Specificity changes in the evolution of type II restriction endonucleases: A biochemical and bioinformatic analysis of restriction enzymes that recognize unrelated sequences

Authors:

• Vera Pingoud,
• Anna Sudina,
• Hildegard Geyer,
• Janusz Marek Bujnicki,
• Rudi Lurz,
• Gerhild Lüder,
• Richard Morgan,
• Elena Kubareva,
• Alfred Pingoud

Abstract

How restriction enzymes with their different specificities and mode of cleavage evolved has been a long standing question in evolutionary biology. We have recently shown that several Type II restriction endonucleases, namely SsoII (↓ CCNGG), PspGI (↓ CCWGG), EcoRII (↓ CCWGG), NgoMIV (G ↓ CCGGC), and Cfr10I (R ↓ CCGGY), which recognize similar DNA sequences (as indicated, where the downward arrows denote cleavage position), share limited sequence similarity over an interrupted stretch of ∼70 amino acid residues with MboI, a Type II restriction endonuclease from Moraxella bovis (Pingoud, V., Conzelmann, C., Kinzebach, S., Sudina, A., Metelev, V., Kubareva, E., Bujnicki, J. M., Lurz, R., Luder, G., Xu, S. Y., and Pingoud, A. (2003) J. Mol. Biol. 329, 913-929). Nevertheless, MboI has a dissimilar DNA specificity (↓ GATC) compared with these enzymes. In this study, we characterize MboI in detail to determine whether it utilizes a mechanism of DNA recognition similar to SsoII, PspGI, EcoRII, NgoMIV, and Cfr10I. Mutational analyses and photocross-linking experiments demonstrate that MboI exploits the stretch of ∼70 amino acids for DNA recognition and cleavage. It is therefore likely that MboI shares a common evolutionary origin with SsoII, PspGI, EcoRII, NgoMIV, and Cfr10I. This is the first example of a relatively close evolutionary link between Type II restriction enzymes of widely different specificities.