Immunological analysis of the polypeptide structure of calf thymus DNA polymerase-primase complex.
Five major polypeptides are found in immunoaffinity-purified calf thymus DNA polymerase-DNA primase complex: 185, 160, 68, 55, and 48 kDa. Individual polypeptides purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were used to produce antibodies in rabbits to aid in identifying the relationships between these polypeptides by immunoblotting and enzyme neutralization procedures. Immunoblot analyses showed that the 160-kDa peptide is derived from the 185-kDa peptide and the 48-kDa peptide is derived from the 68-kDa peptide while antibodies to the 55-kDa peptide do not cross-react with other peptides found in the complex. Direct enzyme neutralization studies demonstrated that antibodies to 185- and 160-kDa peptides inhibit DNA polymerase activity in the complex, confirming earlier suggestions that these peptides are the catalytic peptides for DNA polymerase. DNA primase activity in the complex is inhibited by antibodies to 68-, 55-, and 48-kDa peptides and to a lesser extent by antibodies to the 160-kDa peptide. Free DNA primase isolated from the complex was estimated to have a native molecular weight of about 110,000. The 55- and 48-kDa peptides are found to be associated with the free primase activity. Rabbit antibodies to both 55- and 48-kDa peptides are inhibitory to this primase activity. From these results we suggest that the native calf thymus DNA polymerase-DNA primase complex contains only three unique peptides with the 185-kDa peptide as the catalytic peptide of DNA polymerase and the 55- and 68-kDa peptides constituting the primase peptides. A model illustrating the roles of these peptides in initiation and replication of DNA is presented.
D000818:Animals; D000936:Antigen-Antibody Complex; D002417:Cattle; D002499:Centrifugation, Density Gradient; D019915:DNA Primase; D004591:Electrophoresis, Polyacrylamide Gel; D007074:Immunoglobulin G; D007700:Kinetics; D009097:Multienzyme Complexes / Q000032:analysis* / Q000276:immunology; D010455:Peptides / Q000302:isolation & purification; D012316:RNA Nucleotidyltransferases / Q000032:analysis* / Q000276:immunology; D013950:Thymus Gland / Q000201:enzymology*
D000936:Antigen-Antibody Complex; D007074:Immunoglobulin G; D009097:Multienzyme Complexes; D010455:Peptides; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases