Restriction Enzyme Cleavage: ‘single-site’ enzymes and ‘multi-site’ enzymes

For a list of restriction enzymes from NEB requiring more than one recognition site on the substrate to cleave optimally, please visit this table

Restriction enzymes are proteins used to fragment and clone DNA, but their biological function is to protect bacteria and archaea against viral infections. All bind to double-stranded (ds) DNA at specific sequences of base pairs (the ‘recognition sequence’) and cleave the DNA strands.  Given their catalytic similarities, we might expect all restriction enzymes to be similar to each other, but they are in fact extremely diverse, and vary widely in amino acid sequence, three-dimensional structure, subunit composition, and modes of action. Restriction enzymes of identical specificity (‘isoschizomers’) are sometimes similar, and represent diverged versions of the same ancestral protein, but those of different specificity are often unique, and display no more similarity to one another than to unrelated proteins chosen at random. It seems likely that restriction enzymes arose independently many times during microbial evolution and under varied circumstances.

Accompanying this diversity are subtle differences in the ways that restriction enzymes behave. In one comparison, seven enzymes that cleave the sequence GGCGCC at four different positions were examined, and five distinct reaction pathways were discerned (1). Perhaps the most important mechanistic difference when using restriction enzymes as molecular biology reagents concerns the number of recognition sites a restriction enzyme must bind to in order to cleave. Most restriction enzymes act as simple monomers (one protein chain, e.g. MspI (NEB #R0106) (2)) or homodimers (two identical protein chains, e.g. BamHI (NEB #R3136)), which bind and cleave one recognition site at a time. These enzymes cut substrates with one site as efficiently as they cut substrates with several sites. Others are more complex, and undergo allosteric activation, or form ‘transient’ dimers (e.g. FokI (NEB #R0109) (3-5)), tetramers (e.g. NgoMIV (NEB #R0564) (6)), or even larger assemblages (e.g. BcgI (NEB #R0545)  (7,8)), and these cut only when bound to two, and sometimes more (9), sites at once. In some cases, this ‘multi-site’, behavior might be an adaptation against accidental cleavage of the bacterium’s own DNA. Structurally, it likely stems from the subunit organizations of the enzymes and how their recognition and catalytic domains fit together (8). Irrespective of the why and how, the need to bind to more than one site in order to cleave can make substrates with only one site difficult to cut in vitro.

Restriction enzymes that bind several sites in order to cleave exhibit several characteristics:

  1. Cleavage kinetics. Substrates with single sites are cleaved slowly and in some cases incompletely because enzymes must interact with (‘bridge’) two or more DNA molecules at once. The probability of doing so declines precipitously at low DNA concentrations. Adding more enzyme to try to improve cleavage in this situation can do the opposite and make matters even worse, because increasing enzyme concentration in effect reduces the relative substrate concentration (refer to #2 below). If the contacts between the subunits are fragile, as they are thought to be for transient dimers such as FokI (NEB #R0109) (10), then enzymes bridging sites in trans, in different molecules, can be unstable and ineffective. In contrast, when multiple sites are present in the same DNA molecule, the local concentration of sites is higher, and enzymes bridging sites in cis are more stable, both of which lead to faster, more complete, cleavage. Multi-site enzymes vary in the degree to which they are affected by these and related factors. Some, such as BspMI/BfuAI (NEB #R0502) (11) and NmeAIII (NEB #R0711) barely cleave 1-site substrates at all. Others (e.g. SacII (NEB #R0157) cut partially, and yet others (e.g. PluTI (NEB #R0713)) can cleave to near-completion.

  2. Site-saturation. Even on substrates with multiple recognition sites, when the number of enzyme molecules in the reaction substantially exceeds the number of sites, few enzyme molecules can bind to more than one site at a time. Most sites remain uncut under these circumstances because they are bound by ‘dormant’ enzyme molecules. This situation does not affect enzymes that can cleave at single sites; for these, as a rule, the higher the enzyme concentration, the faster the DNA will cleave to completion. For enzymes that require multiple sites, however, excessive enzyme can result in very incomplete cleavage.

