Welch S G, Williams R A
Department of Biochemistry, Faculty of Basic Medical Sciences, Queen Mary and Westfield College, University of London, U.K.
Biochem J. 1995 Jul 15;309 ( Pt 2)(Pt 2):595-9. doi: 10.1042/bj3090595.
Sixteen isolates of thermophilic bacteria from the genus Thermus, isolated from neutral and alkaline hot water springs in the southwest region of Iceland, were tested for the presence of restriction endonucleases. Extracts from five of the isolates showed evidence of the presence of restriction endonuclease activity by producing discrete nucleotide fragments when incubated at 65 degrees C with lambda phage DNA. Two of the isolates (Tsp4C and Tsp8E) were found to have particularly high levels of restriction endonuclease activity, and the respective enzymes from these two Thermus isolates were partially purified and characterized and their recognition and cleavage sites were determined. Enzyme Tsp4C I is a novel Type II restriction endonuclease recognizing the interrupted palindromic tetranucleotide sequence ACNGT, where N can be any one of the four bases in DNA. Tsp4C I, which retains full enzyme activity when incubated for 10 min at temperatures up to 76 degrees C, hydrolyses the phosphodiester bond in both strands of a double-stranded DNA substrate between the third and fourth bases of the recognition sequence (ACN/GT), generating fragments with a single base 3'-OH overhang. Enzyme Tsp8E I is a thermostable isoschizomer of the mesophilic Type II restriction endonuclease Bgl I (GCCNNNN/NGGC) [Lee, Clanton and Chirikjiam (1979) Fed. Proc. 28, 294], generating fragments with a three base 3'-OH overhang. However, unlike Bgl I, Tsp8E I exhibits considerable thermal stability, retaining full enzyme activity when incubated for 10 min at temperatures up to 78 degrees C. Both Tsp4C I and Tsp8E I represent significant additions to the small but expanding list of the extremely thermostable restriction endonucleases.
从冰岛西南部的中性和碱性温泉中分离出16株嗜热栖热菌属嗜热细菌，对其进行了限制内切酶检测。其中5株菌株的提取物在与λ噬菌体DNA于65℃孵育时产生离散的核苷酸片段，显示出存在限制内切酶活性的证据。发现其中两株菌株（Tsp4C和Tsp8E）具有特别高的限制内切酶活性，对这两株栖热菌分离株的相应酶进行了部分纯化和特性鉴定，并确定了它们的识别和切割位点。酶Tsp4C I是一种新型II型限制内切酶，识别中断的回文四核苷酸序列ACNGT，其中N可以是DNA中四种碱基中的任何一种。Tsp4C I在高达76℃的温度下孵育10分钟时仍保留全部酶活性，它水解双链DNA底物两条链中识别序列（ACN/GT）第三个和第四个碱基之间的磷酸二酯键，产生具有单个3'-OH碱基突出端的片段。酶Tsp8E I是嗜温II型限制内切酶Bgl I（GCCNNNN/NGGC）[Lee、Clanton和Chirikjiam（1979年）《联邦程序》28，294]的热稳定同裂酶，产生具有三个3'-OH碱基突出端的片段。然而，与Bgl I不同，Tsp8E I表现出相当高的热稳定性，在高达78℃的温度下孵育10分钟时仍保留全部酶活性。Tsp4C I和Tsp8E I都是对数量虽少但不断增加的超嗜热限制内切酶列表的重要补充。
