Mechanisms of G-quadruplex Recognition and Unfolding by the FANCJ Helicase and REV1 Polymerase

G-quadruplexes (G4s) are secondary structures formed by guanine-rich nucleic acid sequences. Accumulated G4s in cells can disrupt DNA replication, RNA transcription, and other cellular processes. G4 DNA can adopt parallel, antiparallel, or hybrid conformations depending on solution conditions and base sequence. Moreover, reactive oxygen species can readily convert guanine to 8-oxoguanine (8oxoG) to form 8oxoG4s. However, the precise mechanisms by which repair proteins recognize and remove such lesions remain unclear. The FANCJ DNA helicase possesses an AKKQ motif that functions as a G4-targeting site that binds to G4s and 8oxoG4s. Additionally, FANCJ interacts with the REV1 translesion synthesis polymerase to facilitate replication across G4-forming sites. Although REV1 can also target and disrupt G4s, it is unknown whether the activities of FANCJ and REV1 are redundant or if they act on specific G4 substrates. We hypothesized that the presence of 8oxoG would impact the folding and stability of parallel, antiparallel, and hybrid G4s in specific positions within the G4. Furthermore, we anticipated that FANCJ and REV1 would have distinct 8oxoG4 binding preferences that can be correlated with the physical properties of the 8oxoG4s. We predicted that the collaborative action of FANCJ and REV1 would repair tightly-folded 8oxoG4 structures, whereas REV1 alone may suffice for disrupting the loosely folded 8oxoG4s. Our results revealed that introducing 8oxo1 significantly reduced the thermal stability of both parallel and antiparallel G4 structures. While 8oxo5 destabilized hybrid structures. FANCJ and REV1 both preferred parallel G4 and 8oxoG4s due to their greater stability and tighter folding compared to hybrid or antiparallel conformations. Furthermore, FANCJ had the lowest affinity for antiparallel 8oxo3 G4s, while REV1 had a lower affinity for hybrid 8oxo1 G4s. These findings indicate that the presence of 8oxoG modification in G4 structures may hinder the binding of these proteins. The study revealed that both FANCJ and REV1 have distinct substrate-specific preferences for unfolding G4s. FANCJ demonstrated lower unfolding rates when 8oxo5 was present within the G4 stack, indicating that the position of the lesion plays a critical role in the unfolding efficiency. REV1, on the other hand, showed a lower unfolding rate for more stable G4 structures like the GGGT G4, but faster rates for the oxidized constructs. This research provides significant insights into the specific roles and mechanisms of FANCJ and REV1 in unfolding 8oxoG-damaged G4s, contributing to our understanding of DNA repair mechanisms.