Metal ion dependence of DNA cleavage by SepMI and EhoI restriction endonucleases
| Autor: | Abdelkarim Belkebir, Houssine Azeddoug |
|---|---|
| Rok vydání: | 2012 |
| Předmět: |
DNA
Bacterial Cations Divalent Metal ions in aqueous solution Enterobacter Biology Cleavage (embryo) Microbiology Metal chemistry.chemical_compound Staphylococcus epidermidis Magnesium DNA Cleavage Deoxyribonucleases Type II Site-Specific Manganese Restriction enzyme Crystallography Kinetics Biochemistry chemistry Ionic strength Metals visual_art visual_art.visual_art_medium Calcium Steady state (chemistry) pUC19 DNA Plasmids |
| Zdroj: | Microbiological research. 168(2) |
| ISSN: | 1618-0623 |
| Popis: | Most of type II restriction endonucleases show an absolute requirement for divalent metal ions as cofactors for DNA cleavage. While Mg 2+ is the natural cofactor other metal ions can substitute it and mediate the catalysis, however Ca 2+ (alone) only supports DNA binding. To investigate the role of Mg 2+ in DNA cleavage by restriction endonucleases, we have studied the Mg 2+ and Mn 2+ concentration dependence of DNA cleavage by SepMI and EhoI. Digestion reactions were carried out at different Mg 2+ and Mn 2+ concentrations at constant ionic strength. These enzymes showed different behavior regarding the ions requirement, SepMI reached near maximal level of activity between 10 and 20 mM while no activity was detected in the presence of Mn 2+ and in the presence of Ca 2+ cleavage activity was significantly decreased. However, EhoI was more highly active in the presence of Mn 2+ than in the presence of Mg 2+ and can be activated by Ca 2+ . Our results propose the two-metal ion mechanism for EhoI and the one-metal ion mechanism for SepMI restriction endonuclease. The analysis of the kinetic parameters under steady state conditions showed that SepMI had a K m value for pTrcHisB DNA of 6.15 nM and a V max of 1.79 × 10 −2 nM min −1 , while EhoI had a K m for pUC19 plasmid of 8.66 nM and a V max of 2 × 10 −2 nM min −1 . |
| Databáze: | OpenAIRE |
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