Nucleic Acids Research Advance Access originally published online on April 11, 2007
Nucleic Acids Research 2007 35(8):2777-2786; doi:10.1093/nar/gkm114
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Nucleic Acids Research, 2007, Vol. 35, No. 8 2777-2786
© 2007 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.
Molecular Biology |
R.KpnI, an HNH superfamily REase, exhibits differential discrimination at non-canonical sequences in the presence of Ca2+ and Mg2+
1Department of Microbiology and Cell Biology, 2Department of Biochemistry, Indian Institute of Science, Bangalore 560 012 and 3Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore 560 064, India
*To whom correspondence should be addressed. Tel: +91-80-23600668; Fax: 91-80-23602697; Email: vraj{at}mcbl.iisc.ernet.in
Received January 14, 2007. Revised February 4, 2007. Accepted February 8, 2007.
KpnI REase recognizes palindromic sequence, GGTAC
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 Mg2+. Surprisingly, Ca2+ suppresses the Mg2+-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, Ca2+-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 Mg2+- and Mn2+-mediated reactions and was about three times slower with Ca2+. The enzyme discriminates non-canonical sequences poorly from the canonical sequence in Mg2+-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.
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