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Star Activity (Relaxation of Specificity)

Restriction enzymes recognize specific nucleotide sequences within DNA molecules. However, the recognition specificity of restriction enzymes can be reduced in vitro (1). Under certain conditions, enzymes are able to recognize and cleave nucleotide sequences which differ from the canonical site. At low ionic strength, for example, BamHI (with the recognition sequence GGATCC) is able to cleave the following sequences: NGATCC, GPuATCC and GGNTCC (2, 3). This phenomenon is called “relaxed” or “star” activity (4, 5).

In most practical applications of restriction enzymes, star activity is not desirable. Analysis of several reports (4, 6-10) on the star activity suggests the following causes for this phenomenon:

• prolonged incubation;
• high enzyme concentration in the reaction mixture;
• high glycerol concentration in the reaction mixture;
• presence of organic solvents, such as ethanol or dimethyl sulfoxide, in the reaction mixture;
• low ionic strength of the reaction buffer;
• suboptimal pH values of the reaction buffer;
• substitution of Mg²⁺ for other divalent cations, such as Mn²⁺ or Co²⁺.

In some cases, the termini generated by DNA cleavage with a restriction enzyme at the canonical site have been shown to stimulate the enzyme’s star activity (11).
Star activity and incomplete DNA digestion result in atypical electrophoresis patterns which can be identified by careful examination of gel images (see picture below). Here, incomplete DNA digestion results in additional low-intensity bands above the expected DNA bands on the gel. No additional bands below the smallest expected fragment are observed. These additional bands disappear when the incubation time or amount of enzyme is increased. On the contrary, star activity results in additional DNA bands below the expected bands and no additional bands above the largest expected fragment. These additional bands become more intense with the increase of either the incubation time or the amount of enzyme, while the intensity of the expected bands decreases.
Some restriction enzymes may remain associated with the substrate DNA after cleavage and thus change the mobility of digestion products during electrophoresis. The resulting atypical pattern is not related to star activity. To avoid confusing electrophoresis patterns, use a loading dye with SDS (e.g., the Fermentas 6X DNA Loading Dye & SDS Solution). Then, heat the sample for 10 min at 65°C and place it on ice prior to loading it on the gel.
Any tendency of a restriction enzyme to exhibit star activity is indicated both in the product description and in the Certificate of Analysis supplied with each enzyme.

Enzyme star activity.
1 - Lambda DNA
2 - Lambda DNA incubated 1 hour with 0.15 u of EcoRI (incomplete cleavage)
3 - Lambda DNA incubated 1 hour with 0.4 u of EcoRI (incomplete cleavage)
4 - Lambda DNA incubated 1 hour with 1 u of EcoRI (complete digestion)
5 - Lambda DNA incubated 16 hours with 40 u of EcoRI (star activity)
6 - Lambda DNA incubated 16 hours with 70 u of EcoRI (star activity)

References

1. Nasri, M., Thomas, D., Relaxation of recognition sequence of specific endonuclease HindIII, Nucleic Acids Res., 14, 811-821, 1986.

2. George, J., Chirikjian, J.G., Sequence-specific endonuclease BamHI: Relaxation of sequence recognition, Proc. Natl. Acad. Sci. USA, 79, 2432-2436, 1982.
3. Kolesnikov, V.A., et al., Relaxed specificity of endonuclease BamHI as determined by identification of recognition sites in SV40 and pBR322 DNAs, FEBS Letters, 132, 101-103, 1981.
4. Polisky, B., et al., Specificity of substrate recognition by the EcoRI restriction endonuclease, Proc. Natl. Acad. Sci. USA, 72, 3310-3314, 1975.
5. Woodbury, C.P., et al., DNA site recognition and reduced specificity of the EcoRI endonuclease, J. Biol. Chem., 255, 11534-11546, 1980.
6. George, J., et al., Sequence-specific endonuclease BamHI, J. Biol. Chem., 255, 6521-6524, 1980.
7. Malyguine, E., et al., Alteration of the specificity of restriction endonucleases in the presence of organic solvents, Gene, 8, 163-177, 1980.
8. Hsu, M., Berg, P., Altering the specificity of restriction endonuclease: effect of replacing Mg²⁺ with Mn²⁺, Biochemistry, 17, 131-138, 1978.
9. Mayer, H., Optimization of the EcoRI* activity of EcoRI endonuclease, FEBS Letters, 90, 341-344, 1978.
10. Nasri, M., Thomas, D., Alteration of the specificity of PvuII restriction endonuclease, Nucleic Acids Res., 15, 7677-7687, 1987.
11. Bitinaite, J., Schildkraut, I., Self generated DNA termini relax the specificity of SgrAI restriction endonuclease, Proc. Natl. Acad. Sci. USA, 99, 1164-1169, 2002.

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Updated sausio 31, 2008 16:23