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Nucleic Acids Research 2004 32(20):6129-6135; doi:10.1093/nar/gkh951
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Published online 23 November 2004

Nucleic Acids Research, Vol. 32 No. 20 © Oxford University Press 2004; all rights reserved

Type II restriction endonuclease R.KpnI is a member of the HNH nuclease superfamily

Matheshwaran Saravanan1, Janusz M. Bujnicki4, Iwona A. Cymerman4, Desirazu N. Rao2 and Valakunja Nagaraja1,3,*

1 Microbiology and Cell Biology Department, 2 Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India, 3 Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore 560 064, India and 4 Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology, Trojdena 4, 02-109 Warsaw, Poland

* To whom correspondence should be addressed. Tel: +91 80 23600668; Fax: +91 80 23602697; Email: vraj{at}mcbl.iisc.ernet.in

Received September 27, 2004; Revised October 19, 2004; Accepted November 1, 2004

The restriction endonuclease (REase) R.KpnI is an orthodox Type IIP enzyme, which binds to DNA in the absence of metal ions and cleaves the DNA sequence 5'-GGTAC^C-3' in the presence of Mg2+ as shown generating 3' four base overhangs. Bioinformatics analysis reveals that R.KpnI contains a ßß{alpha}-Me-finger fold, which is characteristic of many HNH-superfamily endonucleases, including homing endonuclease I-HmuI, structure-specific T4 endonuclease VII, colicin E9, sequence non-specific Serratia nuclease and sequence-specific homing endonuclease I-PpoI. According to our homology model of R.KpnI, D148, H149 and Q175 correspond to the critical D, H and N or H residues of the HNH nucleases. Substitutions of these three conserved residues lead to the loss of the DNA cleavage activity by R.KpnI, confirming their importance. The mutant Q175E fails to bind DNA at the standard conditions, although the DNA binding and cleavage can be rescued at pH 6.0, indicating a role for Q175 in DNA binding and cleavage. Our study provides the first experimental evidence for a Type IIP REase that does not belong to the PD...D/EXK superfamily of nucleases, instead is a member of the HNH superfamily.


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