  3. DNA looping. On substrates with multiple sites, enzymes that bind to more than one site at a time cause the intervening DNA to loop out. Looping can be detected by fluorescence resonance energy transfer (12), and visualized by electron microscopy (13,14) and atomic force microscopy (15-18). Enzymes that require multiple sites induce DNA-looping, whereas those that cleave at single sites do not.

  4. DNA tension. Using single-molecule tethered-particle techniques, variable tension can be applied to DNA molecules to relax them so that loops can form, or to stretch them so that loops cannot form. Enzymes that can cleave at single sites are relatively insensitive to the applied tension. Enzymes that require multiple sites, in contrast, can only cleave when the tension is sufficiently low for the DNA to shorten as loops

  5. Activator oligonucleotides. In some circumstances, adding double stranded oligonucleotides that contain a recognition site improves cleavage by multi-site enzymes (11,21-24). The oligos provide the additional binding targets needed to activate substrate-bound, but dormant enzyme molecules. Addition of oligos can also reduce the effective enzyme concentration, however, because they compete with the substrate for enzyme binding-sites. Enhancing cleavage by the addition of oligos requires a delicate stoichiometric balance between enzyme, substrate, and oligo. Outside of this ‘sweet-spot’ range, adding oligos is ineffective or even detrimental. Regardless of the number of sites in a substrate, if the enzyme-to-substrate ratio is at or below optimum, then adding oligos reduces cleavage since it lowers the effective enzyme concentration. If this ratio is somewhat above optimum, then adding oligos can improve cleavage and lead to complete digestion. If the ratio is excessively high, such that site-saturation is occurring, then adding oligos can alleviate this and also improve cleavage. In this latter situation, however, the same improvement can be achieved by adding less of the enzyme and no oligo at all. In general, to use oligos effectively, the stoichiometry of the reaction needs to be established beforehand by performing a series of titrations and identifying the optimum range for the concentration of oligos.

Given the diversity of restriction enzymes, many exceptions occur, but single-site and multi-site enzymes partition fairly well into two distinguishable groups based on positions of cleavage.

  1. Single-site enzymes. The majority of restriction enzymes that cleave within or very close to their recognition sequence are active at single-sites. These enzymes are classified as ‘Type IIP’ if their sequences are palindromic (symmetric), and ‘Type IIT’ if their sequences are asymmetric and two different catalytic sites are used to cleave the DNA strands. They cleave to completion DNA substrates with only one site as efficiently as they cleave substrates with several sites. Their cleavage ability is not inhibited at high enzyme concentration due to site-saturation, and is not enhanced by the addition of specific oligos.

    Type IIT enzymes AciI (NEB #R0551)  and EarI (NEB #R0528) are possible exceptions to this generalization, as too are several Type IIP enzymes. These enzymes cleave 1-site substrates slowly, and in some cases, incompletely; cleavage is inhibited at high enzyme concentrations due to site-saturation; and cleavage can be enhanced by the addition of specific oligos. These Type IIP enzyme exceptions include: AluI (NEB #R0137); BsaWI (NEB #R0567) (25); BsrFI/Cfr10I (NEB #R0562) (14,19,20); Ecl18kI (12,18); EcoRII (9,17,19,20); HpaII (NEB #R0171) (19,20); NaeI (NEB #R0190) (13,24,26,27); NarI/Mly113I (NEB #R0191)  (1,19,20,24,28); NgoMIV ((NEB #R0564) 6); PluTI/BbeI (NEB #R0713) (1); RsrII (NEB #R0501); SacII/Cfr42I (NEB #R0157) (19,29); Sau3AI (NEB #R0169) (19,20); SfiI (NEB #R0123) (16,30-35); SgrAI (NEB #R0603)  (19,20,36-38); SmlI; and XmaI/Cfr9I (NEB #R0180) (19,24), all of which act to one degree or another as multi-site enzymes.

