Cleavage of methylated CCCGGG sequences containing either N4-methylcytosine or 5-methytcytosine with Mspl, Hpall, Smal, Xmal and Cfr9I restriction endonudeases

Abstract

The cleavage specificity of R.Cfr9I was determined to be CCGGG whereas the methylation specificity of H.Cfr9I was C4mCCGGG. The action of HspI, HpaII, SmaI, XmaI and Cfr9l restriction endonudeases on an unmethylated parent d(GGACCCGGGTCC) dodecanucleotide duplex and a set of oligo-nuoleotide duplexes, containing all possible substitutions of either 4mC or 5mC for C in the CCCGGG sequence, was investigated. It was found that 4mC methylation. in contrast to 5mC, renders the CCCGGG site resistant to practically all the investigated endonuoleases. The cleavage of methylated substrates with restriction endonucleases is discussed. © 1987 IRL Press Limited, Oxford, England.