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

• Published in: Nucleic acids research 2005-07, Vol.33 (13), p.e112-e112
• Main Authors: Ishikawa, Ken, Watanabe, Miki, Kuroita, Toshihiro, Uchiyama, Ikuo, Bujnicki, Janusz M., Kawakami, Bunsei, Tanokura, Masaru, Kobayashi, Ichizo
• Format: Article
• Language: English
• Subjects: Base Sequence; Cell-Free System; Computational Biology; DNA Restriction Enzymes - genetics; DNA Restriction Enzymes - isolation & purification; DNA Restriction Enzymes - metabolism; Genomics; Hot Temperature; Methods Online; Molecular Sequence Data; Plant Extracts - chemistry; Protein Biosynthesis; Pyrococcus abyssi - enzymology; Pyrococcus abyssi - genetics; Pyrococcus horikoshii - genetics; Substrate Specificity; Triticum - chemistry
• ISSN: 0305-1048; 1362-4962; 1362-4962
• DOI: 10.1093/nar/gni113

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.