Conferring operator specificity on restriction endonucleases

Michael Koob, Eric Grimes, Waclaw Szybalski

Research output: Contribution to journalArticlepeer-review

53 Scopus citations


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 λ 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 λ o L operator, which was protected by λ repressor from M·Hph I methylation.

Original languageEnglish (US)
Pages (from-to)1084-1086
Number of pages3
Issue number4869
StatePublished - 1988
Externally publishedYes


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