M1.Mboll and M2.Mboll type IIS methyltransferases: different specificities, the same target

Methylation of a base in a specific DNA sequence protects the DNA from nucleolytic cleavage by restriction enzymes recognizing the same sequence. The Mboll restriction-modification (R-M) system of Moraxella bovis ATCC 10900 consists of a restriction endonuclease gene and two methyltransferase genes. The enzymes encoded by this system recognize an asymmetrical sequence 5'-GAAGA-3'/3'-CTTCT-5'. M1.Mboll modifies the last adenine in the recognition sequence 5'-GAAGA-3' to N-6-methyladenine. A second methylase, M2.Mboll, was cloned and purified to electrophoretic homogeneity using a four-step chromatographic procedure. It was demonstrated that M2.Mboll modifies the internal cytosine in the recognition sequence 3'-CTTCT-5', yielding N-4-methylcytosine, and moreover is able to methylate single-stranded DNA. The protein exists in solution as a monomer of molecular mass 30 000 +/- 1000 Da under denaturing conditions, Divalent cations (Ca2+, Mg2+, Mn2+, and Zn2+) inhibit M2.Mboll methylation activity. It was found that the isomethylomer M2.Ncul from Neisseria cuniculi ATCC 14688 behaves in the same manner. Functional analysis showed that the complete Mboll R-M system, consisting of two methyltransferases genes and the mbollR gene, is the most stable and the least harmful to bacterial cells.