Abstract
DNA polymerase was partially purified from mitochondrial extracts of rat liver by phosphocellulose, DEAE-cellulose, heparin-Sepharose CL-6B and DNA-agarose column chromatography. By these purification steps, DNA polymerase and proliferating cell nuclear antigen (PCNA) were completely separated at the step of heparin-Sepharose CL-6B column chromatography. The isolated DNA polymerase was inhibited by ddTTP, but not by aphidicolin. The enzyme sedimented at about 8 S on 5-20 % analytical sucrose density gradient centrifugation. These data showed that the DNA polymerase isolated from mitochondria is γ in type. After the separation of DNA polymerase γ and PCNA, the two fractions were remixed and DNA polymerase γ activity was measured. DNA polymerase γ activity was stimulated about three-fold or more in the presence of the PCNA fraction. This stimulation was inhibited by the addition of anti-PCNA rabbit IgG2a. In addition, highly purified human recombinant PCNA stimulated the DNA polymerase γ activity. These results indicate that DNA polymerase γ, like DNA polymerase δ, is activated by PCNA.
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