Functional Roles of Conserved Amino Acid Residues in DNA Methyltransferases Investigated by Site-directed Mutagenesis of the EcoRV Adenine-N6-methyltransferase*

Markus Roth, Sabine Helm-Kruse, Tatjana Friedrich, and Albert JeltschDagger

From the Institut für Biochemie, Fachbereich Biologie, Justus-Liebig-Universität, Heinrich-Buff-Ring 58, D-35392 Giessen, Germany

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

All DNA methyltransferases (MTases) have similar catalytic domains containing nine blocks of conserved amino acid residues. We have investigated by site-directed mutagenesis the function of 17 conserved residues in the EcoRV alpha -adenine-N6-DNA methyltransferase. The structure of this class of MTases has been predicted recently. The variants were characterized with respect to their catalytic activities and their abilities to bind to DNA and the S-adenosylmethionine (AdoMet) cofactor. Amino acids located in motifs X, I, and II are shown to be involved in AdoMet binding (Lys16, Glu37, Phe39, and Asp58). Some of the mutants defective in AdoMet binding are also impaired in DNA binding, suggesting allosteric interactions between the AdoMet and DNA binding site. Asp78 (motif III), which was supposed to form a hydrogen bond to the AdoMet on the basis of the structure predictions, turned out not to be important for AdoMet binding, suggesting that motif III has not been identified correctly. R128A and N130A, having mutations in the putative DNA binding domain, are unable to bind to DNA. Residues located in motifs IV, V, VI, and VIII are involved in catalysis (Asp193, Tyr196, Asp211, Ser229, Trp231, and Tyr258), some of them presumably in binding the flipped target base, because mutations at these residues fail to significantly interfere with DNA and AdoMet binding but strongly reduce catalysis. Our results are in substantial agreement with the structure prediction for EcoRV alpha -adenine-N6-methyltransferase and x-ray structures of other MTases.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Methylation of DNA is essential in mammals. It is involved in regulation of gene expression, imprinting, development, chromatin structure, DNA replication, and origin of cancer (for reviews, see Refs. 1-5). DNA can be modified by two classes of DNA methyltransferases (MTases),1 viz. C-MTases modifying cytosines at the C5-position and N-MTases transferring a methyl group to cytosine-N4- or adenine-N6-positions. Prokaryotic DNA MTases comprise approximately 250-400 amino acid residues (for reviews, see Refs. 6-9). The C-terminal domain of eukaryotic enzymes (for a review, see Ref. 1) bears homology to the prokaryotic cytosine-C5-MTases, which contain 10 conserved amino acid motifs (for reviews, see Refs. 8 and 10).

So far the structures of two prokaryotic C-MTases are known, M. HhaI (11-14) and HaeIII DNA methyltransferase (15). Both enzymes consist of two domains. One large, catalytic domain forms the binding site for the cofactor S-adenosylmethionine (AdoMet) and the catalytic center and harbors nine of the 10 conserved amino acid motifs. One smaller domain is responsible for DNA recognition. The catalytic mechanism of these enzymes involves flipping out the target base from the DNA helix (12). Then a conserved cysteine (motif IV) performs a nucleophilic attack on the C6-position of the cytosine, forming a covalent enzyme-DNA intermediate. Thereby, the C5-position is activated and accepts the methyl group from AdoMet. Subsequently, the covalent intermediate is deprotonated at C5 leading to the elimination of the cysteine (16, 12, 14).

N-MTases contain nine conserved amino acid motifs. An FXGXG motif, also present in cytosine-C5-MTases, and a DPPY motif (consensus sequence, (S/N/D)PP(Y/F/W)) are moderately conserved (17-20), and the homologies of seven additional motifs are weak. Consequently, these additional motifs only recently could be identified in structure-guided alignments (21). Whereas some amino acid residues within the FXGXG and DPPY motifs have already been investigated by site-directed mutagenesis (22-25), no mutational studies have been carried out so far to test the functional roles of conserved amino acid residues within the other motifs either in C- or N-MTases.

According to the amino acid sequences and order of the conserved motifs, N-MTases can be subdivided into three distinct groups, viz. alpha -N-MTases (e.g. M. EcoRV, an adenine-N6 MTase), beta -N-MTases (e.g. PvuII DNA methyltransferase, a cytosine-N4-MTase), and gamma -N-MTases (e.g. M. TaqI, an adenine-N6-MTase) (20, 21). Eukaryotic enzymes involved in mRNA processing, double-stranded RNA adenosine deaminase (26), and mRNA(adenosine-N6)-methyltransferase (27) are similar to DNA N-MTases. So far, structures are available only for beta - and gamma -type apoenzymes (M. TaqI (28) and PvuII DNA methyltransferase (29)). Like C-MTases, N-MTases are built up of two domains. Surprisingly, the catalytic domain of N-MTases has a similar structure as the catalytic domains of C-MTases, although the chemistry of catalysis is different (30). Interestingly, the conserved DPPY motif structurally corresponds to motif IV in C-MTases, which contains the catalytic cysteine residue. It is likely that N-MTases, like C-MTases, flip their target base out of the DNA double helix (31-34).

The EcoRV DNA methyltransferase specifically methylates the first adenine within the EcoRV recognition sequence GATATC (35). The enzyme is well characterized with respect to its DNA binding and kinetic properties (33, 34, 36-39). Using multiple sequence alignments (Fig. 1) as well as secondary structure predictions the structure of M. EcoRV and other alpha -type N-MTases was predicted to contain a similar catalytic domain as the other MTases (Fig. 2) (21, 40). On the basis of these predictions, putative functional roles for several conserved amino acid residues (Fig. 1) can be derived, e.g. involvement in DNA binding, AdoMet binding, or catalysis (Fig. 2). In particular, a putative binding site for the flipped target base can be derived by comparison with C-MTases (31, 21). Here we report the results of an extensive mutational study of 17 conserved amino acid positions of M. EcoRV that allow us to draw conclusions as to (i) the validity of the structural model for M. EcoRV and alpha -N-MTases, (ii) the functional roles of some amino acid residues within the conserved motifs of N-MTases, and (iii) the catalytic mechanism of N-MTases. To our knowledge, this work represents the first comprehensive analysis of a DNA MTase by site-directed mutagenesis that covers positions located in all conserved motifs as well as in the DNA recognition domain. Thus, it complements the detailed structural information available for this important family of enzymes.


