Differences between Ca2+ and Mg2+ in DNA binding and release by the SfiI restriction endonuclease: implications for...

Many enzymes acting on DNA require Mg(2+) ions not only for catalysis but also to bind DNA. Binding studies often employ Ca(2+) as a substitute for Mg(2+), to promote DNA binding whilst disallowing catalysis. The SfiI endonuclease requires divalent metal ions to bind DNA but, in contrast to many systems where Ca(2+) mimics Mg(2+), Ca(2+) causes SfiI to bind DNA almost irreversibly. Equilibrium binding by wild-type SfiI cannot be conducted with Mg(2+) present as the DNA is cleaved so, to study the effect of Mg(2+) on DNA binding, two catalytically-inactive mutants were constructed. The mutants bound DNA in the presence of either Ca(2+) or Mg(2+) but, unlike wild-type SfiI with Ca(2+), the binding was reversible. With both mutants, dissociation was slow with Ca(2+) but was in one case much faster with Mg(2+). Hence, Ca(2+) can affect DNA binding differently from Mg(2+). Moreover, SfiI is an archetypal system for DNA looping; on DNA with two recognition sites, it binds to both sites and loops out the intervening DNA. While the dynamics of looping cannot be measured with wild-type SfiI and Ca(2+), it becomes accessible with the mutant and Mg(2+).