Altering the specificity of restriction endonuclease: effect of replacing Mg2+ with Mn2+

Abstract

In the presence of 100 mM Tris buffer (pH 7.5) and 1-10 mM Mg2+ EcoRI endonuclease cleaves DNA at a specific nucleotide sequence and in a characteristic way: -GAATTC-. But if Mg2+ is replaced by Mn2+, the specificity of the cleavage is relaxed and cleavages occur at many other sites; moreover, there appears to be a hierarchy of cleavage rates at the pseudo-EcoRI restriction sites. For example, SV40 DNA is cleaved only once in the usual digestion conditions, but with Mn2+ more than ten cleavages are made; the five most rapidly cleaved SV40 DNA map locations are 0/1.0 larger than 0.93 larger than 0.33 approximately equal to 0.42 larger than 0.29 approximately equal to 0.40 larger than 0.25. Mn2+ also alters the restriction specificity of HindIII but not HpaII endonuclease.