View larger version (56K):
[in this window]
[in a new window]
 


View larger version (67K):
[in this window]
[in a new window]
 
Fig. 1.   Alignments of M. EcoRV with other alpha -N-MTases. Amino acid exchanges introduced in M. EcoRV in this work are indicated by vertical arrows, which point to the identity of the new amino acid. Strongly and moderately conserved residues are boxed and shaded darkly and lightly, respectively. The position of the putative DNA binding domain is indicated by vertical arrows. A, alignment of the catalytic domains of all alpha -N-MTases. On the basis of this structure-guided multiple alignment, 10 conserved amino acid regions have been defined (21). B, alignment of M. EcoRV with the seven most closely related alpha -N-MTases (40). The approximate locations of conserved amino acid motifs as defined in Ref. 21 are indicated.


View larger version (53K):
[in this window]
[in a new window]
 
Fig. 2.   Location of mutated amino acid residues in the structural model of M. EcoRV (40). Strongly conserved amino acid residues are given in gray ovals, and less conserved residues are in white ovals. The positions of the putative AdoMet and adenine binding sites are indicated. The structure prediction is taken from Jeltsch et al. (40). It does not differ from that given by Malone et al. (21) in the region of amino acid residues 1-235. alpha -helices and beta -strand are labeled A-F and 1-9. Localizations of the secondary structure elements in primary sequence are as follows: alpha -helix A, 18-26; beta -strand 1, 42-46; alpha -helix B, 44-48; beta -strand 2, 53-58; alpha -helix C, 61-63; beta -strand 3, 64-70; alpha -helix D, 78-86; beta -strand 4, 188-192; alpha -helix E, 211-222; beta -strand 5, 224-229; alpha -helix F, 239-248; beta -strand 6, 250-258; beta -strand 7, 270-276; beta -strand 8, 280-285; beta -strand 9, 292-299.

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Site-directed Mutagenesis-- Site-directed PCR mutagenesis was carried out on pRVMetH6, encoding the N-terminal His6-M. EcoRV fusion protein (34, 40). In this vector, the M. EcoRV gene is controlled by a synthetic promotor-operator sequence obtained by a combination of the T5 promotor and two lac operators. PCR mutagenesis was performed using a megaprimer protocol modified after Ito et al. (41). Briefly, the method employs three PCR primers (Fig. 3): pup binds to the coding strand upstream of the gene of M. EcoRV; plow1 binds to the lower strand downstream of the gene and removes a SalI site 3' to the gene; and plow2 binds downstream of plow1 but leaves the SalI site intact. The desired mutation is introduced into the gene (together with a marker site) by an additional upper primer (pmut) in a PCR together with plow2. The PCR product that consists of a part of the M. EcoRV gene and carries a SalI site 3' to the gene was hybridized to a PCR product produced using pup and plow1, which contains a BamHI-site 5' to the gene but no SalI site 3' to the gene. After three PCR fill in steps, a PCR using pup and plow2 was carried out. After this procedure, only mutated products carry a SalI site on their 3'-ends and can be cloned into the large BamHI/SalI fragment of pRVMetH6. Usually 30-50% of the obtained clones contained the marker site and the desired mutation. All genes were completely sequenced after mutagenesis.


View larger version (7K):
[in this window]
[in a new window]
 
Fig. 3.   Primers used for site-directed mutagenesis of the M. EcoRV gene. Primer pmut introduces a mutation into the gene of M. EcoRV (symbolized by a triangle) along with a marker site (symbolized by a rectangle). Note that different pmut primers were used for the different mutations introduced. Primer plow1 removes a SalI site downstream of the M. EcoRV gene (symbolized by ×).

In Vivo Activity Assays-- Due to background expression, plasmids encoding a DNA MTase are resistant to cleavage by the corresponding restriction endonuclease. This property can be used to assay the catalytic activity of MTases in vivo. To this end, pRVMetHis (34, 40) or pGEXMRV (42) plasmids were grown in Escherichia coli LK111(lambda ) cells containing chromosomally encoded lacIq. The transcription of the M. EcoRV genes was not induced. Plasmid preparations from overnight cultures and from growing cells (at A600) were carried out using DNA minipreparation kits (Qiagen) according to the instructions of the supplier, except that in the case of growing cells 20 ml of cell culture was used instead of 3-4 ml as used in the case of stationary overnight cultures. Plasmid DNA (1-2 µg) was incubated with 100-200 nM R. EcoRV (corresponding to approximately 100 units) in 10 µl of Tris/HCl (pH 7.5), 10 mM MgCl2, 50 mM NaCl for 30-60 min. Subsequently, the samples were analyzed by agarose gel electrophoresis. Under these conditions, an unmethylated control plasmid was completely cleaved after 5 min, but no nonspecific DNA degradation of methylated plasmids was observed after incubation for 60 min.

Construction, Overexpression, and Purification of GST-M. EcoRV and GST-M. EcoRV Variants-- Mutant genes were amplified from pRVMetH6 by PCR and cloned into pGEXMRV using the BamHI and SalI sites (42). In this vector, the M. EcoRV gene is fused in frame to the 3'-end of the gene for the glutathione S-transferase. The GST-M. EcoRV gene is under control of a tac promotor. Purification of the GST fusion proteins was performed by chromatography over GSH-Sepharose and phosphocellulose as described (42) except that cell lysis was carried out under low salt conditions (30 mM KPi, pH 7.2.; 0.1 mM dithioerythritol; 0.01% (v/v) Lubrol; 0.5 mM EDTA, 100 mM NaCl). All mutant proteins purified in this study were obtained at similar concentrations as wild type M. EcoRV (viz. 5-25 µM). The specific activities of different GST-M. EcoRV preparations (50-100 units/µg) were slightly higher than those reported for untagged M. EcoRV (35). His6-M. EcoRV could be purified to a 10-50-fold higher specific activity (34).

