Rational engineering of type II restriction endonuclease DNA binding and cleavage specificity

Abstract

The type II restriction endonucleases are indispensible tools for molecular biology. Although enzymes recognizing nearly 300 unique sequences are known, the ability to engineer enzymes to recognize any sequence of choice would be valuable. However, previous attempts to engineer new recognition specificity have met limited success. Here we report the rational engineering of multiple new type II specificities. We recently identified a family of MmeI-like type II endonucleases that have highly similar protein sequences but different recognition specificity. We identified the amino-acid positions within these enzymes that determine position specific DNA base recognition at three positions within their recognition sequences through correlations between their aligned amino-acid residues and aligned recognition sequences. We then altered the amino acids at the identified positions to those correlated with recognition of a desired new base to create enzymes that recognize and cut at predictable new DNA sequences. The enzymes so altered have similar levels of endonuclease activity compared to the wild-type enzymes. Using simple and predictable mutagenesis in this family it is now possible to create hundreds of unique new type II restriction endonuclease specificities. The findings suggest a simple mechanism for the evolution of new DNA specificity in Nature.

Figure 1

A portion of the amino-acid multiple sequence alignment of the characterized MmeI family enzymes, grouped according to the DNA base recognized at position 6 of the recognition sequence alignment. The two amino-acid alignment positions correlated with base recognition are highlighted in magenta. ‘Consensus ss’ indicates predicted secondary structure, where ‘h’ indicates alpha-helix and ‘e’ indicates beta-strand.

Figure 2

Restriction fragment patterns for wild type and altered MmeI and NmeAIII variants. (A) Agarose gel showing endonuclease digestion fragment patterns of lambda DNA produced by the wild type and altered enzymes. MmeI cuts at TCCRAC(20/18); MmeI(6G) cuts at TCCRAG(20/18); MmeI(4G) cuts at TCCGAC(20/18); MmeI(4C) cuts at TCCCAC(20/18); MmeI(4G6G) cuts at TCCGAG(20/18); MmeI(4C6G) cuts at TCCCAG(20/18); NmeAIII cuts at GCCGAG(21/19); NmeAIII(6C) cuts at GCCGAC(21/19); NmeAIII(3G4R6C) cuts at GCGRAC(21/19). Size standards (M) are lambda-HindIII plus PhiX174-HaeIII. (B) Computer-generated restriction fragment patterns for cleavage of lambda DNA at the wild type and altered recognition sequences.

Figure 3

A portion of the amino-acid multiple sequence alignment of the characterized MmeI family enzymes, grouped according to the DNA base recognized at position 3 of the recognition sequence alignment. The two amino-acid alignment positions correlated with base recognition are highlighted in orange. The boxes indicate the amino-acid pairs observed at the two correlated positions for each DNA base recognized. ‘Consensus ss’ indicates predicted secondary structure, where ‘h’ indicates alpha-helix and ‘e’ indicates beta-strand.

Figure 4

A portion of the amino-acid multiple sequence alignment of the characterized MmeI family enzymes, grouped according to the DNA base recognized at position 4 of the recognition sequence alignment. The two amino-acid alignment positions correlated with base recognition at position 4 are highlighted in green. The boxes indicate the amino-acid pairs observed at the two correlated positions for each DNA base recognized. ‘Consensus ss’ indicates predicted secondary structure, where ‘h’ indicates alpha-helix and ‘e’ indicates beta-strand.

Figure 5

Predicted amino acid–DNA base contacts for MmeI. The MmeI amino-acid residue predicted to contact each individual base at positions 3, 4 and 6 of the double-stranded recognition sequence is shown. ‘Mtase’ indicates the methyltransferase binding pocket for the adenine that is flipped out of the helix and modified by the methyltransferase activity of the enzyme.