Ro. Anarbaev et al., KLENOW FRAGMENT AND DNA-POLYMERASE ALPHA-PRIMASE FROM SERVA CALF THYMUS IN WATER-IN-OIL MICROEMULSIONS, Biochimica et biophysica acta. Protein structure and molecular enzymology, 1384(2), 1998, pp. 315-324
The activity of DNA polymerase alpha-primase complex from calf thymus
and Klenow fragment of E. coli DNA polymerase 1 has been studied in re
verse 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 a
nd its mixture with other surfactants. We have also found the system c
omposed from the Triton X-114, SDS, CTAB, and Brij 58 (concentration o
f 128, 25, 15, and 10 mM, respectively) in hexanol-decane (1:12 v/v),
in which DNA polymerases revealed maximum activity. The above system w
as optically transparent, fluid, and stable during a few hours with a
water-surfactants molar ratio up to 160. The pH dependence of DNA poly
merase activity was not significantly different in comparison with wat
er; however, DNA polymerase was sensitive to ionic strength in microem
ulsions. The dependence of DNA polymerase activity on w(0) was the cur
ve with a few optima. DNA polymerases synthesized more products in wat
er than in reverse microemulsions, and the processivity of Klenow frag
ment decreased. An increase of the water content resulted in an increa
se of DNA polymerase processivity. (C) 1998 Elsevier Science B.V. All
rights reserved.