In Vitro Activity Assays-- Kinetics of restriction protection were carried out using lambda -DNA (21 EcoRV sites) and pAT153-DNA (one EcoRV site) in 50 mM NaCl, 50 mM HEPES (pH 7.5), 1 mM AdoMet at ambient temperature as described (42). 1 unit is defined as the amount of M. EcoRV required to protect 1 µg of DNA from R. EcoRV attack in 1 h. Specific activities (units/µg of protein) for GST fusion proteins were calculated using the molecular mass only of the M. EcoRV part (35,000 g/mol) in order to make the activities comparable with those measured with His6 fusion proteins. Methylation of a 20-mer oligonucleotide d(GATCGACGATATCGTCGATC) was carried out using [14CH3]AdoMet in 50 mM NaCl, 50 mM HEPES (pH 7.5) at ambient temperature as described (34). To determine Km values for DNA and AdoMet, DNA concentrations were varied between 0.2 and 4 µM at a constant AdoMet concentration of 5 µM, AdoMet concentrations were varied between 1 and 10 µM at 1 µM DNA, and the determined rates of methylation of DNA were fit to the Michaelis-Menten model.

DNA Binding-- DNA binding was analyzed by nitrocellulose filter experiments carried out in a dot blot apparatus (Bio-Rad) as described (34) using a PCR product (382 base pairs) at 1 nM and a 20-mer oligonucleotide d(GATCGACGATATCGTCGATC) (5 nM) both containing one EcoRV site as substrates. The oligonucleotide substrate was labeled radioactively using [32P]ATP (Amersham Pharmacia Biotech) and polynucleotide kinase (MBI Fermentas). The DNA was incubated with different amounts of M. EcoRV variants (10-2500 nM) in 50 µl of 50 mM Tris/HCl (pH 7.5), 20 mM NaCl, 1 mM EDTA, and 200 µM sinefungin for 30 min at ambient temperature. The nitrocellulose filter membrane (Macherey & Nagel, Düren, Germany) was prerinsed with 50 mM Tris/HCl (pH 7.5), 20 mM NaCl for 30 min. After membrane was transferred into the dot blot chamber, the slots were washed twice with 100 µl of 50 mM Tris/HCl, pH 7.5, 20 mM NaCl. The samples were transferred into the wells of the dot blot apparatus using a multiple pipette, immediately sucked through the nitrocellulose filter membrane, and washed several times with 100 µl of washing buffer (50 mM Tris/HCl, pH 7.5, 20 mM NaCl). The radioactivity of the spots was analyzed using an Instant Imager (Canberra Packard), and the results were fitted to the equation describing a bimolecular association equilibrium.

AdoMet Binding-- AdoMet binding was analyzed by filter binding experiments using a positively charged nylon membrane (Nytran-plus, Schleicher & Schüll) and [carboxyl-14C]AdoMet (Amersham Pharmacia Biotech) as described (42). M. EcoRV wild type and mutants were incubated at -20 °C overnight with 100 µM [carboxyl-14C]AdoMet (Amersham Pharmacia Biotech) in storage buffer (30 mM potassium phosphate, pH 7.6, 0.1 mM dithioerythritol, 0.01% (v/v) Lubrol, 0.3 M NaCl, 0.5 mM EDTA, 77% glycerol). The nylon membrane was prerinsed with washing buffer for at least 60 min. After membrane was transferred into the dot blot chamber, the slots were washed twice with washing buffer. After the incubation, the reaction mixtures containing AdoMet and M. EcoRV were diluted 10-fold with washing buffer, transferred into the wells of the dot blot apparatus using a multiple pipette, immediately sucked through the nylon membrane, and washed several times with 100 µl of washing buffer. The radioactivity of the spots was analyzed using an Instant Imager (Canberra Packard) or PhosphorImager (Fuji). After background subtraction, the amounts of retained AdoMet were normalized to the concentrations of the enzyme preparations and compared with wild type binding determined in the same experiment.

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

We have used multiple sequence alignments to identify conserved amino acid residues in the EcoRV adenine-N6-MTase. Two alignments were analyzed for conserved residues, one including all alpha -type N-MTases (Fig. 1A) (21) and one in which only a subgroup of enzymes closely related to M. EcoRV are aligned (Fig. 1B) (40). On the basis of these alignments, we have chosen 17 amino acid residues that are strongly (Lys16, Glu37, Phe39, Asp58, Glu104, Arg128, Asn130, Glu178, Asp193, Tyr196, Ser229) or moderately conserved (Asp78, Asp211, Glu217, Glu238, Tyr258) for this mutational study (cf. Fig. 2 for their location in the structural model). All residues were substituted by alanine; at two positions within the conserved DPPY motif (Asp193 and Tyr196) in addition more conservative amino acid replacements were carried out (Asp right-arrow Asn and Tyr right-arrow Phe). Mutations were introduced by site-directed PCR mutagenesis. All M. EcoRV variants were tested for their ability to methylate DNA in vivo, and most of the variants were purified and also characterized in vitro with respect to their catalytic properties as well as DNA binding and AdoMet binding affinities.

Catalytic Activity of the M. EcoRV Variants in Vivo-- We have cloned all mutants as His6 fusion proteins in pRVMetH6. This plasmid contains one EcoRV recognition site. During propagation of the expression plasmids coding for the individual mutants in E. coli cells, residual expression of the MTase variant takes place even under repressing conditions. Thus, the MTase variant can modify the plasmid DNA in vivo, with the result that purified plasmids are resistant against R. EcoRV cleavage to a degree corresponding to the catalytic activity of the MTase variant. We have carried out this in vivo activity test in two ways by isolating plasmids either from stationary E. coli cells or from growing E. coli cells (Fig. 4, Table I). Plasmids encoding wild type M. EcoRV are fully protected against R. EcoRV cleavage under both experimental conditions. Generally, the degree of methylation of EcoRV sites is lower under conditions of active growth, because then DNA replication takes place and, in comparison with stationary E. coli cells, higher M. EcoRV activities are required to achieve the same degree of methylation. Five of the variants fail to methylate the DNA even in stationary E. coli cells (D58A, R128A, D193A, D193N, and Y196A). In growing cells, many variants show a strongly reduced level of DNA methylation indicating a strongly reduced catalytic activity (- or - - in Table I) (K16A, E37A, F39A, D58A, R128A, N130A, D193(A/N), Y196(A/F), S229A, Y258A).


