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

Gao, Yuandi; Guitton-Sert, Laure; Dessapt, Julien; Coulombe, Yan; Rodrigue, Amélie; Milano, Larissa; Blondeau, Andréanne; Larsen, Nicolai Balle; Duxin, Julien P.; Hussein, Samer M.I.; Fradet-Turcotte, Amélie; Masson, Jean‐Yves

doi:10.1038/s41467-023-35802-y

Cited by 10 publications

(7 citation statements)

References 71 publications

(92 reference statements)

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

Section: Articlesupporting

confidence: 88%

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

Carnie,

Acampora,

Bader

et al. 2024

Nat Cell Biol

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Covalent DNA–protein cross-links (DPCs) are toxic DNA lesions that block replication and require repair by multiple pathways. Whether transcription blockage contributes to the toxicity of DPCs and how cells respond when RNA polymerases stall at DPCs is unknown. Here we find that DPC formation arrests transcription and induces ubiquitylation and degradation of RNA polymerase II. Using genetic screens and a method for the genome-wide mapping of DNA–protein adducts, DPC sequencing, we discover that Cockayne syndrome (CS) proteins CSB and CSA provide resistance to DPC-inducing agents by promoting DPC repair in actively transcribed genes. Consequently, CSB- or CSA-deficient cells fail to efficiently restart transcription after induction of DPCs. In contrast, nucleotide excision repair factors that act downstream of CSB and CSA at ultraviolet light-induced DNA lesions are dispensable. Our study describes a transcription-coupled DPC repair pathway and suggests that defects in this pathway may contribute to the unique neurological features of CS.

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

Section: Articlesupporting

confidence: 88%

“…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.…”

Section: Cs Proteins Are Required For Dpc Tolerancesupporting

confidence: 64%

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

Carnie,

Acampora,

Bader

et al. 2024

Nat Cell Biol

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“…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.…”

Section: Section 1 Protein-targeted Methods For Dpc Preparationmentioning

confidence: 99%

Isolation and detection of DNA–protein crosslinks in mammalian cells

Torrecilla,

Ruggiano,

Kiianitsa

et al. 2023

Nucleic Acids Research

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DNA–protein crosslinks (DPCs) are toxic DNA lesions wherein a protein is covalently attached to DNA. If not rapidly repaired, DPCs create obstacles that disturb DNA replication, transcription and DNA damage repair, ultimately leading to genome instability. The persistence of DPCs is associated with premature ageing, cancer and neurodegeneration. In mammalian cells, the repair of DPCs mainly relies on the proteolytic activities of SPRTN and the 26S proteasome, complemented by other enzymes including TDP1/2 and the MRN complex, and many of the activities involved are essential, restricting genetic approaches. For many years, the study of DPC repair in mammalian cells was hindered by the lack of standardised assays, most notably assays that reliably quantified the proteins or proteolytic fragments covalently bound to DNA. Recent interest in the field has spurred the development of several biochemical methods for DPC analysis. Here, we critically analyse the latest techniques for DPC isolation and the benefits and drawbacks of each. We aim to assist researchers in selecting the most suitable isolation method for their experimental requirements and questions, and to facilitate the comparison of results across different laboratories using different approaches.

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

Section: Prpa (S4 and S8) Focimentioning

confidence: 99%