Reactions in vitro of the DNA polymerase-primase from Xenopus laevis eggs. A role for ATP in chain elongation

Eur J Biochem. 1983 Oct 3;135(3):435-42. doi: 10.1111/j.1432-1033.1983.tb07670.x.


A form of DNA polymerase alpha was purified several thousandfold from a protein extract of Xenopus laevis eggs. The enzyme effectively converts, in the presence of ribonucleoside triphosphates, a circular single-stranded phage fd DNA template into a double-stranded DNA form and, therefore, must be associated with a DNA primase. We first show by gel electrophoresis in the presence of sodium dodecyl sulfate that both enzymatic activities, DNA polymerase and primase, most probably reside on a greater than 100 000-Da subunit of the DNA polymerase holoenzyme. We then assayed the polymerase-primase at various template/enzyme ratios and found that the DNA complementary strand sections synthesized in vitro belong to defined size classes in the range of 600-2000 nucleotides, suggesting preferred start and/or stop sites on the fd DNA template strand. We show that the stop sites coincide with stable hairpin structures in fd DNA. We have used a fd DNA template, primed by a restriction fragment of known size, to show that the polymerase-primase stops at the first stable hairpin structure upstream from the 3'-OH primer site when the reaction was carried out at 0.1 mM ATP. However, at 2 mM ATP the enzyme was able to travers this and other stop sites on the fd DNA template strand leading to the synthesis of 2-4 times longer DNA strands. Our results suggest a role for ATP in the polymerase-primase-catalyzed chain-elongation reaction.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / physiology*
  • Animals
  • Chemical Phenomena
  • Chemistry
  • DNA Primase
  • DNA, Circular / metabolism
  • DNA, Single-Stranded / metabolism
  • DNA-Directed DNA Polymerase / metabolism*
  • Electrophoresis, Polyacrylamide Gel
  • Female
  • In Vitro Techniques
  • Ovum / metabolism
  • RNA Nucleotidyltransferases / metabolism*
  • Xenopus laevis


  • DNA, Circular
  • DNA, Single-Stranded
  • Adenosine Triphosphate
  • DNA Primase
  • RNA Nucleotidyltransferases
  • DNA-Directed DNA Polymerase