Immunochemical detection of a primase activity related subunit of DNA polymerase alpha from human and mouse cells using the monoclonal antibody.
A hybrid cell line (HDR-854-E4) secreting monoclonal antibody (E4 antibody) against a subunit of human DNA polymerase alpha was established by immunizing mice with DNA replicase complex (DNA polymerase alpha-primase complex) prepared from HeLa cells. The E4 antibody immunoprecipitates DNA replicase complex from both human and mouse cells. The E4 antibody neutralizes the primase activity as assessed either by the direct primase assay (incorporation of [alpha-32P]AMP) or by assay of DNA polymerase activity coupled with the primase activity using unprimed poly(dT) as a template. The E4 antibody does not neutralize DNA polymerase alpha activity with the activated calf thymus DNA as a template. Western immunoblotting analysis shows that the E4 antibody binds to a polypeptide of 77 kilodaltons (kDa) which is tightly associated with DNA polymerase alpha. The 77-kDa polypeptide was distinguished from the catalytic subunit (160 and 180 kDa) for DNA synthesis which was detected by another monoclonal antibody, HDR-863-A5. Furthermore, it is unlikely that the 77-kDa peptide is the primase, since we found that the E4 antibody also immunoprecipitates the mouse 7.3S DNA polymerase alpha which has no primase activity, and Western immunoblotting analysis shows that the 77-kDa polypeptide is a subunit of the 7.3S DNA polymerase alpha. Furthermore, after dissociation of the primase from mouse DNA replicase by chromatography on a hydroxyapatite column in the presence of dimethyl sulfoxide and ethylene glycol, the 77-kDa polypeptide is associated with DNA polymerase alpha, and not with the primase.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D002286:Carcinoma, Ehrlich Tumor / Q000201:enzymology; D004257:DNA Polymerase II / Q000032:analysis* / Q000276:immunology; D019915:DNA Primase; D000939:Epitopes / Q000032:analysis; D006367:HeLa Cells / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D051379:Mice; D012316:RNA Nucleotidyltransferases / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D013045:Species Specificity
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D000939:Epitopes; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D004257:DNA Polymerase II