DNA methyltransferases from Neisseria meningitidis and Neisseria gonorrhoeae FA1090 associated with mismatch nicking endonucleases

Institute of Microbiology, Warsaw University, Miecznikowa 1, 02-096 Warsaw, Poland Correspondence Andrzej Piekarowicz anpiek{at}biol.uw.edu.pl The genes encoding the DNA methyltransferases M.NmeDI and M.NmeAI from Neisseria meningitidis associated with the genes encoding putative Vsr endonucleases were overexpressed in Escherichia coli . The enzymes were purified to apparent homogeneity on Ni-NTA agarose columns, yielding proteins of 49±1 kDa and 39·6±1 kDa, respectively, under denaturing conditions. M.NmeDI recognizes the degenerate sequence 5'-RCCGGB-3'. It methylates the first 5' cytosine residue on both strands within the core sequence CCGG. The enzyme shows higher affinity with the hemimethylated degenerate sequence than with the unmethylated degenerate sequence. Comparison of the amino acid sequence of the target-recognizing domain of M.NmeDI with the closest neighbours recognizing the sequence 5'-RCCGGY-3' showed the presence of the homologous domain and an additional domain that may be responsible for recognizing the degenerate sequence. M.NmeAI recognizes the sequence 5'-CCGG-3' and methylates the second 5' cytosine residue on both DNA strands. In Neisseria gonorrhoeae strain FA1090 the homologues of these ORFs are truncated due to a variety of mutations. Abbreviations: AdoMet, S -adenosyl- L -methionine; MTase, methyltransferase; m5C-MTase, DNA methyltransferase methylating the endocyclic C-5 of cytosine; ODN, oligonucleotide; R-M, restriction and modification; TRD, target recognition domain; Vsr, very short patch repair