A CRISPR-Cas9 screen identifies EXO1 as a formaldehyde resistance gene

Abstract

Fanconi Anemia (FA) is a rare, genome instability-associated disease characterized by a deficiency in repairing DNA crosslinks, which are known to perturb several cellular processes, including DNA transcription, replication, and repair. Formaldehyde, a by-product of metabolism, is thought to drive FA by generating DNA interstrand crosslinks (ICLs) and DNA-protein crosslinks (DPCs). However, the impact of formaldehyde on global cellular pathways has not been investigated thoroughly. Herein, using a pangenomic CRISPR-Cas9 screen, we identify EXO1 as a critical regulator of formaldehyde-induced DNA lesions. We show that EXO1 knockout cell lines exhibit formaldehyde sensitivity leading to the accumulation of replicative stress, DNA double-strand breaks, and quadriradial chromosomes, a typical feature of FA. After formaldehyde exposure, EXO1 is recruited to chromatin, protects DNA replication forks from degradation, and functions in parallel with the FA pathway to promote cell survival. In vitro, EXO1-mediated exonuclease activity is proficient in removing DPCs. Collectively, we show that EXO1 limits replication stress and DNA damage to counteract formaldehyde-induced genome instability.

Transcription-coupled repair of DNA–protein cross-links depends on CSA and CSB

“…As reported previously [42][43][44]46 , downregulation of TC-NER factors-CSB, CSA, XPA, XPF and XPG-caused formaldehyde sensitivity (Fig. 1b).…”

“…1a and Supplementary Table 1). As observed in other formaldehyde-based CRISPR screens [43][44][45] , interfering with expression of ADH5 or ESD (encoding formaldehyde-detoxifying enzymes 45,46 ; Extended Data Fig. 1b), resulted in severe formaldehyde sensitivity (Fig.…”

Isolation and detection of DNA–protein crosslinks in mammalian cells

“…The analysis of specific crosslinked substrates alongside total DPC isolates has shown a role in DPC repair for the protease SPRTN ( 32 ), the ATPase p97 and its cofactor TEX264 ( 65 ), the proteasome ( 3 ), the DNA break repair factor XRCC1 ( 95 ), the de-ubiquitylating enzyme (DUB) USP11 ( 68 ), and the exonuclease EXO1 ( 96 ) in human cells; and for the protease GCNA in Drosophila and zebrafish ( 97 ). Furthermore, post-translational modifications such as ubiquitylation and SUMOylation can be detected on Western blots of DPCs ( 59 ) or specific adducts ( 63 , 98 ), establishing a role for the ubiquitin/SUMO system in DPC repair.…”

Chemo-Phosphoproteomic Profiling with ATR Inhibitors Berzosertib and Gartisertib Uncovers New Biomarkers and DNA Damage Response Regulators

“…The protocol was adapted from a previous report (54). Cells were treated with or without 10 Gy irradiation and 3 h release.…”