Effect of episomally encoded DNA polymerases on chemically induced mutagenesis at the hisG46 target in Ames test

Abstract

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 mucAB operon. The S. typhimurium test strains carry also another related samAB 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 mucAB and samAB operons. After demonstrating how these enzymes control chemical and UV mutagenesis at the standard hisD3052 and hisG428 Ames test targets, we are now adding the third Ames test target hisG46 to the trilogy. Results: Four new Ames tester strains based on the hisG46 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 lac 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 hisG46 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" error free replication on a damaged DNA.

Yeast grown in continuous culture systems can detect mutagens with improved sensitivity relative to the Ames test

“…To assess the ability of these chemostats to detect lower concentrations of mutagens, we first focused on testing the DNA methylating agent methyl methanesulfonate (MMS) [ 42 ]. Given that the limit of detection for the Ames test is approximately 2.2–4.4 mg MMS/L [ 43 , 44 ], we began by growing yeast in four parallel chemostats with 0, 0.1 1.0, and 10 mg MMS/L ( Fig 2A ). Our lowest concentration of MMS tested, 0.1 mg MMS/L, corresponds to 22 times lower than the Ames test detection limit.…”

“…The blue box represents the normalized average for the number of Can-resistant colonies in the no mutagen control (that is, 1) + normalized standard deviation. The Ames test limits of detection are 2.2-4.4 mg MMS/L[43,44] and 2.0 μg EtBr/L[21]. A Tukey-HSD test was used to determine statistical significance ( � = p < 0.05, �� = p < 0.01, ��� = p < 0.001).https://doi.org/10.1371/journal.pone.0235303.g002…”

Yeast grown in continuous culture systems can detect mutagens with improved sensitivity relative to the Ames test

“…To assess the ability of these chemostats to detect lower concentrations of mutagens, we first focused on testing the DNA methylating agent methylmethane sulfonate (MMS) [31]. Given that the limit of detection for the Ames test is approximately 2.2 - 4.4 mg MMS/L [32,33], we began by growing yeast in four parallel chemostats with 0, 0.1, 1.0, and 10 mg MMS/L ( Fig 2A ). About every three days for a month, yeast from each chemostat were plated on synthetic media plates without arginine and supplemented with canavanine.…”

“…For other conditions, the number of Can-resistant colonies is represented as the normalized average +/-normalized standard deviation. The Ames test limit of detections are 2.2 - 4.4 mg MMS/L [32,33] and 2.0 - 31.5 [33,35] μg EtBr/L.…”