Reactions in vitro of the DNA polymerase-primase from Xenopus laevis eggs. A role for ATP in chain elongation.
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.
D000255:Adenosine Triphosphate / Q000502:physiology*; D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D019915:DNA Primase; D004270:DNA, Circular / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D066298:In Vitro Techniques; D010063:Ovum / Q000378:metabolism; D012316:RNA Nucleotidyltransferases / Q000378:metabolism*; D014982:Xenopus laevis
D004270:DNA, Circular; D004277:DNA, Single-Stranded; D000255:Adenosine Triphosphate; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D004259:DNA-Directed DNA Polymerase