Peakman Luke J, Antognozzi Massimo, Bickle Thomas A, Janscak Pavel, Szczelkun Mark D
DNA-Protein Interactions Group, Department of Biochemistry, University of Bristol, Bristol BS8 1TD, UK.
J Mol Biol. 2003 Oct 17;333(2):321-35. doi: 10.1016/j.jmb.2003.08.042.
DNA cleavage by the type III restriction endonuclease EcoP1I was analysed on circular and catenane DNA in a variety of buffers with different salts. In the presence of the cofactor S-adenosyl methionine (AdoMet), and irrespective of buffer, only substrates with two EcoP1I sites in inverted repeat were susceptible to cleavage. Maximal activity was achieved at a Res2Mod2 to site ratio of approximately 1:1 yet resulted in cleavage at only one of the two sites. In contrast, the outcome of reactions in the absence of AdoMet was dependent upon the identity of the monovalent buffer components, in particular the identity of the cation. With Na+, cleavage was observed only on substrates with two sites in inverted repeat at elevated enzyme to site ratios (>15:1). However, with K+ every substrate tested was susceptible to cleavage above an enzyme to site ratio of approximately 3:1, including a DNA molecule with two directly repeated sites and even a DNA molecule with a single site. Above an enzyme to site ratio of 2:1, substrates with two sites in inverted repeat were cleaved at both cognate sites. The rates of cleavage suggested two separate events: a fast primary reaction for the first cleavage of a pair of inverted sites; and an order-of-magnitude slower secondary reaction for the second cleavage of the pair or for the first cleavage of all other site combinations. EcoP1I enzymes mutated in either the ATPase or nuclease motifs did not produce the secondary cleavage reactions. Thus, AdoMet appears to play a dual role in type III endonuclease reactions: Firstly, as an allosteric activator, promoting DNA association; and secondly, as a "specificity factor", ensuring that cleavage occurs only when two endonucleases bind two recognition sites in a designated orientation. However, given the right conditions, AdoMet is not strictly required for DNA cleavage by a type III enzyme.
在含有不同盐类的多种缓冲液中，对III型限制性内切酶EcoP1I切割环状和连环DNA的情况进行了分析。在辅因子S-腺苷甲硫氨酸（AdoMet）存在的情况下，无论缓冲液如何，只有在反向重复序列中有两个EcoP1I位点的底物才易被切割。当Res2Mod2与位点的比例约为1:1时可达到最大活性，但仅导致两个位点中的一个发生切割。相比之下，在没有AdoMet的反应结果取决于单价缓冲液成分的性质，特别是阳离子的性质。对于Na +，仅在酶与位点比例升高（>15:1）时，在反向重复序列中有两个位点的底物上才观察到切割。然而，对于K +，每个测试的底物在酶与位点比例约为3:1以上时都易被切割，包括具有两个直接重复位点的DNA分子，甚至是具有单个位点的DNA分子。在酶与位点比例为2:1以上时，反向重复序列中有两个位点的底物在两个同源位点都被切割。切割速率表明存在两个独立的事件：一对反向位点的首次切割的快速初级反应；以及对该对的第二次切割或所有其他位点组合的首次切割的慢一个数量级的次级反应。在ATP酶或核酸酶基序中发生突变的EcoP1I酶不会产生次级切割反应。因此，AdoMet似乎在III型核酸内切酶反应中起双重作用：首先，作为变构激活剂，促进DNA结合；其次，作为“特异性因子”，确保仅当两个核酸内切酶以指定方向结合两个识别位点时才发生切割。然而，在合适的条件下，III型酶切割DNA并不严格需要AdoMet。
