The precise specificity of the approximately 3,000 known restriction enzymes for their >200 different target sequences could be considered their most interesting characteristic. Although all restriction enzymes bind DNA nonspecifically, under optimal conditions the difference in cleavage rates at the cognate site and the next best site (single-base substitution) is very high. For example, the rate difference for EcoRI at its cognate site (5´-GAATTC-3´) and next best site (5´-TAATTC-3´) is of the order of 105 (1)
. Similarly, for EcoRV, cleavage at its cognate site (5´-GATATC-3´) is 106 times faster than at the next best site (5´-GTTATC-3´) (2)
However, under nonoptimal conditions, the differences in cleavage rates between cognate and next-best sites change dramatically for many enzymes. This loss of fidelity or increase in cleavage at sites similar to the cognate site is commonly referred to as star activity. A number of reaction parameters can increase the rate of cleavage at star sites relative to cognate sites. These include pH, type of ions present, ionic strength, metal cofactors other than Mg2+, high enzyme:DNA ratios and the presence of volume excluders (glycerol, ethylene glycol, etc.). In conjunction with this increase in star activity, cleavage rates at the cognate site generally decrease. For example, for EcoRI, the rate difference between cognate and star sites approaches zero as ethylene glycol concentration increases up to 4M (3)
, and for EcoRV, the rate difference drops to only sixfold when Mn2+ is substituted for Mg2+ (2)
For more information on the proposed mechanisms for star activity and a list of enzymes that exhibit star activity, see the Restriction Enzymes Resource.