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Analysis of the subunit assembly of the typeIC restriction-modification enzyme EcoR124I
Introduction
Materials And Methods
DNA substrates
Protein preparations
Reconstitution of the endonuclease
Determination of subunit ratio in the endonuclease-DNA complexes
HPLC gel filtration
DNA cleavage and ATPase assays
Gel retardation assay
Results
Gel retardation analysis of EcoR124I endonuclease reveals two molecular species
Analysis of subunit stoichiometry in protein-DNA complexes of EcoR124I
Analysis of interaction between the MTase and HsdR in the presence of DNA
Analysis of interactions between the MTase and HsdR in the absence of DNA
DNA cleavage and ATPase activities of MTase-HsdR complexes
Discussion
Acknowledgements
References
Analysis of the subunit assembly of the typeIC restriction-modification enzyme EcoR124I
ABSTRACT
INTRODUCTION
Type I restriction and modification (R-M) systems protect the bacterial cell against invasion of foreign DNA (such as viruses) by cleaving DNA which lacks a target specific N6-adenine methylation. The second physiological role of these systems is to restore full methylation of the target sites on the host DNA after DNA replication. Type I R-M enzymes are composed of three different subunits (HsdR, HsdM and HsdS) encoded by the three hsd genes (for recent reviews see 1,2). All three subunits are absolutely required for restriction activity, while the HsdM and HsdS subunits are sufficient for modification activity and can also form an independent DNA methyltransferase (MTase). Type I R-M enzymes specifically recognise a non-palindromic DNA sequence (e.g. GAAnnnnnnRTCG for EcoR124I, where n is any base and R is a purine) but cleave DNA at a non-specific site distant from the recognition sequence using Mg2+, S-adenosylmethionine (AdoMet) and ATP as cofactors (3,4). Binding of the endonuclease to a non-modified recognition site activates a powerful ATPase activity (5), which fuels DNA translocation past the DNA-enzyme complex, while the enzyme remains bound to the recognition site (6,7). DNA is cleaved at positions where the DNA translocation stops either due to a collision of two translocating enzyme molecules on two-site, linear DNA substrates (8,9), or due to the build-up of topological strain on circular molecules (10). The endonuclease does not turnover in the cleavage reaction, however, the ATPase activity continues for a long period of time after the cleavage is completed (3,5). DNA methylation activity of the type I R-M systems results in a transfer of a methyl group from AdoMet to the N-6 position of a specific adenine in each strand of the recognition sequence (11,12).
Type I R-M systems are grouped into four families based on allelic complementation, protein homologies and biochemical properties of the enzymes. Types IA, IB and ID R-M systems are chromosomally encoded (13,14) while most type IC R-M systems are carried on large conjugative plasmids (15-17). The type IA family is typified by the EcoKI and EcoBI enzymes, type IB by EcoAI and type IC by EcoR124I. EcoKI forms a stable R2M2S1 complex; the independent EcoKI MTase (M2S1) is, however, a relatively weak complex, dissociating into an inactive M1S1 species and free HsdM subunit (18-20). The purified EcoBI restriction endonuclease has been described as existing in a number of different stoichiometric forms including R2M2S1, R1M2S1 and R1M1S1 (21). The type IB restriction endonuclease EcoAI was found to be a weak complex that dissociated into MTase and HsdR subunit when purified, and the active endonuclease could only be studied following assembly in vitro (22). We have recently purified the EcoR124I restriction endonuclease from a cell culture harbouring a recombinant, two-plasmid system, which over-produces all three subunits. The stoichiometry of this endonuclease preparation appeared to be R1M2S1 (23). The EcoR124I MTase has also been over-produced and purified to homogeneity (12,24). This enzyme was found to exist only in the M2S1 stoichiometry and no M1S1 complex has been detected (12).
