Abstract
In order to study the interactions of the EcoRI restriction endonuclease and modification methylase with DNA, we have synthesized the self-complementary octanucleotide, pT-G-A-A-T-T-C-A, which contains the nucleotide sequence of the EcoRI substrate. This octamer can act as a substrate for both the endonuclease and methylase; the enzymatic alteration of this molecule is the same as that of DNA, with cleavage and methylation both occurring at the same positions on either substrate. The optimum temperature for the reaction of the octanucleotide with the endonuclease is 15°C and that with the methylase is 12·5°C. The optimum temperature for the reactions of both of the enzymes with DNA is 37°C. The low temperatures for reactions with the octanucleotide reflect the conditions under which this molecule can serve as a substrate for the enzymes. The Km values and turnover numbers for the EcoRI endonuclease using both the synthetic octanucleotide and simian virus 40 (SV40) DNA as substrates were determined. These constants are, respectively, 7 × 10−6 m and 4 min−1 for the endonuclease-octamer reaction and 3 × 10−8 m and 3 min−1 with SV40 DNA. Although the Km does not measure affinity directly, the similarity of turnover numbers on both substrates allows us to conclude that the affinity of the enzyme for SV40 DNA is approximately 200 times greater than for the octanucleotide. The synthetic octanucleotide is self-complementary and can form a duplex structure. Computer analysis of initial reaction-rate data shows that in order to act as a substrate the octamer must be a dimer. This observation, together with the correspondence of the temperature optima for both enzymatic reactions to the tm value for the double-stranded form of the octanucleotide (17 to 19°C), lead us to conclude that the substrate for both EcoRI enzymes is a double-helical segment of DNA containing the central hexamer of the octanucleotide. It is unlikely that any cruciform rearrangement is required for enzyme activity.
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