A genetic map of the response to DNA damage in human cells

Abstract

The response to DNA damage is critical for cellular homeostasis, tumor suppression, immunity and gametogenesis. In order to provide an unbiased and global view of the DNA damage response in human cells, we undertook 28 CRISPR/Cas9 screens against 25 genotoxic agents in the retinal pigment epithelium-1 (RPE1) cell line. These screens identified 840 genes whose loss causes either sensitivity or resistance to DNA damaging agents. Mining this dataset, we uncovered that ERCC6L2, which is mutated in a bone-marrow failure syndrome, codes for a canonical non-homologous end-joining pathway factor; that the RNA polymerase II component ELOF1 modulates the response to transcription-blocking agents and that the cytotoxicity of the G-quadruplex ligand pyridostatin involves trapping topoisomerase II on DNA. This map of the DNA damage response provides a rich resource to study this fundamental cellular system and has implications for the development and use of genotoxic agents in cancer therapy.

CX-5461 activates the DNA damage response and demonstrates therapeutic efficacy in high-grade serous ovarian cancer

“…Mechanistically, we demonstrate HRD potentiates CX-5461-mediated DDR identifying compromised HR-dependent resolution of global replication stress as the likely mechanism of CX-5461 synthetic lethal interaction with HRD in HGSOC. Furthermore, in agreement with our data, two recent reports found the sensitivity profile of CX-5461 to most closely resemble a TOP2 poison 21,22 . TOP2a is an essential component of the Pol I pre-initiation complex 23 and while our data clearly demonstrate CX-5461 inhibits Pol I transcription and activates nucleolar DDR, it is plausible that it does so by trapping TOP2 at rDNA and this perhaps influences TOP2 activity across the genome.…”

“…This suggests that additional mechanisms to HR defects underlie sensitivity to CX-5461. Recently, the sensitivity profile of CX-5461 was shown to closely resemble a topoisomerase II (TOP2) poison 21,22 . TOP2a is an essential component of the Pol I pre-initiation complex 23 and while CX-5461 demonstrates highly selective inhibition of Pol I transcription initiation, it is plausible that it does so by trapping TOP2 at rDNA and potentially across the genome.…”

Modular HUWE1 architecture serves as hub for degradation of cell-fate decision factors

“…Moreover, as HUWE1 has recently been demonstrated to be a key sensitizer to commonly used anti-cancer agents 16 , our structure may suggest routes to new therapeutics that shift the apoptotic…”

“…A growing list of HUWE1 substrates has since been reported 9 , including the stress-responsive regulator of mTORC1 signaling, DDIT4 10,11 , and many DNA damage response factors, such as the BRCA1 tumor suppressor, TopBP1, Cdc6 and CHEK1 [12][13][14][15] . Loss of HUWE1 sensitizes cells not only to DNA damage, but also to a variety of other stressors, including both oxidative and hypoxic stress 9,[16][17][18][19] . In addition, HUWE1 has been shown to mediate the destruction of unassembled constituents of multi-protein complexes, contributing to protein quality control 20,21 .…”

A genome-wide screening for DNA repair genes: much more players than hitherto known

“…In a recent paper published in Cell by Olivieri et al this strategy was applied and the authors succeeded in uncovering a number of new DNA repair factors and gained insights into new mechanisms of genotoxic agents. 2 In this work, a CRISPR-Cas9 lentiviral sgRNA library was infected into a telomerase-immortalized human cell line that expressed Flag-tagged Cas9 and a total of 27 genotoxic agents were used for screenings. From gene level depletion scores, genes were identified, whose mutation/deletion led to a selective growth advantage of the exposed cells.…”