Cation Dependence of Restriction Endonuclease EcoRI Activity

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Abstract
Restriction endonuclease EcoRI (EC 3.1.23.13) cleaves the DNA sequence .**GRAPHIC**. under optimum digestion conditions. A variation in pH and ionic strength can result in EcoRI* activity when .**GRAPHIC**. is cut. A divalent cation, usually Mg2+, is required for enzyme activity, though Mn2+ can also be used. Eight different cations with ionic radius/charge ratios similar to Mg2+ were tested and Co2+ and Zn2+ were also found to act as cofactors for EcoRI. A comprehensive study was made of the effect of NaCl and pH on the EcoRI/EcoRI* transition in the presence of the above 4 cations. Generally, a decrease in NaCl and/or an increase in pH caused a decrease in enzyme specificity. The changeover depended on the cation. They may be placed in order of their ability to increase EcoRI specificity thus: Co2+ > Zn2+ > Mg2+ > Mn2+. The Km of EcoRI for ColE1 DNA in the presence of Co2+ was 0.4 nM, compared to 3 nM with Mg2+; the turnover was only 1 double-stranded scission/min with Co2+ compared to 8/min with Mg2+. The implications of these findings on the enzyme''s mechanism are discussed.