Metal‐Dependent TypeIIRestriction Endonucleases

Abstract

Type II restriction endonucleases are phosphodiesterases that bind double‐stranded DNA with high specificity and cleave both strands of the DNA to yield 5′‐phosphate and 3′‐hydroxyl groups as products utilizing Mg 2+ as a cofactor. Owing to their high specificity and extraordinary number (>3500 discovered), type II restriction enzymes have become indispensable tools in molecular biology and have been widely exploited for studying site‐specific protein–DNA interactions. Surprisingly, they share little or no sequence homology among them, though the three‐dimensional structures of the 14 restriction enzymes determined to date reveal a common‐core motif with a central β‐sheet that is flanked by α‐helices on both sides. The active sites occur at one end of the central β‐sheet and contain at least three superimposable residues that are critical for catalysis. The cleavage reaction occurs through an SN2 mechanism with an in‐line displacement of the 3′‐hydroxyl group and an inversion of configuration of the 5′‐phosphate group. However, there is uncertainty about the precise mechanism of cleavage, especially regarding the number of metals involved in catalysis.