search products / site
Activate your account
Log in / Register
Product CatalogueTechnical ResourceCustomer ServiceMy AccountShopping Basket
Home  >Technical Resource  > Restriction Endonucleases > Site Preferences

Site Preferences

Certain restriction endonucleases show preferential cleavage of some sites in the same substrate. In 1975 Thomas and Davis (1) observed that EcoR I cleaves the five sites on lambda (l) DNA nonrandomly. The site nearest the right terminus is cleaved 10 times faster than the sites in the middle of the molecule. Forsblum et al. (2) reported that EcoR I cleaves sites on adenovirus-2 DNA at different rates. Nath and Azzolina (3) reported 10-fold and 14-fold differences among rates of cleavage of EcoR I and Hind III sites respectively on lambda DNA. Brown and Smith found that Hga I cleaves certain sites on fX174 DNA more quickly than others (4). Gingeras and Brooks (5) reported that a CTCGAG site in adenovirus-2 DNA is completely refractory to cleavage by PaeR7 I, yet is readily cleaved by Xho I, an isoschizomer of PaeR7 I. In this particular case the resistance to cleavage is attributed to the surrounding sequence, a CT dinucleotide bordering the 5´ end of the site CTCGAG. Methylation of the recognition site does not appear to be the cause of the differential cleavage rates in any of the above cases.

Some restriction endonucleases have to interact with two copies of their recognition sequence before they can cleave DNA; this can lead to unusual cleavage properties. For instance, BspM I (6), Sfi I (7) and NgoM IV (8) are all homotetrameric proteins that bind two recognition sequences and cleave four phosphodiester bonds concurrently. These enzymes require two recognition sites in cis for optimal activity; consequently, they do not easily cut a molecule containing a single site. The requirement for two sites seems to be particularly common, although not universal, among Type IIs enzymes, which are generally monomers that transiently associate to form dimers to cleave both strands (9-11). These include such enzymes as Fok I, Bsg I, Bpm I and Mbo II (10). Yet others such as EcoR II and Nae I interact with two copies of their recognition sequence, one being the target for cleavage, the other serving as an allosteric effector (12-15). For these enzymes, the second, non-cleavable site can be provided either on the same DNA molecule or in the form of an activator oligonucleotide (12,13). Slow and/or resistant to cleavage recognition sites on certain substrates have also been observed for Hpa II, Nar I and Sac II, however significantly less is known about the mode of action of these enzymes (14).

These properties of restriction endonucleases can result in dramatic site preference. For example, plasmids containing a single site for Nae I, Nar I and BspM I are cleaved very poorly. Nevertheless, pBR322 contains four Nar I recognition sequences. One unit of Nar I (as defined on adenovirus-2 DNA) will cut two of these sites to completion in an hour under standard conditions. An additional 50 units of enzyme will not cleave the remaining two sites to completion, even after incubation for 16 hours. Similarly, of the four Nae I sites in pBR322, two are readily cleaved, one is cleaved moderately slow, and the fourth is cleaved 50-fold more slowly. Nar I and Nae I each has one recognition site on l DNA, but only partial digestion of l can be achieved by many-fold overdigestion with either of these enzymes. Sac II has four sites on l DNA. Three of the sites are clustered near the center of the DNA molecule. These sites are cleaved 50-fold faster than the remaining site near the right terminus (nucleotide coordinate 40,386). Therefore, the unit definition of these enzymes is based on cleavage of adenovirus-2 DNA, which has more than 10 sites for each enzyme. For example, one unit of Nar I or Nae I is the amount of enzyme required to cut 1 µg of adenovirus-2 DNA to completion in one hour in a 50 µl reaction.

References:

  1. Thomas, M. and Davis, R. W. (1975) J. Mol. Biol. 91, 315.
  2. Forsblum, S. et al. (1976) Nucl. Acids Res. 3, 3255.
  3. Nath, K. and Azzolina, B. A. (1981). In J. G. Chirikjian (Ed.), Gene Amplification and Analysis Vol. 1, (p. 113). New York: Elsevier North Holland, Inc.
  4. Brown, N. L. and Smith, M. (1977) Proc. Natl. Acad. Sci. USA, 74, 3213–3216.
  5. Gingeras. T. R. and Brooks, J. E. (1983) Proc. Natl. Acad. Sci. USA, 80, 402–406.
  6. Gormley, N.A., Hillberg, A.L., and Halford, S.E. (2002) J. Biol. Chem. 277, 4034-4041.
  7. Wentzell, L.M., Nobbs, T.J., and Halford, S.E. (1995) J. Mol. Biol. 248, 581-595.
  8. Deibert, M. et al. (2000) Nat. Struct. Biol. 7, 792-799.
  9. Bitinaite, J., Wah, D.A., Aggarwal, A.K., and Schildkraut, I. (1998) Proc. Natl. Acad. Sci. USA 95, 10570-10575.
  10. Bath, A.J., Silsom, S.E., Gormley, N.A. and Halford, S.E. (2002) J. Biol. Chem. 277, 4024-4033.
  11. Soundararajan, M. et al. (2002) J. Biol. Chem. 277, 887–895.
  12. Krüger, D.H. et al. (1988) Nucl. Acids Res. 16, 3997–4008.
  13. Conrad. M. and Topal, M. D. (1989) Proc. Natl. Acad. Sci. USA, 86, 9707–9711.
  14. Oller, A. R. et al. (1991) Biochemistry 30, 2543–2549.
  15. Pein, C.-D. et al. (1991) Nucl. Acids Res. 19, 5139–5142.