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J. Biol. Chem., Vol. 280, Issue 50, 41584-41594, December 16, 2005

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Mva1269I: A Monomeric Type IIS Restriction Endonuclease from Micrococcus Varians with Two EcoRI- and FokI-like Catalytic Domains^*

Elena Armalyte1, Janusz M. Bujnicki2, Jolanta Giedriene, Giedrius Gasiunas, Jan Kosiski3, and Arvydas Lubys4

From the Institute of Biotechnology, Graiciuno 8, Vilnius LT-02241, Lithuania and the International Institute of Molecular and Cell Biology, Trojdena 4, Warsaw PL-02-109, Poland

Type II restriction endonuclease Mva1269I recognizes an asymmetric DNA sequence 5'-GAATGCN-3'/5'-NGCATTC-3' and cuts top and bottom DNA strands at positions, indicated by the "" symbol. Most restriction endonucleases require dimerization to cleave both strands of DNA. We found that Mva1269I is a monomer both in solution and upon binding of cognate DNA. Protein fold-recognition analysis revealed that Mva1269I comprises two "PD-(D/E)XK" domains. The N-terminal domain is related to the 5'-GAATTC-3'-specific restriction endonuclease EcoRI, whereas the C-terminal one resembles the nonspecific nuclease domain of restriction endonuclease FokI. Inactivation of the C-terminal catalytic site transformed Mva1269I into a very active bottom strand-nicking enzyme, whereas mutants in the N-terminal domain nicked the top strand, but only at elevated enzyme concentrations. We found that the cleavage of the bottom strand is a prerequisite for the cleavage of the top strand. We suggest that Mva1269I evolved the ability to recognize and to cleave its asymmetrical target by a fusion of an EcoRI-like domain, which incises the bottom strand within the target, and a FokI-like domain that completes the cleavage within the nonspecific region outside the target sequence. Our results have implications for the molecular evolution of restriction endonucleases, as well as for perspectives of engineering new restriction and nicking enzymes with asymmetric target sites.

Received for publication, June 22, 2005 , and in revised form, September 20, 2005.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) DQ074451 [GenBank] .

^* 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.

¹ Current address: Fermentas UAB, Graiciuno 8, Vilnius LT-02241, Lithuania.

² Supported by the European Molecular Biology Organization & Howard Hughes Medical Institute Young Investigator Programme and by the Polish Ministry of Scientific Research and Information Technology (Grant 3P04A01124).

³ Supported by the National Institutes of Health, Fogarty International Center Grant R03 TW007163-01.

⁴ Current address: Fermentas UAB, Graiciuno 8, Vilnius LT-02241, Lithuania. To whom correspondence should be addressed. Tel.: 370-5-239-4206; Fax: 370-5-260-2142; E-mail: lubys{at}fermentas.lt.

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