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.