Nucleic Acids Research

Protein purification

Ribosomal protein L3 was purified from E.coli B834. Wild-type and mutant PcrA proteins were over-expressed in E.coli B834 and B834 pLysS respectively. L3 co-purified with the mutant protein and the two proteins were separated from each other by gel filtration. The purification protocol for the mutant and L3 proteins was as follows. Plasmid pBSHII, carrying the mutant pcrA gene cloned into a pET22b T7 promoter-based expression vector, was transformed into E.coli B834 pLysS cells. Individual colonies were selected on chloramphenicol/ampicillin Luria agar plates. A single colony was used to inoculate a 2 l culture of Luria broth containing 100 µg/ml ampicillin and 34 µg/ml chloramphenicol. The culture was grown with shaking at 37°C until the OD₆₀₀ reached 0.6 and was induced with 1 mM IPTG. Growth was continued for 3 h and cells were then harvested by centrifugation at 5000 g. The cell pellet was resuspended in 20 ml buffer A (50 mM Tris, pH 7.5, 2 mM EDTA, 1 mM DTT), plus 2% (v/v) Triton X-100, 1 M NaCl, 10% sucrose and lysed by sonication in the presence of 100 µM phenylmethylsulphonyl fluoride. The supernatant was clarified by centrifugation at 20 000 g. Protein in the clarified supernatant was precipitated by addition of an equal volume of ammonium sulphate-saturated solution and pelleted at 20 000 g. The pellet was resuspended in buffer A in a volume such that the conductivity of the solution was equal to the conductivity of buffer A + 300 mM NaCl. This was applied to a 5 ml Hi-Trap heparin-Sepharose column (Pharmacia) pre-equilibrated with buffer A + 100 mM NaCl. Because the protein is less soluble at low salt concentrations, the sample was diluted to a conductivity equal to buffer A + 100 mM NaCl as it was loaded onto the column using a gradient mixer valve. The column was washed with buffer A + 100 mM NaCl and the protein was eluted with a 160 ml gradient of 100-600 mM NaCl in buffer A. The peak fractions were pooled and the conductivity was adjusted to be equal to the conductivity of buffer A + 300 mM NaCl. The sample was applied to a low substitution (13%) 50 ml blue Sepharose column pre-equilibrated with buffer A + 300 mM NaCl. The column was washed with buffer A + 800 mM NaCl and protein was eluted in buffer A + 1.5 M NaCl. Peak fractions were pooled and protein was precipitated by adding ammonium sulphate (29.1 g/100 ml) at room temperature. The precipitate was harvested by centrifugation at 20 000 g and resuspended in 0.5 ml buffer A + 300 mM NaCl. The PcrA and L3 proteins were separated by gel filtration using a Superdex S200 column (Pharmacia). The column was pre-equilibrated with buffer A + 300 mM NaCl. Prior to storage at -80°C glycerol was added to a final concentration of 10% (v/v). The purity of the proteins was monitored by SDS-PAGE on a 12% gel.

The wild-type PcrA protein was purified using the same purification scheme with the following differences. The cell pellet was resuspended in buffer A + 200 mM NaCl, 10% sucrose. The peak fractions from the Hi-Trap heparin-Sepharose column were pooled and the conductivity was adjusted to be equal to the conductivity of buffer A + 200 mM NaCl. The sample was applied to a low substitution (13%) 50 ml blue Sepharose column pre-equilibrated with buffer A + 200 mM NaCl. PcrA was eluted by applying a 200 ml gradient of 200-700 mM NaCl in buffer A. The ammonium sulphate-precipitated protein was redissolved in buffer A + 200 mM NaCl. The Superdex S200 column was equilibrated in buffer A + 200 mM NaCl.

ATPase assays

ATPase assays were carried out in a total volume of 1 ml in a buffer containing 5 mM Tris-acetate, pH 7.5, 50 mM CH₃COONa, 0.02 mM EDTA, 2 mM MgCl₂, 10% (v/v) glycerol, 1 mM ATP, 0.02 mg/ml BSA and 24 nM PcrA or 2.4 µM L3. ATP hydrolysis was monitored spectrophotometrically by linking it to oxidation of NADH as described elsewhere (11). The effect of L3 on the ATPase activity of PcrA in the presence or absence of ssDNA was investigated using a mixture of both proteins (24 nM PcrA and 2.4 µM L3) in the same reaction mixture. The 3[prime]-tailed oligonucleotide (see helicase assays) was used as the ssDNA co-factor. Reactions were carried out at optimal (1.2 µM) or sub-optimal (120 nM) DNA concentrations.

Helicase assays

Untailed and 3[prime]-tailed substrates for helicase assays were prepared by annealing radioactively labelled synthetic oligonucleotides (5[prime]-ACTCTAGAGGATCCCCGGGTACGTTATTGCATGAAAGCCCGGCTG-3[prime] for 3[prime]-tailed and 5[prime]-ACTCTAGAGGATCCCCGGGTAC-3[prime] for untailed) to M13mp18 ssDNA as described elsewhere (12). Annealings were done with a slight excess of M13mp18 DNA over labelled oligo, so that the substrate concentration mentioned below refers to that of labelled oligo. Time course helicase reactions were performed at 37°C in a reaction buffer containing 20 mM Tris, pH 7.5, 50 mM NaCl, 15 mM MgCl₂, 2.5 mM ATP, 1.5 mM DTT. The reactions were carried out in a total volume of 20 µl, using 1 pmol PcrA and 0.02 pmol DNA substrate. This corresponds to 1 molecule PcrA per 145 nt M13mp18 ssDNA substrate. The effects of L3 or L4R proteins on PcrA activity were examined by adding either 15 pmol L3 or 7.5 pmol L4R to the reaction mixture. This corresponds to 1 molecule L3 per 10 nt M13mp18 ssDNA substrate or 1 molecule L4R per 20 nt M13mp18 ssDNA substrate. The reactions were stopped by adding 5 µl stop buffer (0.4% w/v SDS, 40 mM EDTA, 8% v/v glycerol, 0.1% w/v bromophenol blue). Displaced oligonucleotide was separated from annealed oligonucleotide by electrophoresis through a 12% non-denaturing polyacrylamide mini-gel at constant voltage (130 V). Gels were then dried and analysed quantitatively using a phosphorimager.

For reactions involving mixtures of PcrA helicase with L3 or L4R a 2 min pre-incubation of the reaction mixture at 37°C was done prior to addition of the PcrA helicase.

Electrophoretic mobility shift assays

The 3[prime]-tailed and nicked DNA substrates for gel shifts were prepared by annealing radioactively labelled 3[prime]-tailed oligonucleotide (5[prime]-ACTCTAGAGGATCCCCGGGTACGTTATTGCATGAAAGCCCGGCTG-3[prime]; 12) to oligonucleotide 5[prime]-GTACCCGGGGATCCTCTAGAGT-3[prime] and the 5[prime]-tailed oligonucleotide (5[prime]-GTTATTGCATGAAAGCCCGGCTGACTCTAGAGGATCCCCGGGTAC-3[prime]; 12) to oligonucleotides 5[prime]-AGCCGGGCTTTCATGCAATAAC-3[prime] and 5[prime]-GTACCCGGGGATCCTCTAGAGT-3[prime] respectively. The 3[prime]-tailed oligonucleotide itself was used as the ssDNA target for gel shifts. Because of the different affinities of PcrA, L3 and L4R for different substrates the relative ratio of protein to DNA was optimized for each DNA substrate.

Binding reactions of PcrA with 3[prime]-tailed and nicked DNA substrates were performed in a total volume of 20 µl in the same buffer as used for helicase assays supplemented with 2.5 mM ADPNP (a non-hydrolysable analogue of ATP) instead of ATP, using 53 fmol target DNA substrate and 1 pmol PcrA in the presence or absence of 15 pmol L3. Similar reactions with the same DNA substrates were also performed using 66 fmol target DNA and 0.5 pmol PcrA in the presence or absence of 7.5 pmol L4R. In all these reactions the PcrA to L3 or PcrA to L4R molar ratios were 1:15. Control reactions were carried out with 15 pmol L3 or 7.5 pmol L4R in the absence of PcrA.

Because of the higher binding affinities of PcrA and L4R for ssDNA, binding reactions with the ssDNA species were performed using 65 fmol target ssDNA and 0.130 pmol PcrA in the presence or absence of 15 pmol L3 or 0.5 pmol L4R. The PcrA to L3 and PcrA to L4R molar ratios in these reactions were 1:120 and 1:4. The control reactions contained 15 pmol L3 or 0.5 pmol L4R and no PcrA.

Binding reactions were performed at room temperature for 10 min and electrophoresis was performed as described for the helicase assays.

Escherichia coli ribosomal protein L3 stimulates the in vitro unwinding activity of B.stearothermophilus PcrA helicase in a substrate-independent manner

During mutagenesis studies on PcrA helicase a small molecular weight contaminant protein co-purified with a mutant PcrA protein (Fig. 1A). N-Terminal sequencing identified this protein as being E.coli ribosomal protein L3 (Fig. 1B). Escherichia coli L3 is highly homologous to the B.stearothermophilus ribosomal L3 protein (13). Purified E.coli L3 significantly enhances the in vitro unwinding activity of wild-type B.stearothermophilus PcrA helicase on short oligonucleotides annealed to M13mp18 ssDNA (Figs 2-5). This stimulatory effect is independent of the nature of the DNA substrate, i.e. it is evident irrespective of whether untailed or 3[prime]-tailed substrates were used to assay helicase activity. However, this stimulation is dependent upon the number of nucleotides of M13mp18 ssDNA substrate per L3 molecule in the assay reaction mixture. It is evident at [le]10 nt per L3 molecule, but it is not apparent at [le]100 nt per L3 molecule (Fig. 5). L3 has no unwinding activity on its own.

Figure 1. (A) An SDS-PAGE gel showing co-purified PcrA and L3 before gel filtration and purified PcrA and L3 proteins after gel filtration. (B) The N-terminal sequence determined from purified E.coli ribosomal protein L3 compared with that of ribosomal L3 protein from B.stearothermophilus (13).

Figure 2. The effect of L3 and L4R on PcrA helicase using a 3[prime]-tailed DNA substrate (A) An autoradiograph showing a representative helicase reaction time course. Reactions were done in a total volume of 20 µl, using 1 pmol PcrA and 0.02 pmol 3[prime]-tailed DNA substrate. The effect of L3 was examined by adding 15 pmol L3 to the reaction mixture. Reactions were stopped at appropriate time points as described in Materials and Methods and displaced oligo was separated from annealed oligo by gel electrophoresis as described in Materials and Methods. (B) Graph showing the effects of L3 and L4R proteins on the unwinding activity of PcrA helicase with time using as substrate a 3[prime]-tailed oligo annealed to M13mp18 ssDNA. PcrA helicase activity is shown with open circles, whereas the effects of L3 and L4R are shown with filled circles and open squares respectively. The inherent unwinding activities of L3 and L4R are shown with filled squares and filled diamonds respectively.

L3 does not affect the ATPase activity of PcrA

Escherichia coli L3 ribosomal protein has no detectable ATPase activity, either in the presence or absence of ssDNA (data not shown). In the presence of ssDNA B.stearothermophilus wild-type PcrA helicase is an ATPase exhibiting first order kinetics with a k_cat of 25/s and a K_m of 225 µM (L.E.Bird, personal communication). The ATPase activity in the absence of ssDNA is reduced 1200-fold. Escherichia coli ribosomal protein L3 did not affect the ATPase activity of B.stearothermophilus PcrA helicase, either in the absence or in the presence of optimal (1.2 µM) or sub-optimal (120 nM) ssDNA concentrations (Fig. 4). At 1.2 µM ssDNA the k_cat and K_m values for ATPase activity were unaffected in the presence of L3.

