Structural switches: intracellular G-quadruplexes modulate DNA repair and genome maintenance in Deinococcus...

Guanine quadruplex (G4) structures are secondary structures formed in nucleic acids containing guanine-rich stretches. The regulatory role of G4s in various cellular processes has been studied in both eukaryotes and prokaryotes. Deinococcus radiodurans, an extremophile, shows increased sensitivity to ionizing radiation in the presence of G4-binding ligands during the post-irradiation recovery (PIR) period. Here, for the first time, a detailed study is reported on the intracellular formation and resolution of G4 structures in this organism. The potential G-quadruplex-forming sequences, positioned at different locations within the genes, folded into different topologies in vitro. The divalent cation Mn²⁺, which plays a crucial role in radioresistance, destabilized G4 structures, whereas Mg²⁺ supported the G4 formation. Depending on their location at the 5' or 3' end of the gene, the native G4 motifs differentially regulated the endogenous gene expression under both normal and PIR conditions. Thioflavin T and anti-G-quadruplex antibodies detected the in vivo formation and dynamics of G4s in response to various DNA-damaging agents, with the highest levels observed following gamma radiation treatment. The presence of G4-binding drugs during the PIR arrested G4 structure dynamics, increased the cellular levels of 8-oxo guanine, and delayed the DNA repair process. Furthermore, the absence of RecQ resulted in the accumulation of more G4 structures within the cells, leading to genome instability. These results indicate that in D. radiodurans, the in vivo formation of G4 structures varies significantly under normal and cellular stress conditions, depending on the levels of Mn²⁺ and G4 resolving proteins.

Guanine quadruplexes (G4s) can act as both activators and inhibitors of various cellular functions, depending on their genomic position. While G4 functions are increasingly understood in eukaryotes, their specific roles in bacteria, especially in vivo, are still largely unexplored. Deinococcus radiodurans harbors a GC-rich genome with numerous potential G-quadruplex-forming sequences. In this study, the first direct evidence of G4 formation in vivo and its dynamic nature during the DNA damage response to gamma radiation is shown in a bacterium. The significance of G4 structural dynamics in modulating endogenous gene expression, DNA repair, and genome maintenance processes is demonstrated. These findings provide a novel insight into G4-mediated regulatory mechanisms in prokaryotes and expand our understanding of G4's role as a dynamic structural switch.