Abstract
CLASPIN is an essential mediator in the DNA replication checkpoint, responsible for ATR (ataxia telangiectasia and Rad3-related protein)-dependent activation of CHK1 (checkpoint kinase 1). Here we found a dynamic signaling pathway that regulates CLASPIN turn over. Under unperturbed conditions, the E3 ubiquitin ligase HERC2 regulates the stability of the deubiquitinating enzyme USP20 by promoting ubiquitination-mediated proteasomal degradation. Under replication stress, ATR-mediated phosphorylation of USP20 results in the disassociation of HERC2 from USP20. USP20 in turn deubiquitinates K48-linked-polyubiquitinated CLASPIN, stabilizing CLASPIN and ultimately promoting CHK1 phosphorylation and CHK1-directed checkpoint activation. Inhibition of USP20 expression promotes chromosome instability and xenograft tumor growth. Taken together, our findings demonstrated a novel function of HERC2/USP20 in coordinating CHK1 activation by modulating CLASPIN stability, which ultimately promotes genome stability and suppresses tumor growth.
Highlights
Our genome is extremely vulnerable in S-phase when the genetic material is being duplicated and in mitotic phase when the pairs of sister chromatids are being separated into two daughter cells [1,2]
In a candidate screen of ubiquitin-specific processing proteases (USPs) for modulating CHK1 activation, we revealed that inhibition of USP20 expression in 293T cells delayed HU-induced CHK1 activation (Figure 1A and B), whereas expression of siRNA-resistant form of FLAG-USP20res in the endogenous USP20-depleted cells restored HU-induced CHK1 activation kinetics (Figure 1C)
These results indicate that USP20 promotes CHK1 activation in response to HU-induced replication stress or UV irradiation
Summary
Our genome is extremely vulnerable in S-phase when the genetic material is being duplicated and in mitotic phase when the pairs of sister chromatids are being separated into two daughter cells [1,2]. When the replication fork progression is halted, the intra-S-phase checkpoint is activated, promoting structural stability of stalled forks and preventing the replisome components from dissociation [4,5]. This ensures the rapid resumption of replication following DNA repair. The ATR (ataxia telangiectasia and Rad3-related protein)CHK1 (checkpoint kinase 1) pathway plays key roles in activating the intra-S-phase checkpoint and in stabilizing the stalled replication forks [5,6,7,8]. The ATR/ATRIP complex, in coordination with RAD17 and the 9–1–1 (RAD9HUS1-RAD1) complex, phosphorylates CHK1 on serines 317 and 345 and activates it on chromatin in a CLASPINdependent manner. Activated CHK1 is released from chromatin and phosphorylates downstream effectors [3,5,11,12]
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