Molecular characterization of the DNA methyltransferase M1.NcuI from Neisseria cuniculi ATCC 14688

Abstract The methyltransferase M1. Ncu I is a member of the restriction-modification system in Neisseria cuniculi ATCC14688 and recognizes the asymmetric pentanucleotide sequence 5′-GAAGA-3′/3′-CTTCT-5′. We purified M1. Ncu I to electrophoretic homogeneity using a four-step chromatographic procedure. M1. Ncu I is a protein with M r  = 32,000 ± 1000 under denaturing conditions. It modifies the recognition sequence by transferring the methyl group from S -adenosyl- l -methionine to the 3′ adenine of the pentanucleotide sequence 5′-GAAGA-3′. M1. Ncu I, like many other methyltransferases, occurs as a monomer in solution, as determined by gel filtration. Divalent cations inhibit the methylation activity of M1. Ncu I. Optimal enzyme activity was observed at a pH of 8.0. M1. Ncu I cross-reacted with anti-M1. Mbo II serum which reflects the similarity of M1. Ncu I with M1. Mbo II at the amino acid level. The gene coding for the enzyme, designated ncuIM1 , was cloned, sequenced and overexpressed in Escherichia coli . The structural gene is 780 nucleotides in length coding for a protein of 259 amino acids ( M r 30,098). The presence and distribution of nine highly conserved amino acid sequence motifs and a putative target recognition domain in the enzyme structure suggest that M1. Ncu I, similar to M1. Mbo II and M1. Hpy AII, belongs to N 6 -adenine β-class DNA methyltransferases.