Integration host factor (IHF)-mediated protection against enzymatic methylation at ihf-overlapping sites provides the basis for this novel application of the Achilles' cleavage (AC) technique [Koob., Science 241 (1998) 1084-10861 for generating rare natural cleavage sites. When applying IHF-AC to plasmid, phage λ, Escherichia coli and yeast genomes, only a few of the EcoRI, MboI sites (sites overlapped the ihf sites)remained cleavable after prior methylation with the cognate M· EcoRI, M· HinfI, or Dam methyltransferases in the presence of IHF. Thus, IHF-AC essentially converted these enzymes into very rare cutters. The extent of cleavage could be controlled by varying the IHF:DNA ratio and temperature. Moreover, the method permits the genomic location and strength of the ihf sites to be determined.
- DNA-binding protein
- pulsed-field gel electrophoresis
- recombinant DNA)