title: A putative Type IIS restriction endonuclease GeoICI from Geobacillus sp.--A robust, thermostable alternative to mezophilic prototype BbvI.
abstract: Screening of extreme environments in search for novel microorganisms may lead to the discovery of robust enzymes with either new substrate specificities or thermostable equivalents of those already found in mesophiles, better suited for biotechnology applications. Isolates from Iceland geysers' biofilms, exposed to a broad range of temperatures, from ambient to close to water boiling point, were analysed for the presence of DNA-interacting proteins, including restriction endonucleases (REases). GeoICI, a member of atypical Type IIS REases, is the most thermostable isoschizomer of the prototype BbvI, recognizing/cleaving 5'-GCAGC(N8/12)-3'DNA sequences. As opposed to the unstable prototype, which cleaves DNA at 30°C, GeoICI is highly active at elevated temperatures, up to 73°C and over a very wide salt concentration range. Recognition/cleavage sites were determined by: (i) digestion of plasmid and bacteriophage lambda DNA (Λ); (ii) cleavage of custom PCR substrates, (iii) run-off sequencing of GeoICI cleavage products and (iv) shotgun cloning and sequencing of Λ DNA fragmented with GeoICI. Geobacillus sp. genomic DNA was PCR-screened for the presence of other specialized REases-MTases and as a result, another putative REase- MTase, GeoICII, related to the Thermus sp. family of bifunctional REases-methyltransferases (MTases) was detected.
authors.affiliation: Department of Molecular Biotechnology, Institute for Environment and Human Health Protection, Faculty of Chemistry, University of Gdansk, Wita Stwosza 63, 80-308 Gdansk, Poland.
authors.forename: Joanna
authors.initials: J
authors.lastname: Zebrowska
authors.forename: Olga
authors.initials: O
authors.lastname: Zolnierkiewicz
authors.forename: Marta A
authors.initials: MA
authors.lastname: Skowron
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Zylicz-Stachula
authors.forename: Joanna
authors.initials: J
authors.lastname: Jezewska-Frackowiak
authors.forename: Piotr M
authors.initials: PM
authors.lastname: Skowron
mesh_terms: D000595:Amino Acid Sequence / Q000235:genetics; D001483:Base Sequence / Q000235:genetics; D004262:DNA Restriction Enzymes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability; D056506:Geobacillus / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D004268:DNA-Binding Proteins; D004262:DNA Restriction Enzymes; D015252:Deoxyribonucleases, Type II Site-Specific