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 oligonucleotide substrates containing recognition sites which differ in one or two base pairs from GGATG or CATCC. The N-terminal domain of M.FokI interacts very specifically with GGATG-sequences, because only one of the altered sites is modified. In contrast, the C-terminal domain shows lower specificity. It prefers CATCC-sequences but only two of the 12 star sites (i.e. sites 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, GGATGC- and CGATGC-sites are modified which differ at two positions from CATCC. DNA binding experiments show that the N-terminal domain preferentially binds to hemimethylated GGATG/C(m)ATCC sequences whereas the C-terminal domain binds to DNA with higher affinity but without specificity. Protein-protein interaction assays show that both domains of M.FokI are in contact with each other. However, several DNA-binding experiments demonstrate that DNA-binding 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 systems are discussed.
Friedrich
T
T
Institut für Biochemie, Fachbereich 8, Heinrich-Buff-Ring 58, 35392, Giessen, Germany.
Fatemi
M
M
Gowhar
H
H
Leismann
O
O
Jeltsch
A
A
D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D019175:DNA Methylation; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000378:metabolism*; D013379:Substrate Specificity
D004247:DNA; C058668:DNA modification methylase FokI; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific)