Replication Fork Instability at Specific Loci Drives PARP Inhibitor Cytotoxicity in Cells

Distinct replication-fork lesions in specific genomic regions serve as key determinants of poly(ADP-ribose) polymerase inhibitor cytotoxicity in cancer cells, according to research published in Nature Cell Biology. Although poly(ADP-ribose) polymerase inhibitors show promise for treating homologous recombination-deficient cancers, observed hypersensitivity in additional genetic contexts expands their potential use in precision oncology, despite the specific DNA lesions driving this cytotoxicity remaining poorly defined. Using CRISPR screens, investigators identified that the loss of the replication-coupled cohesin acetyltransferase ESCO2 confers hypersensitivity to poly(ADP-ribose) polymerase inhibitors independently of homologous recombination deficiency, sensitizing cells to treatment.

Through quantitative single-molecule imaging, the researchers demonstrated that ESCO2 depletion triggers replication-associated damage signalling and promotes the accumulation of G-quadruplex DNA structures at a subset of replication-associated sites. Experimental analyses showed that ESCO2 depletion specifically increases these replisome-associated G-quadruplex structures during DNA replication without elevating R-loops. In response to this accumulation, replisome-associated G-quadruplex structures activate PARP1 and engage the fork protection factor TIMELESS to mitigate G-quadruplex-induced replication stress.

The investigation further revealed that these G-quadruplex lesions localize specifically to fragile pericentromeric regions and engage the G-quadruplex helicase DDX11. Under conditions of ESCO2 deficiency, this pericentric replisome-associated G-quadruplex fragility becomes sensitive to poly(ADP-ribose) polymerase inhibition. When poly(ADP-ribose) polymerase is inhibited in this setting, the protective response is disrupted because the treatment prevents the PARP1-dependent engagement of both TIMELESS and DDX11, significantly reducing TIMELESS recruitment and exacerbating the cellular G-quadruplex burden.

Disruption of this protective pathway ultimately leads to unresolved G-quadruplex accumulation, DNA damage, and chromosomal instability in treated cells. Overall, the findings define the G-quadruplex–PARP1–TIMELESS–DDX11 axis as a critical safeguard of replication fork stability at hard-to-replicate loci. By demonstrating that positional replication fork instability drives inhibitor cytotoxicity, the cellular findings reveal ESCO2 as a potential therapeutic target for promoting synthetic lethality in cancers treated with poly(ADP-ribose) polymerase inhibitors.

Subject of Research: ESCO2 deficiency and replication fork instability in PARP inhibitor cytotoxicity

Article Title: Positional replication fork instability drives PARP inhibitor cytotoxicity

Article References: Gupta, D., Ding, X., Zhang, L., Coleman, K. E., Tang, G., Lee, W. T. C., Oyer, J., Kang, X., Ramms, D. J., Yu, Z., VanArsdale, T., Fenyo, D., Huang, T. T., & Rothenberg, E. (2026). Positional replication fork instability drives PARP inhibitor cytotoxicity. Nature Cell Biology. https://doi.org/10.1038/s41556-026-02126-0

Image Credits: AI Generated

DOI: 10.1038/s41556-026-02126-0

Keywords: PARP inhibitors, ESCO2, replication fork instability, G-quadruplex, TIMELESS, DDX11, synthetic lethality, CRISPR screens, Positional, replication, fork, instability