THE TYPE-I RESTRICTION-ENDONUCLEASE R.ECOR1241 - OVER-PRODUCTION AND BIOCHEMICAL-PROPERTIES

In this paper we describe a two-plasmid system which allows over-produ ction of the R.EcoR124I restriction endonuclease. The endonuclease has been purified to homogeneity in milligram amounts and has been shown to be fully active for both restriction and modification. Unexpectedly , the enzyme was found to require only ATP and Mg2+ for ATPase activit y and DNA cleavage; S-adenosyl methionine (SAM), which has been descri bed as a cofactor of type I restriction enzymes, is not required by R. EcoR124I. However, SAM was found to stimulate the rate of ATPase activ ity and DNA cleavage. This may occur through an increase in specific D NA binding by R.EcoR124I in the presence of SAM, as indicated by our s urface plasmon resonance experiments. These functional differences fro m the well described R.EcoKI restriction endonuclease are reflected in a possible structural difference between the two enzymes, namely that the stoichiometry of R.EcoR124I appears to be R(1)M(2)S(1) while that of R.EcoKI is R(2)M(2)S(1). Supercoiled DNA with one or two S-R124I r ecognition sites is cleaved by the same mechanism inferring co-operati on between specifically bound and excess enzymes. Nicked-circle DNA is an intermediate of cleavage reaction. Cleavage of DNA was inhibited b y an increased degree of negative supercoiling, which may reflect an i ncreased difficulty for the enzyme to translocate the DNA. Hemi-methyl ated DNA was the preferred substrate for methylation. (C) 1996 Academi c Press Limited