DNA Structure-Specific Cleavage of DNA-Protein Crosslinks by the SPRTN Protease

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. [Display omitted] •DNA-protein crosslink cleavage by SPRTN is coupled to recognition of DNA context•DNA-protein crosslinks are only cleaved in proximity to activating DNA structures•Two distinct interfaces recognize DNA with single- and double-stranded features•Activation of SPRTN depends on simultaneous engagement of both DNA binding interfaces Reinking et al. show that the protease SPRTN degrades DNA-protein crosslinks in a DNA structure-specific manner, which restricts cleavage to biologically relevant scenarios. NMR analysis reveals that specificity is achieved by a bipartite strategy relying on two DNA binding interfaces that recognize single- and double-stranded features within the substrate.