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Nucleic Acids Research Advance Access originally published online on July 13, 2009
Nucleic Acids Research 2009 37(16):5443-5453; doi:10.1093/nar/gkp569
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Nucleic Acids Research, 2009, Vol. 37, No. 16 5443-5453
© 2009 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Nucleic Acid Enzymes

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

Stuart R. W. Bellamy, Yana S. Kovacheva, Ishan Haji Zulkipli and Stephen E. Halford*

The DNA-Protein Interactions Unit, Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, UK

*To whom correspondence should be addressed. Tel: +44 117 331 2156: Fax: +44 117 331 2168; Email: s.halford{at}bristol.ac.uk

Received May 11, 2009. Revised June 5, 2009. Accepted June 19, 2009.

Many enzymes acting on DNA require Mg2+ ions not only for catalysis but also to bind DNA. Binding studies often employ Ca2+ as a substitute for Mg2+, to promote DNA binding whilst disallowing catalysis. The SfiI endonuclease requires divalent metal ions to bind DNA but, in contrast to many systems where Ca2+ mimics Mg2+, Ca2+ causes SfiI to bind DNA almost irreversibly. Equilibrium binding by wild-type SfiI cannot be conducted with Mg2+ present as the DNA is cleaved so, to study the effect of Mg2+ on DNA binding, two catalytically-inactive mutants were constructed. The mutants bound DNA in the presence of either Ca2+ or Mg2+ but, unlike wild-type SfiI with Ca2+, the binding was reversible. With both mutants, dissociation was slow with Ca2+ but was in one case much faster with Mg2+. Hence, Ca2+ can affect DNA binding differently from Mg2+. 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 Ca2+, it becomes accessible with the mutant and Mg2+.


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