In vitro replication slippage by DNA polymerases from thermophilic organisms.
Replication slippage of DNA polymerases is a potential source of spontaneous genetic rearrangements in prokaryotic and eukaryotic cells. Here we show that different thermostable DNA polymerases undergo replication slippage in vitro, during single-round replication of a single-stranded DNA template carrying a hairpin structure. Low-fidelity polymerases, such as Thermus aquaticus (Taq), high-fidelity polymerases, such as Pyrococcus furiosus (Pfu) and a highly thermostable polymerase from Pyrococcus abyssi (Pyra exo(-)) undergo slippage. Thermococcus litoralis DNA polymerase (Vent) is also able to slip; however, slippage can be inhibited when its strand-displacement activity is induced. Moreover, DNA polymerases that have a constitutive strand-displacement activity, such as Bacillus stearothermophilus DNA polymerase (Bst), do not slip. Polymerases that slip during single-round replication generate hairpin deletions during PCR amplification, with the exception of Vent polymerase because its strand-displacement activity is induced under these conditions. We show that these hairpin deletions occurring during PCR are due to replication slippage, and not to a previously proposed process involving polymerization across the hairpin base.
Viguera
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Laboratoire de Génétique Microbienne, Institut National de la Recherche Agronomique, Domaine de Vilvert 78350 Jouy-en-Josas, France. viguerame@inta.es
Canceill
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Ehrlich
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D016477:Artifacts; D001483:Base Sequence; D004261:DNA Replication / Q000187:drug effects; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000494:pharmacology; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004795:Enzyme Stability; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D006801:Humans; D008274:Magnesium / Q000494:pharmacology; D008957:Models, Genetic; D016296:Mutagenesis / Q000187:drug effects / Q000235:genetics*; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction; D019711:Pyrococcus / Q000201:enzymology*; D012337:RNA, Ribosomal, 18S / Q000737:chemistry / Q000235:genetics; D017384:Sequence Deletion / Q000235:genetics; D013698:Templates, Genetic; D019712:Thermococcus / Q000201:enzymology*; D013824:Thermus / Q000201:enzymology*
D004268:DNA-Binding Proteins; D012337:RNA, Ribosomal, 18S; D004259:DNA-Directed DNA Polymerase; D008274:Magnesium