Purification and characterization of two forms of DNA polymerase alpha from mouse FM3A cells: a DNA polymerase alpha-primase complex and a free DNA polymerase alpha.

Abstract:

Two forms of DNA polymerase alpha, alpha 1 and alpha 2, have been partially purified from mouse FM3A cells by discriminating one form from the other on the basis of the association of primase activity. The primase activity in the most purified alpha 1 fraction co-sedimented with the DNA polymerase activity in a glycerol gradient, and almost no primase activity was detected in the most purified alpha 2 fraction. The primase activity associated with DNA polymerase alpha was assayed indirectly by measuring ATP-dependent DNA synthesis with poly (dT) as template. Characterization of the assay system was performed with the purified alpha 1. The system was absolutely dependent on the presence of ATP and a divalent cation. Mn2+ was much more effective than Mg2+, and 5-fold higher activity was observed with Mn2+ than with Mg2+ at their optimal concentrations. The primase activity assayed by the above system showed sensitivity to (NH4)2SO4 very similar to that of free primase reported by Tseng and Ahlem (J. Biol. Chem. 258, 9845-9849, 1983). The activity was inhibited by more than 50% by 20 mM (NH4)2SO4. alpha 1 and alpha 2 were very similar as DNA polymerases in their sensitivity to several inhibitors and their preference for template-primers, except that alpha 1 had a slightly greater preference for poly (dT) X (rA)10 than alpha 2 did. The major difference between the two forms was observed in their S values, 8.2 and 6.4 S for alpha 1 and alpha 2, respectively.

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