Nucleic Acids Research

Bacterial strains

Escherichia coli strains HRS1004 (35) and H124 (1,36) are derived from AB1157. HRS1004 carries a deletion of the ruvAB operon and HI24 contains a ruvB4 mutation.

Sequence comparisons

Protein sequences of E.coli RuvB (37,38) homologues in Haemophilus influenzae (39), Mycobacterium leprae (40), Mycobacterium tuberculosis (40), Mycoplasma genitalium (41), Pseudomonas aeruginosa (42), Synechocystis sp. (GenBank accession no. U38892), Thermotoga maritima (43) and Thermus thermophilus (43) were aligned using PileUp software (Wisconsin Package Version 9.0, Genetics Computer Group, Madison, WI).

Random mutagenesis

Mutations were introduced into the ruvB gene by error-prone PCR (44). Reaction mixtures (100 µl) contained 25 mM Taps-HCl (pH 9.3), 50 mM KCl, 1 mM dithiothreitol, 2 mM MgCl₂, 0.5 mM MnCl₂, 1 mM dGTP, 1 mM dTTP, 1 mM dCTP, 0.1 mM dATP, 0.2 µM of each primer and 10 ng of templateDNA. The template was plasmid pGTI19 containing the wild-type ruvB gene (36). Two regions of ruvB were amplified separately: the 5[prime]-region between 1 and 432 bp (primers: 5[prime]-GCACTGCAGATGAGGTAAAGGATGATTGAAG-3[prime] and 5[prime]-CGCCGGACCTTCACCA-3[prime]) and the 3[prime]-region between 346 and 1055 bp (primers: 5[prime]-CACCGTCTATCGCCAGT-3[prime] and 5[prime]-GGCATATTGCCAGTGC-3[prime]). Taq polymerase reactions were performed at 94°C for 1 min, 50°C for 1 min and 70°C for 4 min, for 30 cycles, followed by 7 min at 70°C. Amplified DNA was gel purified and digested with KpnI and BbsI (for the 5[prime]-region) or BbsI and ClaI (for the 3[prime]-region). DNA fragments were subcloned into pGTI19 to replace the corresponding regions of ruvB. Ligation mixtures were then transformed into HI24 ruvB4 strain and clones were tested for their ability/inability to restore UV resistance.

UV sensitivity

The UV sensitivities of AB1157 ruv⁺ and HI24 ruvB4 containing plasmid pGTI19 (ruvB⁺), pME2 (ruvB^L268S) or pUC19 were measured as described (34).

Proteins and reagents

RecA (13), RuvA and RuvB (45) and wheat germ topoisomerase I (46) were purified as described. RuvB^L268S was purified from E.coli strain HRS1004 using a modified version of the protocol developed for the wild-type RuvB protein. In the first chromatographic step, the phosphocellulose column was omitted and the supernatant of the cell lysate was loaded directly onto a DEAE-Biogel column. RuvB^L268S eluted at a lower salt concentration (170-220 mM KCl) than wild-type RuvB (240-270 mM KCl). The protein was then applied to a hydroxylapatite column in buffer containing 200 mM KCl, 10 mM potassium phosphate and eluted with a 200 mM KCl, 10-150 mM potassium phosphate gradient. Fractions eluting between 35 and 50 mM potassium phosphate were pooled. RuvB^L268S was then purified to homogeneity by Mono Q FPLC as described for wild-type RuvB.

Protein concentrations are expressed in monomers and were determined using the Bradford assay. However, the concentrations of RecA and RuvB (or RuvB^L268S) were determined spectro-photometrically using [epsis]₂₈₀ = 2.7 × 10⁴ (RecA) and1.64 × 10⁴ M^-1 cm^-1 (RuvB). BSA (Gibco BRL), phosphocreatine (Sigma), phosphocreatine kinase (Sigma), proteinase K(Boehringer-Mannheim), glutaraldehyde (Sigma), T4 polynucleotide kinase (Pharmacia), Taq polymerase (Boehringer-Mannheim) and terminal transferase (Amersham) were purchased as indicated. Restriction enzymes were from New England Biolabs.

DNA substrates

Form I Bluescript KS, pDEA-7Z and pAKE-7Z plasmid DNAs, ØX174 single-stranded and form I duplex DNAs, gapped duplex pDEA-7Z DNA (gDNA) and linearised 3[prime]-³²P-labelled pAKE-7Z DNA were prepared as described (34).

