Conferring operator specificity on restriction endonucleases.
Mapping and manipulation of very large genomes, including the human genome, would be facilitated by the availability of a DNA cleavage method with very high site specificity. Therefore, a general method was devised that extends the effective recognition sequences well beyond the present 8-base pair limit by combining the specificity of the restriction endonuclease with that of another sequence-specific protein that binds tightly to DNA. It was shown that the tightly binding lac or lambda repressor protects a restriction site within the operator from specific modification methylases, M.Hha I or M.Hph I, while all other similar sites are methylated and thus rendered uncleavable. A plasmid containing a symmetric lac operator was specifically cleaved by Hha I, only at the site within the operator, after M.Hha I methylation in the presence of the lac repressor, whereas the remaining 31 Hha I sites on this plasmid were methylated and thus not cleaved. Analogous results were obtained with the Hae II site within the lac operator, which was similarly protected by the lac repressor, and with the Hph I site within the phage lambda oL operator, which was protected by lambda repressor from M.Hph I methylation.
Koob
M
M
McArdle Laboratory for Cancer Research, University of Wisconsin, Madison 53706.
Grimes
E
E
Szybalski
W
W
D010582:Bacteriophage lambda / Q000032:analysis / Q000235:genetics; D004247:DNA / Q000378:metabolism*; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D006358:Hot Temperature; D007763:Lac Operon; D008745:Methylation; D009875:Operator Regions, Genetic; D010957:Plasmids; D012097:Repressor Proteins / Q000378:metabolism; D013379:Substrate Specificity
D012097:Repressor Proteins; D004247:DNA; D004262:DNA Restriction Enzymes