Portal de Periódicos da CAPES

Crystal structure of the site-specific recombinase, XerD

1997; Springer Nature; Volume: 16; Issue: 17 Linguagem: Inglês

10.1093/emboj/16.17.5178

1460-2075

Hosahalli S. Subramanya, Lidia K. Arciszewska, Rachel A. Baker, Louise E. Bird, David J. Sherratt, Dale B. Wigley,

DNA Repair Mechanisms

Article1 September 1997free access Crystal structure of the site-specific recombinase, XerD Hosahalli S. Subramanya Hosahalli S. Subramanya Laboratory of Molecular Biophysics, University of Oxford, Oxford, OX1 3QU UK Search for more papers by this author Lidia K. Arciszewska Lidia K. Arciszewska Microbiology Unit, Department of Biochemistry, University of Oxford, Oxford, OX1 3QU UK Search for more papers by this author Rachel A. Baker Rachel A. Baker Microbiology Unit, Department of Biochemistry, University of Oxford, Oxford, OX1 3QU UK Search for more papers by this author Louise E. Bird Louise E. Bird Laboratory of Molecular Biophysics, University of Oxford, Oxford, OX1 3QU UK Search for more papers by this author David J. Sherratt Corresponding Author David J. Sherratt Laboratory of Molecular Biophysics, University of Oxford, Oxford, OX1 3QU UK Search for more papers by this author Dale B. Wigley Dale B. Wigley Laboratory of Molecular Biophysics, University of Oxford, Oxford, OX1 3QU UK Search for more papers by this author Hosahalli S. Subramanya Hosahalli S. Subramanya Laboratory of Molecular Biophysics, University of Oxford, Oxford, OX1 3QU UK Search for more papers by this author Lidia K. Arciszewska Lidia K. Arciszewska Microbiology Unit, Department of Biochemistry, University of Oxford, Oxford, OX1 3QU UK Search for more papers by this author Rachel A. Baker Rachel A. Baker Microbiology Unit, Department of Biochemistry, University of Oxford, Oxford, OX1 3QU UK Search for more papers by this author Louise E. Bird Louise E. Bird Laboratory of Molecular Biophysics, University of Oxford, Oxford, OX1 3QU UK Search for more papers by this author David J. Sherratt Corresponding Author David J. Sherratt Laboratory of Molecular Biophysics, University of Oxford, Oxford, OX1 3QU UK Search for more papers by this author Dale B. Wigley Dale B. Wigley Laboratory of Molecular Biophysics, University of Oxford, Oxford, OX1 3QU UK Search for more papers by this author Author Information Hosahalli S. Subramanya1, Lidia K. Arciszewska2, Rachel A. Baker2, Louise E. Bird1, David J. Sherratt 1 and Dale B. Wigley1 1Laboratory of Molecular Biophysics, University of Oxford, Oxford, OX1 3QU UK 2Microbiology Unit, Department of Biochemistry, University of Oxford, Oxford, OX1 3QU UK *Corresponding author. E-mail: [email protected] The EMBO Journal (1997)16:5178-5187https://doi.org/10.1093/emboj/16.17.5178 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The structure of the site-specific recombinase, XerD, that functions in circular chromosome separation, has been solved at 2.5 Å resolution and reveals that the protein comprises two domains. The C-terminal domain contains two conserved sequence motifs that are located in similar positions in the structures of XerD, λ and HP1 integrases. However, the extreme C-terminal regions of the three proteins, containing the active site tyrosine, are very different. In XerD, the arrangement of active site residues supports a cis cleavage mechanism. Biochemical evidence for DNA bending is encompassed in a model that accommodates extensive biochemical and genetic data, and in which the DNA is wrapped around an α-helix in a manner similar to that observed for CAP complexed with DNA. Introduction In site-specific recombination, DNA molecules are cleaved in both strands at two separate recombination sites, and the ends are rejoined to new partners, without any synthesis or degradation of DNA or hydrolysis of phosphodiesters. The reactions are catalysed by specialized recombinase proteins and may involve other protein accessory factors that have structural and modulatory roles in the nucleoprotein complex that contains synapsed recombination sites. Site-specific recombinases mediate a wide range of microbial programmed DNA rearrangements that include the integration and excision of bacteriophages from bacterial chromosomes, the control of circular replicon inheritance, the processing of the initial products of genetic transposition and the mediation of genetic ‘switches’ through inversion or deletion of specific DNA segments (reviewed in Stark et al., 1992; Landy, 1993; Stark and Boocock, 1995a; Nash, 1996). There are two families of site-specific recombinases; the resolvase/invertase family use a serine nucleophile to mediate a concerted double strand cleavage and rejoining reaction at nucleotide phosphates separated by 2 bp, while the λ integrase family enzymes use a tyrosine nucleophile to mediate sequential pairs of strand exchanges that are positioned 6–8 bp apart. In site-specific recombination reactions mediated by both families, four recombinase molecules bound to two ∼30 bp recombination core sites catalyse the breaking and rejoining of four DNA phosphodiester bonds. The 298 amino acid site-specific recombinase XerD, along with its related partner recombinase, XerC, belong to the λ integrase family of site-specific recombinases. They function in the stable inheritance of the Escherichia coli chromosome and multicopy circular plasmids, apparently by converting to monomers the circular multimers that can arise by homologous recombination (Blakely et al., 1993; Sherratt et al., 1995). Recombinases of the integrase family are highly diverged in primary amino acid sequence, with only four completely conserved amino acids (RHRY) (Argos et al., 1986; Abremski and Hoess, 1992). All four of the conserved residues have been implicated in catalysis (Pargellis et al., 1988; Evans et al., 1990; Chen et al., 1992a; Friesen and Sadowski, 1992; Lee et al., 1992). In XerD and XerC, the two conserved arginines and the tyrosine are required for DNA cleavage, while the conserved histidine is required for DNA rejoining (Blakely et al., 1993; Arciszewska and Sherratt, 1995; Arciszewska et al., 1997; L.K.Arciszewska, R.A.Baker, P.A.Wigge and D.J.Sherratt, unpublished data). Recombination is initiated when the conserved tyrosine hydroxyl attacks the scissile phosphate, forming a 3′ phosphotyrosyl–DNA complex and a free 5′ hydroxyl. In the second step, a 5′ hydroxyl from the adjacent partner duplex attacks the phosphotyrosine to form a Holliday junction intermediate. The recombination reaction is completed by the exchange of the second pair of strands, using the same mechanism, 6–8 bp away from the site of the initial strand exchanges. Xer site-specific recombination exhibits three features that distinguish it from other well characterized members of the family. First, it uses two related recombinases, XerC and XerD, each of which catalyses one specific pair of strand exchanges (Blakely et al., 1993, 1997; Arciszewska and Sherratt, 1995; Colloms et al., 1996, 1997; Arciszewska et al., 1997). This is proving to be a powerful tool in establishing the roles of the two recombinases and for defining the determinants for recombinase binding to DNA. Analysis of recombination site function has also been facilitated by the availability of a wide variety of naturally occuring Xer recombination sites that contain a range of related recombinase-binding DNA sequences. Second, the recombination reaction has different requirements and outcomes depending on whether it occurs at plasmid or chromosomal recombination sites. Recombination at natural plasmid sites is preferentially intramolecular and requires, in addition to the two recombinases and the 28–30 bp recombination core site, additional accessory proteins and ∼200 bp of adjacent accessory DNA sequences. Interaction of the accessory proteins and accessory sequences promotes the formation of a synaptic complex of precise topology, that can only form efficiently on directly repeated recombination sites in the same molecule (Colloms et al., 1996, 1997). In contrast, recombination at the E.coli chromosomal site, dif, requires only a 28 bp recombination core site at which the two recombinases act. Recombination in vivo at dif, present in multicopy plasmids, occurs intermolecularly and intramolecularly (Blakely et al., 1991, Leslie and Sherratt, 1995; Tecklenberg et al., 1995). Third, despite the sequence divergence of integrase family recombinases, conserved Xer-like recombinase sequences