Reactions in vitro of the DNA polymerase‐primase from Xenopus laevis eggs

A form of DNA polymerase α 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 < 1‐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 2mM 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.