A theoretical model of restriction endonuclease NlaIV in complex with DNA, predicted by fold recognition and validated by site-directed mutagenesis and circular dichroism spectroscopy.

Restriction enzymes (REases) are commercial reagents commonly used in DNA manipulations and mapping. They are regarded as very attractive models for studying protein-DNA interactions and valuable targets for protein engineering. Their amino acid sequences usually show no similarities to other proteins, with rare exceptions of other REases that recognize identical or very similar sequences.

Inference of relationships in the 'twilight zone' of homology using a combination of bioinformatics and site-directed mutagenesis: a case study of restriction endonucleases Bsp6I and PvuII.

Thus far, identification of functionally important residues in Type II restriction endonucleases (REases) has been difficult using conventional methods. Even though known REase structures share a fold and marginally recognizable active site, the overall sequence similarities are statistically insignificant, unless compared among proteins that recognize identical or very similar sequences. Bsp6I is a Type II REase, which recognizes the palindromic DNA sequence 5'GCNGC and cleaves between the cytosine and the unspecified nucleotide in both strands, generating a double-strand break with 5'-protruding single nucleotides.

A homology model of restriction endonuclease SfiI in complex with DNA.

Background: Restriction enzymes (REases) are commercial reagents commonly used in recombinant DNA technologies. They are attractive models for studying protein-DNA interactions and valuable targets for protein engineering. They are, however, extremely divergent: the amino acid sequence of a typical REase usually shows no detectable similarities to any other proteins, with rare exceptions of other REases that recognize identical or very similar sequences.