Mechanisms underlying regulation and removal of G-quadruplex/R-loop transcription-replication conflicts

Maintenance of genome stability is challenged by obstacles that interfere with normal progression of essential DNA-associated transactions, such as DNA replication and transcription. One such obstacle is the G-quadruplex (G4) DNA secondary structure, which can form in G-rich repetitive DNA sequences.

Transcribed G4-DNA loci often co-exist with stable RNA-DNA hybrids (R-loops), potentially causing deleterious transcription-replication conflicts (TRCs). Therefore, the ability of cells to mitigate TRCs formation is critical for cell and organismal fitness.

While the pathways involved in repairing other DNA lesions are relatively well understood, the mechanisms by which cells respond to G4/R-loop-induced TRCs remain uncharted, largely due to a lack of efficient approaches for inducing site-specific G4s/R-loops into the genome.

Several studies reported dedicated helicases capable of unwinding G4s/R-loops, yet whether additional factors exist and how different types of TRCs are resolved remains unknown.

In this project, I aim to dissect the fundamental mechanisms that protect cells from TRCs at DNA loci harbouring G4s/R-loops.

To do so, I will establish a novel system with site-specific G4/R-loop substrates in living cells and I will systematically investigate their protein composition to identify novel factors in G4/R-loop metabolism.

I will further examine the genetic vulnerabilities of candidate G4/R-loop helicase-deficient cells and the regulatory mechanisms underlying G4/R-loop unwinding by these helicases.

As mutations in several G4/R-loop helicases predispose to various cancers and genetic disorders, a detailed understanding of the basic mechanisms controlling G4/R-loop removal is of utmost importance, and will shed light on disease aetiology and the rational development of more targeted therapeutic strategies.