Repair of covalent DNA-protein crosslinks (DPCs) by DNA-dependent proteases has emerged as an essential genome maintenance mechanism required for cellular viability and tumor suppression. However, how proteolysis is restricted to the crosslinked protein while leaving surrounding chromatin proteins unharmed has remained unknown. Using defined DPC model substrates, we show that the DPC protease SPRTN displays strict DNA structure-specific activity. Strikingly, SPRTN cleaves DPCs at or in direct proximity to disruptions within double-stranded DNA. In contrast, proteins crosslinked to intact double- or single-stranded DNA are not cleaved by SPRTN. NMR spectroscopy data suggest that specificity is not merely affinity-driven but achieved through a flexible bipartite strategy based on two DNA binding interfaces recognizing distinct structural features. This couples DNA context to activation of the enzyme, tightly confining SPRTN's action to biologically relevant scenarios.
GrantsDeutsche Forschungsgemeinschaft Ludwig-Maximilians-Universität München Alfried Krupp von Bohlen und Halbach-Stiftung Peter and Traudl Engelhorn Foundation LMU -China Scholarship Council Program International Max-Planck Research School for Molecular Life Sciences European Research Council (ERC) Alfried Krupp Prize for Young University Teachers - Alfried-Krupp von Bohlen und Halbach-Stiftung European Research Council (ERC Starting Grant SOLID) Center for Integrated Protein Science Munich (CIPSM)