USP37 prevents premature disassembly of stressed replisomes by TRAIP

The E3 ubiquitin ligase TRAIP associates with the replisome and helps this molecular machine deal with replication stress. While TRAIP promotes DNA inter-strand crosslink repair by triggering the disassembly of CDC45-MCM2-7-GINS (CMG) helicases that have converged on these lesions, disassembly of single CMGs that have stalled temporarily would be deleterious, suggesting that TRAIP must be carefully regulated. Here, we demonstrate that human cells lacking the de-ubiquitylating enzyme USP37 are hypersensitive to topoisomerase poisons and other replication stress-inducing agents. We further show that TRAIP loss rescues the hypersensitivity of USP37 knockout cells to topoisomerase inhibitors. In Xenopus egg extracts depleted of USP37, TRAIP promotes premature CMG ubiquitylation and disassembly when replisomes stall before they have fully converged. Finally, we provide evidence that USP37 binds replisomes via CDC45, and that this interaction mediates USP37’s response to topological stress. Based on these results, we propose that USP37 protects genome stability by preventing TRAIP-dependent CMG unloading when replication stress impedes timely termination.

Methods
For CRISPR/Cas9 screens, biological duplicates of U2OS Cas9 wild-type and USP37 KO cells were transduced at a MOI of 0.3 and 500-fold coverage of the Brunello library (Doench et al., 2016). Afterward, transductants were selected with 1.5 µg/mL puromycin for 12 days. Library preparation and next-generation sequencing of the samples was performed as described previously (Bowden et al., 2020). Guide-enrichment analysis was performed using DrugZ (Colic et al., 2019).