Mechanisms of coupling between DNA recognition specificity and catalysis in EcoRI endonuclease

Proteins that bind to specific sites on DNA often do so in order to carry out catalysis or specific protein-protein interaction while bound to the recognition site. Functional specificity is enhanced if this second function is coupled to correct DNA site recognition. To analyze the structural and energetic basis of coupling between recognition and catalysis in EcoRI endonuclease, we have studied stereospecific phosphorothioate (PS) or methylphosphonate (PMe) substitutions at the scissile phosphate GpAATTC or at the adjacent phosphate GApATTC in combination with molecular-dynamics simulations of the catalytic center with bound Mg2+. The results show the roles in catalysis of individual phosphoryl oxygens and of DNA distortion and suggest that a "crosstalk ring" in the complex couples recognition to catalysis and couples the two catalytic sites to each other.     

Heparin inhibits EcoRI endonuclease cleavage of DNA at certain EcoRI sites.

Studies presented here demonstrate that heparin inhibits EcoRI endonuclease cleavage of DNA whereas related proteoglycans show no effect. The inhibition occurs at particular EcoRI sites that are near or overlap with palindromic sequences in the murine lambda 5 and Lyt-2 genes. Endogenous heparin from peritoneal mast cells co-isolates with DNA and inhibits digestion of peritoneal cell DNA at the inhibitable sites. Digestion of spleen DNA is inhibited at the same sites when commercial heparin is added prior to digestion. In both cases, the inhibition is abolished by pre-treating the DNA with heparinase. Thus, potential artifacts in restriction fragment length analyses could occur with DNA isolated either from cells that are naturally rich in heparin or from cells to which heparin has been added, e.g., as an anticoagulant.     

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.     

The EcoRI restriction endonuclease, covalently closed DNA and ethidium bromide.

The reactions of the EcoRI restriction endonuclease on the covalently closed DNA of plasmid pMB9 were studied in the presence of ethidium bromide. At the concentrations of ethidium bromide tested, which covered the range over which the DNA is changed from negatively to positively supercoiled, the dye caused no alteration to the rate at which this enzyme cleaved the covalently closed DNA to yield the open-circle form, but the rate at which these open circles were cleaved to the linear product could be inhibited. The fluorescence change, caused by ethidium bromide binding with different stoichiometries to covalently closed and open-circle DNA, provided a direct and sensitive signal for monitoring the cleavage of DNA by this enzyme. This method was used for a steady-state kinetic analysis of the reaction catalysed by the EcoRI restriction enzyme. Reaction mechanisms where a complex between DNA and Mg2+ is the substrate for this enzyme were eliminated, and instead DNA and Mg2+ must bind to the enzyme in separate stages. The requisite controls for this fluorimetric assay in both steady-state and transient kinetics studies, and its application to other enzymes that alter the structure of covalently closed DNA, are described.     

EcoRI restriction endonuclease cleavage site map of bacteriophage P22DNA.

The F plasmid is able to co-transfer (mobilize) the small, chimeric R plasmid pBR322 during conjugation only at a very low frequency (Bolivar et al., 1977). Mobilization has been found here to be invariably (> 99%) associated with a structural alteration of pBR322. The alteration was shown, by restriction endonuclease analysis and electron microscopy, to be an insertion of the F attachment sequence λδ (2.8 to 8.5F). λδ is, therefore, an insertion sequence.     

A monomeric mutant of restriction endonuclease EcoRI nicks DNA without sequence specificity

We have mutated the monomer-monomer interface of the restriction endonuclease EcoRI in order to destabilize the homodimer and to stabilize heterodimers. Mutations of Leu158 to charged amino acid residues result in strong destabilization of the dimer. The largest effect was detected for the L158D mutant which is monomeric even at higher concentrations. It unspecifically degrades DNA by cleaving both single strands independently every 15 nucleotides on the average. Although cleavage is reproducible, it is not determined by nucleotide sequence but by general properties like conformation or deformability as has been found for other unspecific nucleases. Mutations of Ile230, which is in direct contact with Leu158 of the other subunit, cause structural changes with the loss of about ten percent alpha-helix content, but interfere only marginally with homodimerization and double strand cleavage. Again the mutation to aspartate shows the strongest effects. Mixtures of single mutants, one containing aspartate at one of the two positions and the other lysine at the corresponding position, form heterodimers. These are mainly stabilized compared to the homodimers by re-establishment of the wild-type hydrophobic interaction at the not mutated residues while an interaction of aspartate and lysine seems energetically unfavorable in this structural context.     