View larger version (52K):
[in this window]
[in a new window]
 
Fig. 4.   In vivo activity of M. EcoRV mutants. pRVMetH6 (mutant) plasmids isolated from stationary (A) or growing E. coli cells (B) are shown prior to (n) and after incubation with 100 units of R. EcoRV (r). Plasmids coding for active M. EcoRV variants are methylated and protected from R. EcoRV cleavage. The positions of superhelical (sc), open circle (oc), and linear (lin) pRVMetH6 are indicated. On the basis of the degree of protection of the plasmids coding for the M. EcoRV variants from R. EcoRV cleavage, the catalytic activities of the variants were assigned as: ++ (fully protected), + (>50% protected), - (<50% protected), and - - (not protected). wt, wild type.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Compilation of in vivo catalytic activities of His6- and GST-M. EcoRV mutants as indicated by the level of protection of plasmids coding for M. EcoRV variants from R. EcoRV cleavage
All experiments were carried out at least in triplicate using different E. coli clones expressing the M. EcoRV variants. ++, fully protected; +, >50% protected; -, <50% protected; - -, not protected.

Catalytic Activity of the M. EcoRV Variants in Vitro-- To investigate the mutant proteins in vitro, we have overexpressed and purified wild type M. EcoRV and 18 of the variants (Table II). E104A and E178A that are fully active in vivo were not purified and further investigated. Since many mutants could not be overexpressed as His6 fusion proteins or remained in the pellet after cell disruption and centrifugation, we decided to express and purify all mutants as GST fusion proteins. To this end, all mutants were also cloned as GST fusion proteins in pGEXMRV. The catalytic activities of these variants in stationary E. coli cells are very similar to those of the His6 fusion proteins (data not shown). Wild type GST-M. EcoRV and the mutants were overexpressed in 500-ml scale and purified by two chromatography steps (glutathione-Sepharose and phosphocellulose) to >90% purity as judged by Coomassie-stained SDS-polyacrylamide gels. The concentrations of all preparations were between 5 and 25 µM. We have determined the catalytic activity of wild type GST-M. EcoRV and all mutants by lambda - and plasmid-DNA protection assays (Fig. 5; Table II). High catalytic activity could be detected only with wild type M. EcoRV and the D78A mutant. The Y196F, E217A, E238A, and D244A mutants displayed a reduced activity in vitro, and all other variants were catalytically inactive in vitro. In general, there is a very good agreement between the in vivo and in vitro data; most mutants with strongly reduced catalytic activities in vivo, (- or - - in growing cells, Table I), are catalytically inactive in vitro, whereas most mutants with good in vivo activity (+ or ++ in growing cells, Table I) also display catalytic activity in vitro. There are only two exceptions to these rules; Y196F has a low catalytic activity in vivo (- in Table I) but is not inactive in vitro, and D211A is inactive in vitro but shows good activity in vivo (+ in Table I). These differences may be caused by different expression rates and/or different in vivo and in vitro stabilities of these two mutants.

                              
View this table:
[in this window]
[in a new window]
 
Table II
Compilation of in vitro properties of GST-M. EcoRV mutants
Catalytic activities were determined using a lambda -DNA cleavage protection assay. DNA binding and AdoMet binding was assayed by filter binding techniques. Errors of the DNA binding constants are estimated to be ±30%.


View larger version (57K):
[in this window]
[in a new window]
 
Fig. 5.   In vitro activity of GST-M. EcoRV mutants. 0.5 µg of lambda -DNA were incubated with 1 µl of purified M. EcoRV-GST fusion proteins (M. EcoRV, 12 µM; D78A, 13 µM; E217A, 22 µM; S229A, 5.7 µM) diluted as indicated in 10 µl of 50 mM NaCl, 50 mM HEPES (pH 7.5), 1 mM AdoMet at ambient temperature for 60 min. After the times indicated, 100 units of R. EcoRV were added to the reaction mixtures, and MgCl2 was added to a final concentration of 10 mM to measure the degree of methylation of EcoRV sites. As controls, native lambda -DNA (n) (48,502 base pairs) and EcoRV-digested lambda -DNA (r) are shown.

According to their catalytic activities, the variants can be divided into three groups: group 1 (catalytically inactive in vivo and in vitro), D58A, R128A, D193A, D193N, and Y196A; group 2 (catalytically inactive in vitro), K16A, E37A, F39A, N130A, D211A, S229A, and Y258A; group 3 (catalytically active in vivo and in vitro), D78A, E104A, E178A, Y196F, E217A, E238A, and D244A.

Kinetic Characterization of Catalytically Active M. EcoRV Variants-- The activities of the Y196F, E217A, E238A, and D244A variants were determined by the restriction protection assay at concentrations of AdoMet between 1 mM and 0.3 µM (data not shown). The catalytic activities of the variants were analyzed according to the Michaelis-Menten model to estimate a Km value for AdoMet. According to this analysis, the Y196F, E217A, and E238A mutants had Km values between 5 and 20 µM, similar to wild type M. EcoRV and the D78A variant (see below). In contrast, the D244A variant shows a significantly higher Km value for AdoMet (>250 µM), indicating a reduced affinity for the cofactor.

In addition to performing the restriction protection assays using macromolecular substrates, we have analyzed the in vitro activity of all mutants by determining incorporation of radioactively labeled methyl groups into a 20-mer oligonucleotide. However, apart from wild type M. EcoRV, only the D78A variant had sufficiently high activity to allow an unequivocal detection of catalytic activity with this assay. We have analyzed the D78A variant in more detail using the 20-mer substrate and determined its Km values for DNA and AdoMet. Both kinetic constants (Km, DNA = 0.4 ± 0.2 µM in the presence of 5 µM AdoMet; Km, AdoMet = 13 ± 5 µM in the presence of 1 µM 20-mer) were very similar to those measured with wild type GST-M. EcoRV (Km, DNA = 0.3 ± 0.1 µM; Km, AdoMet = 12 ± 5 µM) and wild type His6-M. EcoRV (Km, DNA = 0.3 µM; Km, AdoMet = 12 µM (data taken from Ref. 34)) under these conditions.

