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).