Modulating restriction endonuclease activities and specificities using neutral detergents.
It is well known that type II restriction enzyme activities and specificities can be modulated by altering solution conditions. The addition of co-solvents such as dimethyl sulfoxide (DMSO), alcohols and polyols can promote star activity, which is the cleavage of non-cognate sequences. While neutral detergents are often used to control protein aggregation, little is known about the effect of neutral detergents on restriction enzyme activities and specificities. We report here that BamHI, BglI, BglII, EcoRI, EcoRV, HindIII, MluI, PvuII, SalI and XhoI restriction endonucleases are remarkably tolerant of high concentrations of neutral detergents Triton X-100, CHAPS and octyl glucoside. In most cases, lambda DNA cleavage rates were comparable to those observed in the absence of detergent. Indeed, the specific activities of SalI and XhoI were appreciably increased in the presence of Triton X-100. For all enzymes active in the presence of detergents, sequence specificity toward lambda DNA was not compromised. Assays of star cleavage of pUC18 by EcoRI, PvuII and BamHI endonucleases in equimolar concentrations of Triton X-100 and sucrose revealed reduced star activity in the detergent relative to the sucrose co-solvent. Interestingly, under star activity-promoting conditions, PvuII endonuclease displayed greater fidelity in Triton X-100 than in conventional buffer. Taken altogether, these results suggest that in some cases, neutral detergents can be used to manipulate restriction endonuclease reaction rates and specificities.
Conlan
L H
LH
Texas A&M University, College Station, USA.
José
T J
TJ
Thornton
K C
KC
Dupureur
C M
CM
D010582:Bacteriophage lambda; D002793:Cholic Acids / Q000494:pharmacology; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology*; D004587:Electrophoresis, Agar Gel; D005960:Glucosides / Q000494:pharmacology; D007700:Kinetics; D017830:Octoxynol / Q000494:pharmacology; D009994:Osmolar Concentration; D013379:Substrate Specificity
D002793:Cholic Acids; D004279:DNA, Viral; D003902:Detergents; D005960:Glucosides; C018619:octyl-beta-D-glucoside; D017830:Octoxynol; D004262:DNA Restriction Enzymes; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate