Variation in G-quadruplex Sequence and Topology Differentially Impacts Human DNA Polymerase Fidelity


G-quadruplexes (G4s), a type of non-B DNA with the canonical sequence G≥3N1-7G≥3N1-7G≥3N1-7G≥3, 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 genome stability 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 sought to investigate the impact of G4 sequence and structure on the fidelity of human DNA polymerases delta (δ, four-subunit), eta (η), and kappa (κ). We analyzed errors made within G4 motifs representing those in the human genome that differ in total length, loop sequence, topology, and stability to determine whether these characteristics affect DNA polymerase fidelity. We demonstrate that polymerase fidelity hierarchy (δ4>κ>η) is maintained during G4 synthesis. However, intra-G4 error distributions are variable among the G4 motifs in a manner that is polymerase dependent. In addition, G4 motifs influence the frequency of polymerase errors in 3’ flanking sequences, with the precise impact dependent on G4 sequence and topology. Large- scale errors (deletions, insertions, and complex changes) were observed primarily in constructs containing G4s with parallel strands, further suggesting that G4 topology influences polymerase fidelity. We used in silico analyses to show that most polymerase errors are predicted to have minimal effects on predicted G4 stability; however, all three polymerases do create errors that abolish the potential for G4 formation. Our results provide a unique view of G4s not previously elucidated, showing that G4 motif heterogeneity differentially influences polymerase errors within the motif and flanking sequence. Thus, our study advances the understanding of how DNA replication errors contribute to G4 mutagenesis.

DNA Repair