Exposure
to aristolochic acid I and II (AAI and AAII) has been
implicated in aristolochic acid nephropathy and urothelial carcinoma.
The toxicological effects of AAs are attributed to their ability to
form aristolacatam (AL)-purine DNA adducts. Among these lesions, the
AL-adenine (ALI-N6-A and ALII-N6-A) adducts
cause the “signature” A → T transversion mutations
associated with AA genotoxicity. To provide the currently missing
structural basis for the induction of these signature mutations, the
present work uses classical all-atom molecular dynamics simulations
to examine different (i.e., preinsertion, insertion, and postextension)
stages of replication past the most abundant AA adduct (ALI-N6-A) by a representative lesion-bypass DNA polymerase (Dpo4).
Our analysis reveals that, before dNTP incorporation (i.e., preinsertion
step), ALI-N6-A adopts a nearly planar conformation at
the N6-linkage and the ALI moiety intercalates within the
DNA helix. Since this conformation occupies the dNTP binding site,
the same planar lesion conformation results in a significant distortion
of the polymerase active site at the insertion step and therefore
replication will likely not be successful. However, if ALI-N6-A undergoes a small conformational change to introduce non-planarity
at the N6-linkage during the insertion step, minimal distortion
occurs in the Dpo4 active site upon incorporation of dATP. This insertion
and subsequent extension would initially lead to A:A mismatches and
then result in A → T transversion mutations during the second
round of replication. In contrast, if a large conformation flip of
the ALI moiety occurs at the insertion step to reorient the bulky
moiety from an intercalated position into the major groove, dTTP (non-mutagenic)
incorporation will be favored. Molecular dynamics (MD) simulations
on postextension complexes reveal that damaged DNA will likely further
rearrange during later replication steps to acquire a base-displaced
intercalated conformation that is similar to that previously reported
for (unbound) ALI-N6-A adducted DNA, with the exception
of slight non-planarity at the lesion site. Overall, our results provide
a structural explanation for both the successful non-mutagenic lesion
bypass and the preferential misincorporation of dATP opposite ALI-N6-A and thereby rationalize the previously reported induction
of A → T signature transversion mutations associated with AAs.
This work should thereby inspire future biochemical experiments and
modeling studies on the replication of this important class of DNA
lesions by related human translesion synthesis polymerases.