For helicase assays, a 30 nt oligonucleotide, complementary to the region 570-599 of ØX174 ssDNA, was 5[prime]-³²P-end-labelled using T4 polynucleotide kinase and [[gamma]-³²P]ATP (Amersham). It was annealed with ØX174 ssDNA as described (32) and purified by gel filtration through a 3 ml Bio-Gel A-0.5m column equilibrated with 10 mM Tris-HCl (pH 8.0), 1 mM EDTA and 100 mM NaCl.

Concentrations of ³²P-labelled DNA substrates were determined by calculating the specific activity using DE81 filters (47), or by measuring the absorbance at 260 nm. Unless stated otherwise, DNA concentrations are expressed in moles of nucleotide residues.

DNA binding, ATPase and branch migration assays

Reactions were carried out as described (34).

DNA unwinding assay

DNA helicase reactions (20 µl) contained 1 µM annealed DNA substrate in 50 mM Tris-HCl (pH 8.0), 15 mM MgCl₂, 10 mM NaCl, 2 mM ATP, 2 mM DTT and 100 µg/ml BSA. The DNA was premixed with RuvA, and the reactions initiated by addition of RuvB or RuvB^L268S. After 8 min incubation at 37°C, reactions were stopped and the products deproteinised and analysed by PAGE (32). ³²P-labelled DNA was visualised by autoradiography.

The unwinding of covalently closed DNA was carried out as described previously with a few modifications (48). Reactions contained 20 mM Tris-acetate (pH 7.5), 10 mM Mg(OAc)₂, 0.5 mM EDTA, 0.5 mM ATP, 0.5 mM ATP[gamma]S, 2 mM DTT, 100 µg/ml BSA and 60 µM form I ØX174 DNA. After addition of RuvA (8 µM), unwinding was initiated by addition of RuvB or RuvB^L268S. After 15 min at 37°C, 20 U of wheat germ topoisomerase I, an amount sufficient to relax protein-free DNA within 30 s, was added (48) and incubation at 37°C was continued for 5 min. The reactions were then stopped and deproteinised by treatment with SDS (0.8%) and proteinase K (0.8 mg/ml) for 15 min at 37°C. DNA products were purified by extraction with phenol/chloroform followed by ethanol precipitation. Agarose gel electrophoresis was carried out as described (48). Gels were dried on DE81 paper and then washed with distilled water. The DNA was denatured by soaking the gel in 0.5 M NaOH, 150 mM NaCl for 20 min and then neutralised with 0.5 M Tris-HCl (pH 8.0), 150 mM NaCl. The DNA was hybridised with a 5[prime]-³²P-end-labelled 66mer complementary to the ØX174 DNA, and the gel was dried and exposed to Kodak XAR films.

Holliday junction resolution

Synthetic Holliday junctions were prepared by annealing four oligonucleotides (66 nt in length): strand 1, 5[prime]-TATCGAATCCGTCTAGTCAACGCTGCCGAATTCTACCAGTGAGGATGGACTCCTCACCTGCAGGTT-3[prime]; strand 2, 5[prime]-AACCTGCAGGTGAGGAGTCCATGGTCTTCCGTCAAGCTCGATGCCGGTTGTATGCCCACGTTGACC-3[prime]; strand 3, 5[prime]-GGTCAACGTGGGCATACAACCGGCATCGAGCTTGACGGAAGACCATCCCTGTCTAGAGGATCCGAC-3[prime]; and strand 4, 5[prime]-GTCGGATCCTCTAGACAGGGATCCTCACTGGTAGAATTCGGCAGCGTTGACTAGACGGATTCGATA-3[prime]. DNA annealing and junction purification were carried out essentially as described previously (49). Prior to annealing, strand 1 was 5[prime]-³²P-end-labelled using polynucleotide kinase. Concentrations of synthetic junctions are indicated in moles of junctions.

Junctions (1 nM) were incubated with RuvC, in the presence or absence of RuvA and RuvB, in cleavage buffer [50 mM Tris-acetate (pH 8.0), 15 mM Mg(OAc)₂, 1 mM ATP[gamma]S, 1 mM DTT and 100 µg/ml BSA; total volume 20 µl] for 30 min at 37°C. The products were deproteinised and analysed by 8% neutral PAGE (8).

Electron microscopy

Complexes of RuvB and RuvB^L268S on pAKE-7Z linear duplex DNA were visualised as described (34).

Immunoprecipitation

Co-immunoprecipitation experiments with RuvA, RuvB (or RuvB^L268S) and RuvC were carried out using synthetic Holliday junction X0 as described (15). After incubation, protein-DNA complexes were immunoprecipitated using anti-RuvC monoclonal antibodies coupled to protein-G Sepharose beads (Pharmacia). Complexes were analysed by SDS-PAGE followed by western blotting with rabbit anti-RuvA, anti-RuvB and anti-RuvC polyclonal antibodies using ECL detection (Amersham).

Random mutagenesis of the cloned ruvB gene and identification of mutants that confer UV^S phenotype

Error-prone PCR (44) was used to introduce random mutations into the cloned ruvB gene. Two different segments of ruvB were mutated: the 5[prime]-region between 1 and 432 bp and the 3[prime]-region between 346 and 1055 bp. The amplified fragments were cloned into the high copy number plasmid pGTI19 (ruvB⁺), replacing the equivalent segments of the wild-type gene. The resulting plasmids were transformed into a UV-sensitive ruvB strain (HI24) and clones were tested for their UV sensitivity. In total, 40 out of 1070 clones were identified as being unable to complement the UV^S phenotype. Plasmids were recovered from these clones and tested for their ability to over-express soluble RuvB protein in strain HRS1004, which carries a ruvAB deletion. Although many plasmids expressed insoluble RuvB, 12 gave good over-expression of soluble protein and for each, the entire ruvB open reading frame was sequenced. Of these, eight plasmids were found to contain point mutations resulting in single amino acid changes in the RuvB protein sequence (Fig. 1). The mutants were designated F44L, V90A, K93L, T161A, D232G, F261L, F261S and L268S (Fig. 1).

Figure 1. Alignment of the E.coli RuvB sequence with eight homologues. Identical and similar amino acids are boxed in black and grey, respectively. DNA helicase motifs I-VI are indicated above the alignment. The positions and the nature of the substitutions obtained by random mutagenesis are indicated. Their numbering relates to the amino acid sequence of E.coli RuvB.

Sequence analysis of RuvB indicates the presence of a number of motifs that are characteristically found in DNA/RNA helicases (50). Helicase domains I and II (two motifs involved in nucleotide binding and hydrolysis; 51,52) and helicase motifs III and VI are highly conserved whereas motifs IV and V are not (Fig. 1). Surprisingly, only one of the eight mutants obtained (T161A) contained a mutation within a helicase motif (motif III). When the mutant RuvB proteins were purified, we observed that one, RuvB^L268S, was exceptional in that it acts as a wild-type protein in vitro, although strains carrying it exhibit a severe UV^S phenotype.

Dominant negative phenotype of RuvB^L268S

Plasmid pME2, carrying the ruvB^L268S gene under the control of the plac promoter, was transformed into E.coli strains HI24 ruvB4 and AB1157 ruv⁺ to assess how expression of the mutant protein affected their UV sensitivity. Due to the absence of lacI^q, RuvB^L268S was expressed constitutively in both strains. As expected, in view of the selection screen used after mutagenesis, expression of RuvB^L268S in HI24 failed to complement the ruvB4 mutation, even at low UV doses (Fig. 2A). Expression of RuvB^L268S in AB1157 resulted in a dominant negative phenotype, such that the wild-type strain carrying pME2 became sensitive to UV irradiation (Fig. 2A). In contrast, over-expression of wild-type RuvB (pGTI19) in AB1157 did not affect UV sensitivity.

Figure 2. In vivo complementation assays and SDS-PAGE analysis of purified RuvB^L268S. (A) UV survival curves of HI24 and AB1157 following transformation with plasmids pME2 (ruvB^L268S, [Delta]), pGTI19 (ruvB⁺, [squ]) or pUC19 ( [diams]). (B) SDS-PAGE of RuvB and RuvB^L268S proteins. Proteins (3 µg) were analysed on a 10% polyacrylamide gel and visualised by staining with Coomassie Blue. The positions of the molecular mass markers are indicated.