Temperature-sensitive mutants of the EcoRI endonuclease

The EcoRI endonuclease is an important recombinant DNA tool and a paradigm of sequence-specific DNA-protein interactions. We have isolated temperature-sensitive (TS) EcoRI endonuclease mutants (R56Q, G78D, P90S, V97I, R105K, M157I, C218Y, A235E, M255I, T261I and L263F) and characterized activity in vivo and in vitro. Although the majority were TS for function in vivo, all of the mutant enzymes were stably expressed and largely soluble at both 30°C and 42°C in vivo and none of the mutants was found to be TS in vitro. These findings suggest that these mutations may affect folding of the enzyme at elevated temperature in vivo. Both non-conservative and conservative substitutions occurred but were not correlated with severity of the mutation. Of the 12 residues identified, 11 are conserved between EcoRI and the isoschizomer RsrI (which shares 50% identity), a further indication that these residues are critical for EcoRI structure and function. Inspection of the 2.8 Å resolution X-ray crystal structure of the wild-type EcoRI endonuclease-DNA complex revealed that: (1) the TS mutations cluster in one half of the globular enzyme; (2) several of the substituted residues interact with each other; (3) most mutations would be predicted to disrupt local structures; (4) two mutations may affect the dimer interface (G78D and A235E); (5) one mutation (P90S) occurred in a residue that is part of, or immediately adjacent to, the EcoRI active site and which is conserved in the distantly related EcoRV endonuclease. Finally, one class of mutants restricted phage in vivo and was active in vitro, whereas a second class did not restrict and was inactive in vitro. The two classes of mutants may differ in kinetic properties or cleavage mechanism. In summary, these mutations provide insights into EcoRI structure and function, and complement previous genetic, biochemical, and structural analyses.     

Human cytomegalovirus DNA: BamHI, EcoRI and PstI restriction endonuclease cleavage maps

The cloned HindIII fragments of human cytomegalovirus (HCMV) strain AD169 DNA were mapped with respect to the BamHI, EcoRI and PstI restriction endonuclease cleavage sites. Composite restriction endonuclease cleavage maps for the entire virus genome were constructed using the previously established linkages between the HindIII fragments.     

Thermodynamic parameters governing interaction of EcoRI endonuclease with specific and nonspecific DNA sequences

Equilibrium binding of EcoRI endonuclease to DNA has been analyzed by nitrocellulose filter and preferential DNA cleavage methods. Association constants for pBR322 and a 34-base pair molecule containing the EcoRI site of this plasmid in a central position were determined to be 1.9 X 10(11) M-1 and 1.0 X 10(11) M-1 at 37 degrees C, respectively, with the stoichiometry of binding being 0.8 +/- 0.1 mol of endonuclease dimer per mol of DNA. In contrast, the affinity of the enzyme for a pBR322 derivative from which the EcoRI site has been deleted is 3.2 X 10(9) M-1 as judged by competitive binding experiments. If it is assumed that each base pair can define the beginning of a nonspecific binding site, this value corresponds to an affinity for nonspecific sites of 7.4 X 10(5) M-1. Furthermore, the affinity of the endonuclease for the EcoRI-methylated sequence is at least three orders of magnitude less than that for the unmodified recognition site. The dependence on temperature and ionic strength of the equilibrium constant governing specific interactions has also been examined. The temperature dependence of the reaction indicates that entropy increase accounts for 70% of the free energy of specific binding at 37 degrees C. Affinity of the endonuclease for the EcoRI site is highly dependent on NaCl concentration. Analysis of this dependence according to the theory of Record and colleagues (Record, T. M., Jr., Lohman, T. M., and deHaseth, P. (1976) J. Mol. Biol. 107, 145-158) has implicated 8 ion pairs in the stability of specific complexes, a value identical with the number of phosphate contacts determined by ethylation interference analysis (Lu, A. L., Jack, W. E., and Modrich, P. (1981) J. Biol. Chem. 256, 13200-13206). Extrapolation to 1 M NaCl suggests that nonelectrostatic interactions account for 40% of the free energy change associated with specific complex formation.     

The enfolding arms of EcoRI endonuclease: role in DNA binding and cleavage

The N-terminal segments of the EcoRI endonuclease dimer form part of mobile "arms" that encircle DNA in the recognition complex. By treating endonuclease-TCGCGAATTCGCG complexes with proteases, we have prepared a series of deletion derivatives lacking defined segments of the N-terminal region. The 5-12 segment is essential for DNA cleavage and forms one electrostatic interaction (per subunit) with DNA phosphate. These ionic contacts are directly across the double helix from the scissile phosphodiester bonds; they thus may permit the enfolding arms to immobilize DNA in apposition to the catalytic cleft and/or contribute to the unusual "kinked" conformation of DNA in the complex. Sequence specificity is fully retained when 28 residues are deleted from the N-terminus, but the complexes dissociate more rapidly.     

