Du pore nucléaire aux endommagements de l'ADN : la navette Ddx19 médié par ATR pour résoudre des conflits entre la transcription et la réplication
Cells are continuously challenged by DNA damage resulting from external cues as UV light, γ-irradiation and exposure to genotoxic chemicals, as well as from endogenous stress caused by cellular metabolism. Growing evidence points to transcription as a biological process that could adversely affect genome integrity. One currently highly investigated mechanism by which transcription can induce genome instability is through the formation of R-loops, RNA:DNA hybrid structures exposing a displaced single-stranded DNA tract. These aberrant structures occur as byproducts of transcription and/or upon interference between replication and transcription, and more recently were also shown to accumulate upon disruption of mRNA biogenesis and processing. Persistent unresolved R-loops are a potent source of genomic instability as they ultimately generate double strand breaks and promote recombination events. To deal with the deleterious consequences of DNA damage, cells activate elaborate DNA damage response (DDR) pathways to delay cell division and stimulate repair of lesions, thus preserving genome stability. Recently in yeast transient DDR activation has also been proposed to be important in the coordination of transcription and replication, in order to avoid topological constraints and the formation of aberrant structures generated upon collision of their machineries. By means of an in vitro screen aimed at identifying new DDR genes, we isolated Ddx19, a DEAD-Box helicase known to be involved in mRNA export, as a novel DNA damage responsive gene. Ddx19 interacts with the nucleopore complex via nucleoporin CAN/Nup214, and is involved in mRNA remodelling and export through its ATPase and helicase activities, stimulated by IP6 and the Gle1 factor. My present thesis work unravels a novel function of Ddx19 in preserving genome stability in mammalian cells, distinct from its known role in mRNA export. I show that upon UV-induced damage, Ddx19 transiently relocalizes from the cytoplasmic face of the nucleopore to the nucleus in an ATR-dependent manner. Downregulation of Ddx19 gives rise to spontaneous, proliferation-dependent DNA damage, as determined by the specific activation of the ATM-Chk2 pathway and formation of γH2AX and 53BP1 nuclear foci. This is concomitant with the slowing down of replication forks that are unable to restart after being stalled with camptothecin. In addition, cells depleted of Ddx19 display strong accumulation of nuclear R-loops, enriched in the nucleolar compartment, and around the nuclear periphery. Moreover, these cells show low viability and exhibited synthetic lethality when combined with inhibition of topoisomerase I expression. I propose Ddx19 as a second helicase required for R-loops resolution, functioning alongside but independently of Senataxin, the first known RNA helicase to resolve these structures in vivo in mammalian cells. I provide evidence that this new function of Ddx19 does not depend on its interaction with the nuclear pore, but rather on its helicase activity and on a serine residue phosphorylated by Chk1 which promotes its relocalization into the nucleus upon damage. These data put forward Ddx19 as a novel RNA helicase that facilitates ATR-dependent coordination of DNA replication and transcription through R-loops resolution, thus preserving genome integrity.
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A Tribute to Angelos Constantinou
du
10/03/2025
jusqu'au 13/03/2025
Village Club de Carry-Le-Rouet, France
