3.2 Mutagenic potential of AFB1-FapyGua in relation to mutational signature of AFB1
High levels of base substitutions at the C/G sites, predominantly C/G > A/T transversions, is a common feature of DNA isolated from AFB1-exposed cells or animals and human AFB1-driven cancers (COSMIC signature SBS24) (Chawanthayatham et al. 2017; Huang et al. 2017; Zhang et al. 2017; Fedeles and Essigmann 2018; Kucab et al. 2019; Alexandrov et al. 2020; Volkova et al. 2020). AFB1-FapyGua has been implicated as a major contributor to AFB1-induced mutagenesis and carcinogenesis (reviewed in (McCullough and Lloyd 2019)) particularly, based on observed correlation between the mutational spectra of AFB1 and the miscoding properties of AFB1-FapyGua in replication bypass reactions (Lin et al. 2016) and cultured primate cells (Lin et al. 2014). However, a limitation of these site-specific studies was that the analyses were restricted to a single, TXA, local sequence context. Here, we substantiated the hypothesis about a major role of AFB1-FapyGua in AFB1-induced mutagenesis and carcinogenesis by demonstrating its high potential for inducing base substitutions, including high frequency G > T transversions, in four additional sequence contexts (Figure 2).
The G > T transversions that constitute signature SBS24 are not distributed randomly, but preferentially arise in a limited subset of trinucleotide sequences (Chawanthayatham et al. 2017; Huang et al. 2017; Zhang et al. 2017; Fedeles and Essigmann 2018; Kucab et al. 2019; Alexandrov et al. 2020; Volkova et al. 2020). Several mechanisms have been considered through which this signature is manifested, including sequence-dependent DNA adduct formation, stability of the initial AFB1-N7-dG adduct, rates of repair, and fidelity of replication bypass. The present data, in combination with previous analyses (Lin et al. 2014), strongly suggest that differential fidelity of replication bypass of AFB1-FapyGua does not account for the occurrence of these mutational hot-spots since the adduct was highly mutagenic in all five sequence contexts. The difference between the hot-spot CGC sequence and three other sequences analyzed in the present study was insignificant (Figure 2). Thus, mechanism(s) that define sequence-dependent occurrence of AFB1-induced mutations likely operates prior to replication. Consistent with such possibility, we recently have demonstrated that sequence context modulates the catalytic efficiency of human glycosylase NEIL1 acting on AFB1-FapyGua, with rates of reactions being inversely correlated with thermal stability of the adduct-containing DNA (Tomar et al. 2021). We speculate that AFB1-FapyGua in a more thermally-stable local environment would be more likely to escape repair not only by NEIL1, but also by nucleotide excision repair and thus, largely contribute to the final mutational signature of AFB1. In support for a role of pre-replication events in shaping the final mutational signature of AFB1 is the distribution of AFB1-induced mutations throughout genome: these are enriched in the intergenic and late-replicating regions and the signature has a transcriptional strand asymmetry (https://cancer.sanger.ac.uk/signatures/sbs/sbs24/).