A bacterial protein inhibitor of dUTPase disrupts zebrafish embryogenesis through a conserved active-site mechanism

Perey-Simon, Viktória and Tóth, Zoé Sára and Dombovári, Dániel and Kazzazy, Latifa and Varga, Máté and Vértessy G., Beáta and Nagy, Gergely Nándor and Nyíri, Kinga (2026) A bacterial protein inhibitor of dUTPase disrupts zebrafish embryogenesis through a conserved active-site mechanism. PROTEIN SCIENCE. ISSN 0961-8368 (In Press)

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Abstract

Deoxyuridine triphosphatase (dUTPase) is essential for DNA replication fidelity and embryonic development in metazoa, yet whether a protein inhibitor can acutely disrupt dUTPase function in a living vertebrate has not been tested. The Staphylococcus aureus Stl protein inhibits diverse dUTPases in vitro, but its efficacy in a eukaryotic organism was unknown. We determined the crystal structure of zebrafish dUTPase in complex with the N terminal Stl fragment to 2.26 Å resolution, revealing conserved active-site engagement and displacement of the C-terminal arm. Biolayer interferometry confirmed low-nanomolar binding affinity with KD = 0.7 nM, and enzyme assays demonstrated ~50% inhibition of zebrafish dUTPase activity at saturating Stl concentrations. These structural and biophysical data establish that Stl forms a tight inhibitory complex with zebrafish dUTPase, prompting us to test whether this interaction can perturb enzyme function in vivo. Microinjection of Stl or its point mutant, retaining dUTPase-inhibitory activity but unable to bind DNA, into fertilized zebrafish oocytes reduces embryo survival to ~60% within 48 hours compared to ~90% in controls, demonstrating that lethality depends on dUTPase targeting rather than DNA binding. Accelerated mortality relative to genetic dUTPase knockout indicates that maternally deposited dUTPase supports viability during early cleavage. Stl thus acts as an immediate, proteinaceous dUTPase inhibitor in a living vertebrate, providing a tool to dissect dUTPase function and regulation during embryonic development.

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