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

Petr Grúz,Masatomi Shimizu,Masamitsu Honma,Kei-Ichi Sugiyama,Masami Yamada

doi:10.1186/s41021-020-00154-2

Abstract

BackgroundThe 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.ResultsFour 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.ConclusionA 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.

Highlights

The Ames test has been considered a golden standard for the mutagenicity assays and is used worldwide by the government regulatory agencies to assess the safety of chemicals [1]
Portable polymerase expression system We have previously constructed a versatile plasmidbased system for the controllable expression of the activated Y-family DNA polymerases encoded by the episomal mucA + mucB and samA + samB genes in the standard S. typhimurium Ames tester strains [19]
The strain YG9028 expresses the active form of the DNA polymerase RI, sometimes referred to as PolRI*, which has been already biochemically characterized in our and other labs and which is the enzyme responsible for the great sensitivity of the Ames tester strains like TA100, TA104, TA98 or TA97 to various chemical and physical mutagens [6, 25]

Summary

Introduction

The Ames test has been considered a golden standard for the mutagenicity assays and is used worldwide by the government regulatory agencies to assess the safety of chemicals [1]. DNA polymerases play a key role in mutagenesis by deciding whether unrepaired damaged DNA will be tolerated and replicated often at the expense of altering the genetic code and introducing mutations or whether it will arrest cell division leading to death. One of the highest error prone DNA polymerases is DNA polymerase RI encoded by the mucAB operon carried on the plasmid pKM101 [5, 6] It is utilized in the Ames test where the pKM101 plasmid incorporated in the standard battery of strains greatly enhances sensitivity to various chemical and physical mutagens [7]. It is 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 adding the third Ames test target hisG46 to the trilogy

Methods

Results

Discussion

Conclusion