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

Abstract

Methylation of a base in a specific DNA sequence protects the DNA from nucleolytic cleavage by restriction enzymes recognizing the same sequence. The MboII restriction–modification (R–M) system ofMoraxella bovisATCC 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.MboII modifies the last adenine in the recognition sequence 5′-GAAGA-3′ toN6-methyladenine. A second methylase, M2.MboII, was cloned and purified to electrophoretic homogeneity using a four-step chromatographic procedure. It was demonstrated that M2.MboII modifies the internal cytosine in the recognition sequence 3′-CTTCT-5′, yieldingN4-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.MboII methylation activity. It was found that the isomethylomer M2.NcuI fromNeisseria cuniculiATCC 14688 behaves in the same manner. Functional analysis showed that the complete MboII R–M system, consisting of two methyltransferases genes and themboIIRgene, is the most stable and the least harmful to bacterial cells.