A novel method for converting common restriction enzymes into rare cutters: integration host factor-mediated Achilles' cleavage (IHF-AC).
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 et al., Science 241 (1988) 1084-1086] for generating rare natural cleavage sites. When applying IHF-AC to plasmid, phage lambda, Escherichia coli and yeast genomes, only a few of the EcoRI, HinfI, and MboI sites (which 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.
Kur
J
J
McArdle Laboratory for Cancer Research, Madison, WI 53706.
Koob
M
M
Burkiewicz
A
A
Szybalski
W
W
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D015246:Deoxyribonuclease EcoRI; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D016680:Genome, Bacterial; D016681:Genome, Fungal; D034982:Integration Host Factors; D008745:Methylation; D008969:Molecular Sequence Data; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D034982:Integration Host Factors; D015246:Deoxyribonuclease EcoRI; D015252:Deoxyribonucleases, Type II Site-Specific; C032229:GANTC-specific type II deoxyribonucleases