The effects of base analogue substitutions on the cleavage by the EcoRI restriction endonuclease of octadeoxyribonucleotides containing modified EcoRI recognition sequences

It has been proposed that recognition of specific DNA sequences by proteins is accomplished by hydrogen bond formation between the protein and particular groups that are accessible in the major and minor grooves of the DNA. We have examined the DNA-protein interactions involved in the recognition of the hexameric DNA sequence, GAATTC, by the EcoRI restriction endonuclease by using derivatives of an oligodeoxyribonucleotide that contain a variety of base analogues. The base analogues hypoxanthine, 2-aminopurine, 2,6-diaminopurine, N6-methyladenine, 5-bromouracil, uracil, 5-bromocytosine, and 5-methylcytosine were incorporated as single substitutions into the octadeoxyribonucleotide d(pG-G-A-A-T-T-C-C). The effects of the substitutions on the interactions between the EcoRI endonuclease and its recognition sequence were monitored by determining the steady state kinetic values of the hydrolysis reaction. The substitutions resulted in effects that varied from complete inactivity to enhanced reactivity. The enzyme exhibited Michaelis-Menten kinetics with those substrates that were reactive, whereas octanucleotide analogues containing N6-methyladenine at either adenine position, uracil at the second thymine position, or 5-bromocytosine or 5-methylcytosine at the cytosine position were unreactive. The results are discussed in terms of possible effects on interactions between the enzyme and its recognition site during the reaction. An accompanying paper presents the results of a similar study using these oligonucleotides with the EcoRI modification methylase.     

The highly homologous isoschizomers RsrI endonuclease and EcoRI endonuclease do not recognize their target sequence identically

Using a series of decadeoxyribonucleotides containing base analogues as substrates we measured the steady-state kinetic parameters for the reaction catalyzed by RsrI endonuclease and compared the results to those with its isoschizomer EcoRI. The kinetics of RsrI cleavage are affected by each substitution, with the effects being generally more deleterious than with EcoRI, as shown by the greater reduction in the specificity constant kcat/KM. The magnitudes of the effects of several substitutions are consistent with the formation of direct enzyme-nucleobase contacts at the indicated positions. With substrates containing 2-amino-purine or 2,6-diaminopurine at the central adenine or uracil at the outermost thymine in the recognition sequence, cleavage by RsrI was very slow, less than one-tenth the rate of the corresponding EcoRI-catalyzed reaction. The lower tolerance of RsrI endonuclease for functional group changes in its recognition site may reflect differences in the mechanisms of DNA recognition by the two enzymes. Although RsrI and EcoRI endonucleases bind with similar affinities to specific and nonspecific DNA sequences and appear to introduce similar structural distortions in DNA upon binding, the use of substrate analogues reveals significant differences at the level of catalysis in the mechanisms by which these two endonucleases recognize the duplex sequence GAATTC.     

Overmethylation of DNAs by the EcoRI methylase.

EcoRI methylase is able to catalyze methy incorporation into DNA at sequences other than the canonical EcoRI site. At high enzyme concentrations and over a wide range of pH and ionic strengths, EcoRI methylase modifies polyoma DNA (which contains one EcoRI site) at a number of sites. This modification prevents EcoRI endonuclease activity, and thus is presumably at or near the EcoRI sequences (5') NAATTN.     

DNA recognition of base analogue and chemically modified substrates by the TaqI restriction endonuclease.

