Heterogeneity of DNA polymerase a [DNA nucleotidyltransferase (DNA-directed), EC 2.7.7.7] has commonly been observed during its purification from a variety of cell systems (1-5). This has hampered the establishment of the physical structure of DNA polymerase a and there is, as yet, no general agreement on the physical properties ofthe core enzyme (6-10). The physiological significance, if any, of this heterogeneity is not clear at this time. Johnston and coworkers (11) showed that mild treatment of a high molecular weight form (200,000-250,000 daltons) of calf thymus DNA polymerase a with 2.5 M urea converts the enzyme to a 155,000-dalton form with the release of a 50,000-to 70,000-dalton protein(s). McKune and Holmes (12) reconstituted the high molecular weight form of polymerase a from the combination of the 155,000-dalton and the 50,000-to 70,000-dalton protein (s). They report that the 50,000-to 70,000-dalton protein(s) enhances the catalytic activity of the polymerase with synthetic polydeoxyribonucleotide templates. Villani et al. (9) have recently shown a similar conversion of DNA polymerase a from Drosophila melanogaster embryos in the presence of2.8 M urea (9). The catalytic activity of the. 148,000-dalton form of DNA polymerase a from Drosophilalembryos (9) and a 156,000-dalton form from rat liver (8) were enhanced when associated with four separable proteins of 54,000-64,000 daltons.We have previously reported the isolationwof a protein (C1) from HeLa cells thattspecifically stimulates the catalytic activity of HeLa DNA polymerase a 20-fold with DNA templateprimers that contain extended single-strand regions (13). In this paper we report the isolation and purification of three forms of DNA polymerase a from synchronized HeLa cells, one ofwhich has equal catalytic activity with activated DNA and with DNA template-primers that contain extended single-strand regions. Two proteins (Cl and C2) that are necessary for its catalytic activity with the latter template-primer are resolved from the DNA polymerase a.MATERIALS AND METHODS Materials. 3H-Labeled deoxyribonucleoside triphosphates were purchased from New England Nuclear. Unlabeled deoxyribonucleoside triphosphates and poly-and oligodeoxyribonucleotide homopolymers were from P-L

Pancreatic DNase requires both Ca2+ and Mg2+ for its activity as measured by formation of an activated DNA template for in vitro DNA polymerase alpha assay and by the hyperchromic shift. Mn2+ can partially satisfy the Mg2+ requirement of the DNase for activation of DNA but the resulting template is only 50% as active in the DNA polymerase assay. When precautions are taken to avoid divalent ion contamination, pancreatic DNase is not active in the presence of Ca2+ or Mg2+ alone. analysis of the DNA by sucrose gradient centrifugation shows that only in the presence of Ca2+ plus Mg2+ or Mn2+ does pancreatic DNase produce extensive strand breaks in the DNA. The activated DNA template that yields maximal DNA polymerase activity is low molecular weight material of 30,000 to 50,000 daltons.

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