G-quadruplexes (G4s) are functional elements of the human genome, some of which inhibit DNA replication. We investigated replication of G4s within highly abundant microsatellite (GGGA, GGGT) and transposable element (L1 and SVA) sequences. We found that genome-wide, numerous motifs are located preferentially on the replication leading strand and the transcribed strand templates. We directly tested replicative polymerase ϵ and δ holoenzyme inhibition at these G4s, compared to low abundant motifs. For all G4s, DNA synthesis inhibition was higher on the G-rich than C-rich strand or control sequence. No single G4 was an absolute block for either holoenzyme; however, the inhibitory potential varied over an order of magnitude. Biophysical analyses showed the motifs form varying topologies, but replicative polymerase inhibition did not correlate with a specific G4 structure. Addition of the G4 stabilizer pyridostatin severely inhibited forward polymerase synthesis specifically on the G-rich strand, enhancing G/C strand asynchrony. Our results reveal that replicative polymerase inhibition at every G4 examined is distinct, causing complementary strand synthesis to become asynchronous, which could contribute to slowed fork elongation. Altogether, we provide critical information regarding how replicative eukaryotic holoenzymes navigate synthesis through G4s naturally occurring thousands of times in functional regions of the human genome.

• MeSH
• aminochinoliny MeSH
• DNA-polymerasa II * antagonisté a inhibitory   metabolismus   MeSH
• DNA-polymerasa III * antagonisté a inhibitory   metabolismus   MeSH
• DNA chemie   MeSH
• G-kvadruplexy * MeSH
• genom lidský * MeSH
• holoenzymy metabolismus   MeSH
• kyseliny pikolinové farmakologie   MeSH
• lidé MeSH
• mikrosatelitní repetice MeSH
• proteiny vázající poly-ADP-ribosu MeSH
• replikace DNA * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• aminochinoliny MeSH
• DNA-polymerasa II * MeSH
• DNA-polymerasa III * MeSH
• DNA MeSH
• holoenzymy MeSH
• kyseliny pikolinové MeSH
• POLD3 protein, human MeSH Prohlížeč
• POLE protein, human MeSH Prohlížeč
• proteiny vázající poly-ADP-ribosu MeSH
• pyridostatin MeSH Prohlížeč

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.

• Klíčová slova
• DNA polymerase δ, DNA polymerase η, DNA polymerase κ, DNA replication, Error signature, Non-B DNA,
• MeSH
• DNA-dependentní DNA-polymerasy genetika   metabolismus   MeSH
• DNA genetika   MeSH
• G-kvadruplexy * MeSH
• lidé MeSH
• replikace DNA MeSH
• vaskulární endoteliální růstový faktor A genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• DNA-dependentní DNA-polymerasy MeSH
• DNA MeSH
• vaskulární endoteliální růstový faktor A MeSH