对嗜热栖热放线菌KOD1株DNA聚合酶基因中编码的两种内含肽归巢内切酶的表征。
Characterization of two intein homing endonucleases encoded in the DNA polymerase gene of Pyrococcus kodakaraensis strain KOD1.
作者信息
Nishioka M, Fujiwara S, Takagi M, Imanaka T
机构信息
Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
出版信息
Nucleic Acids Res. 1998 Oct 1;26(19):4409-12. doi: 10.1093/nar/26.19.4409.
Two intein endonucleases, denoted PI- Pko I and PI- Pko II, in the DNA polymerase gene of the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 were expressed in Escherichia coli and the recombinant endonucleases were characterized. Both endonucleases were thermostable and cleaved their intein-less DNA sequences leaving four base 3'-hydroxyl overhangs. PI-Pko I exhibited 22 times higher specific activity than PI-Pko II and the activity of PI-Pko II was enhanced at higher potassium ion concentrations (1 M). Recognition sequences were also determined using synthetic oligonucleotides inserted into plasmid pUC19. It was shown that DNA sequences of 19 and 16 bp are needed for cleavage by PI-Pko I and PI-Pko II, respectively. PI-Pko II could cleave the downstream junction region between intein-encoding and mature DNA polymerase regions and cleavage by PI-Pko II could be detected even when chromosomal DNA of P.kodakaraensis KOD1 was used as substrate. Therefore, it is suggested that these endonucleases are switching endonucleases whose function lies in the rearrangement of chromosomal DNA.
在嗜热古菌柯达卡拉热球菌（Pyrococcus kodakaraensis）KOD1的DNA聚合酶基因中，两种内含肽内切酶，分别命名为PI - Pko I和PI - Pko II，在大肠杆菌中表达，并对重组内切酶进行了表征。这两种内切酶都具有热稳定性，能切割不含内含肽的DNA序列，留下4个碱基的3'-羟基突出端。PI - Pko I的比活性比PI - Pko II高22倍，且PI - Pko II的活性在较高钾离子浓度（1 M）下增强。还使用插入质粒pUC19的合成寡核苷酸确定了识别序列。结果表明，PI - Pko I和PI - Pko II切割分别需要19 bp和16 bp的DNA序列。PI - Pko II可以切割内含肽编码区和成熟DNA聚合酶区之间的下游连接区域，甚至当使用柯达卡拉热球菌KOD1的染色体DNA作为底物时，也能检测到PI - Pko II的切割。因此，有人提出这些内切酶是开关内切酶，其功能在于染色体DNA的重排。
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