Investigation of restriction-modification enzymes fromM. variansRFL19 with a new type of specificity toward modification of substrate

Abstract

The characterization of MvaI restriction-modification enzymes, isolated from Micrococous varians RFL19, is reported. Both enzymes recognize the 5′CC′(A/T)GG nucleotide sequence.The endonuclease cleaves the sequence at the position indicated by the arrow, whereas the methylase modifies the internal cytosine, yielding N⁴-methylcytosine. This type ofmodification protects the substrate from E.Mval cleavage. 5–Methylcytosine in the same position of the recognition sequence does not protect the substrate from H.MvaI cleavage. R.Mval proved to be the first example of a restriction endonuolease differentiating the position of the methyl group in the heterocyclic ring of cytosine, located in the same site of the recognition sequence. M.MvaI modifies DHA dcm+ in vitro yielding N⁴, 5–di-methylcytosine, N⁴–methylcytosine cannot be differentiated from cytosine using the Maxam–Gilbert DNA sequencing procedure.