Characterizing the Role of the E3 Ubiquitin Ligase Subunit MAEA in DNA Repair and Replication

Abstract

My PhD thesis describes for the first time the role of the CTLH E3 ubiquitin ligase complex catalytic subunit MAEA in the DNA damage response. I identified MAEA through focused CRISPR- Cas9 screens using the chemotherapeutic agents camptothecin and etoposide. My subsequent investigations demonstrate that MAEA loss leads to a severe homologous recombination (HR) impairment as exemplified by multiple genotoxic hypersensitivities. This HR deficiency is not the result of an inability to perform CtIP-dependent end resection, which is in fact increased in MAEA null cells. Instead, the HR defect seems to arise during recombination, wherein MAEA null cells are unable to form or maintain RAD51 foci at sites of damage. This portends further investigation into the downstream consequences of inefficient RAD51 focus formation, particularly in DNA replication. I have assembled a cohort of seven patients with variants in MAEA from around the world with the help of multiple clinical collaborators. These variants have unknown functional significance but the patients present with characteristics of a consistent developmental disorder. Recapitulating two of these variants in MAEA null cells shows genotoxic hypersensitivity to camptothecin and an impaired ability to form ionizing radiation-induced RAD51 foci, collectively suggesting they confer HR deficiency. MAEA loss also leads to severe impacts on DNA replication. Alongside my colleague, Dr Samah Awwad, we demonstrate how MAEA loss leads to vulnerability to replication stress inducers like hydroxyurea (HU) and ATR inhibitors. Dr Awwad shows how both of these drugs also induce significant replication catastrophe, measured by chromatinized RPA (i.e. bound to single-stranded DNA) and γH2AX accumulation in MAEA KO, RING-dead mutant, and clinical variant cells. ATR- CHK1 cell cycle checkpoints in MAEA null cells are intact, indicating that the replication catastrophe phenotype is not due unregulated ATR-mediated origin firing. Via a collaboration with Dr Satpal Jhujh at the University of Birmingham, UK, extensive DNA fiber analyses on MAEA null, RING-dead, and clinical variant cell lines displayed inefficient DNA replication, increased stalled forks, difficulty restarting replication at fired origins, and a severe susceptibility to degradation following treatment with HU. These results comport with my earlier observations regarding increased DNA resection and decreased RAD51, both of which lead to unprotected replication intermediates. I then designed and coordinated multiple high-throughput proteomics screens to better understand ubiquitylomic landscapes of MAEA and CTLH null cells, ultimately with the goal of identifying putative substrates. These experiments, conducted in part with the help of Dr Thorsten Mosler at IMB Mainz, Germany, converged on the proteins SET and Ku as putative targets of MAEA and could explain several of the phenotypes observed in both DNA repair and replication due to their antagonistic relationship with RAD51. To conclude, I present a model for how MAEA ubiquitylation of Ku may explain the multiple phenotypes of MAEA loss observed throughout this report.