Sears Alice, Peakman Luke J, Wilson Geoffrey G, Szczelkun Mark D
DNA-Protein Interactions Unit, Department of Biochemistry, University of Bristol, Bristol, BS8 1TD, UK.
Nucleic Acids Res. 2005 Aug 24;33(15):4775-87. doi: 10.1093/nar/gki787. Print 2005.
A new Type III restriction endonuclease designated PstII has been purified from Providencia stuartii. PstII recognizes the hexanucleotide sequence 5'-CTGATG(N)(25-26/27-28)-3'. Endonuclease activity requires a substrate with two copies of the recognition site in head-to-head repeat and is dependent on a low level of ATP hydrolysis ( approximately 40 ATP/site/min). Cleavage occurs at just one of the two sites and results in a staggered cut 25-26 nt downstream of the top strand sequence to generate a two base 5'-protruding end. Methylation of the site occurs on one strand only at the first adenine of 5'-CATCAG-3'. Therefore, PstII has characteristic Type III restriction enzyme activity as exemplified by EcoPI or EcoP15I. Moreover, sequence asymmetry of the PstII recognition site in the T7 genome acts as an historical imprint of Type III restriction activity in vivo. In contrast to other Type I and III enzymes, PstII has a more relaxed nucleotide specificity and can cut DNA with GTP and CTP (but not UTP). We also demonstrate that PstII and EcoP15I cannot interact and cleave a DNA substrate suggesting that Type III enzymes must make specific protein-protein contacts to activate endonuclease activity.
一种名为PstII的新型III型限制性内切酶已从斯氏普罗威登斯菌中纯化出来。PstII识别六核苷酸序列5'-CTGATG(N)(25 - 26/27 - 28)-3'。内切酶活性需要底物具有两个首尾相连重复的识别位点，并且依赖于低水平的ATP水解（约40个ATP/位点/分钟）。切割仅在两个位点中的一个发生，导致在顶链序列下游25 - 26个核苷酸处产生交错切割，生成一个2个碱基的5'-突出末端。该位点的甲基化仅发生在5'-CATCAG-3'的第一条链的第一个腺嘌呤上。因此，PstII具有典型的III型限制酶活性，如EcoPI或EcoP15I所示。此外，T7基因组中PstII识别位点的序列不对称性是体内III型限制活性的历史印记。与其他I型和III型酶不同，PstII具有更宽松的核苷酸特异性，并且可以用GTP和CTP（但不能用UTP）切割DNA。我们还证明，PstII和EcoP15I不能相互作用并切割DNA底物，这表明III型酶必须进行特定的蛋白质-蛋白质接触以激活内切酶活性。
