DNA Binding Specificity of MunI Restriction Endonuclease Is Controlled by pH and Calcium Ions:  Involvement of Active Site Carboxylate Residues

Abstract

Gel shift analysis reveals [Lagunavicius, A., & Siksnys, V. (1997) Biochemistry 36 (preceding paper in this issue)] that at pH 8.3 in the absence of Mg2+, MunI restriction endonuclease exhibits little DNA binding specificity, as compared with the D83A and E98A mutants of MunI. This suggests that charged carboxylate residue(s) influence the DNA binding specificity of MunI. In our efforts to establish the determinants of MunI binding specificity, we investigated the possible role of the ionic milieu, and we found that lowering pH or elevating Ca2+ levels per se induces specific DNA recognition by WT MunI. In contrast to the binding experiments at pH 8.3, gel shift analysis at pH 6.5 indicated tight sequence-specific binding of WT MunI in the absence of Mg2+, suggesting that protonation of active site carboxylate residue(s) which manifest anomalously high pKa value(s) control binding specificity. Interestingly, Ca2+ ion concentrations, which did not support DNA cleavage by MunI also induced DNA binding specificity in WT MunI at pH 8.3. To explore possible structural changes upon DNA binding, we then used a limited proteolysis technique. Trypsin cleavage of MunI-DNA complexes indicated that in the presence of cognate DNA the MunI restriction endonuclease became resistant to proteolytic cleavage, suggesting that binding of specific DNA induced a structural change. CD measurements confirmed this observation, suggesting minor secondary structural differences between complexes of MunI with cognate and noncognate DNA. These results therefore suggest that binding of MunI to its recognition sequence triggers a conformational transition that correctly juxtaposes active site carboxylate residues, which then chelate Mg2+ ions. In the absence of Mg2+ ions, at pH 8.3, conditions in which carboxylate groups would be expected to be completely ionized, electrostatic repulsion between charged carboxylates and phosphate oxygens is enhanced such as to interfere with specific DNA binding. Elimination of such repulsive constraints by replacement of carboxylate residues, by lowering pH, or by metal ion binding, then promotes MunI binding specificity.

Neutralizing Mutations of Carboxylates That Bind Metal 2 in T5 Flap Endonuclease Result in an Enzyme That Still Requires Two Metal Ions

“…Thus either lowering the pH or adding divalent metal ions has a large effect on T5FEN-substrate interactions, but these effects are not additive. Similar stimulation of binding of DNA substrates at lowered pH or by Ca 2ϩ ions has been observed with other metallonucleases and been assigned to protonation or binding of Ca 2ϩ ions at the active site carboxylates (28). Here the protonation state of DNA-binding residues could also be a factor (29,30).…”

Mva1269I: A Monomeric Type IIS Restriction Endonuclease from Micrococcus Varians with Two EcoRI- and FokI-like Catalytic Domains

“…8A). Most likely, this band represents a complex, in which one Mva1269I molecule is bound specifically to the target and another one is bound nonspecifically to the same DNA molecule, as observed with some other REases (38). DNA binding studies of the nicking mutants D537A and E554A/ K556A revealed that they form specific DNA complexes at concentrations comparable to those of the wt enzyme (Fig.…”

Alternative arrangements of catalytic residues at the active sites of restriction enzymes

“…Previous mutational studies of the active site residues of restriction enzymes, e.g. EcoRI [24], MunI [25] and BamHI [26], revealed that Ala replacements do not interfere with DNA binding or even enhance binding a⁄nity in comparison to the wt enzymes. Thus, observed DNA binding and cleavage phenotypes of K182A and E195A mutants are in principle consistent with a proposed active site function of K182 and E195 residues in Ecl18kI.…”