Human placental DNA polymerase delta: identification of a 170-kilodalton polypeptide by activity staining and immunoblotting.
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 A 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.
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D004256:DNA Polymerase I / Q000378:metabolism; D004257:DNA Polymerase II / Q000378:metabolism; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase / Q000302:isolation & purification* / Q000378:metabolism; D005260:Female; D006801:Humans; D007106:Immune Sera; D007700:Kinetics; D008970:Molecular Weight; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D013194:Staining and Labeling; D013698:Templates, Genetic
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D007106:Immune Sera; D004256:DNA Polymerase I; D004257:DNA Polymerase II; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase