The domains of a type I DNA methyltransferase. Interactions and role in recognition of DNA methylation.
The DNA methyltransferases of type I restriction-modification systems are trimeric enzymes composed of one DNA specificity (S) subunit and two modification (M) subunits. The S subunit contains two large regions, each of which recognizes one part of the split, asymmetrical DNA target sequence. Each M subunit contains an amino acid motif for binding the methyl group donor and cofactor, S-adenosyl methionine. The EcoKI methyltransferase has a strong preference for methylating a hemimethylated DNA target rather than an unmodified target. We have used partial proteolytic digestion of EcoKI methyltransferase to generate polypeptide domains that we have identified by amino acid sequencing. The S subunit was cut into two large, folded domains each containing one DNA binding region. Binding of DNA partially protected the S subunit from digestion. The M subunit was also cut into two large domains joined together by a short flexible loop, and a C-terminal tail region. The short loop contained part of the S-adenosyl methionine binding motif, and cofactor binding protected the loop and the two large domains from proteolysis. The C-terminal domain of M remained associated with the N-terminal domain of the S subunit even after the rest of the protein had been digested. The conformation of the tail region of the M subunit was sensitive to the methylation state of DNA in ternary complexes also containing S-adenosyl methionine, and could differentiate between unmethylated and hemimethylated DNA substrates.
Cooper
L P
LP
Institute of Cell and Molecular Biology, University of Edinburgh, U.K.
Dryden
D T
DT
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011487:Protein Conformation; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000737:chemistry* / Q000235:genetics; D013379:Substrate Specificity
D010446:Peptide Fragments; D004247:DNA; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific)