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Structural and dynamic characterization of polymerase ?'s minor groove lesion processing reveals how adduct topology impacts fidelity.

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 ? (Pol?), 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 Pol? 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 N(2)-dG covalent adduct derived from the carcinogenic aromatic amine, 2-acetylaminofluorene (dG-N(2)-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 Pol?'s N-clasp facilitates the significant misincorporation of dTTP opposite dG-N(2)-AAF via wobble pairing. Notably, we show that N-clasp flexibility depends on lesion topology, being markedly reduced in the case of the benzo[a]pyrene-derived major adduct to N(2)-dG, whose bypass by Pol? is nearly error-free. Thus, our studies reveal how Pol?'s unique structural and dynamic properties can regulate its bypass fidelity of polycyclic aromatic lesions and how the fidelity is impacted by lesion structures.

SUBMITTER: Lior-Hoffmann L

PROVIDER: S-EPMC4159208 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature

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Project description:G-quadruplexes (G4s), a type of non-B DNA, play important roles in a wide range of molecular processes, including replication, transcription, and translation. Genome integrity relies on efficient and accurate DNA synthesis, and is compromised by various stressors, to which non-B DNA structures such as G4s can be particularly vulnerable. However, the impact of G4 structures on DNA polymerase fidelity is largely unknown. Using an in vitro forward mutation assay, we investigated the fidelity of human DNA polymerases delta (δ4, four-subunit), eta (η), and kappa (κ) during synthesis of G4 motifs representing those in the human genome. The motifs differ in sequence, topology, and stability, features that may affect DNA polymerase errors. Polymerase error rate hierarchy (δ4 < κ < η) is largely maintained during G4 synthesis. Importantly, we observed unique polymerase error signatures during synthesis of VEGF G4 motifs, stable G4s which form parallel topologies. These statistically significant errors occurred within, immediately flanking, and encompassing the G4 motif. For pol δ4, the errors were deletions, insertions and complex errors within the G4 or encompassing the G4 motif and surrounding sequence. For pol η, the errors occurred in 3' sequences flanking the G4 motif. For pol κ, the errors were frameshift mutations within G-tracts of the G4. Because these error signatures were not observed during synthesis of an antiparallel G4 and, to a lesser extent, a hybrid G4, we suggest that G4 topology and/or stability could influence polymerase fidelity. Using in silico analyses, we show that most polymerase errors are predicted to have minimal effects on predicted G4 stability. Our results provide a unique view of G4s not previously elucidated, showing that G4 motif heterogeneity differentially influences polymerase fidelity within the motif and flanking sequences. Thus, our study advances the understanding of how DNA polymerase errors contribute to G4 mutagenesis.

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Structural and dynamic characterization of polymerase ?'s minor groove lesion processing reveals how adduct topology impacts fidelity.

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