The hsd genes of all R-M systems can be transferred into an unmodified host by conjugation, transformation or transfection despite the fact that the presence of a restriction enzyme in the recipient cell would be a lethal event (17,23,25-31). It has been reported that the appearance of restriction activity was delayed after conjugative transfer of EcoKI genes into an unmodified host, while the modification activity was expressed immediately after the conjugation (29). It was proposed that restriction and modification activities of the EcoKI R-M system are regulated by a gene distant from the hsd locus since the conjugative transfer of EcoKI hsd genes into a mutant form of Escherichia coli C (designated as hsdC) had a lethal effect on the cell (29). The same regulation mechanism was found to be employed in the type IB R-M systems (32). However, the conjugal transfer of EcoR124I (IC) hsd genes into the hsdC mutant was not lethal and full restriction activity was detected six generations after the start of conjugation (32). Recently, it has been shown that ClpX and ClpP, components of the ClpXP protease, are necessary for efficient transmission of genes encoding the EcoKI and EcoAI R-M systems, suggesting protease-mediated post-translational control of restriction and modification (33). The involvement of protease control of restriction and modification would support the proposal that post-translational control of restriction activity of EcoKI could be related to subunit assembly (20).
In this paper we show that the purified EcoR124I restriction endonuclease is a mixture of two species, which have a subunit stoichiometry of R2M2S1 and R1M2S1, respectively. Only the former species was found to have endonuclease activity. However, the R2M2S1 complex is relatively weak, dissociating into free HsdR subunit and the restriction-deficient R1M2S1 assembly intermediate, which appears to be a very tight complex. We discuss the relevance of this situation with respect to the propagation of a conjugative plasmid-borne R-M system into an unmodified host cell. We suggest that control of EcoR124I endonuclease activity in vivo is at the level of subunit assembly in a manner similar to that observed for EcoKI (20).
MATERIALS AND METHODS
DNA substrates
Complementary HPLC-purified oligonucleotides used in the gel retardation assays were purchased from Cruachem Ltd. The EcoR124I recognition site is shown in bold (top strand: 5[prime]-CTACGGTACCGAAACGCGTGTCGGGCCCGCGAAGCTTGC-3[prime]), DNA concentration was determined from the absorption at 260 nm. The extinction coefficients of the oligonucleotides and the annealed oligoduplex were calculated as a sum of the contributions from individual nucleotides, taking into account hyperchromicity observed after digestion of the DNA to completion with snake venom phosphodiesterase (34). The oligoduplex (usually 2.5 µg) was 5[prime]-end-labelled using [[gamma]-32P]ATP (25 µCi) and T4 polynucleotide kinase. Unincorporated label was removed using `Nuctrap' columns (Stratagene).
A 2891 bp plasmid containing a single EcoR124I recognition site, pDRM-1R, (23) used in DNA cleavage assays was produced from E.coli HB101 (35) and its covalently closed form was purified by an equilibrium centrifugation in a CsCl-ethidium bromide gradient (36).
Protein preparations
The EcoR124I restriction endonuclease, MTase (M2S1) and HsdR subunit were over-produced and purified as described previously (12,23). Either the plasmid pACR124 or pBGSR124 containing the hsdR gene under the control of the Ptrc promoter (23), were used together with the MTase plasmid pJS4M (24) to over-produce the endonuclease. The latter plasmid combination produced a higher concentration of HsdR than the former, presumably due to the higher copy number of pBGSR124. Both two-plasmid systems produced the same concentrations of the HsdS and HsdM subunits (data not shown). To obtain a stable clone containing the pJS4M-pBGSR124 plasmid-expression system, the cells were first transformed with the pJS4M plasmid (MTase) and then with pBGSR124 plasmid (HsdR). Transformation of pJS4M and pBGSR124 at the same time was found to be lethal. The molar concentration of purified proteins was determined from the absorbance at 280 nm using molar extinction coefficients calculated as a sum of contributions from tyrosines and tryptophans in the predicted amino acid sequences [HsdR subunit;91 900/M/cm, MTase (M2S1); 160 400/M/cm, endonuclease,252 300/M/cm for R1M2S1, 344 200/M/cm for R2M2S1].
Reconstitution of the endonuclease
Fully active reconstituted EcoR124I endonuclease, used in the DNA cleavage and gel retardation assays, was simply produced by mixing of the purified MTase (M2S1) and HsdR subunit in assay reaction buffers (see below). The concentration of active endonuclease was taken as the input concentration of the MTase.
