Survival of the Replication Checkpoint Deficient Cells Requires MUS81-RAD52 Function

Abstract

In checkpoint-deficient cells, DNA double-strand breaks (DSBs) are produced during replication by the structure-specific endonuclease MUS81. The mechanism underlying MUS81-dependent cleavage, and the effect on chromosome integrity and viability of checkpoint deficient cells is only partly understood, especially in human cells. Here, we show that MUS81-induced DSBs are specifically triggered by CHK1 inhibition in a manner that is unrelated to the loss of RAD51, and does not involve formation of a RAD51 substrate. Indeed, CHK1 deficiency results in the formation of a RAD52-dependent structure that is cleaved by MUS81. Moreover, in CHK1-deficient cells depletion of RAD52, but not of MUS81, rescues chromosome instability observed after replication fork stalling. However, when RAD52 is down-regulated, recovery from replication stress requires MUS81, and loss of both these proteins results in massive cell death that can be suppressed by RAD51 depletion. Our findings reveal a novel RAD52/MUS81-dependent mechanism that promotes cell viability and genome integrity in checkpoint-deficient cells, and disclose the involvement of MUS81 to multiple processes after replication stress.

The fork and the kinase: A DNA replication tale from a CHK1 perspective

“…Several lines of evidence support the hypothesis that Chk1 avoids the collapse of ongoing forks: i) Chk1 pharmacological inhibition or depletion by siRNA lead to increased accumulation of replication-associated DSBs (Forment et al, 2011; Murfuni et al, 2013; Sorensen et al, 2005; Syljuasen et al, 2005); ii) CHOC cells treated with the Chk1 inhibitor UCN-01 and Chk1−/− avian B-lymphoma cells show reduced ability to resume replication after an APH-induced arrest (Feijoo et al, 2001; Zachos et al, 2003); iii) Chk1 inactivation reduces the rate of replication fork progression in unperturbed, UV-exposed and CPT-treated cells (Petermann and Caldecott, 2006; Seiler et al, 2007; Speroni et al, 2012). Although these results suggest that Chk1 stabilizes replication forks, impaired fork elongation in Chk1-deficient backgrounds may represent an indirect effect of elevated origin firing (Petermann et al, 2010).…”

“…Particularly, Chk1 phosphorylates Rad51 and BRCA2 to stimulate the recruitment of Rad51 to sites of DNA damage (Bahassi et al, 2008; Sorensen et al, 2005). Nuclease-mediated accumulation of DSBs after Chk1 inhibition or depletion reveals that, apart from promoting HR-dependent processing of DSBs, Chk1 prevents the accumulation of DSBs in the first place (Forment et al, 2011; Murfuni et al, 2013; Syljuasen et al, 2005). Specifically, DSB formation in Chk1-deficient cells is catalyzed by the Mus81 endonuclease (Forment et al, 2011; Murfuni et al, 2013) and the MRE11 nuclease (Thompson et al, 2012).…”

“…Nuclease-mediated accumulation of DSBs after Chk1 inhibition or depletion reveals that, apart from promoting HR-dependent processing of DSBs, Chk1 prevents the accumulation of DSBs in the first place (Forment et al, 2011; Murfuni et al, 2013; Syljuasen et al, 2005). Specifically, DSB formation in Chk1-deficient cells is catalyzed by the Mus81 endonuclease (Forment et al, 2011; Murfuni et al, 2013) and the MRE11 nuclease (Thompson et al, 2012). Moreover, nucleases such as SLX4 and GEN1 cause DSB formation in the absence of both Chk1 and Mus81 (Murfuni et al, 2013).…”

Mammalian RAD52 Functions in Break-Induced Replication Repair of Collapsed DNA Replication Forks

“…A role for Rad52 in BIR can explain why its depletion in cells with DRS leads to increased DNA damage (Wray et al., 2008, Murfuni et al., 2013, Galanos et al., 2016; Figures 3A and 3B), why the RAD52 gene is amplified in human cancers, and why its inactivation curtails cancer development (Treuner et al., 2004, Cramer-Morales et al., 2013, Lieberman et al., 2016; Figure 4). BIR also mediates DNA repair synthesis in mitosis (Minocherhomji et al., 2015), and it is noteworthy that Rad52 is essential also in this context (Bhowmick et al., 2016).…”

MUS81 is associated with cell proliferation and cisplatin sensitivity in serous ovarian cancer

“…These findings have indicated that MUS81 plays a key role in the maintenance of genetic stability. Meanwhile, some works demonstrated that MUS81 interacts with checkpoint proteins, such as Checkpoint kinase 1 [12] and Checkpoint kinase 2 [13], suggesting the association between MUS81 and cell cycle progression.…”