Abstract
The DNA damage response is vigorously activated by DNA double-strand breaks (DSBs). The chief mobilizer of the DSB response is the ATM protein kinase. We discovered that the COP9 signalosome (CSN) is a crucial player in the DSB response and an ATM target. CSN is a protein complex that regulates the activity of cullin ring ubiquitin ligase (CRL) complexes by removing the ubiquitin-like protein, NEDD8, from their cullin scaffold. We find that the CSN is physically recruited to DSB sites in a neddylation-dependent manner, and is required for timely repair of DSBs, affecting the balance between the two major DSB repair pathways—nonhomologous end-joining and homologous recombination repair (HRR). The CSN is essential for the processivity of deep end-resection—the initial step in HRR. Cullin 4a (CUL4A) is recruited to DSB sites in a CSN- and neddylation-dependent manner, suggesting that CSN partners with CRL4 in this pathway. Furthermore, we found that ATM-mediated phosphorylation of CSN subunit 3 on S410 is critical for proper DSB repair, and that loss of this phosphorylation site alone is sufficient to cause a DDR deficiency phenotype in the mouse. This novel branch of the DSB response thus significantly affects genome stability.
Highlights
The DNA damage response (DDR) constitutes a central axis in the maintenance of genome stability [1,2,3]
In order to search for DNA damage-induced phosphorylation of COP9 signalosome (CSN) subunits in cells, we expressed these subunits in HEK293 cells as ectopic HA-tagged proteins, and treated the cells with the radiomimetic drug neocarzinostatin (NCS) concurrently with a protein phospho-labeling pulse
A polyclonal phospho-specific antibody raised to detect this assumed phosphorylation reacted strongly with ectopic wild-type CSN subunit 3 (CSN3) expressed following NCS treatment, but not with an S410A mutant version of this protein (Figure 1C). This result indicated that phosphorylation of CSN3 on Ser410 occurred in cells in response to DNA damage and was detected by the antibody
Summary
The DNA damage response (DDR) constitutes a central axis in the maintenance of genome stability [1,2,3]. Its early phase is marked by the recruitment of a heterogeneous group of proteins to DSB sites, collectively dubbed ‘sensors’ or ‘mediators’ These proteins coalesce into highly ordered structures, visible as nuclear foci at the break sites [4], whose activity leads to chromatin reorganization and transcription arrest at the sites of DNA damage and sets the scene for DSB repair [5]. This activity is regulated by extensive protein post-translational modifications, such as phosphorylation and ubiquitylation, which
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