Determination of subunit ratio in the endonuclease-DNA complexes
The two DNA-protein complexes observed with purified and reconstituted EcoR124I endonucleases (Fig.
HPLC gel filtration
HPLC gel filtration used a Rainin Dynamax 4.6 × 250 mm Hydropore-5-SEC column and a guard column. Samples of 20 or 50 µl, in 20 mM Tris, 20 mM 2-[N-morpholine] ethane sulphonic acid (MES), 0.2 M NaCl, 10 mM MgCl2, 7 mM [beta]-mercaptoethanol, 0.1 mM EDTA, pH 6.5, were injected onto the column. The samples were equilibrated at room temperature for varying time periods up to 24 h prior to injection. The flow rate was 0.5 ml/min and detection was most sensitive and stable at 254 nm. The column was calibrated with a series of globular proteins of known molecular weight giving a linear calibration curve of log (molecular weight) as a function of elution time. Most of our samples after buffer exchange into the column buffer using PD10 Sephadex G50 columns (Pharmacia), contained trace amounts of a small molecule which we believe to be glycerol. This gave rise to a `solvent peak' after ~6.5 min, the elution time of which served as an internal standard to correct for slight run to run variation in protein elution times. The apparent molecular weight of the proteins eluting from the column was calculated. In the titration of MTase with HsdR subunit, the HsdR subunit-MTase complex was present only as a partially resolved shoulder when HsdR subunit was in large excess of the MTase.
DNA cleavage and ATPase assays
DNA cleavage and ATPase activities of EcoR124I restriction endonuclease were assayed using plasmid pDRM-1R (single EcoR124I site) as described previously (23). DNA, usually at a concentration of 200 nM, was incubated either with an equimolar concentration of the MTase and increasing concentration of the HsdR, or with a range of concentrations of reconstituted endonuclease. After addition of ATP, the reactions were incubated for 5 min (sufficient time to reach the reaction endpoint). DNA cleavage activity (concentration of full-length linear plasmid DNA) and ATPase activity (concentration of inorganic phosphate) were measured as described previously (23).
Gel retardation assay
DNA binding reactions were usually performed in a volume of 10 µl in the presence of a buffer consisting of 50 mM Tris-HCl (pH 8.0), 50 mM NaCl, 10 mM MgCl2, 1 mM DTT and 10% (v/v) glycerol. The end-labelled 39mer oligoduplex (see above) of known concentration was incubated with purified endonuclease, MTase and MTase/HsdR or endonuclease (ENase)/HsdR mixtures over a range of various concentrations and/or protein:protein ratios, respectively, at room temperature for 10 min. Unbound DNA and DNA-protein complexes were separated on a 5% non-denaturing polyacrylamide gel run, at 4oC, at 100 V in 40 mM Tris-acetate pH 8.0, 1 mM EDTA buffer (TAE). After electrophoresis, gels were dried and subjected to autoradiography or quantified using a PhosphorImager and Image Quant software.
RESULTS
Gel retardation analysis of EcoR124I endonuclease reveals two molecular species
We have previously purified the EcoR124I endonuclease following over-production of the protein by a recombinant, two-plasmid system consisting of pJS4M (MTase) and pACR124 (HsdR). Using a combination of data from gel filtration and densitometric scanning SDS-polyacrylamide gels stained with Coomassie Blue, we concluded that this enzyme had a stoichiometry of R1M2S1 (23).
Figure 1. Gel retardation analysis of purified EcoR124I restriction endonuclease. DNA binding reactions were carried out with 200 nM 39mer oligoduplexes as described in Materials and Methods. Lane 1, free DNA; lane 2, 200 nM endonuclease over-produced by the pJS4M-pACR124 plasmid system; lane 3, as lane 2 plus 1 µM HsdR; lane 4, 200 nM endonuclease over-produced by the pJS4M-pBGSR124 plasmid system; lane 5, as lane 4 plus 1 µM HsdR; lane 6, as lane 4 plus 200 nM MTase; lane 7, 200 nM MTase; lane 8, 200 nM MTase plus 200 nM HsdR; lane 9, 200 nM MTase plus 1200 nM HsdR. Positions of individual DNA-protein complexes and unbound DNA on the gel are indicated. To investigate the DNA-binding properties of the over-produced EcoR124I endonuclease, the technique of gel retardation, with a 39mer oligoduplex carrying a single EcoR124I recognition site, was utilised. Unexpectedly, two DNA-protein complexes were observed after separation of bound and unbound DNA on a non-denaturing polyacrylamide gel (Fig. 
Analysis of subunit stoichiometry in protein-DNA complexes of EcoR124I
To investigate the stoichiometry of the two species present in the endonuclease preparations, the proteins bound in complexes I and II were extracted from the retardation gel and subjected to an SDS-PAGE analysis together with a series of R/MTase mixtures, including ratios 0.5:1, 1:1, 2:1, 3:1 and 4:1. The relative ratio of the three endonuclease subunits, in each sample, was determined from a densitometer scan of the polyacrylamide gel after staining the gel with Coomassie Blue (23). As can be seen in Figure
Figure 2. Subunit composition of the gel-retarded protein-DNA complexes of EcoR124I. The histograms compare relative ratios of subunits extracted from the two DNA-protein complexes of purified EcoR124I endonuclease produced by the pJS4M-pBGSR124 plasmid system (complexes I and II shown in Fig. 1) to the relative ratios of subunits in a series of mixtures of HsdR and MTase, as determined by densitometric scanning of SDS-PAGE gels stained with Coomassie Blue. White bars, HsdS; grey bars, HsdM; black bars, HsdR. Panels 1-5 on the x-axis correspond to the following HsdR:MTase ratios: 0.5:1, 1:1, 2:1, 3:1, 4:1. Panels 6 and 7 are complex I and complex II, respectively. The data suggest that the stoichiometry of the species in complex I is R1M2S1, and the stoichiometry of the species in complex II is R2M2S1.
Analysis of interaction between the MTase and HsdR in the presence of DNA
To investigate the endonuclease assembly in vitro, we performed a reconstitution titration of the MTase (M2S1) with the HsdR subunit, and followed this by a gel retardation assay using a 39mer oligoduplex carrying the EcoR124I recognition site (Fig.
Figure 3. Titration of EcoR124I MTase with HsdR followed by gel retardation assay. A mixture of 200 nM MTase and 200 nM 39mer oligoduplex, containing one EcoR124I recognition site, was incubated with increasing concentrations of HsdR subunit at room temperature for 10 min. DNA-protein complexes and free DNA were separated on a non-denaturing polyacrylamide gel as described in Materials and Methods. The HsdR:MTase ratio in individual reactions and the position of DNA-protein complexes on the gel are indicated. In order to measure an apparent dissociation constant characterising the equilibrium between R1M2S1 and R2M2S1 species, the subunit complexes present in a series of dilutions of a 1:2 mixture of the MTase and HsdR, starting from a protein concentration of 1 mM, were analysed by gel retardation assay in the presence of 20 nM 39mer oligoduplex. Figure 
Table 1.
