Star activity refers to the aberrant DNA cleavage by type II restriction endonucleases at sites other than their canonical recognition sequences (see Restriction-Modification Systems and Restriction Enzymes). The star activity of an enzyme is usually denoted with an asterisk following the name of the enzyme, such as EcoRI*. Ordinarily, the recognition specificity of restriction endonucleases for the canonical sequence is very high. The canonical site is generally cleaved 106 times more frequently than other DNA sequences under optimal conditions (1). With high enzyme concentration or with long incubation periods, however, cleavage can also occur at sites differing by one or more base pairs (referred to as star sites), although the canonical site continues to be selected preferentially. Presumably star sites are cleaved much more slowly because of the absence of critical interactions of the enzyme with the bases or the phosphate backbone that are needed for optimal binding or catalysis (2).
Specificity can also be altered as a result of suboptimal reaction conditions: high pH, low ionic strength, Mn instead of Mg , or the presence of cosolvents (glycerol, DMSO, or ethanol). Under these conditions, selectivity for the canonical restriction site is reduced as the enzyme relaxes its sequence specificity, and cleavage at degenerate or shortened versions of the canonical sequence occurs more often. The allowable degeneracy may range from (1) a single-base-pair change, (2) loss of recognition of distal base pairs in the canonical segment, to (3) several possible base pair substitutions in the primary recognition site. In the presence of organic solvents, EcoRI, which normally cleaves at the sequence GAATTC, has a star activity, EcoRI*, toward sequences containing the core tetranucleotide sequence AATT (1). XbaI, which cleaves at the canonical sequence TCTAGA, is an example of an extreme star activity; it becomes a nonspecific nuclease under conditions of elevated pH and in the presence of glycerol and DMSO (1). Isoschizomeric enzymes (see Isoschizomer) have been shown to have different cleavage specificities under star reaction conditions (3), so they respond differently to conditions. Star activity can be a problem when restriction enzymes are used as tools for precisely fragmenting DNA. On the other hand, the characteristic changes in specificity patterns of restriction enzymes under conditions inducing star activity has been useful in mechanistic studies (4).
