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Abstract
Coordination of the multiple processes underlying DNA replication is key for maintaining genome stability and preventing tumorigenesis. CLASPIN, a critical player in replication fork stabilization and checkpoint responses, must be tightly regulated during the cell cycle to prevent the accumulation of DNA damage. In this study, we used a quantitative proteomics approach and identified USP9X as a novel CLASPIN-interacting protein. USP9X is a deubiquitinase involved in multiple signaling and survival pathways whose tumor suppressor or oncogenic activity is highly context dependent. We found that USP9X regulated the expression and stability of CLASPIN in an S-phase-specific manner. USP9X depletion profoundly impairs the progression of DNA replication forks, causing unscheduled termination events with a frequency similar to CLASPIN depletion, resulting in excessive endogenous DNA damage. Importantly, restoration of CLASPIN expression in USP9X-depleted cells partially suppressed the accumulation of DNA damage. Furthermore, USP9X depletion compromised CHK1 activation in response to hydroxyurea and UV, thus promoting hypersensitivity to drug-induced replication stress. Taken together, our results reveal a novel role for USP9X in the maintenance of genomic stability during DNA replication and provide potential mechanistic insights into its tumor suppressor role in certain malignancies. Cancer Res; 76(8); 2384-93. ©2016 AACR.
Highlights
The timely and precise duplication of DNA in S-phase of the cell cycle is critical to maintaining genome stability and preventing tumorigenesis [1, 2]
We show that USP9X controls CLASPIN levels, most likely through counteracting its ubiquitinylation in S-phase
The effect of USP9X depletion on DNA replication is almost indistinguishable from the direct downregulation of CLASPIN itself, suggesting that CLASPIN is the main target of USP9X in the process of DNA replication
Summary
The timely and precise duplication of DNA in S-phase of the cell cycle is critical to maintaining genome stability and preventing tumorigenesis [1, 2]. Genome duplication is a tightly coordinated process where mechanisms that regulate replication origin activation lead to the assembly of new replication forks in a defined spatiotemporal program [3]. During S-phase, cells are extremely vulnerable to exogenous and endogenous sources of DNA damage that impede the progression of replication forks causing replication stress. Cellular surveillance mechanisms that overlap with the DNA damage response pathways detect, stabilize, and resolve stalled replication forks to help preserve genome stability [2]. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Gaboriau: Facility for Imaging by Light Microscopy (FILM), National Heart and Lung Institute, Imperial College London, London SW7 2AZ, United Kingdom
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