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Title

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

Authors
Xu, Shuang-Yong
Chan, Siu-Hong
Zheng, Yu
Published in Nucleic Acids Research. 2011, vol. 39, no. 13, p. 5597-610
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/metabolismColiphages/physiologyDNA Restriction Enzymes/chemistry/genetics/metabolismDeoxyadenine Nucleotides/metabolismEscherichia coli/geneticsMetals/chemistryMutagenesis, Site-DirectedPolymerase Chain ReactionProtein Structure, TertiarySodium Chloride/chemistryStaphylococcus aureus/enzymologySubstrate Specificity
Identifiers
PMID: 21421560
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Article (Published version) (13.9 MB) - public document Free access
Structures
Research group Acquisition et expression de facteurs de virulence chez Staphylococcus aureus (86)
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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. 5597-610. https://archive-ouverte.unige.ch/unige:25418

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Deposited on : 2013-01-11

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