Lagunavicius A, Grazulis S, Balciunaite E, Vainius D, Siksnys V
Institute of Biotechnology, Graiciuno 8, Vilnius 2028, Lithuania.
Biochemistry. 1997 Sep 16;36(37):11093-9. doi: 10.1021/bi963126a.
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.
凝胶迁移分析表明[拉古纳维丘斯，A.，& 西克斯尼斯，V.（1997年）《生物化学》第36卷（本期之前的论文）]，在pH 8.3且不存在Mg2+的情况下，与MunI的D83A和E98A突变体相比，MunI限制性内切酶表现出很少的DNA结合特异性。这表明带电荷的羧酸盐残基影响MunI的DNA结合特异性。在我们确定MunI结合特异性决定因素的过程中，我们研究了离子环境的可能作用，并且我们发现降低pH值或提高Ca2+水平本身会诱导野生型MunI对特定DNA的识别。与在pH 8.3下的结合实验不同，在pH 6.5下的凝胶迁移分析表明，在不存在Mg2+的情况下野生型MunI具有紧密的序列特异性结合，这表明表现出异常高pKa值的活性位点羧酸盐残基的质子化控制着结合特异性。有趣的是，不支持MunI进行DNA切割的Ca2+离子浓度在pH 8.3时也能诱导野生型MunI的DNA结合特异性。为了探究DNA结合后可能发生的结构变化，我们随后使用了有限蛋白酶解技术。对MunI-DNA复合物进行胰蛋白酶切割表明，在同源DNA存在的情况下，MunI限制性内切酶对蛋白酶解具有抗性，这表明特定DNA的结合诱导了结构变化。圆二色性测量证实了这一观察结果，表明MunI与同源和非同源DNA的复合物之间存在微小的二级结构差异。因此，这些结果表明，MunI与其识别序列的结合引发了构象转变，使活性位点羧酸盐残基正确并列，然后螯合Mg2+离子。在不存在Mg2+离子的情况下，在pH 8.3时，预计羧酸盐基团会完全电离，带电荷的羧酸盐与磷酸氧之间的静电排斥增强，从而干扰特定DNA的结合。通过取代羧酸盐残基、降低pH值或金属离子结合消除这种排斥限制，进而促进MunI的结合特异性。