are present in the chromosomes of almost all bacteria examined (including the archaebacterium, Methanococcus janaschii), suggesting that there is a strong constraint on how Xer recombination functions in chromosome segregation. These enzymes may be the progenitors of the many integrase-like enzymes found in different microbes. In this study, the substantial body of information that has accumulated on how XerC and XerD interact with their recombination site DNA and mediate recombination is used in order to relate the XerD structure to function. XerC and XerD each bind cooperatively to related 11 bp sites that are separated by a 6–8 bp ‘central’ region; binding of the recombinases to DNA leads to substantial DNA bending (Blakely et al., 1993, 1997; Blakely and Sherratt, 1994, 1996a). XerC-mediated strand exchange of ‘top’ strands occurs at the border of its binding site and the central region, while XerD exchange of ‘bottom’ strands occurs 6–8 bp away at the border of its binding site and the central region. Genetic and biochemical analysis of the recombination core site has identified the nucleotides that provide specificity for recombinase binding (Blake et al., 1997; Hayes and Sherratt, 1997), and has indicated which backbone and base contacts are involved in this interaction (Blakely and Sherratt, 1994, 1996a; Blakely et al., 1997). A deletion and pentapeptide insertion analysis of XerD has revealed parts of the protein involved in DNA binding and interaction with XerC (Spiers and Sherratt, 1997; Y.Cao, B.Hallet, D.J.Sherratt and F.Hayes, unpublished data). XerC and XerD are catalytically autonomous as judged by the demonstration that normal strand exchange by either XerC or XerD does not require the tyrosine nucleophile of the partner recombinase (Arciszewska and Sherratt, 1995; Arciszewska et al., 1997), and that when either XerC or XerD are incubated with supercoiled plasmid containing dif, a XerC or XerD site-specific type I topoisomerase activity is detectable (Cornet et al., 1997; Spiers and Sherratt, 1997). Finally, the substrate requirements for XerC- and XerD-mediated catalysis have been compared and the topological parameters of the recombination reaction determined (Colloms et al., 1996, 1997; Arciszewska et al., 1997). Results and discussion Structure of the protein Details of the structure determination and refinement are presented in Table I and in Materials and methods. Of the 298 amino acids of the protein, 271 were defined in our final model. The missing residues are located at the N- and C-termini and in three disordered surface loops. The enzyme comprises two domains: domain 1 consists of residues 1–107, while domain 2 comprises residues 108–298 (Figure 1). Domain 1 contains four α-helices, arranged such that there are two parallel helix hairpins arranged at 90° to each other. Domain 2 is also mainly α-helical, but with a three-stranded antiparallel β-sheet along one edge. The fold of this domain is similar to that determined recently for λ and HP1 integrases (λ Int and HP1 Int, respectively), and is at present unique to this family of proteins (Hickman et al., 1997; Kwon et al., 1997). Domains 1 and 2 of XerD correspond to domains of λ Int, HP1 Int and FLP identified by limited proteolysis, although in FLP the C-terminal domain has been divided further into three further sub-domains (Moitoso de Vargas et al., 1988; Evans et al., 1990; Chen et al., 1991; Pan and Sadowski, 1993; Sadowski, 1995; Hickman et al., 1997; Kwon et al., 1997). Figure 1.Overall structure of the protein. This stereo figure was prepared using PREPPI. The numbering refers to the beginning and end of secondary structural elements. Residues that are not defined are located at the N- and C-termini and in three disordered loops (residues 64–70, 101–110 and 269–270). Download figure Download PowerPoint Table 1. Summary of crystallographic structure analysis Data collection λ (Å) Resolution (Å) Rsym (%) Redundancy of data Completeness (%) Native 0.89 2.5 4.8 3.0 98.9 NaAuCl4 0.89 3.0 7.8 3.5 96.1 (10 mM, 3 h) Trimethyl lead acetate 0.89 2.5 5.6 2.6 95.4 (100 mM, 18 h) Ethylmercury phosphate 1.54 2.5 7.4 2.9 99.1 Derivative Anomalous data No. of sites MFID Rcullis Phasing power Mean FOM NaAuCl4 