Role of Chromatin and DNA Damage Response Functions in R Loop-Mediated Genome Instability

Coordination of DNA replication with DNA-damage sensing, repair and cell cycle progression ensures with high probability genome integrity during cell divisions. One important type of genome instability is that associated with transcription. R loops, structures formed by a DNA-RNA hybrid and the displaced single-stranded DNA (ssDNA) molecule, are transcriptional by-products that can be formed naturally as key intermediates in specific cellular processes. Nevertheless, they are also a major source of transcription-associated genome instability and compelling evidence supports that this is mainly caused by replication fork impairment. To explore further the role that replication functions and the DNA damage response (DDR) have on R loop-mediated genome instability we have determined the impact that R loops have on replication fork progression in normal cells and in cells accumulating high levels of R loops. Our results show that this transcription- and RNA-mediated genome instability is not necessarily caused or linked to a lower fork velocity, but to a higher level of obstacles that impair RF progression at sites scattered throughout the genome. Then, after a screen searching for new genes involved in R loop-mediated genome instability among a collection of genes involved in DNA metabolism, we selected 21 genes whose depletion in HeLa cells increased R loops apart of the previously reported role of the Fanconi Anemia/BRCA factors. Our in-depth analysis of R loop-mediated instability in human cells depleted of these factors as well as the THO complex involved in RNA biogenesis reveals a new role for chromatin modifications in R loop accumulation and DDR. The implications for the role of chromatin and DDR functions in both R loop formation and transcription-associated genome instability will be discussed.

This research was funded by grants from the European Research Council, the Spanish Ministry of Science and Innovation, the Junta de Andalucía and the FEDER European regional funds.