Structures of the Rare-Cutting Restriction Endonuclease NotI Reveal a Unique Metal Binding Fold Involved in DNA Binding

Abstract

The structure of the rare-cutting restriction endonuclease NotI, which recognizes the 8 bp target 5'-GCGGCCGC-3', has been solved with and without bound DNA. Because of its specificity (recognizing a site that occurs once per 65 kb), 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 protein elements for DNA recognition, and serves a structural role. While recognition of the central six base pairs of the target is accomplished via a saturated hydrogen bond network typical of restriction enzymes, the most peripheral base pairs 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.

On the Divalent Metal Ion Dependence of DNA Cleavage by Restriction Endonucleases of the EcoRI Family

“…9,10 The second and third β-strands harbour the catalytic residues of the PD…D/EXK motif, which is not absolutely conserved: the lysine is substituted by glutamic acid in BamHI and by glutamine in BglII, BstYI, 11 HinP1I, 12 and NotI. 13 Furthermore, in Bse634I, Cfr10I, EcoO109I, EcoRII, Ecl18kI, NgoMIV, and PspGI, the second carboxylate of the PD…D/EXK motif comes from a neighbouring α-helix, highlighting the importance of spatial, rather than sequence, conservation. 14,15 Finally, in SdaI, two amino acid residues separate the E and K of the PD…D/EXK motif.…”

“…16 The crystal structures of 19 members of the EcoRI family (Table 1), representatives of which we have studied in this paper, have been determined, including 13 co-crystal structures of enzyme-DNA complexes: BamHI, BglII, BsoBI, BstYI, Ecl18kI, EcoRI, EcoO109I, FokI, MunI, NgoMIV, NotI, PspGI, 17 and SgrAI. 7,18 Six of these co-crystal structures were determined in the presence of divalent metal ions: BamHI with Ca 2+ (cognate complex: PDB accession code 2BAM) and Mn 2+ (product complex, 3BAM), 19 BglII with Ca 2+ (cognate complex: 1DFM), 20 EcoRI with Mn 2+ (product complex: 1QPS), 21 NgoMIV with Mg 2+ (product complex: 1FIU), 22 NotI with Ca 2+ (cognate complex: 3C25), 13 and SgrAI with Ca 2+ (noncognate complex: 3DPG; cognate complex: 3DVO) and with Mn 2+ (cognate complex: 3DVO). 23 Whereas the structure analyses showed that BamHI, NgoMIV, and NotI have two divalent metal ions bound per active site (subunit), BglII and EcoRI have only one.…”

Engineering BspQI nicking enzymes and application of N.BspQI in DNA labeling and production of single-strand DNA

“…The two zinc metal ions in the KpnI endonuclease seem to play dual roles in both structural folding and specificity [48]. In the NotI:DNA cocrystal structure, however, four Cys residues coordinate the binding of an iron atom in a unique metal binding fold thought to be involved in protein structural stability [49]. Mutation of these individual Cys residues abolishes NotI restriction activity (PZ and SYX, unpublished results).…”

Rescue of the activity of HNH nuclease mutants - towards controlled enzymes for gene therapy

“…Alternative nuclease domains have been applied in few cases in mainly monomeric chimera [53][54][55][56][57][58] and non-FokI-based zinc finger nucleases have also been developed by introducing mutations or small modifications into the zinc finger array, such as the exchange of the metal ion-binding cysteines to histidines [59][60][61]. Trials to establish controlled artificial nucleases through lightactivation [62,63], DNA binding [64], cold-shock technique [65], protein-protein interaction [66], DNA modification [67] or metal ion dependent DNA binding [68] were also executed. Bioinformatics was utilized to predict ZFN and TALEN off-target cleavage [69].…”