| Sample | Apparent molecular weight by HPLC gel filtration (kDa) |
Expected molecular weight (kDa) |
| M2S1 MTase | 220-230 | 162.2 |
| HsdR subunit | 137-148 | 119.6 |
| In vivo assembled R1M2S1a | 280-300 | 281.8 |
| In vivo assembled mixture of R1M2S1 + R2M2S1b | 333-350 | 281.8 + 401.5 |
| In vitro assembled R2M2S1 (freshly prepared) | 290-315 | 401.5 |
| In vitro assembled R2M2S1 (after 24 h incubation) | 380-420 | 401.5 |
Analysis of interactions between the MTase and HsdR in the absence of DNA
To analyse endonuclease assembly in the absence of DNA, we used HPLC gel filtration. The apparent molecular weights of various subunit complexes of EcoR124I from an HPLC gel filtration column were determined from their elution times and are given in Table 1. It can be seen that the MTase and HsdR subunit eluted from the column earlier than expected, giving a slightly higher weight. Similar behaviour was observed for the EcoKI enzyme (20). The endonuclease purified from cells containing the pJS4M-pACR124 plasmid-expression system eluted with an apparent molecular weight similar to that expected for R1M2S1. This is in agreement with previously published data (23). The endonuclease purified from cell culture containing the pJS4M-pBGSR124 plasmid-expression system had an apparent mass intermediate between R1M2S1 and R2M2S1 and formed a slightly asymmetric peak suggesting that it was a mixture of these two species. A freshly prepared mixture of MTase with a 5-fold excess of HsdR subunit, eluted with an apparent weight close to that expected for R1M2S1. However, the same mixture after a 24 h incubation at room temperature showed the formation of a higher molecular weight species with a weight equal to that expected for the R2M2S1 endonuclease. Figure
(a)
 (b)  |
Figure 4. Dissociation of the EcoR124I endonuclease complex. Dissociation of R2M2S1 into R1M2S1 and HsdR was monitored by a gel retardation assay with the 39mer oligoduplex containing one EcoR124I recognition site. The endonuclease was reconstituted by mixing the MTase and HsdR in a molar ratio of 1:2. A series of dilutions of this mixture in a concentration range from 1000 to 5 nM was incubated with 20 nM DNA at room temperature for 10 min and subsequently analysed on a 5% non-denaturing polyacrylamide gel (a). Lanes 1-12 correspond to the following protein concentrations: 1000, 500, 200, 150, 100, 80, 60, 40, 20, 10, 5, 0 nM. The gel was quantified using a PhosphorImager and the percentage of R2M2S1-DNA complex of the total bound DNA was plotted against protein concentration (b).
(a)
 (b)  |
Figure 5. Elution profiles from the HPLC gel filtration column and titration of MTase with HsdR followed by HPLC gel filtration. (a) From left to right the traces show MTase (420 nM), HsdR subunit (1 µM), MTase (420 nM) freshly mixed with a 5-fold excess of HsdR subunit, and MTase (420 nM) incubated with a 5-fold excess of HsdR subunit for 24 h before application to the column. The numbers are the elution times, in minutes, of the peaks. (b) The molecular weight, determined by HPLC gel filtration, of the complex formed between MTase and HsdR subunit as the HsdR subunit concentration is increased. The MTase concentration was 420 nM throughout. The samples were applied to the column immediately after mixing ([open circle]) or after 24 h incubation ([solid circle]). The lines drawn are only to guide the eye.
DNA cleavage and ATPase activities of MTase-HsdR complexes
To investigate enzyme activities of the R1M2S1 and R2M2S1 species, DNA cleavage and ATP hydrolysis were assayed following a reconstitution titration of the MTase with HsdR in the presence of a plasmid DNA substrate. The DNA used contained a single EcoR124I recognition site. This DNA readily undergoes primary cleavage to produce full-length linear DNA. Secondary processing of the linear product is observed only at very high enzyme:DNA ratios (23). Reaction products (linear DNA and inorganic phosphate, respectively) were quantified by densitometric scanning of agarose gels and malachite green assay as described elsewhere (23). A gradually increasing concentration of HsdR subunit was added into a 1:1 mixture of the MTase and DNA, which were present at a concentration of 200 nM to ensure full DNA binding. The same concentration conditions were also used in the reconstitution titration followed by a gel retardation assay (Fig.
DISCUSSION
The gel retardation and DNA cleavage experiments presented in this work revealed that the active form of the type IC EcoR124I restriction endonuclease has a subunit stoichiometry of R2M2S1. It is assembled from the trimeric MTase (M2S1) and the HsdR subunits through a stable intermediate with a stoichiometry of R1M2S1, which does not cleave DNA. While binding between the MTase and the first HsdR subunit is apparently very stable, interaction between the R1M2S1 intermediate and the second HsdR subunit is much weaker. The R2M2S1 complex dissociated into the R1M2S1 complex and the HsdR subunit with an apparent Kd of ~2.4 × 10-7 M (Fig.
In contrast to EcoR124I, the R2M2S1 complex of EcoKI endonuclease, a member of the type IA family, is relatively stable and both HsdR subunits appear to be bound to the MTase with similar affinities (20; L.Powell and N.Murray, personal communication).
