The manipulation of DNA with restriction enzymes in low water systems

• Published in: Biochimica et biophysica acta Vol. 1074; no. 1; pp. 40 - 44
• Main Authors: Bryan Hanley, A., Furniss, Caroline S.M., Kwiatkowska, Christine A., Mackie, Alan R.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 24.05.1991; Elsevier
• Subjects: Analytical, structural and metabolic biochemistry; Bacteriophage lambda - genetics; Biological and medical sciences; Deoxyribonuclease HindIII - metabolism; Diatomaceous Earth; Dioctyl Sulfosuccinic Acid - chemistry; DNA, Viral - chemistry; DNA, Viral - metabolism; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Hydrolases; Lambda DNA; Low water system; Micelles; Restriction enzyme; Restriction Mapping; Solubility; Surface-Active Agents - chemistry; Water - chemistry; Restriction enzyme; Low water system; AOT; Lambda DNA; Enzyme; Micelle; Environmental factor; Surfactant; Virus; Lambda phage group; Styloviridae; DNA; Endodeoxyribonuclease HindIII; Cleavage; Aqueous solution; Phage lambda; Spectrophotometry; Phage
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/0304-4165(91)90036-G

The cleavage of phage lambda (λ) DNA by the restriction enzyme HindIII in low water systems has been investigated. Two types of low water systems have been studied - those which contain a surfactant in a reverse micelle environment and a surfactant-free system in which a solid support (celite) is used. The effect of the surfactants themselves in a normal aqueous environment has also been studied. Charged surfactants were found to greatly inhibit HindIII activity in aqueous buffer, while non-ionic surfactants did not affect either the activity or the specificity of the restriction enzyme. The rate of cleavage by HindIII in a reverse micelle system consisting of sodium dioctylsulphosuccinate is very slow, however, in a Triton B system the expected fragments are observed. In a surfactant-free low water environment, cleavage occurs at the expected sites but in a different order to that observed in normal aqueous systems. These results suggest that DNA tertiary structure in low water systems is different to that in aqueous solution and that this influences cleavage by the restriction enzyme HindIII.