Probing the domain structure of the type IC DNA methyltransferase M.EcoR124I by limited proteolysis.
Limited proteolysis has been used to probe the domain structure of the type I DNA methyltransferase M.EcoR124I. Trypsin digestion of the methyltransferase generates two fragments derived from the HsdS subunit, a 28 kDa N-terminal domain and a 19 kDa C-terminal domain, leaving the HsdM subunit intact. Extensive digestion by chymotrypsin, however, removes 59 amino acid residues from the N terminus of the HsdM subunit to leave a 52 kDa C-terminal domain. Binding of the cofactor S-adenosyl methionine has no appreciable effect on the rate of cleavage, but binding of a 30 bp DNA duplex containing the cognate recognition sequence confers almost total protection. Following trypsin cleavage of the methyltransferase, a stable proteolytic product is produced which has been purified for biochemical characterisation. The trypsinised enzyme is shown to be a multimeric complex containing two intact HsdM subunits and both fragments of the HsdS subunit, consistent with the circular model proposed for the organisation of domains in the specificity subunit in type IC methyltransferases. Gel retardation studies show that the proteolysed enzyme still retains DNA binding activity, but its specificity for the DNA recognition sequence is dramatically reduced.
Webb
M
M
Biophysics Laboratories, School of Biological Sciences, University of Portsmouth, UK.
Taylor
I A
IA
Firman
K
K
Kneale
G G
GG
D000595:Amino Acid Sequence; D001483:Base Sequence; D002918:Chymotrypsin; D004247:DNA / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010449:Peptide Mapping; D017434:Protein Structure, Tertiary; D012436:S-Adenosylmethionine / Q000378:metabolism; D017421:Sequence Analysis; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014357:Trypsin
D010446:Peptide Fragments; D012436:S-Adenosylmethionine; D004247:DNA; C072994:EcoR124 modification methylase type I; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D002918:Chymotrypsin; D014357:Trypsin