Regulation of restriction endonuclease activity with antibiotics

Abstract

The effect of restriction endonucleases in vivo is limited by methylation of recognition sites with appropriate methylases. The action of the same restriction endonucleases in vitro may be regulated with the antibiotics distamycin A, actinomycin D and related compounds. By their sensitivity towards antibiotics the restriction endonucleases can be classified in the following way: (i) sensitive to distamycin A (and its analogs), (ii) sensitive to actinomycin D and (iii) sensitive to both the antibiotics. Sensitivity of restriction endonucleases to the one and the other antibiotic is determined by the composition of the restriction endonuclease recognition site on DNA, and also by the composition of the surrounding sequences. Distamycin A recognizes and protects the sites rich with the d(A/T) base pairs against cleavage by restriction endonucleases, and actinomycin D the sites rich with the d(G/C) base pairs. The effect of the antibiotic is already exerted when it shields 15–30% of the template. The dependence of the degree of inhibition on the antibiotic concentration is expressed with the S-shaped curve which is the evidence of the cooperative character of inhibition. Cleavage of the recognition sites of one and the same endonuclease is sustained by different concentrations of the antibiotic. This allows a differentiated inhibition of individual recognition sites whereas others remain accessible for cleavage. At definite concentrations of antibiotic only 1–2 sites are open for the enzyme. An important feature is that the sites sensitive to distamycin A (and related compounds) are less sensitive to actinomycin D and vice versa. This permits fine regulation of DNA cleavage with restriction endonucleases. It has been shown using the distamycin type compounds that modification of the molecule may change the affinity and selectivity of the action of the antibiotic as the agent blocking the action of restriction endonucleases. Since the studied antibiotics bind in the minor DNA groove one may suggest that the minor groove is needed for interaction of the restriction endonucleases with DNA.