Use of IHF--mediated Achilles' heel cleavage (IHF-AC) method for mapping ihf sites.
We have shown that Integration Host Factor of E. coli can successfully be used in the IHF-mediated Achilles' Heel Cleavage (IHF-AC) technique (Kur et al., 1992b), for generating rare natural cleavage sites. The first step of this procedure is methylation of DNA in the presence of IHF, when the overlapping ihf/restriction sites are protected from methylation, and in the second step the DNA is cut by the cognate restriction enzyme. The extent of cleavage could be controlled by varying the IHF:DNA ratio and temperature. The aim of the present study is to demonstrate that IHF-AC procedure might serve as a useful tool for finding new protein-binding sites which overlap known restriction sites. I have used this approach in conjunction with several MTases to find several other unknown IHF-binding sites.
Kur
J
J
Department of Microbiology, Technical University of Gdańsk, Poland.
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D005821:Genetic Techniques; D034982:Integration Host Factors; D008745:Methylation; D008969:Molecular Sequence Data; D015183:Restriction Mapping; D013696:Temperature
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D034982:Integration Host Factors; D004247:DNA; D015252:Deoxyribonucleases, Type II Site-Specific; C032229:GANTC-specific type II deoxyribonucleases; C024405:GATC-specific type II deoxyribonucleases