yes 8 0.28 0.73 1.4 0.57 Trimethy lead acetate yes 9 0.21 0.77 1.3 Ethylmercury phosphate yes 6 0.23 0.70 1.4 Refinement of native crystal Resolution (Å) 2.5 Final R-factor (all data, 10–2.5Å) 22.4 Rfree (5% of data) 28.7 r.m.s.d. bonds (Å) 0.013 r.m.s.d. angles (Å) 0.041 No. of residues 271 No. of water molecules 105 Ramachandran analysis (most favoured/additional allowed/generous/disallowed)a (%) 89.4/10.2/0.4/0.0 B-factors (lowest/highest/mean) 6.8/134.8/50.4 protein only 6.8/134.8/49.9 water molecules 20.8/97.8/59.9 a Definitions according to Laskowski et al. (1993). The region of structural homology within the C-terminal domains of the XerD, λ Int and HP1 Int spans ∼170 residues (Figure 2). Two conserved sequence motifs, that have been proposed to indicate a conservation of structure across a wide family of integrases that includes XerD and XerC as well as λ Int and HP1 Int (Argos et al., 1986; Abremski and Hoess, 1992; Blakely and Sherratt, 1996b), are located in domain 2 of XerD. The locations of motif I and the N-terminal portion of motif II are similar in the structure of XerD (residues 145–159 and 244–281, respectively) and those of λ and HP1 integrases (Hickman et al., 1997; Kwon et al., 1997). However, the extreme C-terminal portions of the proteins, which include the C–terminal portion of motif II, could hardly be more different (Figure 2). This region of the proteins is of particular interest because it contains the active site tyrosine residue to which the DNA becomes attached covalently during the recombination reaction. Figure 2.Comparison of the structures of the C-terminal domains of XerD, λ Int and HP1 Int. Regions of the C-terminal domains of the proteins that show the greatest structural similarity are shown in grey. The major structural differences (shown in magenta) are located in the polypeptide segments that extend from conserved motif II (Argos et al., 1986) to the C-terminus of the proteins. Download figure Download PowerPoint In λ Int, these C-terminal residues (334–356) form a flexible loop that is disordered in one of the two molecules in the asymmetric unit, but is more ordered in the other, where the final 15 residues form two additional β-strands along one edge of the antiparallel sheet. In HP1 Int, this region (residues 307–337) forms an extended structure which protrudes from the surface of the protein molecule and contains two short helices. This region is involved in crystal contacts which the authors propose to be representative of one of the protein dimer interfaces during the recombination reaction. By contrast, in XerD, this region (residues 271–298) forms a turn followed by a long α-helix, containing the active site tyrosine, that extends almost to the C-terminus (the last six residues of the protein are disordered in the crystal structure). It is intriguing that a region of such vital importance should be so different in the three enzymes. Furthermore, it is likely that this region will differ further in other members of the λ integrase family. For example, in FLP and other yeast recombinases, this region is longer than in the bacterial members of the family, a difference that has been proposed to play a role in determining whether the tyrosine nucleophile of a given enzyme molecule attacks the scissile phosphate bond and is activated by that molecule (cleavage in cis), or whether it attacks the scissile phosphate bond and is activated by a different molecule (cleavage in trans) (discussed in Landy, 1993; Jayaram and Lee, 1995; Stark and Boocock, 1995b; Blakely and Sherratt, 1996b; Jayaram, 1997). Interactions between XerD and DNA The structures of the catalytic domains of λ and HP1 integrases suggested how DNA might interact with the C-terminal region of these proteins (Hickman et al., 1997; Kwon et al., 1997). Calculations of the electrostatic potential of the surface of domain 2 of XerD [using GRASP (Nicholls and Honig, 1991)] reveal an obvious site for interaction with DNA (Figure 3A) that is consistent with those proposed for λ Int and HP1 Int. However, when combined with the extensive biochemical information that is available for the Xer proteins, the structure of XerD provides a more detailed view of this interaction. The