DNA Binding-- We have determined the binding constants of the GST-M. EcoRV variants to a 20-mer oligonucleotide substrate. Wild type GST-M. EcoRV binds to DNA with similar affinity (Ka = 5 × 106 M-1) as His6-M. EcoRV (Ka = 6 × 106 M-1, Ref. 34) and untagged M. EcoRV (Ka to a 30/33-mer oligonucleotide = 2 × 107 M-1; Ref. 37). The D58A, D78A, D193(A/N), Y196(A/F), D211A, E217A, S229A, E238A, D244A, and Y258A variants bind to their DNA substrate with similar binding constants as wild type M. EcoRV (Table II, Fig. 6). Some of the mutants even bind slightly better to DNA than wild type M. EcoRV, which in some of the cases (e.g. D58A, D193N, or D211A) might be caused by the removal of negative charge due to the amino acid exchange. Binding to the 20-mer could not be detected with the K16A, E37A, F39A, R128A, and N130A. In addition, we have analyzed DNA binding using a 382-mer substrate, because we have shown previously that this substrate is bound better by many M. EcoRV mutants (42). Binding to the 382-mer was detectable with all mutants that were able to bind the 20-mer but in addition with the K16A, E37A, and F39A variants. Only the R128A and N130A enzymes did not bind to both substrates (Table II, data not shown). Thus, only amino acid substitutions introduced into the putative DNA binding domain of M. EcoRV prevent DNA binding of the enzyme, whereas all variants carrying mutations in the putative catalytic domain can bind to DNA.


View larger version (29K):
[in this window]
[in a new window]
 
Fig. 6.   DNA binding of the GST-M. EcoRV variants. 5 nM 20-mer was incubated with enzyme amounts as indicated in 50 mM Tris/HCl (pH 7.5), 20 mM NaCl, 1 mM EDTA, and 200 µM sinefungin and passed trough a nitrocellulose membrane under vacuum. Retained radioactivity was quantified, and the data were fitted to a bimolecular association equilibrium. All experiments were carried out at least twice. The Ka values are given in Table II.

AdoMet Binding-- To determine AdoMet binding abilities of the GST-M. EcoRV variants, a filter binding assay was employed. In these experiments, 50 pmol of wild type GST-M. EcoRV retain 0.5 pmol of AdoMet on the nylon membrane. At present, we cannot explain this low retention efficiency. We do not think that most of the protein is inactive, because with His6-M. EcoRV the AdoMet retention efficiency was only 3-fold higher (42), although 80% of the protein in this preparation is active in DNA binding (34). It should be noticed that here a heterophasic assay is employed to analyze a binding equilibrium characterized by a relatively low equilibrium binding constant (Km for AdoMet is 12 µM; Ref. 34). Under such circumstances, often not all bound ligand molecules are retained on the filter, and stoichiometric data cannot be obtained. Consequently, the AdoMet binding efficiencies observed here (Fig. 7, Table II) were only interpreted qualitatively. Four of the GST-tagged variants displayed AdoMet binding comparable with wild type GST-M. EcoRV (D78A, R128A, D193N, and S229A). AdoMet binding was detectable with all other variants except the K16A, E37A, F39A, and D58A variants. It should be noted that all of these four variants carry an amino acid exchange in the putative AdoMet binding site of M. EcoRV. As shown in Fig. 7, the amount of AdoMet binding observed in the filter binding assay is significantly lower with the D244A variant than with D78A, Y196F, E217A, and E238A. This result is in good agreement with the results of the kinetic analyses of the AdoMet dependence of the D78A, Y196F, E217A, E238A, and D244A variants, because D244A has a significantly higher Km for AdoMet than all other variants that are active in vitro.


View larger version (27K):
[in this window]
[in a new window]
 
Fig. 7.   AdoMet binding of the GST-M. EcoRV variants. AdoMet binding was analyzed by filter binding experiments using a positively charged nylon membrane. The amount of retained AdoMet per mol of enzyme is given in relation to the amount of AdoMet bound by wild type M. EcoRV. All experiments were carried out at least in triplicate, and error bars indicate the maximal deviation between all experiments carried out with each mutant.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Implications of the Results on the Structural Model for a-type Adenine MTases-- We have investigated 17 highly or moderately conserved amino acid residues of the EcoRV DNA methyltransferase by site-directed mutagenesis. Most variants were purified and analyzed in vitro with respect to their catalytic activities as well as DNA binding and AdoMet binding affinities. The mutants showing a reduced catalytic activity can be divided into three groups: group A (AdoMet binding-deficient), K16A, E37A, F39A, and D58A; group D (DNA binding-deficient), R128A and N130A; group C (detectable AdoMet and DNA binding, strongly reduced catalytic efficiency), D193(A/N), Y196A, S229A, D211A, and Y258A.

All mutants of groups A and D are catalytically inactive or show a severely reduced catalytic efficiency. The locations of the variants from groups A, D, and C in the structural model are shown in Fig. 8. Obviously, excellent agreement exists between the structural model and the biochemical properties of the mutants, because all variants of group A (no AdoMet binding) are located at the putative AdoMet binding site, all variants of group D (no DNA binding) in the putative DNA binding domain, and all variants of group C (only catalysis affected) at the putative binding site of a flipped target base. These results support the structural model of M. EcoRV. In the following paragraphs, the biochemical properties of the variants will be discussed in more detail.


View larger version (43K):
[in this window]
[in a new window]
 
Fig. 8.   Location of groups of variants in the structural model of M. EcoRV (cf. Fig. 2). A, location of residues exchanged in variants unable to bind AdoMet (group A: Lys16, Glu37, Phe39, and Asp58). B, location of residues exchanged in variants unable to bind to DNA (group D: Arg128 and Asn130). C, location of residues exchanged in variants that bind to AdoMet and DNA but show a strongly reduced catalytic activity (group C: Asp193, Tyr196, Asp211, Ser229, Trp231, and Tyr258).

AdoMet Binding-- Four of the mutants investigated are unable to bind AdoMet in vitro (K16A, E37A, F39A, D58A). All of these mutants are catalytically inactive in vitro and show a strongly reduced catalytic efficiency in vivo. These variants are mutated at the following amino acid residues that are implicated in AdoMet binding of M. EcoRV. Lys16 (motif X) is analogous to Thr23 in M. TaqI and Asn304 in M. HhaI, both of which contact one of the carboxyl oxygens of the AdoMet. Glu37 (motif I) corresponds to Glu45 in M. TaqI and Asp16 in M. HhaI, which contact the peptide backbone within motif I (FXGXG), thereby stereochemically constraining this region. In addition, M. TaqI-Glu45 contacts the amino group of the methionine moiety of the AdoMet. Phe39 (motif I) is equivalent to Phe18 in M. HhaI, which forms a hydrophobic contact to the adenine ring of the AdoMet (the Phe in the motif I absent in M. TaqI). Asp58 (motif II) corresponds to Glu40 in M. HhaI and Glu71 in TaqI, which hydrogen bond to 2'- and 3'-oxygens of the ribose of the AdoMet.