Purification of RuvB^L268S protein

To characterise the biochemical defects associated with the mutation in RuvB^L268S, the mutant protein was over-expressed in the ruvAB deletion strain E.coli HRS1004. The procedure used to purify RuvB^L268S was similar to the protocol developed for wild-type RuvB. However, during its purification, we observed that RuvB^L268S eluted from DEAE-Biogel at a significantly lower salt concentration (170-220 mM KCl) than that of wild-type protein (240-270 mM KCl), suggesting that the L268S mutation may have produced a change in the conformational state of the protein. After purification, RuvB^L268S protein was found to be >95% homogeneous when analysed by SDS-PAGE (Fig. 2B).

DNA binding and ATPase activities of RuvB^L268S

To determine whether RuvB^L268S bound DNA, wild-type and mutant proteins were incubated with supercoiled circular duplex DNA in the presence of 15 mM Mg²⁺ and ATP[gamma]S. The resulting protein-DNA complexes were fixed with glutaraldehyde and analysed by agarose gel electrophoresis. The band-shift patterns obtained with RuvB or RuvB^L268S were similar, such that the degree of DNA retardation was directly proportional to the protein concentration until saturation was reached (Fig. 3A). Similar results were obtained without glutaraldehyde fixation (data not shown). Electron microscopic analysis of glutaraldehyde-fixed complexes, formed between RuvB^L268S and linear duplex DNA, showed that the mutant protein formed double rings on DNA (Fig. 3C) that were indistinguishable from the double hexameric rings made by wild-type protein (Fig. 3B).

Figure 3. Duplex DNA binding by RuvB and RuvB^L268S. (A) DNA binding assays were carried out as described in Materials and Methods using the indicated concentrations of RuvB or RuvB^L268S. (B and C) Electron microscopic visualisation of RuvB and RuvB^L268S, respectively, in the presence of linear duplex DNA.

In previous studies, it was shown that RuvB exhibits a weak affinity for DNA at low Mg²⁺ concentrations ([le]10 mM Mg²⁺) and that under these conditions its interaction with DNA requires RuvA. Analysis of the RuvA-directed binding of RuvB or RuvB^L268S, in the presence of 5 mM Mg²⁺, indicated that wild-type and mutant proteins bound with similar affinities (data not shown).

The DNA-dependent ATPase activities of RuvB and RuvB^L268S were also found to be comparable. In both cases, the amount of ATP hydrolysed in a given time was proportional to protein concentration (Fig. 4A). In the absence of RuvA, rates of hydrolysis in the order of 4 mol ATP/min/mol RuvB were observed (Fig. 4B). The rates of hydrolysis catalysed by RuvB or RuvB^L268S were stimulated to the same extent by the presence of RuvA (Fig. 4B). These results indicate that the DNA binding and DNA-dependent ATPase activities of RuvB and RuvB^L268S are indistinguishable and that both proteins interact with RuvA.

Figure 4. ATPase activity of RuvB^L268S compared to wild-type RuvB. (A) ATPase activity analysed as a function of protein concentration. Reactions contained the indicated concentrations of RuvB ([open square]) or RuvB^L268S ([open circle]) and were carried out as described in Materials and Methods. Incubation was for 20 min at 37°C. (B) Effect of RuvA on RuvB- or RuvB^L268S-mediated ATP hydrolysis. Large scale reactions (100 µl) contained either RuvB (3 µM) with ([closed square]) or without ([open square]) RuvA, or RuvB^L268S with ([closed circle]) or without ([open circle]) RuvA. RuvA (1 µM) was added to the preincubation mixtures immediately before initiation. Aliquots were taken at the indicated times and the percentage of ATP hydrolysed was determined. Background levels of ADP (~3%) have been subtracted.

DNA unwinding properties of RuvB^L268S

Two assays were used to determine whether RuvB^L268S possessed the ability to promote DNA unwinding. First, we analysed its activity in a DNA helicase assay, which measured the displacement of a short (30 nt) ³²P-labelled oligonucleotide annealed to circular single-stranded DNA (32). In the presence of RuvA, we observed that RuvB^L268S displaced the 30mer with the same efficiency as wild-type RuvB (Fig. 5A, compare lanes c-f with h-k). Secondly, we used a sensitive topological assay to measure protein-induced DNA underwinding (48). In this assay, the underwinding of covalently closed duplex DNA by RuvAB results in the introduction of positive superhelical turns that are removed by eukaryotic topoisomerase I. Upon deproteinisation, the underwound DNA is observed after agarose gel electrophoresis as moderately negatively supercoiled DNA (Fig. 5B, lane h). In contrast, DNA that has not been underwound is seen at the position of relaxed DNA (lane b). With both wild-type RuvB and RuvB^L268S, we found that the degree of unwinding was directly proportional to the protein concentration (compare lanes d-h with i-m). We conclude that RuvB^L268S fails to exhibit any defect in its ability to promote DNA unwinding.