It has been proposed that protein-DNA recognition is mediated via specific hydrogen bond, hydrophobic, and/or electrostatic interactions between the protein and DNA surfaces. We have attempted to map and quantitate the energies of these interactions for the TaqI endonuclease by constructing substrates substituted with base or phosphate analogues that either remove or sterically obstruct particular functional groups in the canonical TCGA sequence. The DNA backbone was also modified using a chemical approach (phosphate ethylation) which identified several phosphates in the recognition sequence essential for cleavage. The base analogues, N6-methyl-A, N7-deaza-A, N7-deaza-G, inosine, N4-methyl-C, 5-methyl-C, uracil, 5-bromo-U, and the phosphate analogues, alpha-thio-A, alpha-thio-G, alpha-thio-T, alpha-thio-A, were substituted for their corresponding unmodified counterpart in one strand of the TCGA duplex. The effects of these analogues were monitored by measuring the steady state (Km, kcat) and single-turnover (kst) kinetic constants. Only the N6-methyl-A-substituted DNA, which mimics in vivo methylation, was unreactive while the remaining analogue substitutions exhibited Michaelis-Menten kinetics. In general, the Km was either unchanged or lowered by the analogue substitutions. In contrast, many of the analogues severely reduced kcat, suggesting the modified functional groups served mainly to destabilize the transition state. Single-turnover measurements paralleled the kcat results, pointing to the N7 and N6 of A, the N7 of G, and one of the nonbridging oxygens 3' to T as putative contacts made in achieving the transition state. Substrates with double substitutions displayed simple additivity of delta delta G" implying that these changes behaved independently. The unmodified strand in 10 out of 12 hemisubstituted substrates had a normal kst value suggesting that a particular cleavage center is controlled predominantly by recognition of determinants on the same strand as the scissile bond. These results are discussed in relation to base analogue work from the EcoRI, RsrI, and EcoRV restriction endonucleases.     

EcoRI DNA methyltransferase-DNA interactions.

We present a novel strategy with synthetic hemimethylated DNA substrates containing uracil for thymine and inosine for guanosine replacements and EcoRI DNA methyltransferase to characterize the importance of major groove hydrophobic groups to the sequence-specific modification of DNA. The bacterial Mtase uses S-adenosyl-L-methionine to methylate the double-stranded DNA site 5'GAATTC3' at the N6 position of the central adenosine of each strand. Uracil substitution in either strand at the outer thymine (5'GAATUC3') causes 2.2- and 1.7-fold improvements in specificity (kcat/KmDNA). The fact that the specificity constant for the substrate containing uracil in both strands is identical to the value expected for noninteracting substitutions suggests that no significant methyltransferase-DNA interactions are altered beyond the site of either substitution. Similar analysis of the internal thymine (5'GAAUTC3') also shows these methyl groups to make a negative contribution to specificity, although the observed nonadditivity with the doubly modified substrate clearly shows methyltransferase-DNA interactions beyond the site of substitution to be affected in this case. To further probe the effect of analogue incorporation on methyltransferase-DNA interactions beyond the site of substitution, the relatively "silent" and additive uracil changes (5'GAATUC3') were combined with inosine for guanosine substitutions (e.g., 5'IAATTC3') known to have significant negative effects on specificity. In contrast to the additivity observed with the outer thymines, these studies show significant changes in methyltransferase-DNA interactions caused by the removal of the thymine methyls. Our results implicate a complex and flexible methyltransferase-DNA interface in which subtle structural changes in the substrate are transmitted over the entire canonical site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of the restriction endonuclease RsrI, an isoschizomer of EcoRI

Rhodobacter sphaeroides strain 630 produces restriction enzyme RsrI which is an isoschizomer of EcoRI. We have purified this enzyme and initiated a comparison with the EcoRI endonuclease. The properties of RsrI are consistent with a reaction mechanism similar to that of EcoRI: the position of cleavage within the -GAATTC-site is identical, the MgCl2 optimum for the cleavage is identical, and the pH profile is similar. Methylation of the substrate sequence by the EcoRI methylase protects the site from cleavage by the RsrI endonuclease. RsrI cross-reacts strongly with anti-EcoRI serum indicating three-dimensional structural similarities. We have determined the sequence of 34 N terminal amino acids for RsrI and this sequence possesses significant similarity to the EcoRI N terminus.     

In vitro specificity of EcoRI DNA methyltransferase.

The sequence selectivity of enzyme-DNA interactions was analyzed by comparing discrimination between synthetic oligonucleotides containing the canonical site GAATTC and altered DNA sequences with the EcoRI DNA methyltransferase. The specificities (kcat/KmDNA) are decreased from 5- to 23,000-fold relative to the unmodified site. For several substrates the decrease in kcat makes a disproportionate contribution to the specificity difference, suggesting that discrimination is mediated by the placement of critical catalytic residues rather than binding interactions. This is supported by our observation that specificity changes are generally not followed by changes in the stability of the methyltransferase-DNA complexes. Also, base pair substitutions near the site of methylation result in greater decreases in complex stability, suggesting that recognition and catalytic mechanisms overlap.     

