Site-directed mutagenesis studies with EcoRV restriction endonuclease to identify regions involved in recognition and catalysis

Abstract

Guided by the X-ray structure analysis of a crystalline EcoRV-d(GGGATATCCC) complex (Winkler, in preparation), we have begun to identify functionally important amino acid residues of EcoRV. We show here that Asn70, Asp74, Ser183, Asn185, Thr186, and Asn188 are most likely involved in the binding and/or cleavage of the DNA, because their conservative substitution leads to mutants of no or strongly reduced activity. In addition, C-terminal amino acid residues of EcoRV seem to be important for its activity, since their deletion inactivates the enzyme. Following the identification of three functionally important regions, we have inspected the sequences of other restriction and modification enzymes for homologous regions. It was found that two restriction enzymes that recognize similar sequences as EcoRV (DpnII and HincII), as well as two modification enzymes (M.DpnII and, in a less apparent form, M.EcoRV), have the sequence motif -SerGlyXXXAsnIleXSer- in common, which in EcoRV contains the essential Ser183 and Asn188 residues. Furthermore, the C-terminal region, shown to be essential for EcoRV, is highly homologous to a similar region in the restriction endonuclease SmaI. On the basis of these findings we propose that these restriction enzymes and to a certain extent also some of their corresponding modification enzymes interact with DNA in a similar manner.

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

“…. (D/E)XK motif, typical for many type II restriction endonucleases (53)(54)(55), whereas the other domain of Sau3AI has only a cryptic catalytic center, in which the second carboxylate (D/E) is substituted by Val. The function of the C-terminal domain is not known; it might be involved in DNA binding and/or in protein dimerization.…”

Evolutionary Relationship between Different Subgroups of Restriction Endonucleases

“…. (D/E)XK motif, which characterizes the active site of many type II restriction endonucleases (6,(33)(34)(35), in the sequence of any of the endonucleases represented in Fig. 3.…”

Influence of the Phosphate Backbone on the Recognition and Hydrolysis of DNA by the EcoRV Restriction Endonuclease

“…This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. protein and the GATATC bases, have been probed using alternative sequences (40,42,49), base analogues (13-15, 50 -53), and site-directed mutagenesis (43,54), and many have been shown to be essential for efficient catalysis. The endonuclease also makes extensive contacts to the phosphate backbone (38,39).…”