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Short answer. A nicking endonuclease (nicking enzyme) cuts only one strand of double-stranded DNA, leaving the other strand intact. Most restriction enzymes cut both strands. Nicking enzymes are made by engineering restriction enzymes or isolated from natural sources, and they are used where a single-strand break is needed, such as isothermal amplification and some cloning methods.
Definition and scope
An endonuclease cleaves phosphodiester bonds inside a nucleic-acid chain; an exonuclease removes nucleotides from a chain end. A nicking endonuclease is an endonuclease that cleaves a single strand of a DNA duplex at a defined site, producing a nick rather than a double-strand break. Nicking is distinct from the double-strand cutting described in our restriction endonuclease guide, although many nicking enzymes are closely related to restriction enzymes. The word “nicking” on its own, in this lab context, means this single-strand cut. Nicking enzymes are sold for research use; see the supplier literature for each enzyme.
How nicking enzymes work and are used
Strand-specific nicking and naming
According to New England Biolabs (NEB), strand-specific nicking is only possible with enzymes that recognise an asymmetric (non-palindromic) sequence, because a palindromic site looks identical on both strands. NEB’s naming convention marks the nicked strand in the enzyme name: Nt. means the enzyme nicks the top strand (for example Nt.BstNBI), and Nb. means the bottom strand (for example Nb.BbvCI).
Where the enzymes come from
NEB describes two routes: engineering restriction enzymes into nicking variants, and isolating naturally occurring nicking enzymes. Both routes are documented in the primary literature, compared below.
Typical laboratory uses
NEB lists several applications of nicking enzymes: optical mapping, open-chromatin studies (with Nt.CviPII), isothermal amplification such as strand displacement amplification (SDA) and EXPAR, and creating overhangs for USER cloning (with Nt.BbvCI and XbaI). Our isothermal amplification guide places SDA alongside LAMP, NASBA and HDA.
Types and related concepts: ways to get a single-strand cut
| Route | Example | What the paper reports |
|---|---|---|
| Natural nicking enzyme closely related to a type IIS restriction enzyme | N.BstNBI (recognises GAGTC) | Preferentially nicks the top strand; shares catalytic residues with the type IIS enzymes MlyI and PleI, which cut both strands; remains a monomer with very limited second-strand cleavage (Higgins et al., 2001) |
| Restriction enzyme converted to a nicker by changing its oligomerisation state | MlyI mutant | Mutating Tyr491 and Lys494 disrupted dimerisation, and the mutant cut only one strand (Besnier and Kong, 2001) |
| Nicking by substrate modification | HincII with a hemiphosphorothioate site | HincII nicks the unmodified strand; exo-minus Klenow fragment extends from the nick and displaces the strand, giving about 106-fold amplification in 4 hours at 37 °C (Walker et al., 1992, PNAS) |
| Natural nicking enzyme from Streptomyces | Streptomyces nicking endonuclease (about 71 kDa) | Monomer requiring Mg2+; converts supercoiled plasmid to open circular DNA and then slowly to linear; resembles type IIS enzymes (Somyoonsap et al., 2013) |
The pattern across the first two rows: a nicker works because the enzyme does not form the dimer (or engage the second strand) that a full double-strand cut involves. In the SDA row the enzyme is an ordinary restriction enzyme and the modification of one strand creates the nick. A second SDA paper (Walker et al., 1992, Nucleic Acids Research) reported more than 107-fold amplification in 2 hours at 37 °C without a pre-cutting step.
Where it matters for labs
For a bench scientist the practical question is which strand needs nicking and which enzyme recognises a site in the right place. Because strand-specific nicking needs an asymmetric site, check the recognition sequence and its orientation in your template before choosing; the enzyme name prefix (Nt. or Nb.) then tells you which strand is cut. Buffer, temperature and substrate requirements differ between enzymes, so take them from the supplier datasheet for the specific enzyme. For ordinary double-strand digests, see the restriction enzyme digest setup guide and the restriction enzyme name index.
Common misunderstandings
- “Nicking enzymes are a different family from restriction enzymes.” Many are close relatives: N.BstNBI shares catalytic residues with MlyI and PleI, and MlyI can be converted into a nicker with two mutations.
- “A nick is the same as a break.” A nick leaves the duplex held together by the intact strand; a double-strand break separates it.
- “Any restriction site can be nicked strand-specifically.” Only asymmetric recognition sequences allow it.
- “Nicking enzymes are only for amplification.” Uses also include optical mapping, chromatin accessibility work and USER cloning.
FAQ
What is endonuclease vs exonuclease activity?
Endonucleases cut within a nucleic-acid chain; exonucleases remove nucleotides from the ends. Restriction and nicking enzymes are endonucleases. (General molecular-biology definition.)
What is the role of nicks in DNA replication?
In cells, nicks occur naturally as intermediates, for instance when DNA ligase seals the gaps between lagging-strand fragments. This is general textbook background and is separate from the sequence-specific nicking enzymes used as laboratory reagents above.
What determines where a restriction endonuclease will cut a DNA molecule?
The recognition sequence. Type IIP enzymes cut at a fixed position within the site, while type IIS enzymes such as MlyI and PleI cut at a defined position outside it. Nicking enzymes related to type IIS, such as N.BstNBI, are tied to their recognition site in the same way but cut one strand. Details are in the restriction endonuclease guide.
What are type 4 restriction enzymes?
Type IV enzymes act on modified DNA, for example methylated DNA, rather than on an unmodified recognition sequence. They are covered in the restriction endonuclease guide, and they are not nicking enzymes.
Sources
- New England Biolabs, Nicking endonucleases: the discovery and engineering of restriction enzyme variants, and What are nicking enzymes and how are they used? (naming, asymmetric sites, applications). Read 2026-11-07.
- Higgins LS, Besnier C, Kong H. Nucleic Acids Res 2001;29(12):2492. doi:10.1093/nar/29.12.2492 (PMID 11410656).
- Besnier CE, Kong H. EMBO Rep 2001;2(9). doi:10.1093/embo-reports/kve175 (PMID 11520857).
- Walker GT et al. Proc Natl Acad Sci USA 1992;89(1):392-396. doi:10.1073/pnas.89.1.392; and Nucleic Acids Res 1992;20(7):1691-1696. doi:10.1093/nar/20.7.1691.
- Somyoonsap P et al. ISRN Biochem 2013 (PMID 25937959).
