Differences between Ca2+ and Mg2+ in DNA binding and release by the SfiI restriction endonuclease: implications for DNA looping.
Differences between Ca2+ and Mg2+ in DNA binding and release by the SfiI restriction endonuclease: implications for DNA looping.
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DOI：
10.1093/nar/gkp569
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发表时间：
2009-09
影响因子：
14.9
通讯作者：
Halford SE
中科院分区：
文献类型：
--
作者：
Bellamy SR;Kovacheva YS;Zulkipli IH;Halford SE
作用于DNA的许多酶不仅需要镁离子催化，还需要结合DNA。结合研究经常使用钙离子作为镁离子的替代品，以促进DNA结合，同时不允许催化。SfiI内切酶需要二价金属离子才能与DNA结合，但与许多钙离子模拟镁离子的体系不同，钙离子导致SfiI几乎不可逆转地与DNA结合。野生型SfiI在DNA切割时不能与存在的镁离子进行平衡结合，因此，为了研究镁离子对DNA结合的影响，构建了两个催化失活的突变体。突变体在存在钙离子或镁离子的情况下与DNA结合，但与野生型SfiI与钙离子的结合不同，这种结合是可逆的。在两个突变体中，钙离子的解离速度都很慢，但有一种情况下，镁离子的解离速度要快得多。因此，钙离子对DNA结合的影响不同于镁离子。此外，SfiI是DNA环化的原型系统；在具有两个识别位点的DNA上，它与两个位点结合，并环化中间的DNA。虽然环路的动力学不能用野生型SfiI和钙离子测量，但可以用突变体和镁离子来测量。
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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