DNA duplexes containing methylated bases or non-nucleotide inserts in the recognition site are cleaved by restriction endonuclease R.EcoRII in presence of canonical substrate.

DNA duplexes containing the natural methylated bases N6-methyladenine (m6Ade), N4-methylcytosine (m4Cyt) or C5-methylcytosine (m5Cyt) in one strand of the recognition sequence are resistant to EcoRII restriction endonuclease (R.EcoRII). Hydrolysis of these modified duplexes was observed in the presence of the canonical substrate. Incorporation of m4Cyt or m5Cyt into both strands of the recognition sequence precludes such activation by a canonical substrate. R.EcoRII also fails to cleave substrate analogs in which one of the nucleosides in the recognition site is replaced by the 1,2-dideoxyribose (D) or by 1,3-propanediol (Prd) (modeling DNA with an abasic site). The hydrolysis of DNA duplexes with non-nucleotide inserts is also activated in the presence of canonical substrate. Thus, the two-substrate mechanism of EcoRII-DNA interaction allows hydrolysis of apurinic/apyrimidinic and hemimethylated DNA.