Wu, Colin GPaul, AlyssaAvery, AdamVilla-Diaz, Luis G2026-07-172026-07-172026-01-01https://hdl.handle.net/10323/22129DNA damage repair pathways are essential to continue replication past DNA lesions and secondary structures such as G-quadruplexes (G4s). Unresolved G4s pose a threat to genomic stability due to their formation in common oncogenic promoters and telomeres. The DNA damage repair proteins REV1 polymerase and FANCJ helicase are capable of unfolding G4s to allow their replication. In this study, we examined the G4 unfolding efficiencies of REV1 and FANCJ for parallel (GGGT)4 and hybrid (TTAGGG)4 sequences with and without oxidative damage (8oxoG4) using BMVC fluorescence assays and hemin colorimetric assays. We suspected that REV1 and FANCJ would demonstrate a preference for different G4 conformations, and that 8oxoG4s would be more efficiently unfolded compared to undamaged sequences. Additionally, we investigated the effects of the DNA damage inducers menadione and bleomycin on HEK293T cells with FANCJ upregulation. We expected that increasing treatment concentrations would induce increasing DNA damage and that FANCJ upregulation would suppress this damage. The unfolding data revealed that REV1 preferentially resolved (TTAGGG)4 structures compared to (GGGT)4 and that REV1 unwound these structures with a higher efficiency compared to FANCJ. Conversely, FANCJ preferentially unfolded (GGGT)4 structures, and did so more efficiently than REV1. Both proteins were capable of unfolding 8oxoG4s, with oxidative damage impacting unfolding differently for each G4 sequence and protein pair. REV1 and FANCJ were also capable of unfolding G4s with and without ATP. Damage induction results showed that 0.001-10 µM menadione triggered DNA damage response pathways to suppress damage and did not yield a measurable increase in damage. The transfection of FANCJ caused a similar response in menadione-treated cells, with decreased DNA damage compared to untransfected cells. Bleomycin-treated cells did demonstrate increasing DNA damage, and FANCJ upregulation suppressed damage at higher treatment concentrations compared to low-efficiency transfection or untransfected cells. Overall, these results show that REV1 and FANCJ binding is sufficient to unfold G4s and suggests a model in which FANCJ targets and unfolds hybrid G4s then recruits REV1 to continue DNA replication, while REV1 unfolds parallel G4 structures before continuing replication or recruiting other TLS pols. Additionally, the upregulation of FANCJ may improve the damage suppression response for cells exposed to bleomycin, suggesting that protein upregulation in non-cancerous cells could have a protective effect against DNA damage inducers.FANCJG-quadruplexREV1