Biology subjects

Galanty, Y.

Publications and source records attributed to Galanty, Y..

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↗

bioRxiv2025

UFMylation orchestrates chromatin engagement of core NHEJ components to promote DNA double-strand break repair

DNA double-strand breaks (DSBs) are highly cytotoxic lesions whose misrepair can lead to genomic instability, cancer and developmental disorders. Through systematic screening of understudied ubiquitin-like modifiers (UBLs), we identify UFM1 as a previously unrecognised regulator of non-homologous end-joining (NHEJ). Using a structure-guided chemical biology strategy, we develop a photo-crosslinkable UFM1 probe and, together with high-resolution NMR, uncover non-canonical UFM1-binding regions in core NHEJ components, including XRCC4. Mechanistically, proximity-dependent proteomics reveals Ku70 as a key UFMylation substrate, establishing a functional axis in which XRCC4 engages UFMylated Ku70 to promote the chromatin assembly of NHEJ factors. Perturbation of UFM1 signalling, via UFSP2 depletion or a hypomorphic UBA5 allele in patient-derived fibroblasts, impairs these processes, linking UFMylation defects to altered regulation of DSB repair. Our findings define a complete UFM1 signalling module in genome maintenance and uncover a molecular connection between hereditary UFMylation disorders and dysregulated DSB repair pathways.

molecular biology↗

bioRxiv2018

Mutations generated by repair of Cas9-induced double strand breaks are predictable from surrounding sequence

The exact DNA mutation produced by cellular repair of a CRISPR/Cas9-generated double strand break determines its phenotypic effect. It is known that the mutational outcomes are not random, and depend on DNA sequence at the targeted location. Here, we present a systematic study of this link. We created a high throughput assay to directly measure the edits generated by over 40,000 guide RNAs, and applied it in a range of genetic backgrounds and for alternative CRISPR/Cas9 reagents. In total, we gathered data for over 1,000,000,000 mutational outcomes in synthetic constructs, which mirror those at endogenous loci. The majority of reproducible mutations are insertions of a single base, short deletions, or long microhomology-mediated deletions. gRNAs have a cell-line dependent preference for particular outcomes, especially favouring single base insertions and microhomology-mediated deletions. We uncover sequence determinants of the produced mutations at individual loci, and use these to derive a predictor of Cas9 editing outcomes with accuracy close to the theoretical maximum. This improved understanding of sequence repair allows better design of editing experiments, and may lead to future therapeutic applications.

cell biology↗

bioRxiv2018

ATM orchestrates the DNA-damage response to counter toxic non-homologous end-joining at broken replication forks

Mutations in the ATM tumor suppressor confer hypersensitivity to DNA-damaging agents. To explore genetic resistance mechanisms, we performed genome-wide CRISPR-Cas9 screens in cells treated with the DNA topoisomerase poison topotecan. Thus, we establish that loss of terminal components of the non-homologous end-joining (NHEJ) machinery or the BRCA1-A complex specifically confers topotecan resistance to ATM-deficient cells. We show that hypersensitivity of ATM-mutant cells to topotecan or the poly-(ADP-ribose) polymerase inhibitor olaparib is due to delayed homologous recombination repair at DNA-replication-fork-associated double-strand breaks (DSBs), resulting in toxic NHEJ-mediated chromosome fusions. Accordingly, restoring legitimate repair in ATM-deficient cells, either by preventing NHEJ DNA ligation or by enhancing DSB-resection by BRCA1-A complex inactivation, markedly suppresses this toxicity. Our work suggests opportunities for patient stratification in ATM-deficient cancers and when using ATM inhibitors in the clinic, and identifies additional therapeutic vulnerabilities that might be exploited when such cancers evolve drug resistance.\n\nOne Sentence SummaryATM counteracts toxic NHEJ at broken replication forks

cell biology↗