T4 RNA ligase catalyzed synthesis of base analogue-containing oligodeoxyribonucleotides and a characterization of their thermal stabilities.

Self-complementary oligodeoxyribonucleotides containing the base analogues 2-aminopurine, 2,6-diaminopurine, N6-methyladenine, uracil, and 5-bromouracil were synthesized by a general method that allows incorporation of the analogues at specific positions. The method uses chemically synthesized partial sequences but circumvents the need for protected base analogues by incorporating their unprotected 3',5'-bisphosphate derivatives enzymatically. T4 RNA ligase was used to add the analogues to the oligodeoxyribonucleotides with yields from 54 to greater than 95 percent. Oligodeoxyribonucleotides were joined to the oligodeoxyribonucleotides containing the analogues at their 3'-termini in yields from 22 to 81 percent. The high yields obtained in these joinings suggest that RNA ligase should be of general use for the specific incorporation of other deoxyribonucleotide analogues into oligodeoxyribonucleotides. The oligodeoxyribonucleotides containing the base analogues were characterized by their mobilities during HPLC, nucleoside compositions, sequences, and thermal stabilities.     

The kinetic mechanism of EcoRI endonuclease.

Steady-state parameters governing cleavage of pBR322 DNA by EcoRI endonuclease are highly sensitive to ionic environment, with K(m) and k(cat) increasing 1,000-fold and 15-fold, respectively, when ionic strength is increased from 0.059 to 0.23 M. By contrast, pre-steady-state analysis has shown that recognition, as well as first and second strand cleavage events that occur once the enzyme has arrived at the EcoRI site, are essentially insensitive to ionic strength, and has demonstrated that the rate-limiting step for endonuclease turnover occurs after double-strand cleavage under all conditions tested. Furthermore, processive cleavage of a pBR322 variant bearing two closely spaced EcoRI sites is governed by the same turnover number as hydrolysis of parental pBR322, which contains only a single EcoRI sequence, ruling out slow release of the enzyme from the cleaved site or a slow conformational change subsequent to double-strand cleavage. We attribute the effects of ionic strength on steady-state parameters to nonspecific endonuclease.DNA interactions, reflecting facilitated diffusion processes, that occur prior to EcoRI sequence recognition and subsequent to DNA cleavage.     

Role of thymidine residues in DNA recognition by the EcoRI and EcoRV restriction endonucleases.

We have synthesized a series of oligonucleotides containing the EcoRI (GAATTC) or EcoRV (GATATC) recognition site within which or adjacent to which thymidine was substituted by uridine or derivatives of uridine. The effects of these substitutions on the rate of the EcoRI and EcoRV catalyzed cleavage reaction were investigated. Our results show that most of the substitutions within the site are quite well tolerated by EcoRI, not, however, by EcoRV. We conclude that the thymin residues most likely are not directly involved in the recognition process of the EcoRI reaction. In contrast, they are major points of contact, between substrate and enzyme in the EcoRV reaction. The effects of substitutions in the position adjacent to the recognition site is also markedly different for EcoRI and EcoRV. Here, EcoRI seems to be considerably more selective than EcoRV.     

