DNA-dependent protease activity of human Spartan facilitates replication of DNA–protein crosslink-containing DNA

Mutations in SPARTAN are associated with early onset hepatocellular carcinoma and progeroid features. A regulatory function of Spartan has been implicated in DNA damage tolerance pathways such as translesion synthesis, but the exact function of the protein remained unclear. Here, we reveal the role of human Spartan in facilitating replication of DNA–protein crosslink-containing DNA. We found that purified Spartan has a DNA-dependent protease activity degrading certain proteins bound to DNA. In concert, Spartan is required for direct DPC removal in vivo; we also show that the protease Spartan facilitates repair of formaldehyde-induced DNA–protein crosslinks in later phases of replication using the bromodeoxyuridin (BrdU) comet assay. Moreover, DNA fibre assay indicates that formaldehyde-induced replication stress dramatically decreases the speed of replication fork movement in Spartan-deficient cells, which accumulate in the G2/M cell cycle phase. Finally, epistasis analysis mapped these Spartan functions to the RAD6-RAD18 DNA damage tolerance pathway. Our results reveal that Spartan facilitates replication of DNA–protein crosslink-containing DNA enzymatically, as a protease, which may explain its role in preventing carcinogenesis and aging.