Our results suggest that all of these contacts to the cofactor observed in the x-ray structure analyses of several methyltransferases (M. HhaI (7), catechol-O-methyltransferase (43), M. TaqI (28), HaeIII DNA methyltransferase (15), VP39 vaccinia protein RNA methyltransferase (44), CheR (45), and PvuII DNA methyltransferase (29)) are very important for binding and positioning the cofactor. The data obtained with Glu37 and Phe39 in motif I (FXGXG) complement other mutational studies concerned with this region, because exchanging the first glycine of the FXGXG by other amino acid residues prevents AdoMet binding by EcoKI and EcoP15I (23, 24).

Interestingly, K16A, E37A, and F39A also show a reduced DNA binding capacity. This finding is in good agreement with data obtained for wild type M. EcoRV showing that DNA binding is about 10-fold stronger in the presence than in the absence of cofactor (37) similarly as described for other DNA MTases (EcoRI DNA methyltransferase (46), MspI DNA methyltransferase (47), and HaeIII DNA methyltransferase (48)). It has been suggested that an allosteric effect connecting conformational changes at the DNA and AdoMet binding sites is responsible for this effect (49). In addition to the residues mentioned above, the D244A mutant shows a significantly reduced affinity for AdoMet. If the structural model (Fig. 2) is correct in this region, this effect could only be mediated allosterically because alpha -helix F is far away from the AdoMet binding site in M. TaqI and M. HhaI.

The properties of the D78A variant are very different from what one would have expected on the basis of the structural model for M. EcoRV, because according to the model this residue should correspond to Asp89 in M. TaqI and Asp60 in M. HhaI that contact the adenine N6 of the AdoMet. However, D78A does not show any evidence for a reduced affinity toward AdoMet or a reduced catalytic efficiency; it has high catalytic activity both in vivo and in vitro, it has a similar Km value for AdoMet as wild type M. EcoRV, and it binds as much AdoMet as wild type M. EcoRV in the AdoMet binding assay. We conclude that either the structural model for M. EcoRV is wrong in this detail or this contact is of minor importance for AdoMet binding. Therefore, we have looked for alternative candidate residues to fulfill the function of Asp89 in M. TaqI. Glu104 is the best conserved acidic amino acid residue in putative DNA binding domain, and Glu178 is highly conserved and positioned such that if M. EcoRV had similar structure as observed for PvuII DNA methyltransferase it would be located at the edge of alpha -helix D. However, exchanging these residues to alanine did not provide evidence for an important role of them. Since there are several other alternative candidate residues, e.g. Asp74, Asp111, Asp187, and Asp188, we think that this question is still unresolved.

DNA Binding-- The DNA binding domain of M. EcoRV was predicted to comprise amino acid residues 90-180 (40). In this study, we have investigated two residues within this region (Arg128 and Asn130). R128A and N130A both are not able to bind DNA but can bind the cofactor AdoMet. These are the only residues in this study that are unable to bind to DNA. This finding is comparable with similar results of a random mutagenesis study that identified 13 M. EcoRV single mutants that are catalytically inactive in vivo and in vitro. Five of these variants do not bind to DNA, four of which carry a mutation in the putative DNA binding domain (42).

Catalytic Mechanism-- There is evidence that N-MTases flip out their target adenine prior to methyl group transfer (31-34). Thus, the active site of the enzyme must form a binding site for the flipped out adenine residue, providing a hydrophobic pocket and hydrogen bond partners for the adenine as well as catalytic residues. It should be noted that variants that are not able to flip out the target base are not expected to have a lower affinity toward DNA, because nonspecific binding of M. EcoRV to DNA is almost as strong as specific binding (34). Two of the most conserved mutants investigated here (D193N, S229A) are catalytically inactive but bind to DNA and AdoMet with similar affinities as wild type M. EcoRV. Ser229 and Asp193 consequently are prime candidate residues to be involved in catalysis. This conclusion is not surprising for Asp193, because the aspartic acid of the DPPY motif (motif IV) almost certainly is part of the catalytic site of N-MTases (30, 31). Our results with the D193N and D193A variants are in agreement with mutational data obtained with EcoKI, BcgI, and E. coli Dam (22, 23, 25); a DPPY right-arrow NPPY exchange in E. coli Dam (an alpha -N-MTase) abolishes catalytic activity but leaves DNA binding of the enzyme intact. Interestingly, AdoMet binding could not be demonstrated with this variant (22). In EcoKI, an NPPF right-arrow DPPF exchange results in a catalytically inactive enzyme that still binds the cofactor (23) and variants of BcgI in which the NPPY Asn was exchanged by Ala, Asp, or Gln are catalytically inactive (25).

Ser229, which is highly conserved in alpha -N-MTases is located in motif VI and corresponds to Glu119 in M. HhaI. This residue is involved in acid base catalysis in M. HhaI (13). It should be noted that the reaction catalyzed by M. EcoRV, methylation of an adenine N6, requires the abstraction of one proton from the target base. In contrast to alpha -N-MTases in beta -type enzymes (mostly cytosine-N4-MTases), an acidic residue is located at a corresponding position in motif VI (Asp96 in PvuII DNA methyltransferase) (29). However, motif IV has a SPPF sequence in these enzymes. Thus, it appears as if the active site of alpha -type enzymes (DPPY ... S) is analogous to the SPPF ... D arrangement found in beta -type enzymes. Hence, Ser229 possibly together with Asp193 could be involved in a proton relay system (adenine ... Ser229 ... Asp193) similarly as suggested recently for the beta -type N-MTase PvuII methyltransferase (29). Alternatively, Ser229 may form a hydrogen bond to the flipped adenine.