Inhibition of EcoRI DNA methylase with cofactor analogs

Four analogs of the natural cofactor S-adenosylmethionine (AdoMet) were tested for their ability to bind and inhibit the prokaryotic enzyme, EcoRI adenine DNA methylase. The EcoRI methylase transfers the methyl group from AdoMet to the second adenine in the double-stranded DNA sequence 5'GAATTC3'. Dissociation constants (KD) of the binary methylase-analog complexes obtained in the absence of DNA with S-adenosylhomocysteine (AdoHcy), sinefungin, N-methyl-AdoMet, and N-ethylAdoMet are 225, 43, greater than 1000, and greater than 1000 microM, respectively. In the presence of a DNA substrate, all four analogs show simple competitive inhibition with respect to AdoMet. The product of the enzymic reaction, AdoHcy, is a poor inhibitor of the enzyme (KI(AdoHcy) = 9 microM; KM(AdoMet) = 0.60 microM). Two synthetic analogs, N-methyl-AdoMet and N-ethyl-AdoMet, were also shown to be poor inhibitors with KI values of 50 and greater than 1000 microM, respectively. In contrast, the naturally occurring analog sinefungin was shown to be a highly potent inhibitor (KI = 10 nM). Gel retardation assays confirm that the methylase-DNA-sinefungin complex is sequence-specific. The ternary complex is the first sequence-specific complex detected for any DNA methylase. Potential applications to structural studies of methylase-DNA interactions are discussed.     

On the mechanism of DNA-adenine methylase

Experiments were performed to determine whether EcoRI methylase catalyzes the transfer of the methyl group of S-adenosylmethionine (a) directly to the N6 of adenine in DNA or (b) initially to N1 to give N1-methyladenine followed by isomerization of the N1-methylamino and 6-NH2 to give N6-methyladenine (Dimroth rearrangement). A facile synthesis of highly enriched [6-15N]deoxyadenosine and a dodecamer substrate of EcoRI methylase with [6-15N]adenine in the methylation site are reported. In the product of EcoRI enzymatic methylation, all of the isotope remains at the N6 position of the N6-methyladenine product. It is concluded that, contrary to existing chemical precedent, the methylation occurs by direct transfer from S-adenosylmethionine to the N6 of adenine in DNA.     

Binding of EcoP15I DNA methyltransferase to DNA reveals a large structural distortion within the recognition sequence

EcoP15I DNA methyltransferase, a member of the type III restriction-modification system, binds to the sequence 5'-CAGCAG-3' transferring a methyl group from S-adenosyl-l-methionine to the second adenine base. We have investigated protein-DNA interactions in the methylase-DNA complex by three methods. Determination of equilibrium dissociation constants indicated that the enzyme had higher affinity for DNA containing mismatches at the target base within the recognition sequence. Potassium permanganate footprinting studies revealed that there was a hyper-reactive permanganate cleavage site coincident with adenine that is the target base for methylation. More importantly, to detect DNA conformational alterations within the enzyme-DNA complexes, we have used a fluorescence-based assay. When EcoP15I DNA methyltransferase bound to DNA containing 2-aminopurine substitutions within the cognate sequence, an eight to tenfold fluorescent enhancement resulting from enzymatic flipping of the target adenine base was observed. Furthermore, fluorescence spectroscopy analysis showed that the changes attributable to structural distortion were specific for only the bases within the recognition sequence. More importantly, we observed that both the adenine bases in the recognition site appear to be structurally distorted to the same extent. While the target adenine base is probably flipped out of the DNA duplex, our results also suggest that fluorescent enhancements could be derived from protein-DNA interactions other than base flipping. Taken together, our results support the proposed base flipping mechanism for adenine methyltransferases.     

Facilitated diffusion during catalysis by EcoRI endonuclease. Nonspecific interactions in EcoRI catalysis

The potential for processive EcoRI endonuclease hydrolysis has been examined on several DNA substrates containing two EcoRI sites which were embedded in identical sequence environments. With a 388-base pair circular DNA, in which the two recognition sites are separated by 51 base pairs (shorter distance) or 337 base pairs (longer distance), 77 and 34% of all events involved processive hydrolysis at ionic strengths of 0.059 and 0.13, respectively. However, the frequency of processive action on linear substrates, in which the two sites were separated by 51 base pairs, was only 42 and 17% at these ionic strengths, values half those observed with the circular DNA. Processive action was not detectable on circular or linear substrates at an ionic strength of 0.23. These findings indicate that DNA search by the endonuclease occurs by facilitated diffusion, a mechanism in which the protein locates and leaves its recognition sequence by interacting with nonspecific DNA sites. We suggest that processivity on linear substrates is limited to values half that for small circles due to partitioning of the enzyme between the two products generated by cleavage of a linear molecule. Given such topological effects, measured processivity values imply that the endonuclease can diffuse within a DNA domain to locate and recognize an EcoRI site 50 to 300 base pairs distant from an initial binding site, with minimum search efficiencies being 80 and 30% at ionic strengths of 0.059 and 0.13, respectively. The high efficiency of processive action indicates that a positionally correlated mode of search plays a major role in facilitated diffusion in this system under such conditions. Also consistent with this view was the identification of a striking position effect when two closely spaced EcoRI sites were asymmetrically positioned near the end of a linear DNA. The endonuclease displays a substantial preference for the more centrally located recognition sequence. This preference does not reflect differential sensitivity of the two sites to cleavage per se, but can be simply explained by preferential entry of the enzyme via the larger nonspecific target available to the more centrally positioned recognition sequence. These conclusions differ from those of a previous qualitative analysis of endonuclease processivity over short distances (Langowski, J., Alves, J., Pingoud, A., and Maass, G. (1983) Nucleic Acids Res. 11, 501-513).     

