Specific binding by EcoRV endonuclease to its DNA recognition site GATATC
Specific binding by EcoRV endonuclease to its DNA recognition site GATATC
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DOI：
10.1006/jmbi.1997.1027
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发表时间：
1997-05-30
影响因子：
5.6
通讯作者：
JenJacobson, L
中科院分区：
文献类型：
--
作者：
Engler, LE;Welch, KK;JenJacobson, L
据报道，限制性内切核酸酶EcoRV在不存在催化辅因子Mg 2+的情况下不能区分其特异性DNA位点GATATC与非特异性DNA位点，因此仅在催化步骤中行使序列特异性。与此相反，我们在这里表明，在适当的条件下FH和盐浓度，特定的复合物与寡核苷酸含有GATATC网站可以检测到的过滤器结合或凝胶阻滞。平衡结合常数（K-A）很容易通过直接平衡法和平衡竞争法测定。在不存在二价阳离子的情况下，在pH 7下，"特异性"结合优于"非特异性"结合的优选性为约1000倍（每摩尔寡核苷酸）或12，000倍（每摩尔结合位点）。乙基化干扰足迹法表明，“特定”复合物包括与磷酸基团GpApTpApTC的强接触。特异性DNA结合是强烈的pH依赖性的，在pH 6以上每增加一个pH单位，特异性DNA结合降低约15倍，但非特异性结合不是;因此，结合特异性随着pH的增加而降低。在pH小于或等于7时，凝胶阻滞和过滤器结合产生的特异性位点结合的K-A值基本相同，但在pH> 7时，凝胶阻滞显著低估了k（A）。特异性位点结合被Ca2+刺激约700倍（不是切割的辅因子），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+的反应与母体寡核苷酸相似，Mg2+分别仅刺激结合4倍和2倍。因此，Mg2+不会显著增强结合特异性。考虑到结合和磷酸二酯键断裂的一级速率常数的区分，对不正确位点GTTATC的总体区分约为10（7）倍。因此EcoRV内切核酸酶不是没有结合特异性的位点特异性相互作用的"新范例"，但像其他II型限制性内切核酸酶一样，通过区分DNA结合和催化来实现序列特异性。(C)出版社：Academic Press Limited。
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 FH 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 less than or equal to 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. (C) 1997 Academic Press Limited.