Department of Pathology, The Jake Gittlen Laboratories for Cancer Research, Penn State University College of Medicine, Hershey, PA, USA.
Department of Biology, Penn State University Eberly College of Science, University Park, PA, USA.
DNA Repair (Amst). 2022 Nov;119:103402. doi: 10.1016/j.dnarep.2022.103402. Epub 2022 Sep 9.
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.
四链体（G4s）是一种非 B 型 DNA，在多种分子过程中发挥重要作用，包括复制、转录和翻译。基因组完整性依赖于高效和准确的 DNA 合成，而各种应激源会破坏基因组完整性，非 B 型 DNA 结构如 G4s 特别容易受到影响。然而，G4 结构对 DNA 聚合酶保真度的影响在很大程度上是未知的。我们使用体外正向突变测定法，研究了人类 DNA 聚合酶 δ（δ4，四聚体）、η 和 κ 在合成代表人类基因组中 G4 基序时的保真度。这些基序在序列、拓扑和稳定性上存在差异，这些特征可能影响 DNA 聚合酶错误。在 G4 合成过程中，聚合酶错误率层次结构（δ4 < κ < η）基本保持不变。重要的是，我们观察到在 VEGF G4 基序的合成过程中出现了独特的聚合酶错误特征，这些基序是稳定的平行拓扑结构。这些具有统计学意义的错误发生在 G4 基序内、紧邻 G4 基序的区域以及包含 G4 基序和周围序列的区域。对于 pol δ4，错误是 G4 内或包含 G4 基序和周围序列的 G4 内的缺失、插入和复杂错误。对于 pol η，错误发生在 G4 基序侧翼的 3'序列中。对于 pol κ，错误是 G4 内 G 链中的移码突变。由于这些错误特征在合成反平行 G4 时以及在较小程度上合成杂交 G4 时没有观察到，因此我们认为 G4 拓扑结构和/或稳定性可能会影响聚合酶保真度。我们使用计算机分析表明，大多数聚合酶错误预计对预测的 G4 稳定性的影响最小。我们的结果提供了以前未阐明的 G4s 的独特视角，表明 G4 基序的异质性会在基序内和侧翼序列中对聚合酶保真度产生不同的影响。因此，我们的研究加深了对 DNA 聚合酶错误如何导致 G4 突变的理解。
