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J. Biol. Chem., Vol. 279, Issue 48, 49736-49740, November 26, 2004

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Ca²+-mediated Site-specific DNA Cleavage and Suppression of Promiscuous Activity of KpnI Restriction Endonuclease^*

Siddamadappa Chandrashekaran, Matheshwaran Saravanan, Deshpande R. Radha, and Valakunja Nagaraja¶||

From the Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560 012 and ^¶Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore 560 064, India

The characteristic feature of type II restriction endonucleases (REases) is their exquisite sequence specificity and obligate Mg²⁺ requirement for catalysis. Efficient cleavage of DNA only in the presence of Ca²⁺ ions, comparable with that of Mg²⁺, is previously not described. Most intriguingly, KpnI REase exhibits Ca²⁺-dependent specific DNA cleavage. Moreover, the enzyme is highly promiscuous in its cleavage pattern on plasmid DNAs in the presence of Mn²⁺ or Mg²⁺, with the complete suppression of promiscuous activity in the presence of Ca²⁺. KpnI methyltransferase does not exhibit promiscuous activity unlike its cognate REase. The REase binds to oligonucleotides containing canonical and mapped noncanonical sites with comparable affinities. However, the extent of cleavage is varied depending on the metal ion and the sequence. The ability of the enzyme to be promiscuous or specific may reflect an evolutionary design. Based on the results, we suggest that the enzyme KpnI represents an REase evolving to attain higher sequence specificity from an ancient nonspecific nuclease.

Received for publication, August 18, 2004

^* This work was supported by the Department of Science and Technology, Government of India. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains Figs. 1 and 2.

Present address: AstraZeneca India Pvt. Ltd., Bellary Rd., Hebbal, Bangalore 560 024.

^|| To whom correspondence should be addressed. Tel.: 91-80-23600668; Fax: 91-80-23602697; E-mail: vraj{at}mcbl.iisc.ernet.in.

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