The Flavobacterium okeanokoites adenine-N6-specific DNA-methyltransferase M.FokI is a tandem enzyme of two independent domains with very different kinetic properties.
The Flavobacterium okeanokoites adenine-N6-specific DNA-methyltransferase, M.FokI, modifies both adenine residues within its asymmetric recognition sequence 5'-GGATG/CATCC-3'. It is a fusion protein comprising two independent enzymes. We have cloned, overexpressed and purified full-length M.FokI as well as both individual domains and analyzed their kinetics of DNA methylation using unmethylated and hemimethylated oligodeoxynucleotide substrates. Our data show that both domains of M.FokI methylate DNA independently of each other but cooperate in DNA binding. In agreement to former studies, the N-terminal domain of M.FokI modifies the upper strand of the recognition sequence (GGATG). It strongly prefers hemimethylated (5'-GGATG/CmATCC-3'; mA = N6-methyladenosine) over unmethylated substrates. In contrast, the C-terminal domain prefers unmethylated DNA substrates. Surprisingly, in addition to methylating the lower strand of the recognition sequence (CATCC), M.FokI-(335-647) also modifies the upper strand (GGATG), albeit with a lower activity. In addition, methylation was detected at CACCC sites, but not at sites in which a central AT dinucleotide is flanked by at least one A x T or T x A base pair. These results suggests that M.FokI-(335-647) either has a very degenerate specificity for (G/C)AT(G/C) and sites similar to CATCC or GGATG or, alternatively, that it has a dual specificity for CATCC and GGATG.
Leismann
O
O
Institut für Biochemie, FB Biologie, Justus-Liebig-Universität Giessen, Germany.
Roth
M
M
Friedrich
T
T
Wende
W
W
Jeltsch
A
A
D001483:Base Sequence; D001665:Binding Sites; D019976:Cloning, Organism; D019175:DNA Methylation; D005417:Flavobacterium / Q000201:enzymology*; D007700:Kinetics; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; C058668:DNA modification methylase FokI; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific)