Abstract Thu107: Mechanism of G-Quadruplex Unfolding by REV1 Polymerase and FANCJ Helicase

Abstract

G-Quadruplexes (G4s) are secondary nucleic-acid structures that form through Hoogstein hydrogen bonding of four consecutive guanine runs. G4s can be found in promoters, replication origins, and telomeres; they possess a regulatory role in DNA replication, transcription, and RNA translation. Unresolved G4s can stall essential cellular processes and must be unfolded by specialized motor proteins. G4s are also highly susceptible to oxidative damage (8oxoG4s), which is known to impact their conformation and stability. We examined the human REV1 polymerase and FANCJ helicase to better understand their individual roles in G4 maintenance and their ability to resolve 8oxoG4s. We used BMVC fluorescence assays and hemin colorimetric assays to assess the unfolding of two G4 sequences (GGGT)4 (Tm: 88.4oC) and (TTAGGG)4 (Tm: 62.5oC) by REV1 and FANCJ. We hypothesized that REV1 and FANCJ would unfold different G4 sequences preferentially, as shown by lower Km values (i.e. higher unfolding efficiency). Additionally, we expected that 8oxoG4s would be more efficiently unfolded compared to the undamaged G4s. The unfolding data showed that REV1 preferentially resolved (TTAGGG)4 structures compared to (GGGT)4 and that REV1 unwound these structures with higher efficiency compared to FANCJ. Conversely, FANCJ preferentially unfolded (GGGT)4 structures and did so more efficiently than REV1. 8oxo(TTAGGG)4 structures demonstrated lower Km values when compared to the undamaged G4 resolved by REV1, however, no difference was seen with FANCJ. 8oxo(GGGT)4 structures showed similar Km values to undamaged (GGGT)4 for both REV1 and FANCJ. Overall, our data suggests a model in which FANCJ helicase targets and unfolds 8oxo(GGGT)4 and then recruits REV1 polymerase to continue DNA replication, while REV1 alone can unfold and synthesize DNA across from 8oxo(TTAGGG)4.