Highlights d A CRISPR-Cas9 screen identifies ETAA1 as a determinant of CDC7 inhibitors' potency d CDC7 inhibition causes replication stress and an ATR response requiring ETAA1 d ATR reinforces replication origin firing suppression caused by CDC7 inhibitors d CDC7 and ATR co-inhibition leads to premature and highly defective mitosis
Ubiquitination regulates protein homeostasis and is tightly controlled by deubiquitinases (DUBs). Loss of the DUB UCHL1 leads to neurodegeneration, and its dysregulation promotes cancer metastasis and invasiveness. Small molecule probes for UCHL1 and DUBs in general could help investigate their function, yet specific inhibitors and structural information are rare. Here we report the potent and non-toxic chemogenomic pair of activity-based probes GK13S and GK16S for UCHL1. Biochemical characterization of GK13S demonstrates its stereoselective inhibition of cellular UCHL1. The crystal structure of UCHL1 in complex with GK13S shows the enzyme locked in a hybrid conformation of apo and Ubiquitin-bound states, which underlies its UCHL1-specificity within the UCH DUB family. Phenocopying a reported inactivating mutation of UCHL1 in mice, GK13S, but not GK16S, leads to reduced levels of monoubiquitin in a human glioblastoma cell line. Collectively, we introduce a set of structurally characterized, chemogenomic probes suitable for the cellular investigation of UCHL1.
In order to prevent the deleterious effects of genotoxic agents, cells have developed complex surveillance mechanisms and DNA repair pathways that allow them to maintain genome integrity. The Ubiquitin Specific Protease 9X (USP9X) contributes to genome stability during DNA replication and chromosome segregation. Depletion of USP9X leads to DNA double strand breaks, some of which are triggered by replication fork collapse. Here we identify USP9X as a novel regulator of homologous recombination (HR) DNA repair in human cells. Using a cellular HR reporter assay, irradiation induced focus formation assays, and colony formation assays we show that USP9X is required for efficient HR. Mechanistically, we show USP9X is important to sustain the expression levels of key HR factors, namely BRCA1 and RAD51 through a non-canonical regulation of their mRNA abundance. Intriguingly, we find that USP9X contribution to BRCA1 and RAD51 expression is independent of its known catalytic activity. Thus this work identifies USP9X as a regulator of HR, demonstrates a novel mechanism by which USP9X can regulate protein levels and provides insights in to the regulation of BRCA1 and RAD51 mRNA.
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