DNA protection by type Ⅱ restriction methylases

Abstract

Activities of restriction endonucleases for methylated DNAs by type II restriction methylases in vitro were observed. The cleavage reactions of Sma I, Ava I and Nae I endonucleases were inhibited completely by the methylation of Hpa II methylase but was greatly for Xma I endonuclease. Also the methylation of Hha I methylase inhibited the cleavage reactions of Hae II and Aha II endonucleases completely, greatly for Nar I but not influenced any apparent activity for Ban I. To analyze the cleavage reactions, pDR323 plasmid (323 base pairs of Pvu II fragment was deleted from pUC9), M13mp9 RF and tritium labeled pUC9 plasmid DNAs were used. The degree of the inhibitions against cleavages by the endonucleases were measured. The results imply the specificities of the related hexameric restriction methylases which have not been isolated yet. And now, one could use these tetrameric restriction methylases in gene manipulations instead of related hexameric methylases. Also from above results, specific nicks could be introduced at the site in which a restriction endonuclease was inhibited greatly. Specific nicks are useful in sequence analysis and site specific mutagenesis. Additionally Aha II endonuclease was inhibited by the methylation of one external cytosine in recognition sequence.