Klenow fragment and DNA polymerase alpha-primase fromserva calf thymus in water-in-oil microemulsions.

Abstract:

The activity of DNA polymerase alpha-primase complex from calf thymus and Klenow fragment of E. coli DNA polymerase 1 has been studied in reverse microemulsions formed by sodium bis(2-ethylhexyl) sulfosuccinate (AOT), sodium dodecylsulfate (SDS), cetyl trimethyl ammonium bromide (CTAB), polyoxyethylene 20 cetyl ether (Brij 58), and Triton X-114 in decane. DNA polymerases were not active in AOT, CTAB, and SDS reverse microemulsions, but these enzymes catalyzed DNA synthesis in Brij 58 and its mixture with other surfactants. We have also found the system composed from the Triton X-114, SDS, CTAB, and Brij 58 (concentration of 128, 25, 15, and 10 mM, respectively) in hexanol-decane (1:12 v/v), in which DNA polymerases revealed maximum activity. The above system was optically transparent, fluid, and stable during a few hours with a water-surfactants molar ratio up to 160. The pH dependence of DNA polymerase activity was not significantly different in comparison with water; however, DNA polymerase was sensitive to ionic strength in microemulsions. The dependence of DNA polymerase activity on w0 was the curve with a few optima. DNA polymerases synthesized more products in water than in reverse microemulsions, and the processivity of Klenow fragment decreased. An increase of the water content resulted in an increase of DNA polymerase processivity.

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