title: Differences between Ca2+ and Mg2+ in DNA binding and release by the SfiI restriction endonuclease: implications for DNA looping.
abstract: 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+).
authors: The DNA-Protein Interactions Unit, Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, UK.
authors: Stuart R W
authors: SR
authors: Bellamy
authors: Yana S
authors: YS
authors: Kovacheva
authors: Ishan Haji
authors: IH
authors: Zulkipli
authors: Stephen E
authors: SE
authors: Halford
mesh_terms: D002118:Calcium / Q000737:chemistry*; D020134:Catalytic Domain; D002413:Cations, Divalent / Q000737:chemistry; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004492:Edetic Acid / Q000737:chemistry; D024202:Electrophoretic Mobility Shift Assay; D031541:Fluorescence Resonance Energy Transfer; D008274:Magnesium / Q000737:chemistry*; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D011485:Protein Binding
chemical_list: D002413:Cations, Divalent; D004268:DNA-Binding Proteins; D004247:DNA; D004492:Edetic Acid; C041749:endodeoxyribonuclease SfiI; D015252:Deoxyribonucleases, Type II Site-Specific; D008274:Magnesium; D002118:Calcium