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
• 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
• 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 are noncanonical nucleic acid structures formed by stacked guanosine tetrads. Despite their functional and structural diversity, a single consensus model is typically used to describe sequences with the potential to form G-quadruplex structures. We are interested in developing more specific sequence models for G-quadruplexes. In previous work, we functionally characterized each sequence in a 496-member library of variants of a monomeric reference G-quadruplex for the ability to bind GTP, promote a model peroxidase reaction, generate intrinsic fluorescence, and to form multimers. Here we used NMR to obtain a broad overview of the structural features of this library. After determining the 1H NMR spectrum of each of these 496 sequences, spectra were sorted into multiple classes, most of which could be rationalized based on mutational patterns in the primary sequence. A more detailed screen using representative sequences provided additional information about spectral classes, and confirmed that the classes determined based on analysis of 1H NMR spectra are correlated with functional categories identified in previous studies. These results provide new insights into the surprising structural diversity of this library. They also show how NMR can be used to identify classes of sequences with distinct mutational signatures and functions.

• Klíčová slova
• DNA, G-quadruplex, Multimeric structures, NMR,
• MeSH
• G-kvadruplexy * MeSH
• guanosintrifosfát chemie   metabolismus   MeSH
• magnetická rezonanční spektroskopie metody   MeSH
• mutace MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• guanosintrifosfát MeSH

G-quadruplexes (G4s) formed within RNA are emerging as promising targets for therapeutic intervention in cancer, neurodegenerative disorders and infectious diseases. Sequences containing a succession of short GG blocks, or uneven G-tract lengths unable to form three-tetrad G4s (GG motifs), are overwhelmingly more frequent than canonical motifs involving multiple GGG blocks. We recently showed that DNA is not able to form stable two-tetrad intramolecular parallel G4s. Whether RNA GG motifs can form intramolecular G4s under physiological conditions and play regulatory roles remains a burning question. In this study, we performed a systematic analysis and experimental evaluation of a number of biologically important RNA regions involving RNA GG motifs. We show that most of these motifs do not form stable intramolecular G4s but need to dimerize to form stable G4 structures. The strong tendency of RNA GG motif G4s to associate may participate in RNA-based aggregation under conditions of cellular stress.

• MeSH
• dimerizace MeSH
• G-kvadruplexy * MeSH
• genetická transkripce MeSH
• lidé MeSH
• nukleotidové motivy * MeSH
• RNA * chemie   metabolismus   genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• RNA * MeSH

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

We have identified seven putative guanine quadruplexes (G4) in the RNA genome of tick-borne encephalitis virus (TBEV), a flavivirus causing thousands of human infections and numerous deaths every year. The formation of G4s was confirmed by biophysical methods on synthetic oligonucleotides derived from the predicted TBEV sequences. TBEV-5, located at the NS4b/NS5 boundary and conserved among all known flaviviruses, was tested along with its mutated variants for interactions with a panel of known G4 ligands, for the ability to affect RNA synthesis by the flaviviral RNA-dependent RNA polymerase (RdRp) and for effects on TBEV replication fitness in cells. G4-stabilizing TBEV-5 mutations strongly inhibited RdRp RNA synthesis and exhibited substantially reduced replication fitness, different plaque morphology and increased sensitivity to G4-binding ligands in cell-based systems. In contrast, strongly destabilizing TBEV-5 G4 mutations caused rapid reversion to the wild-type genotype. Our results suggest that there is a threshold of stability for G4 sequences in the TBEV genome, with any deviation resulting in either dramatic changes in viral phenotype or a rapid return to this optimal level of G4 stability. The data indicate that G4s are critical elements for efficient TBEV replication and are suitable targets to tackle TBEV infection.

• MeSH
• antivirové látky * farmakologie   terapeutické užití   MeSH
• G-kvadruplexy * MeSH
• klíšťová encefalitida farmakoterapie   genetika   MeSH
• lidé MeSH
• ligandy MeSH
• RNA virová genetika   MeSH
• RNA-dependentní RNA-polymerasa genetika   MeSH
• viry klíšťové encefalitidy * účinky léků   genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antivirové látky * MeSH
• ligandy MeSH
• RNA virová MeSH
• RNA-dependentní RNA-polymerasa MeSH