Vaccinia virus L4R protein affects the PcrA helicase activity in a substrate-dependent manner

Vaccinia virus ssDNA (and RNA) binding protein L4R also affects in vitro PcrA helicase activity. However, its effect is dependent upon the nature of the DNA substrate. It does not affect the activity of PcrA helicase on an untailed substrate (Fig. 3) but strongly inhibits the reaction upon a 3[prime]-tailed substrate (Fig. 2). In common with L3, L4R also has neither ATPase nor unwinding activity of its own.

Figure 3. The effect of L3 and L4R on PcrA helicase using an untailed DNA substrate. The effect with time of L3 and L4R on PcrA helicase activity on an untailed oligo annealed to M13mp18 ssDNA is shown. PcrA helicase activity is shown with open circles, whereas the effects of L3 and L4R are shown with filled circles and open squares respectively. The inherent unwinding activities of L3 and L4R are shown with filled squares and filled diamonds respectively. Reactions were done as described in Figure 2.

L3 enhances the DNA binding affinity of PcrA

The effect of L3 on PcrA helicase activity could be the result of either stimulation of the ATPase activity of PcrA or modulation of its interaction with the DNA substrate. L3 has no intrinsic ATPase activity and does not stimulate the ATPase activity of wild-type PcrA protein. However, L3 binds to ss and dsDNA (Fig. 6A). PcrA binds to different DNA substrates (3[prime]-tailed, nicked ds and ssDNAs) with different affinities. Its ability to bind to all of these substrates is enhanced markedly in the presence of L3 (Fig. 6A). In contrast, L4R has no effect upon the DNA binding affinity of PcrA (Fig. 6B). Although the results are complicated, as all of the proteins bind all of the different DNA substrates to some extent, it is evident that the proportion of DNA shifted in the presence of L3 and PcrA is greater than the sum of their individual contributions. This is not the case for the L4R/PcrA mixture.

Figure 4. The effect of L3 on the ATPase activity of PcrA helicase. A graph showing that E.coli ribosomal protein L3 does not affect the ATPase activity of B.stearothermophilus PcrA helicase. ATPase reactions were in the presence of 24 nM PcrA plus 2.4 µM L3 protein (filled symbols) or 24 nM PcrA protein (open symbols). The effect of L3 was investigated at optimal (1.2 µM, squares) and sub-optimal (120 nM, diamonds) ssDNA concentrations as well as in the absence of ssDNA (circles). In the absence of ssDNA the ATPase activities were very small and therefore the graphs in the presence or absence of L3 protein are indistinguishable.

Figure 5. The stimulatory effect of L3 on PcrA helicase activity is concentration dependent. (A) An autoradiograph showing that the stimulatory effect of L3 on PcrA helicase activity is concentration dependent. All reactions were for 10 min at 37°C in a total reaction volume of 20 µl, using 1 pmol PcrA and 0.02 pmol 3[prime]-tailed DNA substrate. Lanes a and b show the annealed oligo and displaced oligo (after heating at 95°C for 10 min) respectively. Lane c shows PcrA helicase activity in the absence of L3. Lanes d-f show the effect of adding increasing amounts of L3 to the reaction mixture: lane d, 0.15 pmol; lane c, 1.5 pmol; lane e 15 pmol. These are equivalent to 1 molecule PcrA per 1000, 100 and 10 nt M13mp18 ssDNA substrate respectively. (B) An autoradiograph showing a comparison of the effects of L3 and L4R at optimal concentrations on PcrA helicase activity. Lanes a and b show annealed and displaced (heated at 95°C for 10 min) oligos respectively. Lane c shows PcrA helicase activity in the absence of L3 or L4R. Lane d shows the stimulatory effect of L3, whereas lane e shows the inhibitory effect of L4R. All reactions were as desribed in (A). The effects of L3 and L4R were examined by adding 15 pmol L3 (lane d) or 15 pmol L4R (lane e) to the reaction mixture.

