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A type IV modification-dependent restriction enzyme SauUSI from Staphylococcus aureus subsp. aureus USA300
Shuang-Yong Xu1, Anna R Corvaglia, Siu-Hong Chan
1New England Biolabs, Inc. 240 County Road, Ipswich, MA 01938, USA. xus@neb.com
Nucleic Acids Research
|March 23, 2011
Summary
Staphylococcus aureus USA300 enzyme SauUSI cleaves modified DNA containing 5-methylcytosine (5mC) and 5-hydroxymethylcytosine. This type IV restriction endonuclease lacks a companion methylase, indicating its role in defending against foreign DNA.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Bacterial restriction-modification systems are crucial for genome defense.
- Type IV restriction endonucleases are ATP-dependent enzymes that target modified DNA.
- Staphylococcus aureus USA300 is a significant pathogen with a complex genome.
Purpose of the Study:
- To characterize a novel DNA helicase from Staphylococcus aureus USA300.
- To determine the DNA cleavage specificity and enzymatic properties of the purified protein, named SauUSI.
- To elucidate the biological role and classification of SauUSI within restriction-modification systems.
Main Methods:
- Gene cloning and protein expression in Escherichia coli.
- Protein purification using chromatography techniques.
- DNA cleavage assays with modified and unmodified DNA substrates.
- Enzyme activity assays under varying buffer conditions and cofactor presence.
- Bioinformatic analysis to identify homologous genes and assess evolutionary distribution.
Main Results:
- The purified enzyme, SauUSI, exhibits endonuclease activity predominantly on DNA containing 5-methylcytosine (5mC) and 5-hydroxymethylcytosine.
- SauUSI preferentially cleaves at S5mCNGS sites (S = C or G) and requires adenosine triphosphate (ATP) or dATP for activity.
- The enzyme is inhibited by ethylenediaminetetraacetic acid and is most active in Mg(++) buffers.
- No associated methylase gene was identified, supporting its classification as a type IV restriction endonuclease.
- SauUSI demonstrated biological function in restricting 5mC-modified plasmid DNA transformation and phage λ infection in E. coli.
Conclusions:
- SauUSI is a novel type IV restriction endonuclease from Staphylococcus aureus USA300.
- Its ability to cleave modified DNA in the absence of a cognate methylase highlights its role in innate bacterial immunity.
- SauUSI represents a conserved family of restriction enzymes found across various bacterial and archaeal species.
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