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

SRW Bellamy, YS Kovacheva, I Haji Zulkipli, SE Halford

Research output: Contribution to journalArticle (Academic Journal)peer-review

15 Citations (Scopus)

Abstract

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+.
Translated title of the contributionDifferences between Ca2+ and Mg2+ in DNA binding and release by the Sfil restriction endonuclease: implications for DNA looping
Original languageEnglish
Pages (from-to)5443 - 5453
Number of pages11
JournalNucleic Acids Research
Volume37 (16)
DOIs
Publication statusPublished - Sep 2009

Bibliographical note

Other: Author Haji Zulkipli was a final year undergraduate project student. First published on-line 13th July 2009

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