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The telomeric DNA, a distal region of eukaryotic chromosome containing guanine-rich repetitive sequence of (TTAGGG)n, has been shown to adopt higher-order structures, specifically G-quadruplexes (G4s). Previous studies have demonstrated the implication of G4 in tumor inhibition through chromosome maintenance and manipulation of oncogene expression featuring their G-rich promoter regions. Besides higher order structures, several regulatory roles are attributed to DNA epigenetic markers. In this work, we investigated how the structural dynamics of a G-quadruplex, formed by the telomeric sequence, is affected by inosine, a prevalent modified nucleotide. We used the standard (TTAGGG)ntelomere repeats with guanosine mutated to inosine at each G position. Sequences (GGG)4, (IGG)4, (GIG)4, (GGI)4, (IGI)4, (IIG)4, (GII)4,and (III)4, bridged by TTA linker, are studied using biophysical experiments and molecular modeling. The effects of metal cations in quadruplex folding were explored in both Na+and K+containing buffers using CD and UV-melting studies. Our results show that antiparallel quadruplex topology forms with the native sequence (GGG)4and the terminal modified DNAs (IGG)4and (GGI)4in both Na+and K+containing buffers. Specifically, quadruplex hybrid was observed for (GGG)4in K+buffer. Among the other modified sequences, (GIG)4, (IGI)4and (GII)4show parallel features, while (IIG)4and (III)4show no detectable conformation in the presence of either Na+or K+. Our studies indicate that terminal lesions (IGG)4and (GGI)4may induce certain unknown conformations. The folding dynamics become undetectable in the presence of more than one inosine substitution except (IGI)4in both buffer ions. In addition, both UV melting and CD melting studies implied that in most cases the K+cation confers more thermodynamic stability compared to Na+. Collectively, our conformational studies revealed the diverse structural polymorphisms of G4 with position dependent G-to-I mutations in different ion conditions.
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This content will become publicly available on August 1, 2026
8-Oxoguanine Disrupts G-Quadruplex DNA Stability and Modulates FANCJ AKKQ Peptide Binding
Guanine-rich nucleic acid sequences can adopt G-quadruplex (G4) structures, which pose barriers to DNA replication and repair. The FANCJ helicase contributes to genome stability by resolving these structures, a function linked to its G4-binding site that features an AKKQ amino acid motif. This site is thought to recognize oxidatively damaged G4, specifically those containing 8-oxoguanine (8oxoG) modifications. We hypothesize that FANCJ AKKQ recognition of 8oxoG-modified G4s (8oxoG4s) depends on the sequence context, the position of the lesion within the G4, and overall structural stability. Using fluorescence spectroscopy, we measured the binding affinities of a FANCJ AKKQ peptide for G4s formed by (GGGT)4, (GGGTT)4, and (TTAGGG)4 sequences. G4 conformation and thermal stability were assessed by circular dichroism spectroscopy. Each sequence was modified to include a single 8oxoG at the first (8oxo1), third (8oxo3), or fifth (8oxo5) guanine position. In potassium chloride (KCl), the most destabilized structures were (GGGT)4 8oxo1, (GGGTT)4 8oxo1, and (TTAGGG)4 8oxo5. In sodium chloride (NaCl), the most destabilized were (GGGT)4 8oxo1, (GGGTT)4 8oxo5, and (TTAGGG)4 8oxo5. FANCJ AKKQ binding affinities varied according to damage position and sequence context, with notable differences for (GGGT)4 in KCl and (TTAGGG)4 in NaCl. These findings support a model in which FANCJ binding to G4 and 8oxoG4 structures is modulated by both the oxidative damage position and the G4 local sequence environment.
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- Award ID(s):
- 2142839
- PAR ID:
- 10640450
- Publisher / Repository:
- MDPI
- Date Published:
- Journal Name:
- Molecules
- Volume:
- 30
- Issue:
- 16
- ISSN:
- 1420-3049
- Page Range / eLocation ID:
- 3424
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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