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

Biochemistry. 1978 Jan 10;17(1):131-38. doi: 10.1021/bi00594a019.

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • DNA Restriction Enzymes / metabolism*
  • DNA, Circular
  • DNA, Viral
  • Escherichia coli / enzymology
  • Magnesium / pharmacology*
  • Manganese / pharmacology*
  • Molecular Weight
  • Simian virus 40
  • Substrate Specificity

Substances

  • DNA, Circular
  • DNA, Viral
  • Manganese
  • DNA Restriction Enzymes
  • Magnesium