Nucleoside Analogues Are Potent Inducers of Pol V-mediated Mutagenesis

Abstract

Drugs targeting DNA and RNA in mammalian cells or viruses can also affect bacteria present in the host and thereby induce the bacterial SOS system. This has the potential to increase mutagenesis and the development of antimicrobial resistance (AMR). Here, we have examined nucleoside analogues (NAs) commonly used in anti-viral and anti-cancer therapies for potential effects on mutagenesis in Escherichia coli, using the rifampicin mutagenicity assay. To further explore the mode of action of the NAs, we applied E. coli deletion mutants, a peptide inhibiting Pol V (APIM-peptide) and metabolome and proteome analyses. Five out of the thirteen NAs examined, including three nucleoside reverse transcriptase inhibitors (NRTIs) and two anti-cancer drugs, increased the mutation frequency in E. coli by more than 25-fold at doses that were within reported plasma concentration range (Pl.CR), but that did not affect bacterial growth. We show that the SOS response is induced and that the increase in mutation frequency is mediated by the TLS polymerase Pol V. Quantitative mass spectrometry-based metabolite profiling did not reveal large changes in nucleoside phosphate or other central carbon metabolite pools, which suggests that the SOS induction is an effect of increased replicative stress. Our results suggest that NAs/NRTIs can contribute to the development of AMR and that drugs inhibiting Pol V can reverse this mutagenesis.

The antiretroviral agents azidothymidine, stavudine, and didanosine have the identical mutational fingerprint in the rpoB region of Escherichia coli

“…The frequencies are given in Table 1. The increases over background are significant, but not as high as those reported by Sumabe et al (2021).…”

“…AZT, STAV, and DIDA are important antiretroviral agents that are employed as part of combination therapy against HIV and other diseases, as they have a high affinity for reverse transcriptases (Furman et al, 1986; Olivero, 2007). However, they can also be incorporated into cellular DNA (Ellwell et al, 1987; Scruggs & Dirks Naylor, 2008; Seier et al, 2012; Sumabe et al, 2021). These compounds act as chain breakers, which leads to single and double‐strand gaps that ultimately lead to cell killing (Cooper & Lovett, 2011).…”

“…Lovett and coworkers have found that recombination functions are involved in the protection against cell killing (Brown et al, 2015; Cooper & Lovett, 2011). These compounds also induce mutations in cellular DNA (Seier et al, 2012; Sumabe et al, 2021). The mechanism of much of this mutagenesis is unclear.…”

“…With that in mind, we can state that a significant finding of this work is that DIDA shows the same mutational fingerprint in the rpoB/Rifr system as both AZT and STAV, despite the former being an adenylate analog, and the latter two being thymidine analogs. Because AZT has been shown to induce the SOS system (Seier et al, 2012) and to require Pol V for mutagenesis (Sumabe et al, 2021), one might consider that the A:T→C:G changes observed result not from directly targeted lesion, but rather as a result of the translesion polymerase PolV making errors across from undamaged bases. However, analysis of mutations in rpoB occurring as a result of Pol IV and Pol V induction do not show a preference for A:T→C:G substitutions (Curti et al, 2009; Wolff et al, 2004).…”

“…Azidothymidine (AZT; Zidovudine; 3′‐azido‐3′‐deoxythymidine), stavudine (STAV; 2′,3′‐dideoxythymidine), and didanosine (DIDA; 2′,3′‐dideoxyinosine), depicted in Figure 1, are reverse transcriptase inhibitors that are widely used in the treatment of HIV‐1 and other retroviral infections. Although they have higher affinity for reverse transcriptases than cellular DNA polymerases (Furman et al, 1986; Olivero, 2007), still, they are incorporated into cellular DNA and cause mutations and toxicity in both higher cells and bacteria (Ellwell et al, 1987; Scruggs & Dirks Naylor, 2008; Seier et al, 2012; Sumabe et al, 2021). All three compounds act as chain terminators, since they lack the OH group on the 3′ position of the deoxyribose moiety (Figure 1), preventing the extension of the growing DNA chain (Olivero, 2007; Thompson & Lamont, 2019).…”