine mutant (M106C) on the interface caused hyperactive behaviour in vitro (M.R.Boocock and N.D.F.Grindley, unpublished results). Some models for strand exchange by resolvase require disruption of the dimer interface; mutations of residues contributing to the interface might make it easier to separate the subunits, or reduce the degree of control on the disruption. Figure 6.The structure of γδ resolvase bound to a 34 bp site I fragment (Yang and Steitz, 1995) is shown (peptide backbone only; the subunits of the dimer are coloured green and yellow), with the sidechains of residues relevant to this work in spacefill representation. S10 is dark blue; E102 is red; M103 is pale blue; E124 is magenta. The DNA is in spacefill representation (CPK colours; nitrogen, blue; oxygen, red; phosphorous, yellow; carbon, grey). The peptide backbone of the E-helices is shown thicker. The image was created with RasMol2. Download figure Download PowerPoint However, the activating mutations at D102 and M103, identified in the in vivo screen, were not predicted by this reasoning. In γδ resolvase, E102 (the residue equivalent to Tn3 resolvase D102) does not contribute to the crystallographic dimer interface in any of the structures solved (Sanderson et al., 1990; Rice and Steitz, 1994; Yang and Steitz, 1995). E102 has very different conformations in the two subunits of the resolvase dimer–site I complex (Figure 6). In one, the glutamate sidechain extends out from the subunit surface, and does not contact any other parts of the protein. The other glutamate sidechain is folded back to interact with a lysine residue (K105). In Tn3 resolvase, residue 105 is a glutamine. Both E102 sidechains are very distant from the DNA of site I (Figure 6). In the structures of γδ resolvase without DNA, the conformations of E102 are quite similar to those seen in the co-crystal. In no case does E102 interact directly with part of another subunit. M103 is the N-terminal residue of the E-helix, but also makes no trans-interactions. In one subunit, the M103 sidechain makes a hydrophobic interaction with the M106 sidechain of the same E-helix. The region around E102 is apparently quite mobile, the residues having high B-coefficients and showing somewhat different conformations in five different observed crystallographic forms of γδ resolvase (Rice and Steitz, 1994; Yang and Steitz, 1995). Note that since our method of mutagenesis created random point mutations, not all possible amino acid changes at D102 and M103 will have been screened. Single point mutations of the codon for D102 (GAT) can cause substitution by E, N, H, Y, V, A or G; M103 (ATG) can be substituted by I, L, V, T, K or R. The hyperactive mutant sidechains (Y, V, I) are large and rather hydrophobic; the significance of this is not yet clear. Currently we have two hypotheses for the mode of stimulation of activity by the D102 and M103 mutants. One is that this region of the resolvase surface is involved in dimer–dimer contacts required for formation of the productive catalytic assembly at site I. The mutations might have disrupted a control feature, which prevents productive interaction unless preceded by formation of a full synaptic complex involving sites II/III. A second hypothesis is that these mutants change the properties of a 'hinge' between the E-helix and the main part of the N-terminal domain. The globular part of the N-terminal domain (residues 1–100) is held in place by interactions with residues of the E-helix of its own subunit, and residues of the E-helix of the partner subunit. We speculate that a significant movement of the 1–100 region relative to the E-helix of the same subunit might be integral to the catalytic mechanism. Mutations in other parts of the interface between the 1–100 region and the rest of the dimer (for example, at E124) could increase its freedom of movement. This hypothesis is therefore consistent with the increased activation in the double mutant resolvase. We note that some activating mutations of Gin invertase are at residues predicted to be contributing to the equivalent interface (Klippel et al., 1993). The mutation at E124 was introduced by design, because in one subunit of the co-crystal structure this residue hydrogen bonds to S10 (the active site nucleophile) and R68 (another residue at the catalytic site) of the partner subunit (Yang and Steitz, 1995). Maybe this interaction sequesters the sidechains of these active site residues, thus preventing catalytic activity in inappropriate situations. The γδ resolvase E124Q mutant showed activated behaviour, consistent with this idea (Fi
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