Belotserkovskaya, R. | bioRxiv Science

bioRxiv2026

Insights into mechanisms of ATM activation via constitutively active mutants

The Ser/Thr kinase ATM orchestrates cellular responses to DNA double-strand breaks (DSBs) and promotes DSB repair by homologous recombination. In this process, ATM is activated by DNA and the MRN (MRE11, RAD50, and NBS1) complex. Here we show that mutations of the conserved PIKK regulatory domain (PRD) within ATMs kinase domain can confer a maximally active state that no longer requires MRN/DNA. In ATM-knockout human cells, the PRD mutants display substantially higher phosphorylation of histone H2AX, KAP1, and CHK2 than wild-type ATM, with or without IR-induced DNA damage. Cryo-EM structures of two PRD mutants each revealed basal or activated conformations depending on bound ligands, suggesting that disrupting the ordered portion of the PRD results in an enzyme poised to transition to the active conformation. However, the identity of the active-site nucleotide is a key driver of the conformational switching. We speculate that this plasticity might be exploited to develop small-molecule ATM modulators for therapeutic applications.

molecular biology↗

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↗