Human placental DNA polymerase .delta.: identification of a 170 kilodalton polypeptide by activity staining and immunoblotting

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Abstract
DNA polymerase .delta. was isolated from human placenta and identified as such on the basis of its association with a 3''- to 5''-exonuclease activity. The association of the polymerase and exonuclease activities was maintained throughout purification and attempted separations by physical or electrophoretic methods. Moreover, ratios of the two activities remained constant during the purification steps, and both activities were inhibited by aphidicolin, oxidized glutathione, and N-ethylmaleimide. The purified enzyme had an estimated molecular weight of 172,000, on the basis of a Stokes radius of 53.6 .ANG. and a sedimentation coefficient of 7.8 S. On sodium dodecyl sulfate (SDS) gel electrophoresis, polymerase .delta. preparations contained a band of ca. 170 kilodaltons (kDa) as well as several smaller polypeptides. The 170-kDa polypeptide was identified as the largest polypeptide component in the preparation possessing DNA polymerase activity by an activity staining procedure following gel electrophoresis in the presence of SDS. Western blotting of DNA polymerase .delta. with polyclonal antisera also revealed a single 170-kDa immunoreactive polypeptide. Monoclonal antibodies to KB cell polymerase .alpha. inhibited placental polymerase .alpha. but did not inhibit DNA polymerase .delta., while the murine polyclonal antisera to polymerase .delta. inhibited .delta. but not .alpha.. These findings establish the existence of DNA polymerase .delta. in a human tissue and support the view that both its polymerase and its exonuclease activities may be associated with a single protein.

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