Influence of enzyme-substrate contacts located outside the EcoRI recognition site on cleavage of duplex oligodeoxyribonucleotide substrates by EcoRI endonuclease.

A complete understanding of the sequence-specific interaction between the EcoRI restriction endonuclease and its DNA substrate requires identification of all contacts between the enzyme and substrate, and evaluation of their significance. We have searched for possible contacts adjacent to the recognition site, GAATTC, by using a series of substrates with differing lengths of flanking sequence. Each substrate is a duplex of non-self-complementary oligodeoxyribonucleotides in which the recognition site is flanked by six base pairs on one side and from zero to three base pairs on the other. Steady-state kinetic values were determined for the cleavage of each strand of these duplexes. A series of substrates in which the length of flanking sequence was varied on both sides of the hexamer was also examined. The enzyme cleaved both strands of each of the substrates. Decreasing the flanking sequence to fewer than three base pairs on one side of the recognition site induced an asymmetry in the rates of cleavage of the two strands. The scissile bond nearest the shortening sequence was hydrolyzed with increasing rapidity as base pairs were successively removed. Taken together, the KM and kcat values obtained may be interpreted to indicate the relative importance of several likely enzyme-substrate contacts located outside the canonical hexameric recognition site.     

How the EcoRI endonuclease recognizes and cleaves DNA.

One popular recombinant DNA tool is the EcoRI endonuclease, which cleaves DNA at GAATTC sites and serves as a paradigm for sequence specific DNA-enzyme interactions. The recently revised X-ray crystal structure of an EcoRI-DNA complex reveals EcoRI employs novel DNA recognition motifs, a four alpha-helix bundle and two extended chains, which project into the major groove to contact substrate purines and pyrimidines. Interestingly, pyrimidine contacts had been predicted based on genetic and biochemical studies. Current work focuses on the EcoRI active site structure, enzyme and substrate conformational changes during catalysis, and host-restriction system interactions.     

Thermodynamic and kinetic basis for the relaxed DNA sequence specificity of "promiscuous" mutant EcoRI endonucleases

Promiscuous mutant EcoRI endonucleases produce lethal to sublethal effects because they cleave Escherichia coli DNA despite the presence of the EcoRI methylase. Three promiscuous mutant forms, Ala138Thr, Glu192Lys and His114Tyr, have been characterized with respect to their binding affinities and first-order cleavage rate constants towards the three classes of DNA sites: specific, miscognate (EcoRI*) and non-specific. We have made the unanticipated and counterintuitive observations that the mutant restriction endonucleases that exhibit relaxed specificity in vivo nevertheless bind more tightly than the wild-type enzyme to the specific recognition sequence in vitro, and show even greater preference for binding to the cognate GAATTC site over miscognate sites. Binding preference for EcoRI* over non-specific DNA is also improved. The first-order cleavage rate constants of the mutant enzymes are normal for the cognate site GAATTC, but are greater than those of the wild-type enzyme at EcoRI* sites. Thus, the mutant enzymes use two mechanisms to partially bypass the multiple fail-safe mechanisms that protect against cleavage of genomic DNA in cells carrying the wild-type EcoRI restriction-modification system: (a) binding to EcoRI* sites is more probable than for wild-type enzyme because non-specific DNA is less effective as a competitive inhibitor; (b) the combination of increased affinity and elevated cleavage rate constants at EcoRI* sites makes double-strand cleavage of these sites a more probable outcome than it is for the wild-type enzyme. Semi-quantitative estimates of rates of EcoRI* site cleavage in vivo, predicted using the binding and cleavage constants measured in vitro, are in accord with the observed lethal phenotypes associated with the three mutations.     

