|
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 non-optimal 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 DNA:enzyme 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 6-fold when Mn2+ is substituted for Mg2+ (2).
Several plausible explanations for star activity are based on the proposed mechanisms
for target site identification and hydrolysis (see Structure and
Mechanism of Action for more information). During nonspecific binding, a large number
of water molecules are present at the protein-DNA interface. When tighter binding and
positioning of the catalytic site occurs upon recognition of the target sequence, the
number of these interface water molecules is significantly reduced. The higher osmotic
pressure caused by volume excluders results in the same reduction in the amount of
interface water molecules and allows easier active complex formation at star sites (3). At
alkaline pH, higher OH- concentrations may reduce the need for an activated
water molecule, which normally initiates nucleophilic attack on the scissile phosphorous.
Mn2+ has a higher affinity for oxygen ligands than Mg2+and may bind
more easily to a catalytic site in a partially active conformation at a star site. Also,
it is possible that Mn2+-bound water is better able to protonate the leaving
group since it has a lower pKa than Mg2+ bound water (4).
Although all restriction enzymes probably exhibit some decrease in the cleavage rate
difference between cognate and near-cognate sites under such extreme conditions as 4M
ethylene glycol, most are not significantly affected under common usage conditions. Those
that are susceptible to star activity are induced to different degrees by variations in
reaction conditions or by combinations of the conditions listed above. Table 1.4 lists the
enzymes sold by Promega that may exhibit star activity, especially under reaction
conditions that deviate from those recommended. In multiple enzyme digests or multiple
step applications, it is advisable to stay at or near the optimal conditions for these
enzymes whenever possible.
Table 1.4. Promega Enzymes That May Exhibit Star Activity.
|