footprinting data indicate clearly that there are contacts between the protein and DNA that encircle the DNA duplex in the region around the cleavage site, an observation that is not explained by the interactions between domain 2 and DNA alone. Consequently, it is likely that domain 1 also contributes to the DNA-binding site, a view supported by genetic and biochemical studies of XerD and other recombinases (Hoess et al., 1990; Panigrahi et al., 1992; Panigrahi and Sadowski, 1994; Sadowski, 1995; Spiers and Sherratt, 1997). While the proposed DNA-binding sites in the structures of the catalytic core fragments of λ and HP1 integrases are exposed, in the XerD structure, access to the active site is blocked by the positioning of domain 1 over this region (Figure 1). Hence, in order for XerD to bind to DNA, there has to be a large conformational change to allow access of the DNA to the active site region. The electrostatic potential of the surface of domain 1 also reveals a likely candidate for a DNA-binding surface (Figure 3B), supporting the view that both domain 1 and 2 contribute to the DNA-binding surface of the protein. Because of the conformational changes of the protein required to bind to DNA, we have restricted our detailed discussion of the DNA binding and recombinase–recombinase interactions to the C-terminal catalytic domain of XerD, which we shall refer to as domain 2. Figure 3.Electrostatic surface potential of domains of XerD. (A) Domain 2 and (B) domain 1. Regions of negative potential are coloured in red and positive potential in blue. The surface is transparent to reveal the underlying Cα backbone of the protein (shown in green). This figure was prepared using GRASP (Nicholls and Honig, 1991). Download figure Download PowerPoint Initial attempts to model the XerD domain 2–DNA complex were based upon those proposed for the interactions of catalytic core fragments of λ and HP1 integrases with linear B-form DNA (Hickman et al., 1997; Kwon et al., 1997). However, it was evident immediately that the application of the λ Int and HP1 models to XerD did not take into account the biochemical evidence which shows that the DNA binding of XerD induces an ∼40° bend in the DNA (Blakely and Sherratt, 1996a). Furthermore, these simple models were inconsistent with the footprinting and binding interference data which show that XerD binding to its DNA target involves major and minor groove interactions that encircle the DNA (Blakely and Sherratt, 1994, 1996a; Blakely et al., 1997). For example, the DNA footprinted by XerD extends over a much larger region than would be possible by docking the XerD structure onto linear duplex B-form DNA, but could be accommodated by wrapping the DNA around the protein. The degree of bending of the DNA in the XerD–DNA complex has been shown to be comparable with that induced by each of the molecules of the catabolite activator protein (CAP) dimer, as observed in the crystal structure of the CAP–DNA complex and measured in biochemical experiments (Schultz et al., 1991). This led us to compare the structures to seek any similarities that might help to understand the XerD–DNA complex. In the CAP–DNA complex, there is a sharp 40° kink in the bound DNA as it wraps around a helix–turn–helix motif on the protein surface. Comparison of the DNA-binding surface of CAP with that of XerD revealed a striking similarity in the spatial arrangement of the helix–turn–helix motif in CAP and the positions of two helices (helices αG and αJ) in the XerD structure (Figure 4A), although in the XerD structure these two helices are separated by ∼65 residues rather than the tight turn found in CAP. Furthermore, the positions of residues which are involved in contacts with the phosphodiester backbone of the DNA, in the CAP structure, are conserved in the XerD structure (Figure 4). These residues also occur in other helix–turn–helix motifs (Brennan and Matthews, 1989) and, perhaps more importantly, the sequences and structures of the comparable regions of the λ and HP1 integrases are also consistent with them being DNA recognition helices (Figure 4B and C; Hickman et al., 1997; Kwon et al., 1997). This latter point may be particularly significant given the low level of sequence