Sequences spanning the EcoRI substrate site.

Substrate recognition by the EcoRI restriction endonuclease was investigated by analysis of the nucleotide sequences at the sites of enzymatic cleavage in various DNA molecules. 5'-end labeling and homochromatographic fingerprinting led to the determination of a 17-base-pair sequence spanning the EcoRI site of simian virus 40 DNA and a 15-base-pair sequence overlapping the EcoRI site of Col El plasmid DNA. Three other DNAs were similarly tested, although extended sequences were not determined in these cases. The EcoRI site was shown to be symmetric double-stranded equivalent of -N-G-A-A-T-T-C-N-.     

Role of the 2-amino group of deoxyguanosine in sequence recognition by EcoRI restriction and modification enzymes.

The dG residues within the EcoRI recognition sequence of ColE1 DNA have been selectively replaced with dI. Methylation of the altered sequence by the EcoRI modification enzyme is extremely slow as compared with methyl transfer to the natural recognition site. Since the affinity of the modification enzyme for the dI-containing sequence is considerably less than that for the natural sequence, we have concluded that the 2-amino group of dG has an important role in DNA site recognition by this enzyme. In contrast, the altered site is subject to cleavage by EcoRI endonuclease at rates essentially identical with those observed with the natural sequence. These results strongly suggest that the two enzymes utilize different contacts within the EcoRI site and are consisted with our conclusion (Rubin, R. A., and Modrich, P. (1977) J. Biol. Chem. 252, 7265-7272) that the two proteins interact with their common recognition sequence in different ways.     

Non-additivity of sequence-specific enzyme-DNA interactions in the EcoRI DNA methyltransferase.

We describe a novel strategy to characterize protein-DNA interactions involving monomeric enzymes such as DNA methyltransferases (Mtases). This strategy is applied to our investigation of the EcoRI DNA Mtase, which binds its double stranded recognition site 5'-G-AATTC-3' and methylates the central adenosine of each strand using S-adenosyl-L-methionine as the methyl donor. We show that prior methylation of adenosine in either strand does not perturb catalysis. In contrast, substrates substituted with deoxyinosine at either guanosine position (T-BMI5 and TI5-BM) show the minor groove residing N2 amino group of both guanosines contribute to DNA recognition since specificity constants for the modified substrates are reduced 13 and 39 fold. Similar analysis of a substrate containing deoxyinosine at both positions (TI5-BMI5) clearly shows that some communication occurs between the sites. To determine the extent to which structural changes in the DNA alone contribute to this lack of additivity, we performed DNA melting analysis of the singly and doubly substituted substrates, and also found non-additivity. Although our functional and structural analyses suggest that deoxyinosine incorporation causes long range conformational effects, the similarity of KmAdoMet for all substrates suggests that no large-scale structural changes occur in the Mtase-DNA-AdoMet complex. Our results support the following conclusions: 1) The non-additivity shown in this system contrasts with the widespread demonstration of additivity involving repressors [Lehming et al., 1990; Takeda et al., 1989; Ebright et al., 1987], suggesting that sequence discrimination by enzymes may involve more complex mechanisms. Further, this non-additivity precludes quantitative assignment of individual interactions and we suggest that future analyses of this and related enzyme systems with base analogs include detailed information about the long range structural consequences of individual substitutions. 2) Although TI5-BM and T-BMI5 are shown to be radically different by thermodynamic analysis, the similar specificity constants with the Mtase suggest that the underlying structural differences (e.g., altered helical parameters of the DNA) are not critical for sequence-recognition. 3) The significance of minor groove Mtase-DNA interactions to specificity is confirmed.