Structural and Dynamic Characterization of Polymerase
κ’s Minor Groove Lesion Processing Reveals How Adduct
Topology Impacts Fidelity
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Abstract
DNA
lesion bypass polymerases process different lesions with varying
fidelities, but the structural, dynamic, and mechanistic origins of
this phenomenon remain poorly understood. Human DNA polymerase κ
(Polκ), a member of the Y family of lesion bypass polymerases,
is specialized to bypass bulky DNA minor groove lesions in a predominantly
error-free manner, by housing them in its unique gap. We have investigated
the role of the unique Polκ gap and N-clasp structural features
in the fidelity of minor groove lesion processing with extensive molecular
modeling and molecular dynamics simulations to pinpoint their functioning
in lesion bypass. Here we consider the <i>N</i><sup>2</sup>-dG covalent adduct derived from the carcinogenic aromatic amine,
2-acetylaminofluorene (dG-<i>N</i><sup>2</sup>-AAF), that
is produced via the combustion of kerosene and diesel fuel. Our simulations
reveal how the spacious gap directionally accommodates the lesion
aromatic ring system as it transits through the stages of incorporation
of the predominant correct partner dCTP opposite the damaged guanine,
with preservation of local active site organization for nucleotidyl
transfer. Furthermore, flexibility in Polκ’s N-clasp
facilitates the significant misincorporation of dTTP opposite dG-<i>N</i><sup>2</sup>-AAF via wobble pairing. Notably, we show that
N-clasp flexibility depends on lesion topology, being markedly reduced
in the case of the benzo[<i>a</i>]pyrene-derived major adduct
to <i>N</i><sup>2</sup>-dG, whose bypass by Polκ is
nearly error-free. Thus, our studies reveal how Polκ’s
unique structural and dynamic properties can regulate its bypass fidelity
of polycyclic aromatic lesions and how the fidelity is impacted by
lesion structures