3.3 Replication bypass of AFB1-FapyGua: modulation by sequence context and proposed mechanisms
Although AFB1-FapyGua was nearly equally mutagenic in all sequence contexts tested, the spectra of mutations were partially affected by the identities of neighboring nucleotides. Specifically, there was a trend for elevated frequencies of G > A transitions in the AXC sequence context. The frequency of G > C transversions was increased in the CXA context. These observations suggest that replication bypass of AFB1-FapyGua can proceed via the primer-template misalignment/realignment mechanism, with the 5’ adjacent nucleotide being utilized as a template instead of modified nucleotide. It is possible that at least a fraction of G > T transversions generated in either the TXC context or the previously tested TXA context (Lin et al. 2014), is formed via a similar mechanism.
Our data demonstrate that even in the absence of a T at neighboring positions, dAMP is preferentially inserted opposite AFB1-FapyGua in primate cells. The mechanism accounting for such a mutagenic event is currently unknown. It could be formation of a mispair between the modified nucleotide and incoming dATP, as was proposed for unsubstituted FapyGua (Gehrke et al. 2013), or non-templated dAMP incorporation, according to the “A rule” (Strauss 2002), as previously proposed for α-anomers of nitrogen mustard-induced FapyGua (Minko et al. 2017). With regard to the later proposal, AFB1-FapyGua exists as a complex mixture of various isomeric species that include interconvertible α- and β-anomers (Brown et al. 2009; Li et al. 2015). The fraction of α-anomeric species is substantial in single-stranded DNA (Brown et al. 2009).
The identity of DNA polymerase(s) responsible for bypass of AFB1-FapyGua has been addressed by biochemical studies with recombinant human DNA polymerases (Lin et al. 2014). The majority of these enzymes could not synthesize DNA past AFB1-FapyGua. The only polymerase that could, pol ζ, did it in an extremely error-prone manner by preferentially inserting a dAMP opposite the adduct and extending exclusively from this mispair. Further validation of the role of pol ζ in the bypass of the AFB1-FapyGua adduct was obtained from differential cytotoxicity, cell cycle progression, and chromosomal abnormalities when cells proficient or deficient in pol ζ were challenged with the AFB1 epoxide (Lin et al. 2016).