Replication-Coupled DNA-Protein Crosslink Repair by SPRTN and the Proteasome in Xenopus Egg Extracts

• Published in: Molecular cell 2019-02, Vol.73 (3), p.574-588.e7
• Main Authors: Larsen, Nicolai B., Gao, Alan O., Sparks, Justin L., Gallina, Irene, Wu, R. Alex, Mann, Matthias, Räschle, Markus, Walter, Johannes C., Duxin, Julien P.
• Format: Article
• Language: English
• Subjects: Animals; DNA - biosynthesis; DNA - chemistry; DNA - genetics; DNA Repair; DNA Replication; DNA-portein crosslink (DPC); Female; Male; Nucleic Acid Conformation; Proteasome; Proteasome Endopeptidase Complex - genetics; Proteasome Endopeptidase Complex - metabolism; Protein Interaction Domains and Motifs; Proteolysis; Sf9 Cells; SPRTN; Structure-Activity Relationship; TRAIP; translesion synthesis (TLS); Ubiquitin; Ubiquitination; Xenopus laevis - genetics; Xenopus laevis - metabolism; Xenopus Proteins - genetics; Xenopus Proteins - metabolism
• ISSN: 1097-2765; 1097-4164
• DOI: 10.1016/j.molcel.2018.11.024

DNA-protein crosslinks (DPCs) are bulky lesions that interfere with DNA metabolism and therefore threaten genomic integrity. Recent studies implicate the metalloprotease SPRTN in S phase removal of DPCs, but how SPRTN is targeted to DPCs during DNA replication is unknown. Using Xenopus egg extracts that recapitulate replication-coupled DPC proteolysis, we show that DPCs can be degraded by SPRTN or the proteasome, which act as independent DPC proteases. Proteasome recruitment requires DPC polyubiquitylation, which is partially dependent on the ubiquitin ligase activity of TRAIP. In contrast, SPRTN-mediated DPC degradation does not require DPC polyubiquitylation but instead depends on nascent strand extension to within a few nucleotides of the lesion, implying that polymerase stalling at the DPC activates SPRTN on both leading and lagging strand templates. Our results demonstrate that SPRTN and proteasome activities are coupled to DNA replication by distinct mechanisms that promote replication across immovable protein barriers. [Display omitted] •The proteasome and SPRTN can each degrade DPCs during DNA replication•Efficient DPC ubiquitylation and proteasome targeting requires TRAIP•SPRTN can degrade DPCs that are not ubiquitylated•SPRTN-mediated DPC degradation is a post-replicative process DNA-protein crosslinks (DPCs) pose major threats to genomic integrity. Using a cell-free system, Larsen et al. show that SPRTN and the proteasome operate as independent replication-coupled DPC proteases. The existence of two DPC proteases with distinct modes of action facilitates the degradation of chemically diverse DPCs.