Figure 5. DNA unwinding activity of RuvB^L268S compared to wild-type RuvB. (A) Displacement of a 30 nt fragment annealed to circular single-stranded DNA. Reactions were carried out as described in Materials and Methods, using the indicated amounts of RuvA and RuvB/RuvB^L268S. Products were analysed by 8% PAGE. Lane m, control in which the DNA was heat-denatured at 100°C for 3 min prior to loading. (B) Unwinding of circular duplex DNA. Assays were carried out as described in Materials and Methods using the indicated concentrations of RuvA and RuvB proteins. Topoisomerase was added where indicated. DNA products were deproteinised and analysed on a 1% agarose gel.

Effect of RuvB^L268S on RuvB- and RuvAB-mediated branch migration

Using ³²P-labelled recombination intermediates ([alpha]-structures) prepared by RecA-mediated strand exchange reactions (13) between gapped circular pDEA-7Z plasmid DNA and ³²P-labelled PstI linearised duplex pAKE-7Z DNA which contains a heterologous block, we observed that wild-type RuvB catalysed branch migration to produce ³²P-labelled linear duplex products (Fig. 6A, lanes b-g). As shown previously (24,53), the wild-type protein promoted branch migration in the absence of RuvA with an efficiency directly proportional to its concentration. Under the same conditions, similar results were obtained with RuvB^L268S (lanes h-m).

Figure 6. Branch migration activities of RuvB and RuvB^L268S. (A) ³²P-labelled recombination intermediates were incubated with the indicated concentrations of RuvB or RuvB^L268S. Reactions were stopped and DNA products were visualised by agarose gel electrophoresis followed by autoradiography. (B) Assays were carried out as in (A), except that RuvA (30 nM) was added immediately before RuvB or RuvB^L268S.

Branch migration experiments were also carried out in the presence of RuvA. Because RuvA promotes the specific targeting of RuvB to the Holliday junction, these experiments were performed at low RuvB concentrations. RuvB^L268S was found to promote efficient branch migration, such that >90% of the recombination intermediates were processed into branch migration products (Fig. 6B). In a series of experiments, the efficiency of branch migration by RuvB^L268S and wild-type RuvB were compared over a range of protein concentrations. The two proteins were found to promote branch migration with similar efficiencies (data not shown).

RuvABC complex formation

Since RuvB^L268S failed to show any obvious defect in its ability to promote branch migration, we next tested to see whether it was capable of forming functionally-active RuvABC complexes. First, we used immunoprecipitation assays to analyse whether RuvB^L268S was able to form RuvABC-Holliday junction complexes in vitro (Fig. 7A), and secondly, we tested whether RuvB^L268S was capable of stimulating Holliday junction resolution catalysed by the RuvC protein (Fig. 7B).

Figure 7. Formation of functional RuvABC complexes by RuvB^L268S. (A) Co-immunoprecipitation of RuvABC-Holliday junction complexes using wild-type RuvB or RuvB^L268S, as indicated. Complexes were prepared and pulled down using anti-RuvC MAbs as described in Materials and Methods. Aliquots were analysed by SDS-PAGE and the gels were immunoblotted with a mixture of anti-RuvA and anti-RuvB polyclonal antibodies (upper panel) or anti-RuvC antibodies (lower panel). Proteins were detected by ECL. (B) Stimulation of RuvC-mediated Holliday junction resolution by RuvAB or RuvAB^L268S. Resolution reactions contained ³²P-labelled synthetic Holliday junctions and were carried out as described in Materials and Methods using RuvA, RuvB and RuvC as indicated. Resolution products (³²P-labelled nicked duplex DNA) were analysed by 8% neutral PAGE and visualised by autoradiography.

Immunoprecipitation experiments were carried out by mixing RuvA, RuvB^L268S (or RuvB as a control) and RuvC with Holliday junction DNA, and the resulting complexes were immunoprecipitated using monoclonal antibodies (MAbs) raised against RuvC (15). As shown in Figure 7A, the anti-RuvC MAbs pulled down a complex that contained all three Ruv proteins. Similar results were obtained using either RuvB or mutant RuvB^L268S.

The efficiency of Holliday junction resolution by RuvC (Fig. 7B, lane b) can be stimulated by the presence of RuvB (lane c), or more efficiently by RuvAB (lane d). Stimulation is thought to involve formation of a functional RuvABC-Holliday junction resolvase complex (8,9). When reactions were carried out using RuvB^L268S, we found that the mutant protein stimulated RuvC-mediated Holliday junction resolution (lanes f and g). The sites of RuvC-mediated resolution were essentially the same in the presence of RuvB or RuvB^L268S (data not shown). RuvB^L268S was also found to stimulate the resolution of supercoiled figure-8 DNA molecules containing Holliday junctions (data not shown). We conclude that RuvB^L268S forms RuvABC-junction complexes and that these complexes exhibit normal resolution activities in vitro.

Conclusions

In this paper we have described the isolation of a mutant ruvB allele that encodes a protein, RuvB^L268S, which exhibits wild-type RuvB activities in vitro. The ruvB^L268S mutation results in a substitution of an aliphatic amino acid (leucine 268) for a polar amino acid (serine) positioned between the poorly-conserved helicase domain V and the well-conserved helicase domain VI. In eight out of the nine RuvB sequences shown in the alignment in Figure 1, an aliphatic amino acid is found at position 268. The surrounding amino acids are relatively well-conserved, with 66% similarity in the 57 amino acids between the helicase domains V and VI. Since no crystallographic data are available with RuvB, the structure and function of this region is presently unknown.

Although RuvB^L268S exhibits wild-type biochemical activities in vitro with regard to DNA binding, ATPase activity, ring formation, DNA unwinding, branch migration and ability to form functional RuvABC-Holliday junction complexes, the L268S substitution has a dramatic effect in vivo. Indeed, the ruvB^L268S allele was unable to complement the ruvB4 mutation, and over-expression of RuvB^L268S exerted a dominant negative effect in wild-type cells. Since associations between mutant (RuvB^D113N) and wild-type RuvB proteins have been observed previously (34), it is possible that RuvB^L268S interacts with wild-type RuvB to form a hetero-hexamer that is inactive for some critical RuvB function.

Although RuvB^L268S appeared normal when assayed in vitro, during its purification RuvB^L268S eluted from the DEAE-Biogel column at lower salt concentrations than the wild-type protein, suggesting that the mutation caused a conformational change that might affect protein-protein or protein-DNA interactions. Since we have not observed any defects in self-association of the RuvB hexamer, interactions with RuvA or RuvC, nor assembly of the RuvABC-Holliday junction complex, one possibility to be considered is that RuvB associates with some other protein in E.coli and that leu268 is critical for this interaction.

Alternatively, subtle changes in the way that RuvB^L268S interacts with RuvA and/or RuvC, may lead to the observed cellular defect. Because our extensive in vitro analysis of RuvB^L268S failed to identify the biochemical defect in this protein, the data expose the limitations of such in vitro assays which, by their necessary simplicity, can only partially reproduce the complexity of reactions that take place in vivo. For example, recombination intermediates made in vitro by RecA are naked, relaxed DNA molecules that possess free ends, whereas the natural substrates for RuvABC are likely to be supercoiled and have many associated proteins. Indeed, target DNAs may be actively undergoing DNA replication, transcription and repair, and it is possible that RuvB^L268S is unable to act within the context of a DNA processing machine.

ACKNOWLEDGEMENTS

We thank David Adams, Angela Eggleston, Irina Tsaneva and other members of the laboratory for their interest and providing protein reagents. This work was supported by the Imperial Cancer Research Fund, the Human Frontiers Science Program, and the Swiss-British Joint Research Program and the Swiss National Foundation. C.M. and A.J.v.G. were supported in part by fellowships from the European Molecular Biology Organisation, and D.Z. by the Centre National de la Recherche Scientifique.

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*To whom correspondence should be addressed. Tel: +44 171 269 3868; Fax: +44 171 269 3811; Email: s.west@icrf.icnet.uk
Present addresses: ⁺Laboratoire de Génétique Moléculaire de la Recombinaison, UMR 144, Institut Curie, Section de Recherche, 26 rue d’Ulm, 75248 Paris 05, France and ^§Institute de Pharmacologie et Biologie Structurale du CNRS, 205 Route de Narbonne, 31077 Toulouse, France

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