In addition, Asp211 is important for the catalytic activity of M. EcoRV, because the D211A variant is inactive in vitro, although this variant shows considerable activity in vivo. This result is in agreement with the structural model, according to which this residue is located in motif V, that participates in creating the adenine binding pocket. Asp211 is located at a position corresponding to Phe146 in M. TaqI that forms a hydrophobic contact to the AdoMet. However, the Phe146-AdoMet interaction is characteristic for gamma -type N-MTases, and our data do not suggest involvement of the corresponding residue in M. EcoRV (Asp211) in AdoMet binding.

Adenine Binding Site-- In addition to Asp193, Tyr196 is the second conserved amino acid residue in motif IV investigated in this study. In M. TaqI, a hydrogen bond is formed between the hydroxyl group of this tyrosine and the conserved asparagine constraining the conformation of the NPPY motif (motif IV). Perhaps, in M. EcoRV Tyr196 serves to position Asp193 by having a buttressing role in catalysis. In agreement with this observation, we show here that the hydroxyl group of Tyr196 contributes to catalysis but is much less important than the carboxylate group of Asp193. However, our results obtained with the Y196A variant demonstrate that an aromatic residue is essential at this position, presumably because it is involved in binding the flipped adenine. These findings are in agreement with data obtained with EcoKI where the Phe residue of the NPPF motif was replaced by Tyr and Trp and the resulting NPPY and NPPW variants still had catalytic activity (1/4 of wild type in the case of NPPY, much less in the case of NPPW) (23). In BcgI, an NPPY right-arrow NPPA exchange abolishes catalytic activity but does not severely affect DNA binding and AdoMet binding. Similarly as found here, NPPY right-arrow NPPF or NPPW variants of BcgI retain catalytic activity (25). In contrast, a DPPY right-arrow DPPW exchange in EcoP15I inactivates this enzyme (24).

A second candidate residue to contribute to the adenine binding pocket is Trp231, which has been identified in a random mutagenesis approach (42). A W231R mutant binds to DNA and AdoMet similar to wild type M. EcoRV but is catalytically inactive in vivo and in vitro. According to the structure prediction for M. EcoRV, Trp231 is located at a position corresponding to Val121 in M. HhaI (40). This residue forms a hydrophobic contact to the flipped cytosine (12), suggesting that Trp231 in M. EcoRV may have a similar function in binding the flipped adenine.

Finally, Tyr258 (motif VIII) could participate in binding the flipped out adenine. This residue could provide an aromatic ring and/or hydroxyl group to interact with the adenine base. It corresponds to Phe196 in M. TaqI, which has been suggested to be involved in hydrophobic contacts to the flipped adenine ring (21, 31). Our data demonstrate that Tyr258 indeed is an important residue for catalysis, because the Y258A variant is catalytically almost inactive but binds to DNA and AdoMet, albeit with reduced affinity for the cofactor.

Conclusions-- In this site-directed mutagenesis study covering 17 positions of M. EcoRV, we have provided evidence that a model for the structure of alpha -type N-MTases is largely correct. We could demonstrate the importance of amino acid residues in motifs X, I, and II in AdoMet binding. In addition, residues in motifs IV, V, VI, and VIII are important for catalysis and/or might contribute to the binding site for a flipped adenine. These data considerably extend our knowledge on DNA MTases, because so far results of site-directed mutagenesis studies were only published for some residues located in motifs I and IV. Together with more structural information, they will help us to understand better structure-function relationships and mechanism of DNA N-MTases.

    ACKNOWLEDGEMENTS

We are grateful to Drs. X. Cheng, B. A. Connolly, N. O. Reich, W. Saenger, and E. Weinhold for discussions and/or communication of results prior to publication. Thanks are due to Dr. A. Pingoud for insightful comments and support as well as to H. Büngen for excellent technical assistance. We acknowledge the generous gift of sinefungin by Dr. W. Guschlbauer.

    FOOTNOTES

* This work was supported by Deutsche Forschungsgemeinschaft Grant Pi 122/15-1 and a grant from the Justus-Liebig-Universität.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom all correspondence should be addressed: Institut für Biochemie, Fachbereich Biologie, Justus-Liebig-Universität, Heinrich-Buff-Ring 58, D-35392 Giessen, Germany. Tel.: 49 641 99 35404; Fax: 49 641 99 35409; E-mail: Albert.Jeltsch{at}chemie.bio.uni-giessen.de.