Studies on the cleavage of bacteriophage lambda DNA with EcoRI restriction endonuclease

The five EcoRI² restriction sites in bacteriophage lambda DNA have been mapped at 0.445, 0.543, 0.656, 0.810, and 0.931 fractional lengths from the left end of the DNA molecule. These positions were determined electron-microscopically by single-site cleavage of hydrogen-bonded circular λ DNA molecules and by cleavage of various DNA heteroduplexes between λ DNA and DNA from well defined λ mutants. The DNA lengths of the EcoRI fragments are in agreement with their electrophoretic mobility on agarose gels but are not in agreement with their mobilities on polyacrylamide gels. These positions are different from those previously published by Allet et al. (1973). Partial cleavage of pure λ DNA by addition of small amounts of EcoRI endonuclease does not lead to random cleavage between molecules. Also, the first site cleaved is not randomly distributed among the five sites within a molecule. The site nearest the right end is cleaved first about ten times more frequently than either of the two center sites.     

The EcoRI restriction endonuclease with bacteriophage lambda DNA. Kinetic studies.

The kinetics of the reactions of the EcoRI restriction endonuclease at individual recognition sites on the DNA from bacteriophage lambda were found to differ markedly from site to site. Under certain conditions of pH and ionic strength, the rates for the cleavage of the DNA were the same at each recognition site. But under altered experimental conditions, different reaction rates were observed at each recognition site. These results are consistent with a mechanism in which the kinetic stability of the complex between the enzyme and the recognition site on the DNA differs among the sites, due to the effect of interactions between the enzyme and DNA sequences surrounding each recognition site upon the transition state of the reaction. Reactions at individual sites on a DNA molecule containing more than one recognition site were found to be independent of each other, thus excluding the possibility of a processive mechanism for the EcoRI enzyme. The consequences of these observations are discussed with regard to both DNA-protein interactions and to the application of restriction enzymes in the study of the structure of DNA molecules.     

Substrate recognition by the EcoRI endonuclease

The EcoRI restriction endonuclease is one of the most widely used tools for recombinant DNA manipulations. Because the EcoRI enzyme has been extremely well characterized biochemically and its structure is known at 3 A resolution as an enzyme-DNA complex, EcoRI also serves as a paradigm for other restriction enzymes and as an important model of DNA-protein interactions. To facilitate a genetic analysis of the EcoRI enzyme, we devised an in vivo DNA scission assay based on our finding that DNA double-strand breaks induce the Escherichia coli SOS response and thereby increase beta-galactosidase expression from SOS::lacZ gene fusions. By site-directed mutagenesis, 50 of 60 possible point mutations were generated at three amino acids (E144, R145, and R200) implicated in substrate recognition by the crystal structure. Although several of these mutant enzymes retain partial endonuclease activity, none are altered in substrate specificity in vivo or in vitro. These findings argue that, in addition to the hydrogen bond interactions revealed by the crystal structure, the EcoRI enzyme must make additional contacts to recognize its substrate.     

The EcoRI restriction endonuclease with bacteriophage lambda DNA. Equilibrium binding studies.

The EcoRI restriction endonuclease was found by the filter binding technique to form stable complexes, in the absence of Mg2+, with the DNA from derivatives of bacteriophage lambda that either contain or lack EcoRI recognition sites. The amount of complex formed at different enzyme concentrations followed a hyperbolic equilibrium-binding curve with DNA molecules containing EcoRI recognition sites, but a sigmoidal equilibrium-binding curve was obtained with a DNA molecule lacking EcoRI recognition sites. The EcoRI enzyme displayed the same affinity for individual recognition sites on lambda DNA, even under conditions where it cleaves these sites at different rates. The binding of the enzyme to a DNA molecule lacking EcoRI sites was decreased by Mg2+. These observations indicate that (a) the EcoRI restriction enzyme binds preferentially to its recognition site on DNA, and that different reaction rates at different recognition sites are due to the rate of breakdown of this complex; (b) the enzyme also binds to other DNA sequences, but that two molecules of enzyme, in a different protein conformation, are involved in the formation of the complex at non-specific consequences; (c) the different affinities of the enzyme for the recognition site and for other sequences on DNA, coupled with the different protein conformations, account for the specificity of this enzyme for the cleavage of DNA at this recognition site; (d) the decrease in the affinity of the enzyme for DNA, caused by Mg2+, liberates binding energy from the DNA-protein complex that can be used in the catalytic reaction.     

Structural and thermodynamic basis for enhanced DNA binding by a promiscuous mutant EcoRI endonuclease

Promiscuous mutant EcoRI endonucleases bind to the canonical site GAATTC more tightly than does the wild-type endonuclease, yet cleave variant (EcoRI(*)) sites more rapidly than does wild-type. The crystal structure of the A138T promiscuous mutant homodimer in complex with a GAATTC site is nearly identical to that of the wild-type complex, except that the Thr138 side chains make packing interactions with bases in the 5'-flanking regions outside the recognition hexanucleotide while excluding two bound water molecules seen in the wild-type complex. Molecular dynamics simulations confirm exclusion of these waters. The structure and simulations suggest possible reasons why binding of the A138T protein to the GAATTC site has DeltaS degrees more favorable and DeltaH degrees less favorable than for wild-type endonuclease binding. The interactions of Thr138 with flanking bases may permit A138T, unlike wild-type enzyme, to form complexes with EcoRI(*) sites that structurally resemble the specific wild-type complex with GAATTC.     

Polypeptide sequences involved in the cleavage of DNA by the restriction endonuclease EcoRI

We have prepared a variety of fragments of the restriction endonuclease EcoRI by partial or total CNBr or acid cleavage of the protein. These fragments were isolated by preparative polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. They were analyzed in a qualitative manner for phosphodiesterase activity. Antibodies against these fragments were elicited in rats and tested for binding to native EcoRI in an enzyme-linked immunoassay. We conclude from these experiments that the DNA binding site of EcoRI is located in the COOH-terminal half of the molecule, close to and probably comprising amino acid residues 137 to 157. This conclusion is reinforced by the observation that this sequence shows homology to the sequences of the recognition helix of other gene-regulatory proteins.     

The specific binding, bending, and unwinding of DNA by RsrI endonuclease, an isoschizomer of EcoRI endonuclease

To determine whether RsrI endonuclease recognizes and cleaves the sequence GAATTC in duplex DNA similarly to its isoschizomer EcoRI we initiated a functional comparison of the two enzymes. Equilibrium binding experiments showed that at 20 degrees C RsrI endonuclease binds to specific and nonspecific sequences in DNA with affinities similar to those of EcoRI. At 0 degrees C the affinity of RsrI for its specific recognition sequence is reduced 7-fold whereas the affinity for noncanonical sequences remains relatively unchanged. Unlike EcoRI, incubation of RsrI endonuclease with N-ethylmaleimide inactivates the enzyme; however, preincubation with DNA prevents the inactivation. The N-ethylmaleimide-treated enzyme fails to bind DNA as assayed by gel mobility shift assays. Comparison of the deduced amino acid sequences of RsrI and EcoRI endonucleases suggests that modification of Cys245 is responsible for the inactivation. Fe(II). EDTA and methidiumpropyl-EDTA.Fe(II) footprinting results indicate that RsrI, like EcoRI, protects 12 base pairs from cleavage when bound to its specific recognition sequence in the absence of Mg2+. RsrI bends DNA by approximately 50 degrees, as determined by measuring the relative electrophoretic mobilities of specific RsrI-DNA complexes with the binding site in the center or near the end of the DNA fragment. This value is similar to that reported for EcoRI. RsrI also unwinds the DNA helix by 25 degrees +/- 5 degrees, a value close to that reported for EcoRI endonuclease. Collectively, these results indicate that the overall structural changes induced in the DNA by the binding of RsrI and EcoRI endonucleases to DNA in the absence of Mg2+ are similar. In the accompanying paper (Aiken, C. R., McLaughlin, L. W., and Gumport, R. I. (1991) J. Biol. Chem. 266, 19070-19078) we present results of studies of RsrI endonuclease using oligonucleotide substrates containing base analogues which suggest differences in the ways the two enzymes cleave DNA.     

Cation dependence of restriction endonuclease EcoRI activity

Restriction endonuclease EcoRI cleaves the DNA sequence 5'd(-G-A-A-T-T-C-) under optimum digestion conditions. A variation in pH and ionic strength can result in EcoRI activity when 5'd(-A-A-T-T-) is cut. A divalent cation, usually Mg2+, is required for enzyme activity, though Mn2+ can also be used. Eight different cations with ionic radius/charge ratios similar to Mg2+ were tested and Co2+ and Zn2+ were also found to act as cofactors for EcoRI. A comprehensive study has been made of the effect of NaCl and pH on the EcoRI/EcoRI transition in the presence of the above four cations. Generally, a decrease in NaCl and/or an increase in pH caused a decrease in enzyme specificity. The changeover depended on the cation. They may be placed in order of their ability to increase EcoRI specificity thus: Co2+ greater than Zn2+ greater than Mg2+ greater than Mn2+. The Km of EcoRI for ColE1 DNA, in the presence of Co2+, was found to be 0.4 nM, compared to 3 nM with Mg2+, whereas the turnover was only one double-stranded scission/min with Co2+ compared to eight/min with Mg2+. The implications of all these findings on the enzyme's mechanism are discussed.     

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.