Localization of the type I restriction-modification enzyme EcoKI in the bacterial cell
Language English Country United States Media print
Document type Journal Article, Research Support, Non-U.S. Gov't
PubMed
10733903
DOI
10.1006/bbrc.2000.2375
PII: S0006-291X(00)92375-1
Knihovny.cz E-resources
- MeSH
- Bacterial Proteins isolation & purification MeSH
- Cytoplasm enzymology MeSH
- DNA Restriction-Modification Enzymes isolation & purification MeSH
- Endonucleases metabolism MeSH
- Escherichia coli enzymology MeSH
- Cell Compartmentation MeSH
- Membrane Proteins isolation & purification MeSH
- Site-Specific DNA-Methyltransferase (Adenine-Specific) isolation & purification MeSH
- Escherichia coli Proteins * MeSH
- Deoxyribonucleases, Type I Site-Specific * MeSH
- DNA Restriction Enzymes isolation & purification MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Bacterial Proteins MeSH
- DNA modification methylase EcoKI MeSH Browser
- DNA Restriction-Modification Enzymes MeSH
- endodeoxyribonuclease EcoKI MeSH Browser
- Endonucleases MeSH
- HsdM protein, Bacteria MeSH Browser
- HsdR protein, E coli MeSH Browser
- HSDS protein, Bacteria MeSH Browser
- Membrane Proteins MeSH
- Site-Specific DNA-Methyltransferase (Adenine-Specific) MeSH
- Escherichia coli Proteins * MeSH
- Deoxyribonucleases, Type I Site-Specific * MeSH
- DNA Restriction Enzymes MeSH
To localise the type I restriction-modification (R-M) enzyme EcoKI within the bacterial cell, the Hsd subunits present in subcellular fractions were analysed using immunoblotting techniques. The endonuclease (ENase) as well as the methylase (MTase) were found to be associated with the cytoplasmic membrane. HsdR and HsdM subunits produced individually were soluble, cytoplasmic polypeptides and only became membrane-associated when coproduced with the insoluble HsdS subunit. The release of enzyme from the membrane fraction following benzonase treatment indicated a role for DNA in this interaction. Trypsinization of spheroplasts revealed that the HsdR subunit in the assembled ENase was accessible to protease, while HsdM and HsdS, in both ENase and MTase complexes, were fully protected against digestion. We postulate that the R-M enzyme EcoKI is associated with the cytoplasmic membrane in a manner that allows access of HsdR to the periplasmic space, while the MTase components are localised on the inner side of the plasma membrane.
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