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R.KpnI, an HNH superfamily REase, exhibits differential discrimination at non-canonical sequences in the presence of $Ca^{2+}$ and $Mg^{2+}$

Saravanan, Matheshwaran and Vasu, Kommireddy and Kanakaraj, Radhakrishnan and Rao, Desirazu N. and Nagaraja, Valakunja (2007) R.KpnI, an HNH superfamily REase, exhibits differential discrimination at non-canonical sequences in the presence of $Ca^{2+}$ and $Mg^{2+}$. In: Nucleic Acids Research, 35 (8). pp. 2777-2786.

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Abstract

KpnI REase recognizes palindromic sequence, $GGTAC\downarrow C$, and forms complex in the absence of divalent metal ions, but requires the ions for DNA cleavage. Unlike most other REases, R.KpnI shows promiscuous DNA cleavage in the presence of $Mg^{2+}$. Surprisingly, $Ca^{2+}$ suppresses the $Mg^{2+}$-mediated promiscuous activity and induces high fidelity cleavage. To further analyze these unique features of the enzyme, we have carried out DNA binding and kinetic analysis. The metal ions which exhibit disparate pattern of DNA cleavage have no role in DNA recognition. The enzyme binds to both canonical and non-canonical DNA with comparable affinity irrespective of the metal ions used. Further, $Ca^{2+}$-imparted exquisite specificity of the enzyme is at the level of DNA cleavage and not at the binding step. With the canonical oligonucleotides, the cleavage rate of the enzyme was comparable for both $Mg^{2+}$- and $Mn^{2+}$-mediated reactions and was about three times slower with $Ca^{2+}$. The enzyme discriminates non-canonical sequences poorly from the canonical sequence in $Mg^{2+}$-mediated reactions unlike any other Type II REases, accounting for the promiscuous behavior. R.KpnI, thus displays properties akin to that of typical Type II REases and also endonucleases with degenerate specificity in its DNA recognition and cleavage properties.

Item Type: Journal Article
Additional Information: Copyright of this article belongs to Valakunja Nagaraja.
Department/Centre: Division of Biological Sciences > Biochemistry
Division of Biological Sciences > Microbiology & Cell Biology
Date Deposited: 10 Jul 2008
Last Modified: 19 Sep 2010 04:46
URI: http://eprints.iisc.ernet.in/id/eprint/14599

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