| Abstract: | Using a modified system to measure fidelity at an amber site in phi X174, we have employed DNA polymerase alpha to test different mechanisms for proofreading. DNA polymerase alpha does not exhibit the characteristics of "kinetic proofreading" seen with procaryotic polymerases. Polymerase alpha shows no evidence for a "next nucleotide" effect, and added deoxynucleoside monophosphates do not alter fidelity. Pyrophosphate, which increases error rates with a procaryotic polymerase, appears to weakly improve polymerase alpha fidelity. DNA polymerase alpha does exhibit a dramatic increase in error rate in the presence of a deoxycytidine thiotriphosphate (dCTP alpha S), but this enhanced mutagenesis also occurs under conditions where kinetic proofreading should be otherwise defeated. This particular effect with dCTP alpha S appears specific for DNA polymerase alpha and is not seen with the other polymerases tested. |
