Gla I

A bacterial strain of Microbacterium oxydans was identified as a producer of a 5-methylcytosine-dependent, site-specific DNA endonuclease, MoxI. The enzyme recognizes and cleaves the DNA sequence 5′-R(5mC)↓GY-3′ / 3′-YG↓(5mC)R-5′. The moxI gene was cloned and expressed in Escherichia coli, and the recombinant enzyme was purified and biochemically characterized. The recognition sequence of MoxI was shown to be identical to that of the previously described 5mC-directed site-specific DNA endonuclease GlaI. In contrast to GlaI, however, MoxI exhibits maximal catalytic activity at 37 °C and demonstrates higher cleavage efficiency toward substrates containing two 5-methylcytosine residues within the recognition site

cloning of GlaI site-specific methyl-directed DNA endonuclease gene and the comparison of the substrate specificity of recombinant and native enzymes is described. The analysis of recombinant GlaI endonuclease properties showed that the substrate specificity of native and recombinant enzymes didn't differ, but the concentration of recombinant enzyme exceeded 10 times of native enzyme