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Scientific article
Open access
English

A type IV modification-dependent restriction enzyme SauUSI from Staphylococcus aureus subsp. aureus USA300

Published inNucleic acids research, vol. 39, no. 13, p. 5597-5610
Publication date2011
Abstract

A gene encoding a putative DNA helicase from Staphylococcus aureus USA300 was cloned and expressed in Escherichia coli. The protein was purified to over 90% purity by chromatography. The purified enzyme, SauUSI, predominantly cleaves modified DNA containing 5mC and 5-hydroxymethylcytosine. Cleavage of 5mC-modified plasmids indicated that the sites S5mCNGS (S = C or G) are preferentially digested. The endonuclease activity requires the presence of adenosine triphosphate (ATP) or dATP whereas the non-hydrolyzable γ-S-ATP does not support activity. SauUSI activity was inhibited by ethylenediaminetetraacetic acid. It is most active in Mg(++) buffers. No companion methylase gene was found near the SauUSI restriction gene. The absence of a cognate methylase and cleavage of modified DNA indicate that SauUSI belongs to type IV restriction endonucleases, a group that includes EcoK McrBC and Mrr. SauUSI belongs to a family of highly similar homologs found in other sequenced S. aureus, S. epidermidis and S. carnosus genomes. More distant SauUSI orthologs can be found in over 150 sequenced bacterial/archaea genomes. Finally, we demonstrated the biological function of the type IV REase in restricting 5mC-modified plasmid DNA by transformation into clinical S. aureus strain SA564, and in restricting phage λ infection when the endonuclease is expressed in E. coli.

Keywords
  • Adenosine Triphosphate/metabolism
  • Coliphages/physiology
  • DNA Restriction Enzymes/chemistry/genetics/metabolism
  • Deoxyadenine Nucleotides/metabolism
  • Escherichia coli/genetics
  • Metals/chemistry
  • Mutagenesis, Site-Directed
  • Polymerase Chain Reaction
  • Protein Structure, Tertiary
  • Sodium Chloride/chemistry
  • Staphylococcus aureus/enzymology
  • Substrate Specificity
Citation (ISO format)
XU, Shuang-Yong et al. A type IV modification-dependent restriction enzyme SauUSI from Staphylococcus aureus subsp. aureus USA300. In: Nucleic acids research, 2011, vol. 39, n° 13, p. 5597–5610. doi: 10.1093/nar/gkr098
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Article (Published version)
accessLevelPublic
Identifiers
ISSN of the journal0305-1048
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