Biology subjects

Carnie, C. J.

Publications and source records attributed to Carnie, C. J..

4 recordsLinked to original sources

bioRxiv2025

Loss of CTLH component MAEA impairs DNA repair and replication and leads to developmental delay

Ubiquitin E3 ligases play crucial roles in the DNA damage response (DDR) by modulating the turnover, localization, activation, and interactions of DDR and DNA replication proteins. To gain further insight into how the ubiquitin system regulates the DDR, we performed a CRISPR-Cas9 knockout screen focused on E3 ligases and related proteins with the DNA topoisomerase I inhibitor, camptothecin. This uncovered the CTLH ubiquitin E3 ligase complex -- and particularly one of its core subunits, MAEA -- as a critical regulator of the cellular response to single-ended DNA double-strand breaks (seDSBs) and replication stress. In tandem, we identified patients with variants in MAEA who present with neurodevelopmental deficits including global developmental delay, dysmorphic facial features, brain abnormalities, intellectual disability, and abnormal movement. Analysis of patient-derived cell lines and mutation modeling reveal an underlying defect in HR-dependent DNA repair and replication fork restart as a likely cause of disease. We propose that MAEA dysfunction hinders DNA repair by reducing the efficiency of RAD51 loading at sites of DNA damage, which compromises genome integrity and cell division during development.

genetics↗

bioRxiv2024

USP37 prevents premature disassembly of stressed replisomes by TRAIP

The E3 ubiquitin ligase TRAIP associates with the replisome and helps this molecular machine deal with replication stress. Thus, TRAIP promotes DNA inter-strand crosslink repair by triggering the disassembly of CDC45-MCM2-7-GINS (CMG) helicases that have converged on these lesions. However, disassembly of single CMGs that have stalled temporarily would be deleterious, suggesting that TRAIP must be carefully regulated. Here, we demonstrate that human cells lacking the de-ubiquitylating enzyme USP37 are hypersensitive to topoisomerase poisons and other replication stress-inducing agents. We further show that TRAIP loss rescues the hypersensitivity of USP37 knockout cells to topoisomerase inhibitors. In Xenopus egg extracts depleted of USP37, TRAIP promotes premature CMG ubiquitylation and disassembly when converging replisomes stall. Finally, guided by AlphaFold-Multimer, we discovered that binding to CDC45 mediates USP37s response to topological stress. In conclusion, we propose that USP37 protects genome stability by preventing TRAIP-dependent CMG unloading when replication stress impedes timely termination.

molecular biology↗

bioRxiv2023

The dCMP deaminase DCTD and the E3 ligase TOPORS are central mediators of decitabine cytotoxicity

The nucleoside decitabine (5-aza-dC) is used to treat several hematological cancers. Upon triphosphorylation and incorporation into DNA, 5-aza-dC induces covalent DNMT1 DNA-protein crosslinks (DPCs) and DNA hypomethylation. However, 5-aza-dC treatment success varies, and relapse is common. Using genome-scale CRISPR/Cas9 screens, we map factors determining 5-aza-dC susceptibility. Unexpectedly, we find that loss of the dCMP deaminase DCTD causes 5-aza-dC resistance, suggesting that 5-aza-dUMP generation underlies most 5-aza-dC cytotoxicity in wild-type cells. Combining results from a subsequent genetic screen in DCTD-deficient cells with identification of the proximal proteome of DNMT1-DPCs, we uncover the ubiquitin/SUMO1 E3 ligase, TOPORS, as a new DPC repair factor. TOPORS is recruited to DNMT1-DPCs in a SUMO-dependent manner and promotes their degradation. Our study suggests that 5-aza-dC-induced DPCs cause cytotoxicity when DPC repair is compromised, while cytotoxicity in wild-type cells arises from perturbed nucleotide metabolism and lays the foundations for the development of predictive biomarkers for decitabine treatment.

cell biology↗

bioRxiv2023

The eEF2 kinase coordinates the DNA damage response to cisplatin by supporting p53 activation

Eukaryotic elongation factor 2 (eEF2) kinase (eEF2K) is a stress-responsive hub that inhibits the translation elongation factor eEF2, and consequently mRNA translation elongation, in response to hypoxia and nutrient deprivation. EEF2K is also involved in the response to DNA damage but its role in response to DNA crosslinks, as induced by cisplatin, is not known. Here we found that eEF2K is critical to mediate the cellular response to cisplatin. We uncovered that eEF2K deficient cells are more resistant to cisplatin treatment. Mechanistically, eEF2K deficiency blunts the activation of the DNA damage response associated ATM and ATR pathways, in turn preventing p53 activation and therefore compromising induction of cisplatin-induced apoptosis. We also report that loss of eEF2K delays the resolution of DNA damage triggered by cisplatin, suggesting that eEF2K contributes to DNA damage repair in response to cisplatin. In support of this, our data shows that eEF2K promotes the expression of the DNA repair protein ERCC1, critical for the repair of cisplatin-caused DNA damage. Finally, using Caenorhabditis elegans as an in vivo model, we find that deletion of efk-1, the worm eEF2K ortholog, mitigates the induction of germ cell death in response to cisplatin. Together, our data highlight that eEF2K represents an evolutionary conserved mediator of the DNA damage response to cisplatin which promotes p53 activation to induce cell death, or alternatively facilitates DNA repair, depending on the extent of DNA damage.

molecular biology↗