As a rule, when restriction endonucleases bind to their recognition sequences in DNA, they hydrolyze both strands of the duplex at the same time. Two independent hydrolytic reactions proceed in parallel, most often driven by the presence of two catalytic sites within each enzyme, one for hydrolyzing each strand. We have begun to engineer altered restriction enzymes that hydrolyze only one strand of the duplex, to produce DNA molecules that are “nicked”, rather than cleaved. These conventional nicks (3´-hydroxyl, 5´-phosphate) can serve as initiation points for a variety of further enzymatic reactions such as replacement DNA synthesis, strand-displacement amplification (1), exonucleolytic degradation or the creation of small gaps (2).

Eight nicking endonucleases are now available from New England Biolabs. Nt.BstNBI is a naturally occuring thermostable nicking endonuclease cloned from Bacillus Stereothermophilus (3,4). Nb.BsrDI and Nb.BtsI are naturally occurring large subunits of thermostable heterodimeric enzymes (5). Nt.AlwI, a derivative of the restriction enzyme AlwI, has been engineered to behave in the same way (6). Both nick just outside their recognition sequences. Nb.BbvCI and Nt.BbvCI are alternative derivatives of the ­heterodi­meric restriction enzyme BbvCI, each engineered to possess only one functioning catalytic site (7). These two enzymes nick within the recognition sequence but on opposite strands. Nb.BsmI is a bottom-strand specific variant of BsmI discovered from a library of random mutants (8,9)

Nicking endonucleases are as simple to use as restriction endonucleases. Since the nicks generated by 6- or 7-base nicking endonucleases 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 double-stranded cut can be used instead.

We are continuing to engineer more nicking enzymes, particularly in response to specific customer needs and applications.

Please refer to our feature article about nicking endonucleases: The Discovery and Engineering of Restriction Enzyme Variants.

Nicking Endonucleases Available at NEB

References:

1. Walker, G.T. et al. (1992) Proc. Natl. Acad. Sci. USA, 89, 392–396.
2. Wang, H. and Hays, J.B. (2000) Mol. Biotechnol., 15, 97–104.
3. Higgins, L.S. et al. (2001) Nucleic Acids Res., 29, 2492–2501.
4. Morgan, R.D. et al. (2000) Biol. Chem., 381, 1123–1125.
5. Zhu, Z. and Xu, S.Y. unpublished results.
6. Xu, Y. et al. (2001) Proc. Natl. Acad. Sci. USA, 98, 12990–12995.
7. Heiter, D.F. et al. (2005) J. Mol. Biol., 348, 631–40.
8. Samuelson, J.C., Zhu, Z. and Xu, S.Y. (2004) Nucleic Acids Res., 32, 3661–3671.
9. Zhu, Z. et al. (2004) J. Mol. Biol., 337, 573–583.