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What is restriction enzyme star activity?

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 EcoR I at its cognate site (5′-GAATTC-3′) and next best site (5´-TAATTC-3´) is of the order of 10⁵ (1). Similarly, for EcoR V, 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 EcoR I, the rate difference between cognate and star sites approaches zero as ethylene glycol concentration increases up to 4M (3), and for EcoR V, 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.

1. Lesser, D.R., Kurpiewski, M.R. and Jen-Jacobson, L. (1990) The energetic basis of specificity in the EcoR I endonuclease--DNA interaction. Science 250, 776–86.
2. Vermote, C.L. and Halford, S.E. (1992) EcoR V restriction endonuclease: Communication between catalytic metal ions and DNA recognition. Biochemistry 31, 6082–9.
3. Robinson, C.R. and Sligar, S.G. (1998) Changes in solvation during DNA binding and cleavage are critical to altered specificity of the EcoR I endonuclease. Proc. Natl. Acad. Sci. USA 95, 2186–91.

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