Mutagen Synergy: Hypermutability Generated by Specific Pairs of Base Analogs

Abstract

We tested pairwise combinations of classical base analog mutagens in Escherichia coli to study possible mutagen synergies. We examined the cytidine analogs zebularine (ZEB) and 5-azacytidine (5AZ), the adenine analog 2-aminopurine (2AP), and the uridine/thymidine analog 5-bromodeoxyuridine (5BrdU). We detected a striking synergy with the 2AP plus ZEB combination, resulting in hypermutability, a 35-fold increase in mutation frequency (to 53,000 ؋ 10 ؊8 ) in the rpoB gene over that with either mutagen alone. A weak synergy was also detected with 2AP plus 5AZ and with 5BrdU plus ZEB. The pairing of 2AP and 5BrdU resulted in suppression, lowering the mutation frequency of 5BrdU alone by 6.5-fold. Sequencing the mutations from the 2AP plus ZEB combination showed the predominance of two new hot spots for A·T¡G·C transitions that are not well represented in either single mutagen spectrum, and one of which is not found even in the spectrum of a mismatch repair-deficient strain. The strong synergy between 2AP and ZEB could be explained by changes in the dinucleoside triphosphate (dNTP) pools. IMPORTANCEAlthough mutagens have been widely studied, the mutagenic effects of combinations of mutagens have not been fully researched. Here, we show that certain pairwise combinations of base analog mutagens display synergy or suppression. In particular, the combination of 2-aminopurine and zebularine, analogs of adenine and cytidine, respectively, shows a 35-fold increased mutation frequency compared with that of either mutagen alone. Understanding the mechanism of synergy can lead to increased understanding of mutagenic processes. As combinations of base analogs are used in certain chemotherapy regimens, including those involving ZEB and 5AZ, these results indicate that testing the mutagenicity of all drug combinations is prudent. Mutagen-induced mutations have been the subject of intensive investigation for decades (e.g., see references 1 to 4; see reviews in references 5 to 7). However, there are far fewer studies of combinations of mutagens. We are studying possible synergies between mutagens in Escherichia coli and initially examined a set of base analog mutagens ( Fig. 1): the cytidine analogs zebularine (ZEB) and 5-azacytidine (5AZ), the adenine analog 2-aminopurine (2AP), and the uridine/thymidine analog 5-bromodeoxyuridine (5BrdU). ZEB lacks the amino group of cytidine (8) and causes C·G¡T·A changes in the rpoB/rifampin resistance (Rif r ) system in E. coli (9). It is used in chemotherapy as a demethylating agent (8, 10, 11) to reverse the effects of gene-silenced tumor suppressor gene (12-15) and because the hydrated form is a potent inhibitor of cytidine deaminase (16). 5AZ, another cytidine analog that is used as a demethylating agent in chemotherapy (12, 13), possesses a unique mutagenic specificity, stimulating only C·G¡G·C changes (17-19). 5AZ and ZEB have been used in combination in chemotherapy (20). 2-Aminopurine results principally in G·C¡A·T and A·T¡G·C changes (e.g., see references 18 and 21 to 25), as...

Epigenetic inhibitor zebularine activates ear pinna wound closure in the mouse

“…Zebularine mutagenicity has not been extensively investigated, except for tests using bacterial cells [56,57]. Zebularine is mainly incorporated instead of cytidine and mispairs with adenine [57].…”

Cytogenetic anomalies of winter wheat cells, induced by chemical contamination of the territory of Kalush industrial district

“…Synergetic responses of biological systems are observed when factors different in their nature act together: gamma-rays and chemical mutagens, heavy metals and radio nuclides, heavy metals and hypothermia, chemical mutagens and ultra-violet rays, laser radiation and magnetic field, etc. (Ang et al, 2016;Song et al, 2016;Kayalvizhi et al, 2017). The existence of a synergetic effect, under the impact of chemical environmental factors, which are particularly seen in small concentrations, was proved on lentils (Lens culinaris Medik.)…”

The Antibiotic Trimethoprim Displays Strong Mutagenic Synergy with 2-Aminopurine

“…It is important to identify increased mutagenesis that results from combinations of antibiotics and/or chemotherapeutics to allow improved risk assessment in employing these combinations and to possibly uncover new pathways of mutagenesis. In a recent study, we looked for synergies between different base analog mutagens and found a very strong synergy between 2-aminopurine (2AP) and zebularine (ZEB), and a strong antagonistic effect of 2AP on 5-bromodeoxyuridine (5BrdU) (16). We attributed the interaction of base analogs to the resulting changes in the ratios of deoxynucleoside triphosphates (dNTPs) that cause increased mutagenesis, in some cases at specific sequences (16).…”

“…In a recent study, we looked for synergies between different base analog mutagens and found a very strong synergy between 2-aminopurine (2AP) and zebularine (ZEB), and a strong antagonistic effect of 2AP on 5-bromodeoxyuridine (5BrdU) (16). We attributed the interaction of base analogs to the resulting changes in the ratios of deoxynucleoside triphosphates (dNTPs) that cause increased mutagenesis, in some cases at specific sequences (16). There is extensive literature from studies of both microbial and higher cells showing that alterations of the dNTP pools can affect mutation rates (17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29).…”

“…We analyzed the mutational spectra resulting from these pairwise combinations by using the rpoB-Rif r system (30), an excellent system for examining the base substitution distribution of mutagens or mutators. It offers 92 mutations to examine that are fairly evenly distributed among the 6 possible base substitutions (16,(30)(31)(32). This is a large enough set to offer a number of sequence contexts for each base substitution but a small enough set to allow one to determine repeated occurrences of mutations at the same site and, thus, define hotspots (favored sites) and in so doing provide in many cases a fingerprint of a specific process.…”