Specificity of DNA binding and methylation by the M.FokI DNA methyltransferase

The M.FokI adenine-N-6 DNA methyltransferase recognizes the asymmetric DNA sequence GGATG/CATCC. It consists of two domains each containing all motifs characteristic for adenine-N-6 DNA methyltransferases. We have studied the specificity of DNA-methylation by both domains using 27 hemimethylated oli gonucleotide substrates containing recognition sites which differ in one or two base pairs from GGATG or CATCC. The N-terminal domain of M.FokI intera cts very specifically with GGATG-sequences, because only one of the altered sites is modified. In contrast, the C-terminal domain shows lower specific ity. It prefers CATCC-sequences but only two of the 12 star sites (i.e. sit es that differ in 1 bp from the recognition site) are not accepted and some star sites are modified with rates reduced only 2-3-fold. In addition, GGA TGC- and CGATGC-sites are modified which differ at two positions from CATCC . DNA binding experiments show that the N-terminal domain preferentially bi nds to hemimethylated GGATG/C(m)ATCC sequences whereas the C-terminal domai n binds to DNA with higher affinity but without specificity. Protein-protei n interaction assays show that both domains of M.FokI are in contact with e ach other. However, several DNA-binding experiments demonstrate that DNA-bi nding of both domains is mutually exclusive in full-length M.FokI and both domains do not functionally influence each other. The implications of these results on the molecular evolution of type IIS restriction/modification sy stems are discussed. (C) 2000 Elsevier Science B.V. All rights reserved.