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  • The Discovery and Engineering of Restriction Enzyme Variants

    The Discovery and Engineering of Restriction Enzyme Variants

    Restriction endonucleases (REases) recognize specific nucleotide sequences in double-stranded DNA and generally cleave both strands. Some sequence-specific endonucleases, however, cleave only one of the strands. These endonucleases are known as nicking endonucleases (NEases). At NEB, we have been developing nicking endonucleases through the discovery of naturally occurring enzymes, as well as genetic engineering of existing restriction enzymes.

    Double-stranded cleavage usually results from binding of the two half sites of a palindromic sequence by a homodimeric REase (e.g. Type IIP REases). Within the homodimer, each monomer makes a cut on one of the strands such that both strands of the DNA are cleaved. Strand-specific nicking, however, is achievable only when the recognition sequences are asymmetric. In addition, some of the REases that recognize asymmetric sequences are heterodimeric. 

    Thus, one can envision that manipulating the catalytic activity of individual monomers or the dimerization state of restriction endonucleases that recognize asymmetric sequences can result in nicking endonucleases. That is how NEB scientists developed the strand-specific NEases Nb.BbvCI and Nt.AlwI.

    BbvCI is a heterodimeric Type IIS REase. It recognizes the 7 base-pair asymmetric sequence CCTCAGC and cleaves the DNA at (CC↓TCAGC† and CCTCA↑GC) [CCTCAGC (-5/-2)]. It was discovered that each of the two subunits (R1 and R2) contains its own catalytic site. Each of these subunits cleaves the bottom and the top strands of the target sequence, respectively (1). To utilize this property, cleavage-deficient mutants of each subunit were engineered. Heterodimers of functional R1 and cleavage-deficient R2 reconstitute a nicking endonuclease that cleaves only the bottom strand (Nb.BbvCI), whereas functional R2 and cleavage-deficient R1 reconstitute Nt.BbvCI, which cleaves the top strand only (2). The nicking enzyme Nt.AlwI (GGATCNNNN↓) was also successfully engineered to cleave only the top strand of the AlwI target sequence [GGATC(4/5)] (3). This NEase was created by swapping the dimerization domain of AlwI with a non-functional dimerization domain of the natural NEase, Nt.BstNBI, such that the resulting chimeric enzyme, Nt.AlwI, is rendered monomeric.

    Other nicking enzyme engineering projects are less straightforward. Screening libraries of random mutants has enabled us to isolate variants of restriction endonucleases that nick one of the strands specifically (4,5). The engineered enzymes obtained are the bottomstrand specific Nb.BsmI (GAATG↑C) from BsmI [GAATGC(1/-1)] and top-strand specific Nt.SapI (GCTCTTCN↓) from SapI [GCTCTTC(1/4)] (4). Nicking variants have also been generated from BsaI [GGTCTC(1/5)], BsmBI [CGTCTC(1/5)], and BsmAI [GTCTC(1/5)] (5).

    In addition to protein engineering, we are also developing products from natural nicking endonucleases. Nt.BstNBI (GAGTCNNNN↓) is a naturally occurring thermostable NEase cloned from Bacillus stereothermophilus (6). Nt.CviPII (↓CCD), originally identified in a Chlorella virus isolate as a frequent DNA nickase that recognizes 3-base target sequences (7), is also under development at NEB.

    Some nicking endonucleases were discovered quite unexpectedly. Nb.BsrDI (GCAATG↑) is the large subunit of BsrDI [GCAATG(2/0)], a thermostable heterodimeric enzyme identified in Bacillus stearothermophilus. The large subunit was found to be a bottom-strand specific NEase when cloned separately in E. coli (Xu, unpublished observations). A similar observation has been made in BtsI where the large subunit makes a strand-specific nick at the target sequence (Zhu and Xu, unpublished results). The top-strand cleavage activity of BfiI [ACTGGG(5/4)] has also been reported to be inhibited at low pH, resulting in a bottom-strand specific nicking enzyme (8).

    The uses of nicking endonucleases are still being explored. NEases can generate nicked or gapped duplex DNA for studies of DNA mismatch repair and for diagnostic applications. The long overhangs that nicking enzymes make can be used in DNA fragment assembly. Nt.BbvCI has been used to generate long and non-complementary overhangs when used with XbaI in the USER* cloning protocol from NEB. Nicking endonucleases are also useful for isothermal DNA amplifications, which rely on the production of site-specific nicks. Isothermal DNA amplification using Nt.BstNBI in concert with Vent (exo–) DNA Polymerase (NEB #M0257) (EXPAR) has been reported for detection of a specific DNA sequence in a sample (9). Another isothermal DNA amplification technique has also been described using the 3-base cutter Nt.CviPII and Bst DNA Polymerase I [Nicking Endonuclease Mediated- DNA Amplification (NEMDA)] (7). Frequentcutting NEases can generate short partial duplex DNA fragments from genomic DNA. These fragments can be used for cloning or used as probes for hybridization-based applications.

    Nicking endonucleases are simple to use. Since the nicks generated by 6- or 7- base NEases do not fragment DNA, their activities are monitored by conversion of supercoiled plasmids to open circles. Alternatively, substrates with nicking sites close enough on opposite strands to create a doublestranded cut can be used instead.

    Alternatively, REBASE offers a comprehensive database of enzyme properties and useful resources of restriction-modification systems. REBASE also includes citations of all relevant literature as well as links to resources such as structural data and genomic sequences when they are available.

    † Down-arrows (↓) indicate cleavage at the top strand; up-arrows (↑) indicate cleavage at the bottom strand.
    * The USER™ (Uracil-Specific Excision Reagent) Friendly Cloning Kit offers an extremely fast, simple and efficient method for the cloning of PCR products.

    Nicking Endonucleases Available at NEB

    References

    1. Bellamy, S.R.W. et al. (2005) J. Mol. Biol. 345, 641–653.
    2. Heiter, D.F., Lunnen, K.D. and Wilson, G.G. (2005) J. Mol. Biol. 348, 631–640.
    3. Xu, Y. et al. (2001) Proc. Natl. Acad. Sci. USA 98, 12990–12995.
    4. Samuelson, J.C., Zhu, Z. and Xu, S.Y. (2004) Nucl. Acids Res. 32, 3661–3671.
    5. Zhu, Z. et al. (2004) J. Mol. Biol. 337, 573–583.
    6. Morgan, R.D. et al. (2000) Biol. Chem. 381, 1123–1125.
    7. Chan, S.H. et al. (2004) Nucl. Acids Res. 32, 6187–6199.
    8. Sasnauskas, G. et al. (2003) Proc. Natl. Acad. Sci. USA 100, 6410–6415.
    9. Van Ness, J. et al. (2003) Proc. Natl. Acad. Sci. USA 100, 4504–4509.

    From NEB expressions July 2006, vol 1.2
    By Siu-hong Chan, Ph.D. and Shuang-yong Xu, Ph.D., New England Biolabs, Inc.