A novel class of FokI restriction endonuclease mutants that cleave hemi-methylated substrates.
A genetic screen was used to identify amino acid substitutions that enable the FokI restriction endonuclease to cleave DNA in cells that express the cognate methyltransferase activity. Missense mutations that give rise to this phenotype were isolated at eight different positions (G188K, P196S, T343I, S388N, S395F, E407K, E410K, D421N), clustered in two regions of the polypeptide sequence of FokI. Two of the mutant endonucleases (P196S and D421N) were purified to homogeneity and analyzed in detail. Both mutants cleave FokI target sites (5'-GGATG-3') in a manner similar to the wild-type enzyme. Neither mutant cleaved noncanonical sequences, but both efficiently cleaved DNA substrates containing hemi-methylated FokI sites. This class of mutations has not been observed with other restriction enzymes.
Waugh
D S
DS
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Sauer
R T
RT
D001483:Base Sequence; D003001:Cloning, Molecular; D015252:Deoxyribonucleases, Type II Site-Specific / Q000096:biosynthesis / Q000378:metabolism*; D004926:Escherichia coli; D005417:Flavobacterium / Q000201:enzymology* / Q000235:genetics; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000633:toxicity; D008745:Methylation; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010957:Plasmids / Q000378:metabolism*; D017354:Point Mutation; D015183:Restriction Mapping; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000096:biosynthesis / Q000378:metabolism*; D013379:Substrate Specificity
D006898:Hydroxylamines; D019811:Hydroxylamine; C058668:DNA modification methylase FokI; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); C035042:endodeoxyribonuclease FokI; D015252:Deoxyribonucleases, Type II Site-Specific