Our results indicate that the two HsdR subunits interact differently with the MTase in the assembly of the EcoR124I endonuclease. A simple model for assembly would be that there are two equivalent HsdR-binding sites on the MTase, but that the presence of one bound HsdR subunit affects the binding of the second HsdR subunit perhaps by partially blocking the second site, or by means of a conformational change at the second site. This model would require that the R1M2S1 complex should be a 1:1 mixture of two species with occupation of either of the two HsdR-binding sites. A second model would require that the two sites are not equivalent and that the assembly is inherently asymmetric with only one type of R1M2S1 complex being formed. However, the data presented in this work are not sufficient to definitely determine which model is applicable.
Figure 6. Titration of EcoR124I MTase with HsdR, followed by measurement of DNA cleavage and ATPase activity. A mixture of 200 nM MTase and 200 nM pDRM-1R DNA was incubated with increasing concentrations of HsdR in the presence of 0.2 mM AdoMet and 10 mM ATP. Concentration of linear DNA produced after 5 min incubation (a) and the rate of ATP hydrolysis (b) was plotted against HsdR:MTase molar ratio. Reaction products were quantified as described elsewhere (23). The lines drawn are only to guide the eye. Since binding of the first HsdR subunit to the MTase is much stronger than binding the second HsdR subunit to the R1M2S1 intermediate, the in vivo formation of the R2M2S1 complex will be dependent on the ratio between the HsdR subunit and the MTase in the cell. At an HsdR:MTase ratio of <1, only the R1M2S1 complex will be present. The R2M2S1 complex will be formed only when the HsdR:MTase ratio is >1. Indeed, when the ratio between MTase and HsdR was varied in vivo by using different recombinant two-plasmid systems, the resulting endonuclease preparations differed in ratio between the species (Fig. Why should the restriction endonuclease of the EcoR124I R-M system be such a weak complex and the stable assembly intermediate R1M2S1 not be able to cleave DNA? We suggest this may reflect a very sensitive mechanism for regulation of restriction activity following conjugative transfer of the hsd genes into a non-modified recipient cell. The EcoR124I R-M system was originally found on the conjugative plasmid R124 (16) and this presents an unusual problem for the recipient cell. If the R124 plasmid entered a new host and immediately produced an active endonuclease, the unmodified host DNA would be restricted and the host cell would not survive. However, this does not happen and the host chromosome is modified following conjugation. Despite various attempts to show transcriptional or post-transcriptional control of this phenomenon for the EcoR124I R-M system (32; K.Firman, unpublished observations), there is no evidence of such control. Nor is there any evidence of such control for the other type I R-M systems. Therefore, we propose that this control is exercised at the level of subunit assembly. The M2S1MTase is a very tight complex and it will be assembled first in the cell. Since the MTase can form a tight complex with one HsdR subunit, which does not cleave DNA, all the HsdR subunit will initially be trapped in this inactive complex. Therefore, the MTase (and possibly the R1M2S1 complex) can modify and protect the host DNA. The weak endonuclease complex R2M2S1 can be formed only when a large amount of HsdR has built up in the cell, which is unlikely during the early stage of establishing the R-M system in a new host. When cellular levels of individual subunits were altered by using different promoters and expression vectors, it was observed that excess HsdR over MTase was lethal, unless the MTase was already established in the cell (23). This implies that the function of the EcoR124I endonuclease in the cell is finely balanced and absolutely dependent upon subunit concentration. Control of restriction and modification via subunit assembly has also been recently proposed for the EcoKI restriction endonuclease (20), suggesting this mechanism may be common to all type I systems. In addition, the EcoR124I system shows a relatively high methylation activity with non-modified DNA (23) when compared to that of EcoKI (11). This will also ensure that a non-modified chromosome in the recipient cell can be readily modified following conjugal transfer of the hsd genes.
ACKNOWLEDGEMENTS
The authors would like to thank Lynn Powell and Noreen Murray (University of Edinburgh) for communicating results prior to publication. P.J. was supported by an International Fellowship from the Wellcome Trust. D.D. is supported by a Royal Society University Research Fellowship.
REFERENCES
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