A new species of DNA polymerase has been purified more than 10 000-fold from the cytoplasm of erythroid hyperplastic bone marrow. This DNA polymerase, in contrast to previously described eukaryotic DNA polymerases, is associated with a very active 3' to 5' exonuclease activity. Similar to the 3' to 5' exonuclease activity associated with prokaryotic DNA polymerases, this enzyme catalyzes the removal of 3'-terminal nucleotides from DNA, as well as a template-dependent conversion of deoxyribonucleoside triphosphates to monophosphates. The exonuclease activity is not separable from the DNA polymerase activity by chromatography on DEAE-Sephadex or hydroxylapatite, and upon sucrose density gradient centrifugation the two activities cosediment at 7 S or at 11 S depending on the ionic strength. Both exonuclease and polymerase activities have identical rates of heat inactivation and both are equally sensitive to hemin and Rifamycin AF/013, inhibitors of DNA synthesis that act by binding to DNA polymerase and causing its dissociation from its template/primer. These results are consistent with the coexistence of two enzyme activities in a single protein.

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DNA polymerases alpha and delta from rabbit bone marrow were purified to specific activities greater than 30 000 nM TMP incorporated (mg of protein)(-1)h(-1). alpha is quantitatively predominant. alpha and delta have the same reaction requirements and are both similarly sensitive to N-ethylmaleimide. The primary functional distinction is the association of 3' to 5' exonuclease activity with delta. Sedimentation coefficients obtained from zone sedimentation in glycerol gradients and Stokes radii values from gel filtration allow the calculation of true molecular weight and frictional ratios. alpha exhibits a bimodal pattern, sedimenting at 6 and 8 S on glycerol gradients and demonstrating components corresponding to 40.5- and 65-A Stokes radii upon gel filtration. The calculated molecular weights of the two forms of alpha are 100 000 and 215 000; the frictional ratios are 1.34 and 1.65. This and other data suggest a possible monomer-dimer relation. In contrast, delta sediments uniformly at 6.5 S and also behaves uniformly upon gel filtration at 4595 A. The molecular weight of delta is distinct at 122 000; its frictional ratio is 1.39. Because of similarities of the DNA polymerizing activities of both forms of alpha and of delta, it is postulated that alpha is derived from delta by structural modification, resulting in a decrease in molecular weight, the tendency to aggregate as dimers, and a concomitant loss of 3' to 5' exonuclease activity.