Two forms of DNA polymerase α, αl and α2, 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 α1 fraction co-sedimented with the DNA polymerase activity in a glycerol gradient, and almost no primase activity was detected in the most purified α2 fraction. The primase activity associated with DNA polymerase α was assayed indirectly by measuring ATP-dependent DNA synthesis with poly (dT) as template. Characterization of the assay system was performed with the purified α1. 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.
α1 and α2 were very similar as DNA polymerases in their sensitivity to several inhibitors and their preference for template-primers, except that al had a slightly greater preference for poly (dT)•(rA)₁₀ than α2 did. The major difference between the two forms was observed in their S values, 8.2 and 6.4 S for α1 and α2, respectively.