similarity in these regions of the proteins. Figure 4.Model for interactions between XerD and DNA. (A) The helix–turn–helix motif (residues 166B–194B) in the CAP–DNA complex is shown in cyan, with the DNA around the kink site shown in green. The corresponding helices of XerD (residues 146A–155A and 217A–234A) are overlaid in magenta. Only the Cα positions of the main chain are shown, together with the side chains that interact directly with the DNA. (B) Amino acid sequence alignment of the helix–turn–helix DNA recognition α-helix of E.coli CAP with the putative recognition helix of XerD and the comparable regions present in Xer recombinase sequences of other bacteria, λ Int and HP1 Int. The three CAP residues that make base-specific contacts in the major groove are shown in magenta (Schulz et al., 1991). Other helix–turn–helix proteins also often make DNA contacts through these positions (Brennan and Matthews, 1989). Two amino acids of XerD helix αJ that could make base-specific contacts are also coloured magenta. In XerC enzymes, we propose that the conserved R at the position corresponding to 221 of XerD is important for sequence-specific binding. Amino acids of CAP that make DNA backbone contacts are coloured green. In XerD, these equivalent amino acids may contact the DNA backbone, as may 226R (all coloured green). Recombinases described as XerC or XerD have been shown to possess that specific function (G.Blakely, L.Neilson and D.J.Sherratt, unpublished data). Amino acid sequences in this region of Salmonella typhimurium XerD and XerC are identical to those in E.coli. (Hayes et al., 1997). Recombinases described as ‘Xer’ are presumed to be XerD and XerC homologues based on sequence comparison; residues at position 220 and 221 are used for classification. The accession numbers for the recombinase sequences are as follows: E.coli (Ec) XerD, P21891a; S.typhimurium (St) XerD 492525b; Haemophilus influenzae (Hi) XerD P44630a; Mycobacterium tuberculosis (Mt) Xer Q10815a; M. leprae (Ml) Xer 467161b; Bacillus subtilis (Bs) RipX (XerD), P46352a; E.coli XerC, P22885a; S.typhimurium XerC, 492524b; H.influenzae XerC, P44818a; Pseudomonas aeruginosa Sss (XerC), X78478c; B.subtilis CodV (XerC), P39776a; Lactobacillus leichmannii Xer, X84261c; E.coli λ Int, P03700a; H.influenzae HP1 Int, P21442a; Saccharomyces cerevisiae FLP, P03870a. aSWISS-PROT, bNCIB, cGenEMBL. (C) Comparison of CAP recognition helix interactions with DNA (Schultz et al., 1991), and those that our model predicts will be involved in XerD–DNA interactions. Base-specific contacts are in magenta and backbone contacts in green. The sequences are oriented so that the kinks induced by recombinase binding are in the same direction. The relative positions of the contacts are remarkably similar. The XerD-binding site consists of residues 4–14. The nucleotides in deep magenta are implicated in binding specificity, whereas those in blue are important for binding (see text). The position of the scissile phosphate bond is indicated with an arrow. Download figure Download PowerPoint Taken together, these data allow us to construct a model for the interaction between domain 2 of XerD and its recognition sequence based upon the CAP–DNA complex (Schultz et al., 1991). The two helices of the helix–turn–helix motif of one subunit of the CAP–DNA complex were superimposed upon helices αG and αJ of XerD (Figure 4A) and allowed us to position the DNA relative to these helices in XerD. The resulting model is presented in Figure 5, and is in remarkably good agreement with the DNA bending, footprinting and binding interference data (Blakely et al., 1993, 1997; Blakely and Sherratt, 1994, 1996a; summarized in Figure 5). The proposed backbone contacts (Figure 4B and C) compare well with the phosphates whose ethylation prevents binding (Figure 5). The proposed base-specific contacts between the XerD recognition helix, αJ and recombination site DNA are very similar to the comparable CAP–DNA contacts, with both the CAP and the XerD recognition helices being oriented in the same way (Figure 4B and C). In the model, XerD residues 220R and 221Q could make base-specific contacts at precisely the positions that we have identified as being important for