Use of Specific Oligonucleotide Duplexes to Stimulate Cleavage of Refractory DNA Sites by Restriction Endonucleases

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.

Section: Resultssupporting

confidence: 68%

Specificity Changes in the Evolution of Type II Restriction Endonucleases

Pingoud

Sudina

Geyer

et al. 2005

“…Indeed, the activation of Eco57I by oligoduplexes requires the duplex to contain the recognition site, but the downstream cleavage site is not needed (35). Nevertheless, they seem to differ from each other with respect to their responses to AdoMet.…”

Section: Discussionmentioning

confidence: 99%

“…For example, BspMI has a very low activity on a plasmid that has one BspMI site, but its cleavage of this plasmid is stimulated by the addition of DNA molecules that have multiple BspMI sites (14). Similarly, Eco57I can be stimulated to cleave refractory sites by the addition of an oligonucleotide duplex that carries its recognition sequence (35). In these cases, the activating DNA may provide in trans a second site for the enzyme, which then allows the enzyme to cleave the refractory site, as noted with several type IIe and type IIf nucleases (13)(14)(15)(16)(17)(18)(19)(20)(21)(22).…”

mentioning

confidence: 99%

Many Type IIs Restriction Endonucleases Interact with Two Recognition Sites before Cleaving DNA

Bath

Milsom

Gormley

et al. 2002

114

168

Type IIs restriction endonucleases recognize asymmetric DNA sequences and cleave both DNA strands at fixed positions, typically several base pairs away from the recognition site. These enzymes are generally monomers that transiently associate to form dimers to cleave both strands. Their reactions could involve bridging interactions between two copies of their recognition sequence. To examine this possibility, several type IIs enzymes were tested against substrates with either one or two target sites. Some of the enzymes cleaved the DNA with two target sites at the same rate as that with one site, but most cut their two-site substrate more rapidly than the one-site DNA. In some cases, the two sites were cut sequentially, at rates that were equal to each other but that exceeded the rate on the onesite DNA. In another case, the DNA with two sites was cleaved rapidly at one site, but the residual site was cleaved at a much slower rate. In a further example, the two sites were cleaved concertedly to give directly the final products cut at both sites. Many type IIs enzymes thus interact with two copies of their recognition sequence before cleaving DNA, although via several different mechanisms.Over 3000 type II restriction endonucleases have been identified to date (1). These enzymes recognize short DNA sequences, 4 -8 bp long and cleave both strands of the DNA at fixed locations in or near their recognition sites (2). With one exception, BfiI (3), they require Mg 2ϩ or a similar divalent metal ion to cleave DNA (4), although a few also require AdoMet 1 for maximal activity (5). Many of the type II enzymes are homodimeric proteins that interact symmetrically with palindromic DNA sequences, so that one active site in the dimer is placed to cleave one strand of the DNA and the other active site the equivalent phosphodiester bond in the opposite strand: for example, EcoRV, BamHI, and BglI (6 -8). Enzymes of this sort cleave DNA with multiple target sites by means of separate reactions at each site (9), although they can act processively and migrate from one site to another by intramolecular processes (10).Several type II enzymes that recognize palindromic sequences differ from the orthodox enzymes, because they have to interact with two copies of their target sequence before they can cleave DNA (11,12). The latter include the type IIe enzymes, such as EcoRII, NaeI, and Sau3AI (13-17), and the type IIf enzymes, such as SfiI, SgrAI, Cfr10I,. Both the type IIe and IIf enzymes bind two sites concurrently but the former cleave only one site per turnover, whereas the latter cleave both sites concertedly within a single turnover (22). Except for Sau3AI, a monomer in free solution (17), the type IIe enzymes are dimers with two distinct DNA-binding clefts, both of which bind the cognate DNA sequence but one is an allosteric locus that activates DNA cleavage in the other cleft (15,16). In contrast, the type IIf enzymes are generally tetramers with two identical surfaces for binding their palindromic sites, each made from two subun...

“…This can be interpreted to mean that, as proposed for the type IIe enzyme mechanism, the second site functions as an effector site, whose occupation by the enzyme is required for efficient cleavage to occur. This stimulation is optimal when the effector-binding site is on the same molecule as the site to be cleaved (57). Sau3AI shares the property of being inactive as a monomer with the type IIs enzyme FokI, which is converted to an active enzyme by dimerization in the presence of a DNA substrate.…”

Section: Discussionmentioning

confidence: 99%

Sau3AI, a Monomeric Type II Restriction Endonuclease That Dimerizes on the DNA and Thereby Induces DNA Loops

Friedhoff

Lurz

Lüder

et al. 2001

Here, we report that Sau3AI, an unusually large type II restriction enzyme with sequence homology to the mismatch repair protein MutH, is a monomeric enzyme as shown by gel filtration and ultracentrifugation. Structural similarities in the N-and C-terminal halves of the protein suggest that Sau3AI is a pseudo-dimer, i.e. a polypeptide with two similar domains. Since Sau3AI displays a nonlinear dependence of cleavage activity on enzyme concentration and a strong preference for substrates with two recognition sites over those with only one, it is likely that the functionally active form of Sau3AI is a dimer of a pseudo-dimer. Indeed, electron microscopy studies demonstrate that two distant recognition sites are brought together through DNA looping induced by the simultaneous binding of two Sau3AI molecules to the DNA. We suggest that the dimeric form of Sau3AI supplies two DNA-binding sites, one that is associated with the catalytic center and one that serves as an effector site.