In vitro replication slippage by DNA polymerases from thermophilic organisms.

Abstract:

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.

Polymerases:

Topics:

Fidelity, Nucleotide Incorporation, Exonuclease Activity, Source / Purification

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
Pfu Viguera E2001 Overall Error Rate 1.3E-06 errors/bp
Pfu Viguera E2001 Full length or truncated Full length
Vent Viguera E2001 3-5' Exonuclease (proofreading) Yes
Vent Viguera E2001 Full length or truncated Full length
Vent Viguera E2001 Strand Displacement Yes
Bst Viguera E2001 Strand Displacement Yes
Tfu Viguera E2001 3-5' Exonuclease (proofreading) Yes
Tfu Viguera E2001 Strand Displacement Yes
Pyra exo- Viguera E2001 3-5' Exonuclease (proofreading) No
Pyra exo- Viguera E2001 Strand Displacement No

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