title: A type IV modification-dependent restriction enzyme SauUSI from Staphylococcus aureus subsp. aureus USA300.
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.
authors.affiliation: New England Biolabs, Inc. 240 County Road, Ipswich, MA 01938, USA. xus@neb.com
authors.forename: Shuang-Yong
authors.initials: SY
authors.lastname: Xu
authors.forename: Anna R
authors.initials: AR
authors.lastname: Corvaglia
authors.forename: Siu-Hong
authors.initials: SH
authors.lastname: Chan
authors.forename: Yu
authors.initials: Y
authors.lastname: Zheng
authors.forename: Patrick
authors.initials: P
authors.lastname: Linder
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D003090:Coliphages / Q000502:physiology; D004262:DNA Restriction Enzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003838:Deoxyadenine Nucleotides / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D008670:Metals / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D017434:Protein Structure, Tertiary; D012965:Sodium Chloride / Q000737:chemistry; D013211:Staphylococcus aureus / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D003838:Deoxyadenine Nucleotides; D008670:Metals; D012965:Sodium Chloride; D000255:Adenosine Triphosphate; D004262:DNA Restriction Enzymes; C026600:2'-deoxyadenosine triphosphate