The Crystal Structure of the Rare-Cutting Restriction Enzyme SdaI Reveals Unexpected Domain Architecture

Abstract

Rare-cutting restriction enzymes are important tools in genome analysis. We report here the crystal structure of SdaI restriction endonuclease, which is specific for the 8 bp sequence CCTGCA/GG ("/" designates the cleavage site). Unlike orthodox Type IIP enzymes, which are single domain proteins, the SdaI monomer is composed of two structural domains. The N domain contains a classical winged helix-turn-helix (wHTH) DNA binding motif, while the C domain shows a typical restriction endonuclease fold. The active site of SdaI is located within the C domain and represents a variant of the canonical PD-(D/E)XK motif. SdaI determinants of sequence specificity are clustered on the recognition helix of the wHTH motif at the N domain. The modular architecture of SdaI, wherein one domain mediates DNA binding while the other domain is predicted to catalyze hydrolysis, distinguishes SdaI from previously characterized restriction enzymes interacting with symmetric recognition sequences.

The Crystal Structure of D212 from Sulfolobus Spindle-Shaped Virus Ragged Hills Reveals a New Member of the PD-(D/E)XK Nuclease Superfamily

“…While structure-based sequence alignments do not show a lysine residue in the equivalent position in D212, Lys123 is in close proximity to the three acidic active site residues and might serve a similar role. This would be consistent with reports on the migration of active site lysine residues in type II restriction endonucleases (9,16,46,53). Interestingly, HincII also shows a lysine at this position, which is also strictly conserved in the fuselloviral orthologs (Fig.…”

Restriction endonuclease BpuJI specific for the 5′-CCCGT sequence is related to the archaeal Holliday junction resolvase family

“…However, one cannot exclude that dimerization of the BpuJI NTD is required for cognate DNA binding. For example, NTDs of SdaI REase dimerize to bind a single copy of cognate DNA (34). Therefore, the stoichiometry of the BpuJI NTD binding to the cognate oligoduplex was analysed by the size exclusion chromatography.…”

Structural Analysis of the Heterodimeric Type IIS Restriction Endonuclease R.BspD6I Acting as a Complex between a Monomeric Site-specific Nickase and a Catalytic Subunit

“…Migration of the active site lysine to a position two residues away from the second acidic residue in the active site motif was reported for SdaI. 32 Migration over a much greater distance in the protein sequence was observed for other residues in the motif; e.g. for the second acidic residue in crystal structures of Ngo-MIV-like enzymes.…”