Effect of episomally encoded DNA polymerases on chemically induced mutagenesis at the target in Ames test.

BACKGROUND

The standard Ames test strains owe their high sensitivity to chemical and physical mutagens to the episomal Y-family DNA polymerase RI encoded by the operon. The test strains carry also another related operon on a 60-kDa cryptic plasmid. In contrast to the chromosomally encoded Y-family DNA polymerases V and IV, these plasmid born polymerase genes have no direct counterpart in mammalian cells. By replicating damaged templates, DNA polymerases play a central role in mutagenesis and genome stability. It is therefore imperative to investigate their specificity to understand differences in mutagenesis between the prokaryotic versus eukaryotic (mammalian) systems. To this end we have isolated and separately expressed the DNA polymerase subunits encoded by the and operons. After demonstrating how these enzymes control chemical and UV mutagenesis at the standard and Ames test targets, we are now adding the third Ames test target to the trilogy.

RESULTS

Four new Ames tester strains based on the target have been constructed expressing the activated DNA polymerase MucA' and SamA' accessory subunits combined with the MucB and SamB catalytical subunits under the control of promoter. These polymerase assemblies were substituted for the endogenous PolRI, PolV and SamAB polymerases present in the standard TA100 strain and tested for their abilities to promote chemically induced mutagenesis. SamA' + SamB has been able to promote mutagenesis induced by AF-2 and 1,8-DNP to higher extent than SamA' + MucB. The MucA' + MucB (PolRI*) more efficiently promoted MMS as well as spontaneous mutagenesis than its wild type counterpart but was less efficient for other mutagens including AFB1. Strikingly azide mutagenesis was inhibited by PolRI and also SamA'B.

CONCLUSION

A new system for SOS-independent overexpression of the activated DNA polymerases RI and SamA'B and their chimeras in the Ames test background has been established and validated with several representative mutagens. Overall, the TA100 strain showed the highest sensitivity towards most tested mutagens. The observed inhibition of azide mutagenesis by PolRI* suggests that this type of Y-family DNA polymerases can perform also "corrective" replication on a damaged DNA.