The FokI restriction-modification system. II. Presence of two domains in FokI methylase responsible for modification of different DNA strands.
Based on the previous findings that the FokI methylase (MFokI) consists of 647 amino acid residues and contains two copies of the segment specific for adenine methylase, Asp-Pro-Pro-Tyr, at amino acid positions 218-221 and 548-551, the role of these copies in the methylation reaction was investigated by introduction of a mutation into each segment. The MFokI gene was inserted into M13 vectors, and the Asp residues in the two segments were converted to Gly and Ala by oligonucleotide-directed mutagenesis. The wild-type and mutant genes were recloned into an expression vector, from which gene products were purified. A short DNA fragment carrying the FokI recognition site was treated with each of these enzymes, and after separation of the two strands by duplex formation with M13 viral DNAs carrying the respective strands, the presence or absence of modification was judged from susceptibility to FokI endonuclease. The results of analysis showed that different strands were modified in an asymmetric way by the introduction of mutations into one of the two segments, and that the segments at the N-terminal and C-terminal moieties participated in modification of the strands carrying 5'-GGATG-3' and 3'-CCTAC-5', respectively. We concluded that MFokI contained two functional domains each of which was responsible for modification of different strands in the target DNA.
Sugisaki
H
H
Institute for Chemical Research, Kyoto University, Japan.
Kita
K
K
Takanami
M
M
D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005417:Flavobacterium / Q000201:enzymology / Q000235:genetics; D005822:Genetic Vectors; D008745:Methylation; D009154:Mutation; D010957:Plasmids; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D013379:Substrate Specificity
D011994:Recombinant Proteins; C035042:endodeoxyribonuclease FokI; D015252:Deoxyribonucleases, Type II Site-Specific