  2. Multi-site enzymes. The majority of enzymes that recognize asymmetric sequences and cleave some distance away on one or both sides of the recognition sequence require two or more sites in order to cleave. Many of these restriction enzymes fall into either class Type IIS or Type IIC. The former comprises two domains: one for cleavage and the other for sequence-recognition, and only restricts DNA. The latter contains a third domain, for DNA-methylation, and can also modify DNA in addition to restricting it. Cleavage by enzymes of both groups is inhibited if DNA looping is prevented by applied tension (19,20). They tend to cleave 1-site substrates slowly, and usually incompletely (39,40); cleavage is suppressed at high enzyme concentrations due to site-saturation; and, under some conditions, cleavage can be enhanced by the addition of specific oligos. Possible exceptions to this multi-site generalization include BplI (40); BseRI (NEB #R0581) and BtgZI (NEB #R0703); and FauI (NEB #R0651) and MnlI (NEB #R0163), which appear to behave more like single-site enzymes.


If you are using an enzyme that may require more than one recognition site on the substrate to cleave optimally, we suggest two possible optimization methods:  1) Titrate the units of enzyme used in the reaction to determine the optimal enzyme to substrate ratio. As a starting point, we recommend using 1-2 μl of restriction enzyme (at the supplied units/μl) per microgram of substrate and performing 2-fold serial dilutions of the enzyme, keeping the DNA concentration constant.  2) If that is not possible, add duplex oligonucleotides that contain the recognition site. The oligos provide the additional recognition site needed to activate substrate-bound, but dormant enzyme monomers. Addition of duplex DNA containing a recognition site can compete for binding and reduce the effective enzyme concentration. To use oligos effectively, the stoichiometry of the reaction needs to be established beforehand by performing a series of titrations and identifying the optimum range for the concentration of oligos. We recommend keeping the enzyme concentration constant at 2-4 fold above the optimum established in step 1, above, while performing 2-fold serial dilutions of the oligo. As a starting point, we recommend a ratio of 4:1 (oligo sites:substrate sites) and performing a 2-fold serial dilution of the oligo, keeping the enzyme and substrate concentrations constant.


