Abstract
A protein that stimulates DNA polymerase alpha/primase many-fold on unprimed poly(dT) was purified to homogeneity from extracts of cultured mouse cells. The protein contains polypeptides of approximately 132 and 44 kDa, and the total molecular mass of 150 kDa calculated from Stokes radius (54 A) and sedimentation coefficient (6.7 S) indicates that it contains one each of the two subunits. The purified "alpha accessory factor" (AAF) also stimulates DNA polymerase alpha/primase in the self-primed reaction with unprimed single-stranded DNA. In addition to these effects on the coordinate activities of DNA polymerase alpha and DNA primase, stimulatory effects were also demonstrated separately on both the polymerase and primase activities of the enzyme complex. However, there was no stimulation with DNase-treated ("activated") DNA under normal conditions for assay of DNA polymerase alpha. The stimulatory activity of mouse AAF is highly specific for DNA polymerase alpha/primase; no effect was observed with mouse DNA polymerases beta, gamma, or delta, nor with retroviral, bacteriophage, or bacterial DNA polymerases. Mouse AAF stimulated human DNA polymerase alpha/primase with several different templates, similar to results with the mouse enzyme. However, it had very little effect on the DNA polymerase/primase from either Drosophila embryo or from yeast.
Highlights
The column was washed with 200 ml of column equilibration buffer and eluted with 400 ml of the same buffer except 0.2 M KC1 and containing 10 mM KP04, pH 7.6, collecting 20-ml fractions. (The column was subsequently eluted with higher concentrations of phosphate to recover other DNA replication enzymes, including DNA polymerases 6 [21] and Lu/primase [20].) The fractions with the highest AAF activity were pooled and made
The greatest stimulatory effects of AAF on DNA polymerase cu/primase were seen with unprimed poly(dT) (Tables II and III)
No stimulation resulted from AAF when preformed primers, either oligo(dA) or oligo(rA), were supplied for poly(dT), and rATP was omitted; there was no effect on the activity with singly primed single-stranded circular DNA (Table II)
Summary
Cells-Mouse lymphoblast cell line (L1210) was grown in spinner flasks in 5% fetal bovine serum (or 1% fetal bovine and 4% newborn calf serum) and frozen in a mixture containing protease inhibitors, as described elsewhere [20]. Enzymes-Preparation of mouse DNA polymerase cu/primase followed a published procedure [20]. Purification of DNA polymerase cY/primase from cultured HeLa cells was carried through step IV (DEAE-cellulose) of the procedure for purification of mouse DNA polymerase ollprimase. 1 unit of DNA polymerase y is 1 nmol of dTMP in 1 h assayed with poly(rA) . 6 = 1 nmol of total dNMP in 1 h assayed on poly(dAdT) For both the Klenow fragment of E. coli DNA polymerase I (Bethesda Research Laboratories) and bacteriophage T4 DNA polymerase [22], 1 unit = 10 nmol of total dNMP in 30 min at 37 “C; for the E. coli enzyme this was determined with poly(dA-dT), and for the. Reverse transcrintase from a;ian myeloblastosis virus was from Seikagaku; 1 unit = 1 nmol dTMP in 10 min at 35 “C with oolv(rA) .oligo(dT).
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