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Nucleic Acids Research 2005 33(13):e112; doi:10.1093/nar/gni113
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Published online 21 July 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
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Discovery of a novel restriction endonuclease by genome comparison and application of a wheat-germ-based cell-free translation assay: PabI (5'-GTA/C) from the hyperthermophilic archaeon Pyrococcus abyssi

Ken Ishikawa1,2, Miki Watanabe1, Toshihiro Kuroita3, Ikuo Uchiyama4, Janusz M. Bujnicki5, Bunsei Kawakami3, Masaru Tanokura6 and Ichizo Kobayashi1,2,*

1Department of Medical Genome Sciences, Graduate School of Frontier Science, University of Tokyo 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan 2Graduate Program in Biophysics and Biochemistry, Graduate School of Science, University of Tokyo Tokyo, Japan 3TSURUGA Institute of Biotechnology, Toyobo Co., Ltd Tsuruga-shi, Japan 4National Institute for Basic Biology Okazaki, Japan 5Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology Warsaw, Poland 6Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Science, University of Tokyo Tokyo, Japan

*To whom correspondence should be addressed. Fax: +81 3 5449 5422; Email: ikobaya{at}ims.u-tokyo.ac.jp

Received April 7, 2005. Revised June 18, 2005. Accepted June 30, 2005.

To search for restriction endonucleases, we used a novel plant-based cell-free translation procedure that bypasses the toxicity of these enzymes. To identify candidate genes, the related genomes of the hyperthermophilic archaea Pyrococcus abyssi and Pyrococcus horikoshii were compared. In line with the selfish mobile gene hypothesis for restriction–modification systems, apparent genome rearrangement around putative restriction genes served as a selecting criterion. Several candidate restriction genes were identified and then amplified in such a way that they were removed from their own translation signal. During their cloning into a plasmid, the genes became connected with a plant translation signal. After in vitro transcription by T7 RNA polymerase, the mRNAs were separated from the template DNA and translated in a wheat-germ-based cell-free protein synthesis system. The resulting solution could be directly assayed for restriction activity. We identified two deoxyribonucleases. The novel enzyme was denoted as PabI, purified and found to recognize 5'-GTAC and leave a 3'-TA overhang (5'-GTA/C), a novel restriction enzyme-generated terminus. PabI is active up to 90°C and optimally active at a pH of around 6 and in NaCl concentrations ranging from 100 to 200 mM. We predict that it has a novel 3D structure.


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