  1. Gowers, D.M., Bellamy, S.R.W. and Halford, S.E. (2004) One recognition sequence, seven restriction enzymes, five reaction mechanisms. Nucleic Acids Res., 32, 3469-3479.
  2. Xu, Q.S., Roberts, R.J. and Guo, H.C. (2005) Two crystal forms of the restriction enzyme MspI-DNA complex show the same novel structure. Protein Sci., 14, 2590-2600.
  3. Bitinaite, J., Wah, D.A., Aggarwal, A.K. and Schildkraut, I. (1998) FokI dimerization is required for DNA cleavage. Proc. Natl. Acad. Sci. U. S. A., 95, 10570-10575.
  4. Wah, D.A., Bitinaite, J., Schildkraut, I. and Aggarwal, A.K. (1998) Structure of FokI has implications for DNA cleavage. Proc. Natl. Acad. Sci. U. S. A., 95, 10564-10569.
  5. Vanamee, E.S., Santagata, S. and Aggarwal, A.K. (2001) FokI requires two specific DNA sites for cleavage. J. Mol. Biol., 309, 69-78.
  6.  Deibert, M., Grazulis, S., Sasnauskas, G., Siksnys, V. and Huber, R. (2000) Structure of the tetrameric restriction endonuclease NgoMIV in complex with cleaved DNA. Nat. Struct. Biol., 7, 792-799.
  7. Marshall, J.J., Smith, R.M., Ganguly, S. and Halford, S.E. (2011) Concerted action at eight phosphodiester bonds by the BcgI restriction endonuclease. Nucleic Acids Res., 39, 7630-7640.
  8. Smith, R.M., Marshall, J.J., Jacklin, A.J., Retter, S.E., Halford, S.E. and Sobott, F. (2013) Organization of the BcgI restriction-modification protein for the cleavage of eight phosphodiester bonds in DNA. Nucleic acids research, 41, 391-404.
  9. Tamulaitis, G., Sasnauskas, G., Mucke, M. and Siksnys, V. (2006) Simultaneous Binding of Three Recognition Sites is Necessary for a Concerted Plasmid DNA Cleavage by EcoRII Restriction Endonuclease. J. Mol. Biol.
  10. Catto, L.E., Ganguly, S., Milsom, S.E., Welsh, A.J. and Halford, S.E. (2006) Protein assembly and DNA looping by the FokI restriction endonuclease. Nucleic Acids Res., 34, 1711.
  11. Gormley, N.A., Hillberg, A.L. and Halford, S.E. (2002) The type IIs restriction endonuclease BspMI is a tetramer that acts concertedly at two copies of an asymmetric DNA sequence. J. Biol. Chem., 277, 4034-4041.
  12. Rutkauskas, D., Petkelyte, M., Naujalis, P., Sasnauskas, G., Tamulaitis, G., Zaremba, M. and Siksnys, V. (2014) Restriction Enzyme Ecl18kI-Induced DNA Looping Dynamics by Single-Molecule FRET. J. Phys. Chem. B, 118, 8575-8582.
  13. Topal, M.D., Thresher, R.J., Conrad, M. and Griffith, J. (1991) NaeI endonuclease binding to pBR322 DNA induces looping. Biochemistry, 30, 2006-2010.
  14. Siksnys, V., Skirgaila, R., Sasnauskas, G., Urbanke, C., Cherny, D., Grazulis, S. and Huber, R. (1999) The Cfr10I restriction enzyme is functional as a tetramer. J. Mol. Biol., 291, 1105-1118.
  15. Yokokawa, M., Yoshimura, S.H., Naito, Y., Ando, T., Yagi, A., Sakai, N. and Takeyasu, K. (2006) Fast-scanning atomic force microscopy reveals the molecular mechanism of DNA cleavage by ApaI endonuclease. IEE proceedings. Nanobiotechnology, 153, 60-66.
  16. Krasnoslobodtsev, A.V., Shlyakhtenko, L.S. and Lyubchenko, Y.L. (2007) Probing Interactions within the synaptic DNA-SfiI complex by AFM force spectroscopy. Journal of Molecular Biology, 365, 1407-1416.
  17. Shlyakhtenko, L.S., Gilmore, J., Portillo, A., Tamulaitis, G., Siksnys, V. and Lyubchenko, Y.L. (2007) Direct visualization of the EcoRII-DNA triple synaptic complex by atomic force microscopy. Biochemistry, 46, 11128-11136.
  18. Zaremba, M., Owsicka, A., Tamulaitis, G., Sasnauskas, G., Shlyakhtenko, L.S., Lushnikov, A.Y., Lyubchenko, Y.L., Laurens, N., van den Broek, B., Wuite, G.J. et al. (2010) DNA synapsis through transient tetramerization triggers cleavage by Ecl18kI restriction enzyme. Nucleic Acids Res., 38, 7142-7154.
  19. Gemmen, G.J., Millin, R. and Smith, D.E. (2006) Tension-dependent DNA cleavage by restriction endonucleases: Two-site enzymes are "switched off" at low force. Proc. Natl. Acad. Sci. U. S. A.
  20. Gemmen, G.J., Millin, R. and Smith, D.E. (2006) DNA looping by two-site restriction endonucleases: heterogeneous probability distributions for loop size and unbinding force. Nucleic Acids Res., 34, 2864-2877.