XerD binding and XerD–XerC binding specificity. For example, oxidation of any of three adjacent Ts (positions 11–13; Figures 4C and 5) prevents XerD binding to its site, while nucleotides at positions 10, 11 and 13 contribute to XerC–XerD binding discrimination, and the presence of a T or G, but not a C, at position 9 correlates with tight XerD binding (Blake et al., 1997; Hayes and Sherratt, 1997). Moreover, examination of known XerD and XerC recombinases shows that all XerD recombinases have the equivalent of 220R and 221Q, whereas XerC recombinases have a conserved R in place of Q at the equivalent of position 221 and a non-conserved residue in the preceding position. Other putative Xer recombinase sequences present in the databases have either RQ at the positions corresponding to 220 and 221, respectively, or a conserved R at the position corresponding to 221, preceded by a non-conserved residue. This indicates that these presumptive recombinases can be classified as either XerC or XerD proteins on the basis of the amino acid sequence at positions corresponding to 220 and 221, and that these amino acids may provide much of the discrimination that directs XerC and XerD to their specific DNA-binding sites. The weaker binding of XerC, and the reduced bending it appears to induce, may be a consequence of fewer base-specific contacts. Furthermore, the high conservation of amino acid residues at these two positions in XerD recombinases from different bacteria (and at the one position in different XerC enzymes) suggests a very strong functional selection for the maintenance of specific recombinase–DNA contacts in these enzymes. This is supported by our demonstration that the Bacillus subtilis XerC and XerD homologues mediate strand exchange on an E.coli dif-containing Holliday junction (G.B.Blakely and D.J.Sherratt, unpublished data). Figure 5.Model of XerD bound to DNA. Model of XerD domain 2 bound at its recognition sequence, derived from the CAP–DNA complex. The protein is shown in green as a ribbon, while the DNA is shown as a space-filling representation. Residues of the DNA within the OP-Cu footprint are shown in cyan, and those outside of the footprint in beige. Specific contacts, as shown by interference binding analysis, are overlaid in orange (phosphates), blue (adenine minor groove contacts) and magenta (thymine, major groove). The scissile phosphate is shown in red. The view of the active site residues in relation to the scissile phosphate is as in Figure 6. This figure was prepared using RIBBONS (Carson, 1991). Download figure Download PowerPoint The DNA-binding properties of deletion and pentapeptide insertion mutants also agree well with the model in which helix αJ of domain 2 is the recognition helix that interacts with DNA. A truncated XerD derivative containing residues 1–233 is proficient in DNA binding and retains all but the last residue of helix αJ. In contrast, an even shorter XerD derivative, that is deleted for the six C-terminal residues of helix αJ, is binding-deficient (Spiers and Sherratt, 1997). Insertions of proline-containing pentapeptides into this same helix also abolish detectable DNA binding (Y.Cao, B.Hallet, D.J.Sherratt and F.Hayes, unpublished data). The positioning of the scissile phosphate adjacent to the active site residues adds further credence to the model (Figure 6). Figure 6.The active site region of XerD. The positions of residues in the active site of XerD, that have been shown to be important for the cleavage reaction, are consistent with a cis cleavage mechanism for the enzyme. The Cα backbone is shown in yellow, the side chains of Arg148 and Arg247 in blue and the side chain of Tyr279 in purple. A simple rotation about the Cα−Cβ bond of Tyr279 (now depicted in green) would position the side chain appropriately for in-line attack of the scissile phosphate (coloured red). Download figure Download PowerPoint The 11 bp XerD- and XerC-binding sites can be sub-divided into two regions; the inner four nucleotides, that are dyad symmetrical in the XerC- and XerD-binding sites, and the outer seven nucleotides, at least four of which contribute to specific XerD binding (Figure 4C; Blakely and Sherratt, 1994, 1996a; Blake et al., 1997; Bla