The core element of the EcoRII methylase as defined by protease digestion and deletion analysis.
The core element of the EcoRII methylase as defined by protease digestion and deletion analysis.
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DOI：
10.1093/nar/19.19.5403
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发表时间：
1991-10
影响因子：
14.9
通讯作者：
S. Friedman;S. Som;Lian-Fu Yang
中科院分区：
文献类型：
--
作者：
S. Friedman;S. Som;Lian-Fu Yang
EcoRII DNA甲基转移酶与含氮杂胞嘧啶的DNA的结合保护酶免受蛋白酶的消化。极限消化产生的产物在SDS-PAGE上的Mr比完整蛋白低20%。对胰蛋白酶消化产物的N末端进行测序，发现缺失N末端82个氨基酸。在所用条件下，未结合的酶被消化成小肽。保护酶免受蛋白酶消化意味着酶在与DNA结合时经历主要构象变化。EcoRII甲基转移酶的胰蛋白酶敏感区出现在与其他原核DNA（胞嘧啶-5）甲基转移酶共享的第一恒定区之前。为了确定该区域是否在底物结合或特异性中起作用，研究了N-末端缺失突变体。97个氨基酸的缺失导致酶活性降低。进一步的删除导致活性完全丧失。纯化通过氨基酸85缺失的酶，发现其具有与野生型相同的特异性，然而S-腺苷甲硫氨酸（SNAMet）和DNA的Km分别增加27和18倍。EcoRII甲基化酶的N-末端，虽然在许多原核DNA（胞嘧啶-5）甲基化酶中存在的可变区，但在确定酶特异性方面不起作用，尽管它确实有助于与DNA甲基化酶和DNA的相互作用。
Binding of the EcoRII DNA methyltransferase to azacytosine-containing DNA protects the enzyme from digestion by proteases. The limit digest yields a product having a Mr on SDS-PAGE 20% less than the intact protein. The N terminus of the tryptic digestion product was sequenced and found to be missing the N terminal 82 amino acids. Under the conditions used unbound enzyme was digested to small peptides. Protection of the enzyme from protease digestion implies that the enzyme undergoes major conformational changes when bound to DNA. The trypsin sensitive region of the EcoRII methyltransferase occurs prior to the first constant region shared with other procaryotic DNA(cytosine-5)methyltransferases. To determine if this region played a role in substrate binding or specificity, N-terminal deletion mutants were studied. Deletion of 97 amino acids resulted in a decrease of enzyme activity. Further deletions caused a complete loss of activity. Enzyme deleted through amino acid 85 was purified and found to have the same specificity as wild type however there was an increase in Km for both S-adenosylmethionine (AdoMet) and DNA of 27 and 18 fold respectively. The N-terminus of the EcoRII methylase, although a variable region present in many procaryotic DNA(cytosine-5)methylases, plays no role in determining enzyme specificity, although it does contribute to the interaction with both AdoMet and DNA.