Engler L E, Welch K K, Jen-Jacobson L
Department of Biological Sciences, University of Pittsburgh, PA 15260, USA.
J Mol Biol. 1997 May 30;269(1):82-101. doi: 10.1006/jmbi.1997.1027.
Restriction endonuclease EcoRV has been reported to be unable to distinguish its specific DNA site, GATATC, from non-specific DNA sites in the absence of the catalytic cofactor Mg2+, and thus to exercise sequence specificity solely in the catalytic step. In contrast, we show here that under appropriate conditions of pH and salt concentration, specific complexes with oligonucleotides containing the GATATC site can be detected by either filter-binding or gel-retardation. Equilibrium binding constants (K(A)) are easily measured by both direct equilibrium and equilibrium-competition methods. The preference for "specific" over "non-specific" binding at pH 7 in the absence of divalent cations is about 1000-fold (per mole of oligonucleotide) or 12,000-fold (per mole of binding sites). Ethylation-interference footprinting shows that the "specific" complex includes strong contacts to the phosphate groups GpApTpApTC. Specific DNA binding is strongly pH-dependent, decreasing about 15-fold for each increase of one pH unit above pH 6, but non-specific binding is not; thus, binding specificity decreases with increasing pH. Gel retardation and filter-binding at pH < or = 7 yield essentially identical values of K(A) for specific-site binding, but at pH > 7 gel retardation significantly underestimates K(A). Specific-site binding is stimulated about 700-fold by Ca2+ (not a cofactor for cleavage), but with non-cleavable 3'-phosphorothiolate and 4'-thiodeoxyribose derivatives whose response to Ca2+ is similar to that of the parent oligonucleotide, Mg2+ stimulates binding only fourfold and twofold, respectively. Thus, binding specificity is not dramatically enhanced by Mg2+. Taking into account discrimination in binding and in the first-order rate constant for phosphodiester bond scission, the overall discrimination exercised against the incorrect site GTTATC is about 10(7)-fold. EcoRV endonuclease is thus not a "new paradigm" for site-specific interaction without binding specificity, but like other type II restriction endonucleases achieves sequence specificity by discriminating both in DNA binding and in catalysis.
据报道，限制性内切酶EcoRV在没有催化辅因子Mg2+的情况下，无法将其特定的DNA位点GATATC与非特定的DNA位点区分开来，因此仅在催化步骤中行使序列特异性。相比之下，我们在此表明，在适当的pH和盐浓度条件下，可以通过滤膜结合或凝胶阻滞检测到与含有GATATC位点的寡核苷酸形成的特异性复合物。平衡结合常数（K(A)）可以通过直接平衡法和平衡竞争法轻松测量。在不存在二价阳离子的情况下，pH 7时对“特异性”结合相对于“非特异性”结合的偏好约为1000倍（每摩尔寡核苷酸）或12000倍（每摩尔结合位点）。乙基化干扰足迹法表明，“特异性”复合物包括与磷酸基团GpApTpApTC的强烈接触。特异性DNA结合强烈依赖于pH，在pH 6以上每增加一个pH单位，结合力下降约15倍，但非特异性结合则不然；因此，结合特异性随pH升高而降低。在pH≤7时，凝胶阻滞和滤膜结合对于特定位点结合产生的K(A)值基本相同，但在pH>7时，凝胶阻滞显著低估了K(A)。Ca2+（不是切割的辅因子）可使特定位点结合增强约700倍，但对于不可切割的3'-硫代磷酸酯和4'-硫代脱氧核糖衍生物，其对Ca2+的反应与亲本寡核苷酸相似，Mg2+分别仅使结合增强4倍和2倍。因此，Mg2+不会显著增强结合特异性。考虑到结合中的区分以及磷酸二酯键断裂的一级速率常数，对错误位点GTTATC的总体区分约为10^7倍。因此，EcoRV内切酶不是没有结合特异性的位点特异性相互作用的“新范例”，而是像其他II型限制性内切酶一样，通过在DNA结合和催化过程中进行区分来实现序列特异性。
