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The type II restriction endonuclease SsoII shows sequence similarity with 10 other restriction endonucleases, among them the type IIE restriction endonuclease EcoRII, which requires binding to an effector site for efficient DNA cleavage, and the type IIF restriction endonuclease NgoMIV, which is active as a homotetramer and cleaves DNA with two recognition sites in a concerted reaction. We show here that SsoII is an orthodox type II enzyme, which is active as a homodimer and does not require activation by binding to an effector site. Nevertheless, it shares with EcoRII and NgoMIV a very similar DNA-binding site and catalytic center as shown here by a mutational analysis, indicative of an evolutionary relationship between these three enzymes. We suggest that a similar relationship exists between other orthodox type II, type IIE, and type IIF restriction endonucleases. This may explain why similarities may be more pronounced between members of different subtypes of restriction enzymes than among the members of a given subtype.More than 3000 different type II restriction endonucleases are known and characterized with respect to their cleavage specificities (1). This group of enzymes probably constitutes one of the largest families of enzymes with the same basic function. This makes type II restriction endonucleases (like DNA methyltransferases) ideal objects to study evolutionary relationships. Moreover, in principle, the family relationships should eventually allow predicting structural and functional features of an individual restriction endonuclease, solely on the basis of sequence comparisons. This was not obvious for a long time since only 10 years ago restriction endonucleases, because of their little sequence conservation, were not considered to be related in evolution (2, 3). But this changed due to the progress made in the analysis of sequence similarities (4 -6) and, in particular, due to the increasing number of crystal structures of restriction enzymes (reviewed in Refs. 7 and 8), which made it obvious that these enzymes have very similar structural cores. Nevertheless, it has been shown recently that some genuine restriction enzymes belong to distinct nuclease superfamilies (Nuc, HNH, and GIY-YIG), which are unrelated and structurally dissimilar to each other and to the "archetypal" (P)D . . . (D/E)XK superfamily (9 -11). These findings make comparative studies using sequences of restriction enzymes even more challenging.We have begun to study the restriction endonuclease SsoII recently (12-15). It is a type II enzyme (reviewed in Refs. 8 and 16) composed of identical subunits each consisting of 305 amino acid residues (17). It cleaves the palindromic sequence 2CC-NGG in the presence of Mg 2ϩ as indicated (18). Our interest in studying this enzyme is due to its sequence similarity to Eco-RII, a type IIE enzyme (reviewed in Ref. 19), which being somewhat larger than SsoII is a dimer of identical subunits each consisting of 404 amino acid residues (20, 21). As a type IIE enzyme EcoRII has two DNA-binding...

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 (2CCNGG), PspGI (2CCWGG), Eco-RII (2CCWGG), NgoMIV (G2CCGGC), and Cfr10I (R2CCGGY), which recognize similar DNA sequences (as indicated, where the downward arrows denote cleavage position), share limited sequence similarity over an interrupted stretch of ϳ70 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 (2GATC) 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 ϳ70 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.

Properties of 2'-aldehyde-containing double stranded DNAs (dsDNAs) have been studied for the first time as substrate analogs of the restriction endonuclease SsoII. These reactive oligonucleotides were successfully cross-linked to the restriction endonuclease SsoII by reductive amination, and conditions for DNA-protein conjugate trypsinolysis followed by the oligonucleotide-peptide conjugate purification were optimized. Use of MALDI-TOF mass spectrometry revealed that covalent linkage forms between the sugar moiety of the central pyrimidine nucleoside of the SsoII recognition site and Lys173 of the enzyme. The latter is probably involved in initial steps of enzyme-substrate recognition during dsDNA readout.

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