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Site Preferences by Restriction Enzymes

In 1975, Thomas and Davis noticed that EcoRI cleaves its five recognition sites on lambda DNA at rates differing by an order of magnitude (1). Similar examples have been documented for other restriction enzymes. Factors, such as flanking sequence and the number of cleavage sites, appear to influence the cleavage efficiency. There are numerous restriction enzymes (EcoRII, NaeI, NarI, Ksp632I, BspMI, Eco57I et al.), which are known to never achieve complete cleavage of certain unmethylated target DNAs, even when using an excess of enzyme or a prolonged incubation (3-6). Most of these enzymes are members of the expanding group of type II restriction enzymes which require simultaneous interaction with two copies of the target site for effective cleavage (7). These enzymes cleave DNA molecules with one recognition site are cleaved very slowly. In the case of type IIE enzymes (EcoRII, NaeI) one of the target sequences serves as an allosteric effector for the effective cleavage of the other recognition site (3, 8-10). Type IIF enzymes (SfiI, Cfr10I, NgoMIV, BspMI) cleave both recognition sequences in a concerted reaction (11-14).Type IIS enzymes, such as FokI, BpmI, BsgI, MboII, also interact with two copies of their recognition sequence before cleaving DNA by different mechanisms (15).

Cleavage of resistant sites was found to be significantly enhanced by the addition of cleavable DNA, recognition site containing oligodeoxyribonucleotide or spermidine (4, 6, 14, 16, 17).

Different restriction enzymes recognizing the same nucleotide sequence (isoschizomers) often do not cleave the same resistant recognition site( e.g., Fermentas' enzymes BveI, Cfr42I, Eam1104I and PdiI, and their prototypes BspMI, SacII, Ksp632I and NaeI). However, some isoschizomers cleave "resistant" sites at the same rate as other normal sites. For example, EheI cleaves the target DNA more efficiently than its prototype NarI. Thus, one recognition site of NarI on lambda DNA and two sites on pBR322 are not cleaved to completion, even after incubation with 50 units of NarI for 16 hours. Unlike NarI, Fermentas' neoschizomer EheI cleaves lambda DNA and pBR322 DNA completely under standard conditions. Site preferences are a characteristic feature of the following Fermentas prototype enzymes: AarI, AjuI, AlfI, AloI, BdaI, BplI, BseMII, Eco57I, Eco57MI, PpiI, TsoI and TstI. The properties of these enzymes differ significantly from other type II enzymes.

References

  1. Thomas M., Davis R.W., Studies on the cleavage of bacteriophage lambda DNA with EcoRI restriction endonuclease, J. Mol. Biol., 91, 315-328, 1975.
  2. Sapienza P.J., et al., Thermodynamic and Kinetic Basis for the relaxed DNA Sequence Specificity of “Promiscuous” Mutant EcoRI endonuclease, J. Mol. Biol., 348, 307-324, 2005.
  3. Kruger, D.H., et al., EcoRII can be activated to cleave refractory DNA recognition sites, Nucleic Acids Res., 16, 3997-4008, 1988.
  4. Oller, A. R., et al., Ability of DNA and spermidine to affect the activity of restriction endonucleases from several bacterial species, Biochemistry, 30, 2543-2549, 1991.
  5. Bolton, B.J., et al., Ksp632I: a novel class IIS restriction endonuclease from Kluyvera species 632 with the asymmetric hexanucleotide recognition sequence: 5'-CTCTTCN^-3' 3'-GAGAAGNNNN^-5', Gene, 66, 31-43, 1988.
  6. Reuter, M., et al., Use of specific oligonucleotide duplexes to stimulate cleavage of refractory DNA sites by restriction endonucleases, Anal. Biochem., 209, 232-237, 1993
  7. Halford, S.E., Hopping, jumping and looping by restriction endonucleases, Biochem. Soc. Trans, 29, 363-373, 2001.
  8. Gabbara, S., Bhagwat, A.S., Interaction of EcoRII endonuclease with DNA substrates containing single recognition sites, J.Biol.Chem.,267, 18623-18630, 1992.
  9. Yang, C.C., Topal, M.D., Nonidentical DNA-binding sites of endonuclease NaeI recognize different families of sequences flanking the recognition site, Biochemistry, 31, 9657-9664, 1992.
  10. Huai, Q., et al., Crystal structure of NaeI - an evolutionary bridge between DNA endonuclease and topoisomerase, EMBO J., 19, 3110-3118, 2000.
  11. Wentzell, L.M., et al., The SfiI restriction endonuclease makes a four-strand DNA break at two copies of its recognition sequence, J. Mol. Biol.,248, 581-595, 1995.
  12. Siksnys, V., et al., The Cfr10I restriction enzyme is functional as a tetramer, J. Mol. Biol., 291, 1105-1118, 1999.
  13. Deibert, M., et al., Structure of the tetrameric restriction endonuclease NgoMIV in complex with cleaved DNA, Nat. Struct. Biol., 7, 792-799, 2000.
  14. Gormley, N.A., et al., 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, 2002.
  15. Bath, A.J., et al., Many type IIs restriction endonucleases interact with two recognition sites before cleaving DNA, J. Biol. Chem., 277, 4024-4033, 2002.
  16. Conrad, M., Topal, M.D., DNA and spermidine provide a switch mechanism to regulate the activity of restriction enzyme NaeI, Proc. Natl.Acad Sci. USA, 86, 9707-9711, 1989.
  17. Grigaite, R.J., et al., AarI, a restriction endonuclease from Arthrobacter aurescens SS2-322, which recognizes the novel non-palindromic sequence 5'-CACCTGC(N)4/8-3', Nucleic Acids Res., 30, e123, 2002.
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Updated lapkričio 08, 2006 11:06