Mechanism of Action of Restriction Endonuclease EcoRV
Type II restriction/modification (R/M) systems, such as EcoRV, consist of two enzymes that act at the same DNA sequence; a modification methyltransferase and a restriction endonuclease (Bennett and Halford 1989; Wilson and Murray 1991). For EcoRV, the recognition sequence is GATATC (Kholmina et al. 1980). The EcoRV modification enzyme methylates the first adenine within this sequence (Nwosu et al. 1988) while, in the presence of Mg2+ ions, the EcoRV restriction enzyme cleaves DNA specifically at this site (Schildkraut et al. 1984). The endonuclease cuts both strands at the centre of the sequence, to leave blunt-ended DNA fragments. However, prior methylation of the recognition site blocks restriction activity. The basic tenet of R/M systems is that, in vivo, the restriction enzyme cuts only DNA molecules that have not been exposed previously to the methyltransferase. Hence, they enable the cell to degrade foreign DNA entering the cell without destroying host DNA. E. coli strains carrying the EcoRV system show this phenotype (Bougueleret et al. 1984).
KeywordsHydrolysis Crystallization Phosphorus Manganese Catalysis
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