Mutagenesis in vitro by DNA polymerase from an RNA tumour virus

Abstract

THE fidelity of DNA synthesis has usually been determined from polynucleotide templates of restricted base composition^1–3. Mis-matched nucleotides were simply identified as those non-complementary to the template. Error rates estimated in this way are less than were predicted on the basis of Watson–Crick base pairing, 10⁻² (refs 4–6), but greater than were inferred from spontaneous mutation frequencies, 10⁻⁸–10⁻¹¹ (ref. 7). However, in vitro error rates measured for the same enzyme vary from one synthetic template to another^3,8,9. In addition, the repeating structure of homopolymers and copolymers may, by slippage and looping-out of mis-matched bases¹⁰, allow a higher frequency of mis-pairing than natural DNA of random sequence. To avoid these problems, we have recently developed a method for measuring the accuracy of in vitro DNA synthesis using a natural DNA template¹¹. We now report that a DNA polymerase which has a high frequency of mis-incorporation with synthetic polynucleotides¹ is also mutagenic when copying natural DNA in vitro.