DNA primase isolated from the yeast DNA primase-DNA polymerase complex. Immunoaffinity purification and analysis of RNA primer synthesis

J Biol Chem. 1989 Feb 25;264(6):3602-10.

Abstract

We have utilized immunoaffinity chromatography as a means of efficiently isolating a stable yeast DNA primase from the DNA primase-DNA polymerase complex, allowing identification of the polypeptides associated with this DNA primase activity and comparison of its enzymatic properties with those of the larger protein complex. A mouse monoclonal antibody specifically recognizing the DNA polymerase subunit was used to purify the complex. Stable DNA primase was subsequently separated from the complex in high yield. The highly purified protein fraction which bound to the DNA polymerase antibody column consisted of polypeptides with apparent molecular masses of 180, 86, 70, 58, 49, and 47 kDa. DNA primase activity eluted with a fraction containing only the 58-, 49-, and 47-kDa polypeptides. Partial chemical cleavage analysis of these three proteins demonstrated that the 49- and 47-kDa polypeptides are structurally related while the 58-kDa protein is unrelated to the other two. A DNA primase inhibitory monoclonal antibody was able to inhibit the activity of the purified DNA primase as well as the activity of the enzyme in the larger complex. In immunoprecipitation experiments, all three polypeptides were found in the immune complex. Thus, these three polypeptides are sufficient for DNA primase activity. In reactions using ribonucleotide substrates and natural as well as synthetic DNA templates, the purified DNA primase exhibited the same precise synthesis of unit length oligomers as did the larger protein complex and was able to extend these RNA oligomers by one additional unit length. An examination of the effects of deoxynucleotides on these DNA primase-catalyzed reactions revealed that the yeast DNA primase is an RNA-polymerizing enzyme and lacks significant DNA-polymerizing activity under the conditions tested.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Animals
  • Antibodies, Monoclonal
  • Chromatography, Affinity
  • DNA Primase
  • DNA-Directed DNA Polymerase / metabolism
  • Deoxyribonucleotides / pharmacology
  • Electrophoresis, Polyacrylamide Gel
  • Immunoassay
  • Immunosorbent Techniques
  • Magnesium
  • Magnesium Chloride
  • Mice
  • Molecular Weight
  • Peptide Fragments
  • Peptides / isolation & purification
  • RNA / biosynthesis*
  • RNA Nucleotidyltransferases / isolation & purification*
  • RNA Nucleotidyltransferases / metabolism
  • RNA, Fungal / biosynthesis*
  • Saccharomyces cerevisiae / enzymology*
  • Templates, Genetic

Substances

  • Antibodies, Monoclonal
  • Deoxyribonucleotides
  • Peptide Fragments
  • Peptides
  • RNA primers
  • RNA, Fungal
  • Magnesium Chloride
  • RNA
  • Adenosine Triphosphate
  • DNA Primase
  • RNA Nucleotidyltransferases
  • DNA-Directed DNA Polymerase
  • Magnesium