title: Polypeptide structure of DNA primase from a yeast DNA polymerase-primase complex.
abstract: An immunoaffinity chromatographic procedure was developed to purify DNA polymerase-DNA primase complex from crude soluble extracts of yeast cells. The immunoabsorbent column is made of mouse monoclonal antibody to yeast DNA polymerase I covalently linked to Protein A-Sepharose. Purification of the complex involves binding of the complex to the immunoabsorbent column and elution with concentrated MgCl2 solutions. After rebinding to the monoclonal antibody column free primase activity is selectively eluted with a lower concentration of MgCl2. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed the presence of five major peptides, p180, p140, p74, p58, and p48 in the immunoaffinity-purified DNA polymerase-DNA primase complex. Free primase and free polymerase fractions obtained by fractionation on the immunoabsorbent column were analyzed on activity gels and immunoblots. These analyses showed that p180 and p140 are DNA polymerase peptides. Two polypeptides of 58 and 48 kDa co-fractionated with the free yeast DNA primase. From sucrose gradient analysis we estimate a molecular weight of 110 kDa for the native DNA primase.
mesh_terms: D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity; D019915:DNA Primase; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D011071:Poly T / Q000378:metabolism; D011487:Protein Conformation; D012316:RNA Nucleotidyltransferases / Q000032:analysis*; D012441:Saccharomyces cerevisiae / Q000201:enzymology
chemical_list: D011071:Poly T; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D004259:DNA-Directed DNA Polymerase