Figure 6. The effects of L3 and L4R on the DNA binding affinity of PcrA. (A) Electrophoretic mobility shift assays showing the effect of E.coli ribosomal protein L3 on the DNA binding affinity of B.stearothermophilus PcrA helicase on ss, nicked and 3[prime]-tailed DNA ligands. All reactions shown in this panel were as described in Materials and Methods, in a total volume of 20 µl, using 1 pmol PcrA and 53 fmol target DNA in the presence (15 pmol) or absence of L3. (B) Similar gel shift assays as in (A), showing that vaccinia virus protein L4R does not affect the DNA binding affinity of B.stearothermophilus PcrA helicase on ss, nicked and 3[prime]-tailed DNA ligands. All reactions shown in this panel were as described in Materials and Methods, in a total volume of 20 µl, using 0.5 pmol PcrA and 66 fmol target DNA in the presence (7.5 pmol) or absence of L4R.

DISCUSSION

Helicases are ubiquitous enzymes which catalyse the unwinding and strand separation of nucleic acid duplexes. They use the energy from hydrolysis of nucleoside triphosphates to translocate along the nucleic acid duplex, catalysing strand separation. These enzymes are involved in almost every aspect of nucleic acid metabolism. They share certain amino acid sequence similarities which denote `helicase signature motifs'. Our laboratory has solved the crystal structure of B.stearothermophilus helicase PcrA. (9). This is a helicase homologous to Rep and UvrD helicases from E.coli. It also shows structural similarities to the recombination protein RecA, implying that both proteins may employ certain similar mechanistic aspects during catalysis. Despite elucidation of the crystal structure of PcrA, its molecular mechanism of action and physiological role in the cell are not known. In an attempt to understand the molecular mechanism of helicase action we have produced and characterized a series of site-specific mutants of PcrA (manuscript in preparation). During purification of one of our mutants we co-purified a low molecular weight protein, which we identified by N-terminal sequencing as the 23 kDa E.coli ribosomal protein L3. It has been suggested that L3 plays a major role as a ribosomal assembly initiator protein under unfavourable growth conditions where the rRNA is produced in high excess over ribosomal proteins (14). The mutant PcrA is toxic to cells and can only be introduced and over-expressed in E.coli B834 pLysS cells (manuscript in preparation). Even then the cells grow very slowly. Such unfavourable growth conditions may cause a concomitant over-expression of L3, which then co-purifies with the mutant protein during the purification process. Wild-type PcrA does not co-purify with L3 when purified by either protocol (data not shown).

The L3 gene from B.stearothermophilus has been cloned and sequenced (13). The corresponding L3 protein was found to be highly homologous to E.coli L3 protein. L3 protein is known to bind to the 11S 3[prime]-fragment of 23S rRNA and is essential in the ribosomal assembly process (15). L3 was found to stimulate the in vitro helicase activity of PcrA on short oligonucleotides annealed to M13mp18 ssDNA. Untailed and 3[prime]-tailed substrates were used and L3 was shown to activate PcrA helicase activity irrespective of the nature of the DNA substrate, i.e in a substrate-independent manner. L3 has no ATPase activity and no unwinding activity on short oligonucleotides annealed to M13mp18 ssDNA. Although primarily an RNA binding protein, it can bind both ss and dsDNA, as shown by electrophoretic mobility shift assays (Fig. 6). Interestingly, another ribosomal protein (L14) has also been shown to stimulate the unwinding activity of E.coli Rep and UvrD helicases, which are highly homologous to PcrA (2,16). However, it should be emphasized that L3 has no homology to L14. L14 is thought to enhance the processivity of Rep by interacting with the M13 ssDNA substrate (2). However, specific protein-protein interactions between L14 and Rep and/or UvrD cannot be ruled out in view of its inability to affect helicase IV or helicase I (16). An analogous protein-protein interaction has been suggested during homologous DNA strand exchange reactions between the product of gene 2.5 of T7 phage, which is a ssDNA binding protein, and gene 4 protein, a hexameric helicase (5).

Vaccinia virus RNA binding protein L4R, which also binds ssDNA (17) and stimulates the unwinding activity of vaccinia virion helicase I8R (10), inhibited the helicase activity of PcrA when a 3[prime]-tailed substrate was used. In contrast, L4R did not affect PcrA helicase activity on an untailed substrate.

The exact molecular mechanism of the effects of the L3 and L4R proteins on the helicase activity of PcrA is unclear. The simplest explanation is that L3 interacts directly with PcrA via a protein-protein interaction and enhances the helicase activity of PcrA. This may be brought about partly by increasing the DNA binding ability of PcrA, as is suggested by our gel shift experiments. In the presence of L3 PcrA binds significantly better to a number of different DNA substrates, including 3[prime]-tailed, nicked ds and ssDNA (Fig. 6A). In the presence of L4R no such enhancement of PcrA binding to DNA is observed, despite the fact that L4R binds better than L3 to all DNA substrates used in the gel shift experiments (Fig. 6B). A protein-protein interaction is compatible with the substrate-independent nature of the L3 effect on PcrA and is also supported by the failure of L3 to stimulate other helicases, for example the hexameric helicase DnaB from B.stearothermophilus (L.E.Bird, personal communication) or vaccinia virion DNA helicase I8R (data not shown). If the unwinding activity of PcrA was stimulated non-specifically by the ssDNA binding activity of L3 then other ssDNA binding proteins should have a similar effect on PcrA helicase activity. We show that this is not the case for the ssDNA binding protein L4R. Suggestions as to the molecular mechanisms of such effects can be found elsewhere (16). Since such mechanisms operate primarily via interactions of the stimulatory protein with the DNA substrate they are likely to be substrate dependent. This is indeed the case of the L4R effect on PcrA helicase. Although it does not affect the helicase activity on an untailed oligonucleotide annealed to M13mp18 ssDNA, it inhibits the helicase activity on a 3[prime]-tailed substrate.

In summary, we have isolated a low molecular weight protein from E.coli which stimulates the helicase activity of B.stearo-thermophilus PcrA helicase. We identified this protein by N-terminal sequencing as ribosomal protein L3. We compared its effect on B.stearothermophilus PcrA helicase with that of vaccinia virus L4R protein (a ssDNA binding protein) and showed that the L3 protein enhances the DNA binding affinity of PcrA. Furthermore, we showed that this effect is specific for PcrA, since L3 failed to affect the helicase activity of other helicases (B.stearothermophilus DnaB and vaccinia virus I8R proteins).

In order to show that ribosomal protein L3 does interact with PcrA helicase by a protein-protein interaction we will clone the L3 ribosomal protein from the same strain of B.stearothermophilus from which PcrA was cloned. This will enable us to try and co-crystallize the two proteins in order to identify the protein-protein interaction interface.

ACKNOWLEDGEMENTS

We wish to thank Val Cooper for synthesis of the oligonucleotides, Tony Willis for N-terminal peptide sequencing, Chris D.Bayliss and Geoff L.Smith for kindly providing the L4R and I8R proteins and Louise E.Bird for checking the effect of L3 on DnaB. This work was supported by the Wellcome Trust.

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*To whom correspondence should be addressed. Tel: +44 1865 285479; Fax: +44 1865 275515; Email: wigley@eric.path.ox.ac.uk

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