[Cytosine DNA-methylase of a SsoII-type from Shigella sonnei 47 cells]
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Abstract:
Shigella sonnei 47 cells were found to contain DNA-methylase SsoII which is a modifying component of the system of host specificity of SsoII. The recognition sequence (RS) of methylase SsoII is represented by a five-member palyndromic structure--5'...CCNGG...3'--with a degenerated central nucleotide. Modification of SsoII affords protection of acceptor DNA not only from SsoII type restriction, but also from other restrictases, e. g., Eco RII having an analogous RS but with a less degenerated central nucleotide pair. A simple and rapid procedure for isolation and purification of DNA-methylase ScoII, which employs hydrophobic chromatography on phenyl-Sepharose, has been developed. The enzyme preparation does not contain trace amounts of specific and nonspecific endonucleases and keeps stable on storage in 30% glycerol over a period of one year.
Insights
Shigella sonnei DNA-methylase SsoII protects DNA from restriction enzymes by modifying a specific recognition sequence. A new purification method yields a stable, pure enzyme preparation.
Area of Science:
- Molecular Biology
- Enzymology
- Bacteriology
Background:
- Shigella sonnei 47 harbors DNA-methylase SsoII, a key component of its host specificity system.
- The enzyme modifies DNA at a specific palindromic recognition sequence (5'...CCNGG...3') with a degenerate central nucleotide.
Purpose of the Study:
- To characterize DNA-methylase SsoII and its protective capabilities.
- To develop an efficient method for isolating and purifying active DNA-methylase SsoII.
Main Methods:
- Hydrophobic chromatography on phenyl-Sepharose was employed for enzyme purification.
- Enzyme activity and purity were assessed, including testing for contaminating endonucleases.
Main Results:
- DNA-methylase SsoII modification protects DNA from both SsoII restriction and other restrictases like Eco RII.
- A simple, rapid purification procedure yielded a highly pure enzyme preparation.
- The purified enzyme is free of specific and nonspecific endonucleases and remains stable for one year when stored in 30% glycerol.
Conclusions:
- DNA-methylase SsoII plays a significant role in bacterial defense mechanisms.
- The developed purification protocol provides a reliable source of high-quality DNA-methylase SsoII for further research.