Engineering of Variants of the Restriction Endonuclease EcoRV That Depend in Their Cleavage Activity on the Flexibility of Sequences Flanking the Recognition Site

Abstract

The present work describes mutants of the restriction enzyme EcoRV that discriminate very efficiently between oligodeoxynucleotide substrates with an EcoRV recognition sequence in different sequence context. All of these EcoRV variants harbor substitutions at position 226, where in the cocrystal structure of the specific EcoRV/DNA complex an arginine contacts the backbone of the DNA substrate upstream of the recognition sequence, and cleave an oligodeoxynucleotide with an EcoRV site in a nonflexible sequence context (the recognition site being flanked by runs of A and T) with much higher catalytic efficiency (kcat/Km) than an oligodeoxynucleotide with an EcoRV site in a flexible sequence context (the recognition site being flanked by runs of AT), in contrast to the wild-type enzyme, that cleaves both substrates with the same catalytic efficiency. Steady-state and single-turnover kinetics indicate that the enhanced selectivity of the mutants is due to the catalytic step of the reaction. It is possible to enhance the discriminatory power of these EcoRV variants through the choice of appropriate reaction conditions, in particular low salt concentration and low reaction temperatures. It must be emphasized that the enhanced selectivity of these EcoRV variants toward EcoRV sites in a flexible and nonflexible sequence context, respectively, is not only seen with oligodeoxynucleotides, but also with plasmid substrates.

Factors influencing resistance of UV-irradiated DNA to the restriction endonuclease cleavage

“…For example, the HindIII site in pBR322 DNA shows a more than two-fold photoreactivity than the HindIII site in pUC19 DNA, which should be caused by the flanking regions because the target site and the restrictase are the same in both cases. This is in line with observations of the influence of the flanking regions in restrictase interactions with unirradiated DNA [41]. Though there is not a general consent about the length of flanking sequences necessary for proper binding and cleavage of restrictases, spanning from no extra base pair to more than four base pairs flanking both sides of recognition site [38,42,43], it can be inferred that it depends on the nature of the respective enzyme.…”

Modulation of Escherichia coli DNA Methyltransferase Activity by Biologically Derived GATC-flanking Sequences

“…(31). Our results suggest that these conserved phosphate interactions may be mediating higher order specificity similar to that seen with EcoRV (54,57). Mutagenic studies to determine the contribution of these residues to site preference and processivity are under way.…”

“…8). Similar to EcoDam, the affinity of EcoRV for substrates varying in the DNA sequences flanking its target site 5Ј-GATATC-3Ј is not altered, whereas the rate of cleavage for the same substrates does vary (54,57 ) may be important for positioning EcoDam properly onto its target so that methylation can occur (58) and may contribute to the high level of processivity observed with this enzyme FIGURE 7. Flanking sequence conservation of preferred, semipreferred, and nonpreferred GATCs.…”

An EM View of the FokI Synaptic Complex by Single Particle Analysis

“…Another strategy would be to modify the FokI recognition domain to target other (or extended) DNA sites, but this is also less than straightforward, as restriction enzymes have proven resilient to alteration in DNA binding specificities. 10,[23][24][25][26][27][28][29][30] FokI is not the only restriction endonuclease that requires two DNA sites for successful cleavage. A number of other restriction enzymes have now been shown to bind to two DNA sites simultaneously.…”