1 The abbreviations used are: MTase, DNA methyltransferase; M. HhaI, HhaI DNA methyltransferase; M. EcoRV, EcoRV DNA methyltransferase; M. TaqI, TaqI DNA methyltransferase; AdoMet, S-adenosylmethionine; PCR, polymerase chain reaction; GST, glutathione S-transferase; R. EcoRV, EcoRV restriction endonuclease.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Adams, R. L. P. (1995) BioEssays 17, 139-145[Medline] [Order article via Infotrieve]
  2. Counts, J. L., and Goodman, J. I. (1995) Cell 83, 13-15[Medline] [Order article via Infotrieve]
  3. Tate, P. H., and Bird, A. P. (1993) Curr. Opin. Genet. Dev. 3, 226-231[Medline] [Order article via Infotrieve]
  4. Razin, A., and Cedar, H. (1994) Cell 77, 473-476[Medline] [Order article via Infotrieve]
  5. Yoder, J. A., and Bestor, T. H. (1996) Biol. Chem. 377, 605-610
  6. Noyer-Weidner, M., and Trautner, T. A. (1993) in DNA Methylation: Molecular Biology and Biological Significance (Jost, J. P., and Saluz, H. P., eds), pp. 40-108, Birkhauser Verlag, Basel
  7. Hornby, D. P. (1993) Methods Mol. Biol. 16, 201-211
  8. Cheng, X. (1995) Annu. Rev. Biophys. Biomol. Struct. 24, 293-318[CrossRef][Medline] [Order article via Infotrieve]
  9. Ahmad, I., and Rao, D. N. (1996) Crit. Rev. Biochem. Mol. Biol. 31, 361-380[Abstract]
  10. Kumar, S., Cheng, X., Klimasauskas, S., Sha, M., Posfai, J., Roberts, R. J., and Wilson, G. G. (1994) Nucleic Acids Res. 22, 1-10[Abstract]
  11. Cheng, X., Kumar, S., Posfai, J., Pflugrath, J. W., and Roberts, R. J. (1993) Cell 74, 299-307[Medline] [Order article via Infotrieve]
  12. Klimasauskas, S., Kumar, S., Roberts, R. J., and Cheng, X. (1994) Cell 76, 357-369[Medline] [Order article via Infotrieve]
  13. O'Gara, M., Klimasauskas, S., Roberts, R. J., and Cheng, X. (1996) J. Mol. Biol. 261, 634-645[CrossRef][Medline] [Order article via Infotrieve]
  14. O'Gara, M., Roberts, R. J., and Cheng, X. (1996) J. Mol. Biol. 263, 597-606[CrossRef][Medline] [Order article via Infotrieve]
  15. Reinisch, K. M., Chen, L., Verdine, G. L., and Lipscomb, W. N. (1995) Cell 82, 143-153[Medline] [Order article via Infotrieve]
  16. Wu, J. C., and Santi, D. V. (1987) J. Biol. Chem. 262, 4778-4786[Abstract/Free Full Text]
  17. Lauster, R., Kriebardis, A., and Guschlbauer, W. (1987) FEBS Lett. 220, 167-176[CrossRef][Medline] [Order article via Infotrieve]
  18. Klimasauskas, S., Timinskas, A., Menkevicius, S., Butkiene, D., Butkus, V., and Janulaitis, A. A. (1989) Nucleic Acids Res. 17, 9823-9832[Medline] [Order article via Infotrieve]
  19. Smith, H. O., Annau, T. M., and Chandrasegaran, S. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 826-830[Abstract]
  20. Wilson, G. G. (1992) Methods Enzymol. 216, 259-279[Medline] [Order article via Infotrieve]
  21. Malone, T., Blumenthal, R. M., and Cheng, X. (1995) J. Mol. Biol. 253, 618-632[CrossRef][Medline] [Order article via Infotrieve]
  22. Guyot, J.-P., Grassi, J., Hahn, U., and Guschlbauer, W. (1993) Nucleic Acids Res. 21, 3183-3190[Abstract]
  23. Willcock, D. F., Dryden, D. T. F., and Murray, N. E. (1994) EMBO J. 13, 3902-3908[Abstract]
  24. Ahmad, I., and Rao, D. N. (1996) J. Mol. Biol. 259, 229-240[CrossRef][Medline] [Order article via Infotrieve]
  25. Kong, H., and Smith, C. L. (1997) Nucleic Acids Res. 25, 3687-3692[Abstract/Free Full Text]
  26. Hough, R. F., and Bass, B. L. (1997) RNA 3, 356-370[Abstract]
  27. Bokar, J. A., Shambaugh, M. E., Polayes, D., Matera, A. G., and Rottman, F. M. (1997) RNA 3, 1233-1247[Abstract]
  28. Labahn, J., Granzin, J., Schluckebier, G., Robinson, D. P., Jack, W. E., Schildkraut, I., and Saenger, W. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 10957-10961[Abstract/Free Full Text]
  29. Gong, W., O'Gara, M., Blumenthal, R. M., and Cheng, X. (1997) Nucleic Acids Res. 25, 2702-2715[Abstract/Free Full Text]
  30. Schluckebier, G., O'Gara, M., Saenger, W., and Cheng, X. (1995) J. Mol. Biol. 247, 16-20[CrossRef][Medline] [Order article via Infotrieve]
  31. Schluckebier, G., Labahn, J., Granzin, J., Schildkraut, I., and Saenger, W. (1995) Gene (Amst.) 157, 131-134[CrossRef][Medline] [Order article via Infotrieve]
  32. Allan, B. W., and Reich, N. O. (1996) Biochemistry 35, 14757-14762[CrossRef][Medline] [Order article via Infotrieve]
  33. Cal, S., and Connolly, B. A. (1997) J. Biol. Chem. 272, 490-496[Abstract/Free Full Text]
  34. Jeltsch, A., Friedrich, T., and Roth, M. (1998) J. Mol. Biol. 275, 747-758[CrossRef][Medline] [Order article via Infotrieve]
  35. Nwosu, V. U., Connolly, B. A., Halford, S. E., and Garnett, J. (1988) Nucleic Acids Res. 16, 3705-3720[Abstract]
  36. Newman, P. C., Nwosu, V. U., Williams, D. M., Cosstick, R., Seela, F., and Connolly, B. A. (1990) Biochemistry 29, 9891-9901[Medline] [Order article via Infotrieve]
  37. Szczelkun, M. D., and Connolly, B. A. (1995) Biochemistry 34, 10724-10733[Medline] [Order article via Infotrieve]
  38. Szczelkun, M. D., Jones, H., and Connolly, B. A. (1995) Biochemistry 34, 10734-10743[Medline] [Order article via Infotrieve]
  39. Cal, S., and Connolly, B. A. (1996) J. Biol. Chem. 271, 1008-1015[Abstract/Free Full Text]
  40. Jeltsch, A., Sobotta, T., and Pingoud, A. (1996) Protein Eng. 9, 413-423[Abstract]
  41. Ito, W., Ishiguro, H., and Kurosawa, Y. (1991) Gene (Amst.) 102, 67-70[CrossRef][Medline] [Order article via Infotrieve]
  42. Friedrich, T., Roth, M., Helm-Kruse, S., and Jeltsch, A. (1998) Biol. Chem. 379, 475-480[Medline] [Order article via Infotrieve]
  43. Vidgren, J., Svensson, L. A., and Liljas, A. (1994) Nature 368, 354-358[CrossRef][Medline] [Order article via Infotrieve]
  44. Hodel, A. E., Gershon, P. D., Shi, X., and Quiocho, F. A. (1996) Cell 85, 247-256[Medline] [Order article via Infotrieve]
  45. Djordjevic, S., and Stock, A. M. (1997) Structure 5, 545-558[Medline] [Order article via Infotrieve]
  46. Reich, N. O., and Mashhoon, N. (1990) J. Biol. Chem. 265, 8966-8970[Abstract/Free Full Text]
  47. Dubey, A. K., and Roberts, R. J. (1992) Nucleic Acids Res. 20, 3167-3173[Abstract]
  48. Chen, L., MacMillan, A. M., and Verdine, G. L. (1993) J. Am. Chem. Soc. 115, 5318-5319
  49. Schluckebier, G., Kozak, M., Bleimling, N., Weinhold, E., and Saenger, W. (1997) J. Mol. Biol. 265, 56-67[CrossRef][Medline] [Order article via Infotrieve]


Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.