When presented with multiple recognition sites that differ in their flanking sequences, most restriction enzymes exhibit slight preferences and cleave the sites at different rates. These rate differences are such that the addition of a small excess of enzyme will avoid any problems due to incomplete digestion. As always, however, one must be aware of the experimental molar concentration of recognition sites and digest conditions relative to that of the unit definition. See Substrate Considerations for further information.

A. Type IIe Restriction Enzymes

A few restriction enzymes have considerably greater difficulty in cleaving some of their recognition sites. Original experiments with these enzymes led to designation of their site preferences as shown in Table 1.5:

Table 1.5. Restriction Enzyme Site Preferences.

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B. Affector Sequences

Investigation revealed that binding of a second recognition sequence, in cis or trans, to a distal, non-catalytic site on the enzyme allows slow and resistant sites to become cleavable. This affector sequence alters the kinetics in one of two ways. In the K class (Nar I, Hpa II, Sac II), activator DNA binding decreases the K_m without altering the V_max of cleavage, indicating that cooperative binding induces a conformational shift that increases the affinity of the enzyme for its substrate. In the V class (Nae I, BspM I), binding of activator DNA increases the V_max without changing the K_m, indicating that the increased catalytic activity is not related to the affinity of the enzyme for its substrate (1). It is assumed that the flanking sequences of a recognition site influence the kinetics of cleavage at that site, but at this time the interaction is not understood. Considerable differences also exist in the ability of affector sequences to stimulate cleavage. Generally, a recognition site flanked by the sequence from a site that is cleaved easily is a useful starting point for designing good affector sequences.

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C. Turbo™ Restriction Enzymes

Incomplete cleavage by Type IIe enzymes can be problematic in common molecular biology applications. Addition of a simple DNA affector sequence to the digest, in the form of either an oligonucleotide or naturally occurring DNA is of marginal use. Eventually, it will also be cleaved and will no longer be able to stimulate complete cleavage of the desired substrate. For EcoR II, this problem was circumvented by use of affector oligonucleotides modified with specific methylation or nucleotide analogs (3). For Nae I, an oligonucleotide was used in which sulfur replaced the phosphorous at the scissile bond (4). Because of these modifications, the oligonucleotides remain unhydrolyzed and are able to stimulate complete cleavage of the desired substrate.

This technology forms the basis of Promega’s Turbo™ Nae I^(a) and Turbo™ Nar I^(a). These products contain an optimized, thiol-substituted activating oligonucleotide premixed in the 10X Reaction Buffer provided. This oligonucleotide does not interfere with subsequent ligations or random primer labeling. A one-step purification yields a cleaved DNA suitable for end-labeling (5).

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D. References

1. Oller, A.R. et al. (1991) Ability of DNA and spermidine to affect the activity of restriction endonucleases from several bacterial species. Biochem. 30, 2543.
2. Reuter, M. et al. (1993) Use of specific oligonucleotide duplexes to stimulate cleavage of refractory DNA sites by restriction endonucleases. Anal. Biochem. 209, 232.
3. Pein, C.D. et al. (1991) Activation of restriction endonuclease EcoR II does not depend on the cleavage of stimulator DNA. Nucl. Acids Res. 19, 5139.
4. Conrad, M. and Topal, M. (1992) Modified DNA fragments activate Nae I cleavage of refractory DNA sites. Nucl. Acids Res. 20, 5127.
5. Senesac, J.H. and Allen, J.R. (1995) Oligonucleotide activation of the Type IIe restriction enzyme Nae I for digestion of refractory sites. BioTechniques 19, 990.

^(a)Turbo™ Nae I and Turbo™ Nar I are the subjects of U.S. Pat. Nos. 5,248,600 and 5,418,150 and licensed exclusively to Promega Corporation.

Turbo is a trademark of Promega Corporation.