ReXeG · Deciphering molecular mechanisms that resolve mutagenic DNA G-quadruplex structures

Deciphering molecular mechanisms that resolve mutagenic DNA G-quadruplex structures

The genetic information encoded in our genome is mainly stored in double helical form in our DNA. However, increasing biological interest has focused the attention on other nucleic acid conformations such as G-quadruplexes (G4). G4s are stable nucleic acids assemblies that can form in guanine-rich sequences and regulate crucial cellular processes such as replication or transcription. Importantly, these stable structures come also at a cost as they are able to induce DNA damage and provoke genomic instability. This DNA damage induction potential is especially relevant and has increased the interest of using G4s as potential therapeutic targets. Using G4 stabilizing drugs has been shown to affect gene expression and kill cancer cells. However, the development of drugs that target G4 structures has been obstructed by the lack of knowledge on G4s regulation in vivo. Currently, very little is known about the molecular mechanisms that form and resolve G4 structures. Several helicases such as FANCJ, WRN, BLM, Pif1 and DNA2 have been described to unwind these stable assemblies in vitro, however, whether and how they resolve G4s in vivo is poorly understood. In this project, my overall objective was to decipher the molecular mechanisms that unwind G4s using a unique assay developed in the host laboratory. This assay is based on using the Xenopus egg extract system. This uses DNA replication stalling and bypass at defined G4 structures as a direct readout for G4 stability and unwinding under physiological conditions. This is the only system that supports vertebrate DNA replication outside a cell which has generated groundbreaking discoveries in the mechanism of DNA replication and repair. To decipher the molecular details of G4s unwinding, single-stranded plasmids with preformed G4 structures at specific locations are incubated in the extract and DNA replication starts. Upon stalling at the G4, DNA replication is blocked transiently at the G4 after which the G4 is efficiently unwound and the sequence replicated. Therefore, this system enables us to follow the kinetics of G4 unwinding during DNA replication and to determine which proteins are recruited during this process. Conclusions of the action From this work the main achievement has been setting up a biochemical assay that combines the Xenopus egg extract and mass spectrometry to identify specific G4 regulatory factors. Thus, I was able to identify several proteins that selectively bind to G4s during DNA replication. Currently, the biochemical function of these proteins in G4 regulation is being validated in Xenopus egg extract in the host laboratory.

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