Updated project metadata. DNA-protein crosslinks (DPCs) are abundant DNA lesions, which constantly challenge genome stability by interfering with DNA replication. SPARTAN (SPRTN) protease plays a central role in DPCs repair in proliferative vertebrate cells. SPRTN is a constitutive part of the DNA replication machinery and its protease activity is essential for DNA replication fork progression. How this essential protease is activated and regulated during DNA replication is not known. By using biochemical, cell biological and genetic approaches in human cell lines and Zebrafish model system, we identified that SPRTN cleaves covalently crosslinked Checkpoint kinase 1 (CHK1) from chromatin and releases CHK1 during physiological DNA replication. Proteolysed CHK1 is activated and in turn phosphorylates SPRTN. Phosphorylated SPRTN is further recruited to chromatin to proteolyse DPCs that ensures unperturbed DNA replication. Our data suggest SPRTN-CHK1 cross-activation loop is essential for genome stability.