Structures of the rare-cutting restriction endonuclease NotI reveal a novel metal binding fold involved in DNA recognition

摘要

The structure of the rare-cutting restriction endonuclease NotI, which recognizes the 8 basepair target 5′-GCGGCCGC-3′, has been solved with and without bound DNA. Because of its specificity (recognizing a site that occurs once per 65kb), NotI is used to generate large genomic fragments and to map DNA methylation status. NotI contains a unique metal-binding fold, found in a variety of putative endonucleases, occupied by an iron atom coordinated within a tetrahedral Cys4 motif. This domain positions nearby structural elements for DNA recognition, and serves a structural role. While recognition of the central six basepairs of the target is accomplished via a saturated hydrogen bond network typical of restriction enzymes, the most peripheral basepairs are engaged in a single direct contact in the major groove, reflecting reduced pressure to recognize those positions. NotI may represent an evolutionary intermediate between mobile endonucleases (which recognize longer target sites) and canonical restriction endonucleases.