title: A new prototype IIS/IIC/IIG endonuclease-methyltransferase TsoI from the thermophile Thermus scotoductus, recognising 5'-TARCCA(N11/9)-3' sequences.
abstract: The Thermus sp. family of IIS/IIG/IIC enzymes includes the thermostable, bifunctional, fused restriction endonuclease (REase)-methyltransferases (MTase): TaqII, Tth111II/TthHB27I, TspGWI, TspDTI and TsoI. The enzymes are large proteins (approximately 120kDa), their enzymatic activities are affected by S-adenosylmethionine (SAM), they recognise similar asymmetric cognate sites and cleave at a distance of 11/9 nucleotides (nt). The enzymes exhibit similarities of their amino acid (aa) sequences and DNA catalytic motifs. Thermus sp. enzymes are an example of functional aa sequence homologies among REases recognising different, yet related DNA sequences. The family consists of TspGWI- and TspDTI-subfamilies. TsoI appears to be a non-identical 'triplet', related to TspDTI and Tth111II/TthHB27I. The discovery of TsoI, purified from Thermus scotoductus, is described. This prototype, displaying a novel specificity, which was determined by: (i) cleavage of a reference plasmid and bacteriophage DNA, (ii) cleavage of custom PCR DNA substrates, (iii) run-off sequencing of cleavage products and (iv) shotgun cloning and sequencing of bacteriophage lambda (λ) DNA digested with TsoI. The enzyme recognises a degenerated 5'-TARCCA-3' sequence, whereas DNA strands are cut 11/9 nt downstream. The discovery of the TsoI prototype is of practical importance in biotechnology, as it extends the palette of cleavage specificities for gene cloning.
authors.affiliation: Department of Molecular Biotechnology, Institute for Environmental and Human Health Protection, Division of Chemistry, University of Gdansk, Wita Stwosza 63, 80-308 Gdansk, Poland. Electronic address: j.jezewska-frackowiak@ug.edu.pl.
authors.forename: Joanna
authors.initials: J
authors.lastname: Jezewska-Frackowiak
authors.affiliation: Thermo Fisher Scientific Baltics UAB, V.A. Graiciuno 8, LT-02241 Vilnius, Lithuania; Department of Botany and Genetics, Vilnius University, M.K. Ciurlionio 21/27, LT-03101 Vilnius, Lithuania. Electronic address: arvydas.lubys@thermofisher.com.
authors.forename: Arvydas
authors.initials: A
authors.lastname: Lubys
authors.affiliation: Thermo Fisher Scientific Baltics UAB, V.A. Graiciuno 8, LT-02241 Vilnius, Lithuania. Electronic address: jolanta.vitkute@thermofisher.com.
authors.forename: Jolanta
authors.initials: J
authors.lastname: Vitkute
authors.affiliation: Thermo Fisher Scientific Baltics UAB, V.A. Graiciuno 8, LT-02241 Vilnius, Lithuania. Electronic address: laimute.zakareviciene@thermofisher.com.
authors.forename: Laimute
authors.initials: L
authors.lastname: Zakareviciene
authors.affiliation: Department of Molecular Biotechnology, Institute for Environmental and Human Health Protection, Division of Chemistry, University of Gdansk, Wita Stwosza 63, 80-308 Gdansk, Poland. Electronic address: renata.zebrowska@wp.pl.
authors.forename: Joanna
authors.initials: J
authors.lastname: Zebrowska
authors.affiliation: Department of Molecular Biotechnology, Institute for Environmental and Human Health Protection, Division of Chemistry, University of Gdansk, Wita Stwosza 63, 80-308 Gdansk, Poland. Electronic address: daria.krefft@phdstud.ug.edu.pl.
authors.forename: Daria
authors.initials: D
authors.lastname: Krefft
authors.affiliation: Department of Molecular Biology, Division of Biology, University of Gdansk, Wita Stwosza 59, 80-308 Gdansk, Poland. Electronic address: marta.a.skowron@gmail.com.
authors.forename: Marta A
authors.initials: MA
authors.lastname: Skowron
authors.affiliation: Department of Molecular Biotechnology, Institute for Environmental and Human Health Protection, Division of Chemistry, University of Gdansk, Wita Stwosza 63, 80-308 Gdansk, Poland. Electronic address: a.zylicz-stachula@ug.edu.pl.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Zylicz-Stachula
authors.affiliation: Department of Molecular Biotechnology, Institute for Environmental and Human Health Protection, Division of Chemistry, University of Gdansk, Wita Stwosza 63, 80-308 Gdansk, Poland. Electronic address: piotr.skowron@ug.edu.pl.
authors.forename: Piotr M
authors.initials: PM
authors.lastname: Skowron
mesh_terms: D004247:DNA / Q000378:metabolism; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D008780:Methyltransferases / Q000378:metabolism*; D013379:Substrate Specificity; D013824:Thermus / Q000201:enzymology*
chemical_list: D004247:DNA; D008780:Methyltransferases; D004262:DNA Restriction Enzymes
keywords: Methyltransferase; Restriction endonuclease; Thermus; Type IIG; Type IIS