S-adenosyl-L-methionine is required for DNA cleavage by type III restriction enzymes

The requirement of S-adenosyl-L-methionine (AdoMet) in the cleavage reactio n carried out by type III restriction-modification enzymes has been investi gated. We show that DNA restriction by EcoPI restriction enzyme does not ta ke place in the absence of exogenously added AdoMet. Interestingly, the clo sely related EcoP15I enzyme has endogenously bound AdoMet and therefore doe s not require the addition of the cofactor for DNA cleavage. By employing a variety of AdoMet analogs, which differ structurally from AdoMet, this stu dy demonstrates that the carboxyl group and any substitution at the epsilon carbon of methionine is absolutely essential for DNA cleavage. Such analog s could bring about the necessary conformational change(s) in the enzyme, w hich make the enzyme proficient in DNA cleavage. Our studies, which include native polyacrylamide gel electrophoresis, molecular size exclusion chroma tography, W, fluorescence and circular dichroism spectroscopy, clearly demo nstrate that the holoenzyme and apoenzyme forms of EcoP15I restriction enzy me have different conformations. Furthermore, the Res and Mod subunits of t he EcoP15I restriction enzyme can be separated by gel filtration chromatogr aphy in the presence of 2 M NaCl. Reconstitution experiments, which involve mixing of the isolated subunits, result in an apoenzyme form, which is res triction proficient in the presence of AdoMet. However, mixing the Res subu nit with Mod subunit deficient in AdoMet binding does not result in a funct ional restriction enzyme. These observations are consistent with the fact t hat AdoMet is required for DNA cleavage. In vivo complementation of the def ective mod allele with a wild-type mod allele showed that an active restric tion enzyme could be formed. Furthermore, we show that while the purified c 2-134 mutant restriction enzyme is unable to cleave DNA, the c2-440 mutant enzyme is able to cleave DNA albeit poorly. Taken together, these results s uggest that AdoMet binding causes conformational changes in the restriction enzyme and is necessary to bring about DNA cleavage. (C) 2001 Academic Pre ss.