  21. Pein, C.D., Reuter, M., Cech, D. and Kruger, D.H. (1989) Oligonucleotide duplexes containing CC(A/T)GG stimulate cleavage of refractory DNA by restriction endonuclease EcoRII. FEBS Lett., 245, 141-144.
  22. Conrad, M. and Topal, M.D. (1992) Modified DNA fragments activate NaeI cleavage of refractory DNA sites. Nucl. Acids Res., 20, 5127-5130.
  23. Senesac, J.H. and Allen, J.R. (1995) Oligonucleotide activation of the type IIe restriction enzyme NaeI for digestion of refractory sites. Biotechniques, 19, 990-993.
  24. Reuter, M., Kupper, D., Pein, C.D., Petrusyte, M., Siksnys, V., Frey, B. and Kruger, D.H. (1993) Use of specific oligonucleotide duplexes to stimulate cleavage of refractory DNA sites by restriction endonucleases. Anal. Biochem., 209, 232-237.
  25. Tamulaitis, G., Rutkauskas, M., Zaremba, M., Grazulis, S., Tamulaitiene, G. and Siksnys, V. (2015) Functional significance of protein assemblies predicted by the crystal structure of the restriction endonuclease BsaWI. Nucleic Acids Res., 43, 8100-8110.
  26. Conrad, M. and Topal, M.D. (1989) DNA and spermidine provide a switch mechanism to regulate the activity of restriction enzyme NaeI. Proc. Natl. Acad. Sci. USA, 86, 9707-9711.
  27. Huai, Q., Colandene, J.D., Chen, Y., Luo, F., Zhao, Y., Topal, M.D. and Ke, H. (2000) Crystal structure of NaeI - an evolutionary bridge between DNA endonuclease and topoisomerase. EMBO J., 19, 3110-3118.
  28. Kruger, D.H., Barcak, G.J., Reuter, M. and Smith, H.O. (1988) EcoRII can be activated to cleave refractory DNA recognition sites. Nucleic Acids Res., 16, 3997-4008.
  29. Gasiunas, G., Sasnauskas, G., Tamulaitis, G., Urbanke, C., Razaniene, D. and Siksnys, V. (2007) Tetrameric restriction enzymes: expansion to the GIY-YIG nuclease family. Nucleic Acids Res., 36, 938-949.
  30. Wentzell, L.M., Nobbs, T.J. and Halford, S.E. (1995) The SfiI restriction endonuclease makes a four-strand DNA break at two copies of its recognition sequence. J. Mol. Biol., 248, 581-595.
  31. Nobbs, T.J. and Halford, S.E. (1995) DNA cleavage at two recognition sites by the SfiI restriction endonuclease: Salt dependence of Cis and Trans interactions between distant DNA sites. J. Mol. Biol., 252, 399-411.
  32. Szczelkun, M.D. and Halford, S.E. (1996) Recombination by resolvase to analyse DNA communications by the SfiI restriction endonuclease. EMBO J., 15, 1460-1469.
  33. Kupper, D., Moncke-Buchner, E., Reuter, M. and Krueger, D.H. (1999) Oligonucleotide stimulators allow complete cleavage of agarose-embedded DNA by particular type II restriction endonucleases. Anal. Biochem., 272, 275-277.
  34. Milsom, S.E., Halford, S.E., Embleton, M.L. and Szczelkun, M.D. (2001) Analysis of DNA looping interactions by type II restriction enzymes that require two copies of their recognition sites. J. Mol. Biol., 311, 515-527.
  35. Suzuki, Y., Gilmore, J.L., Yoshimura, S.H., Henderson, R.M., Lyubchenko, Y.L. and Takeyasu, K. (2011) Visual Analysis of Concerted Cleavage by Type IIF Restriction Enzyme SfiI in Subsecond Time Region. Biophys. J., 101, 2992-2998.
  36. Bilcock, D.T., Daniels, L.E., Bath, A.J. and Halford, S.E. (1999) Reactions of type II restriction endonucleases with 8-base pair recognition sites. J. Biol. Chem., 274, 36379-36386.
  37. Daniels, L.E., Wood, K.M., Scott, D.J. and Halford, S.E. (2003) Subunit assembly for DNA cleavage by restriction endonuclease SgrAI. J. Mol. Biol., 327, 579-591.
  38. Lyumkis, D., Talley, H., Stewart, A., Shah, S., Park, C.K., Tama, F., Potter, C.S., Carragher, B. and Horton, N.C. (2013) Allosteric Regulation of DNA Cleavage and Sequence-Specificity through Run-On Oligomerization. Structure, 21, 1848-1858.
  39. Bath, A.J., Milsom, S.E., Gormley, N.A. and Halford, S.E. (2002) Many type IIs restriction endonucleases interact with two recognition sites before cleaving DNA. J. Biol. Chem., 277, 4024-4033.
  40. Marshall, J.J., Gowers, D.M. and Halford, S.E. (2007) Restriction Endonucleases that Bridge and Excise Two Recognition Sites from DNA. J. Mol. Biol., 367, 419-431.