DNA polymerase .delta.: one polypeptide, two activities

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Abstract
DNA polymerase .delta. from rabbit bone marrow has an associated 3''-5''-exonuclease. Previous studies demonstrated a Stokes radius of 45.5 .ANG. by gel filtration and a sedimentation coefficient of 6.5 S by zone sedimentation. Thus, a MW of 122,000 and a frictional coefficient of 1.39 were calculated. Several problems obstructed further purification and definition of DNA polymerase .delta.. The small amount of protein obtained limited further purification as the nonspecific loss of enzyme in subsequent procedures was excessive. Furthermore, the amount of protein recovered was insufficient for conventinal analysis. These difficulties have been overcome, and DNA polymerase .delta. was purified to apparent homogeneity. Under conditions of nondenaturing microgel electrophoresis, DNA polymerase .delta. aggregates to MW species of 300,000 and higher. In situ assays for DNA polymerase and exonuclease in these gels generated concordant activity profiles. Upon sodium dodecyl sulfate gel electrophoresis, .delta. is a single polypeptide of 122,000 apparent MW. The DNA polymerase incorporates between 250,000 and 300,000 nmol of dTMP into poly(dA)/oligo(dT) (mg of protein)-1 h-1 at 37.degree. C; the exonuclease simultaneously hydrolyzes 13% of the newly synthesized DNA. Aphidicolin, considered to be a specific inhibitor of DNA polymerase .alpha., inhibits both the DNA polymerase and 3''-5''-exonuclease activities of .delta.. DNA polymerase .alpha. from rabbit bone marrow does not share a common subunit with .delta.. Therefore, aphidicolin binding is not specific for .alpha., and conclusions based upon the supposition that it is must be reconsidered.