RNF4 and PLK1 are required for replication fork collapse in ATR-deficient cells

DNA replication is a significant source of double-strand breaks (DSBs) that promote cancer and age-associated pathologies. Accordingly, increased cancer risk and stem cell exhaustion directly correlate with conditions that increase replicative demand in various tissues. These occurrences have been more recently characterized as forms of replication stress, which at the molecular level is defined by an increased frequency of the uncoupling of DNA unwinding and synthesis. The ssDNA that results from this uncoupling activates the ATR–CHK1 pathway as well as other proreplicative responses (Cimprich and Cortez 2008; Branzei and Foiani 2010). In addition to regulating cell cycle phase progression, the ATR–CHK1 (Mec1–Rad53 in Saccharomyces cerevisiae) pathway has been shown to prevent replication fork collapse, which is generally defined as the inability to reinitiate DNA replication after fork stalling (Lopes et al. 2001; Tercero and Diffley 2001; Cimprich and Cortez 2008; Branzei and Foiani 2010). The generation of replication fork-associated recombination structures (e.g., reversed replication forks) and DSBs is increased in ATR/Mec1 pathway-deficient metazoan and yeast cells (Brown and Baltimore 2000; Lopes et al. 2001; Myung et al. 2001; Tercero and Diffley 2001; Cimprich and Cortez 2008; Branzei and Foiani 2010). However, substantial defects in replication restart appear to take place earlier than the generation of recombinatorial structures in S. cerevisiae mec1 mutants (Lopes et al. 2001; Tercero and Diffley 2001; Cobb et al. 2003; Tercero et al. 2003; Raveendranathan et al. 2006). Along these lines, it has been shown in yeast that loss of replication reinitiation coincides with the loss of replisome components from chromatin in mec1 cells (Cobb et al. 2003; Raveendranathan et al. 2006). These findings suggest an association between replisome disassembly and the recombinatorial processes that lead to DSB generation. Indeed, remodeling of the replication apparatus has been proposed to be necessary for replication fork repair and damage bypass in Escherichia coli (Goldfless et al. 2006). In support of the existence of such a mechanism, several replisome components in yeast and metazoans are known to be targeted for protein degradation after lengthy fork stalling. Mrc1 (S. cerevisiae) and CLSPN (metazoans) are orthologous replication factors associated with the MCM2–7 helicase complex that have additional functions in the transmission of Mec1–Rad53 and ATR–CHK1 signaling in response to replication stress (Cimprich and Cortez 2008). Degradation of these proteins is stimulated by their phosphorylation by the Cdc5 (S. cerevisiae) and PLK1 (metazoans) kinases, an event that promotes K48-linked polyubiquitination of CLSPN via recruitment of the β-TRCP ubiquitin ligase in metazoan cells (Yoo et al. 2004; Mailand et al. 2006; Mamely et al. 2006; Peschiaroli et al. 2006). Notably, because CLSPN is required as a docking site to recruit CHK1 for phosphorylation by ATR, the degradation of CLSPN causes a downward spiral driven by the combined effects of replication factor removal and the loss of fork stabilization by the ATR–CHK1 axis (Yoo et al. 2004; Cimprich and Cortez 2008). The same PLK1–β-TRCP pathway regulates the degradation of FANCM, another replisome-associated factor that functions as part of the Fanconi anemia group of repair proteins (Kee et al. 2009). PLK1 and orthologs in yeast (S. cerevisiae Cdc5) have also been shown to regulate the activation of MUS81–EME1 and stimulate its association with the SLX4–endonuclease complex (Matos et al. 2011; Gallo-Fernandez et al. 2012; Munoz-Galvan et al. 2012; Schwartz et al. 2012; Szakal and Branzei 2013). Although it is not clearly essential for fork collapse in all cell types, the Mus81–Eme1 complex in Schizosaccharomyces pombe plays a crucial role in DSB generation at stalled replication forks (Froget et al. 2008), and a similar relationship has been observed in mammalian cells when CHK1 is inhibited (Forment et al. 2011). These findings in aggregate suggest that PLK1 could play an active facilitative role in replication fork collapse at the level of both the replisome and cleaving aberrant fork structures. The S. cerevisiae ubiquitin ligase complex Slx5–Slx8 has also been implicated in replication restart after prolonged stalling (Mullen et al. 2001; Prudden et al. 2007; Nagai et al. 2008). These studies indicated that yeast Slx5–Slx8 and RNF4 in mammalian cells play facilitative roles in the reformation of the replication fork structure by homologous recombination (HR) (Nagai et al. 2008; Galanty et al. 2012; Yin et al. 2012). However, in aggregate, these and other studies also suggest that this complex may affect replication restart through regulation of replication factor stability (Branzei et al. 2006; Bruderer et al. 2011; Plechanovova et al. 2011; Cremona et al. 2012). Slx5–Slx8 and the orthologous mammalian RNF4 dimer are targeted to sites of polysumoylation through four contiguous SUMO-interacting motifs (Bruderer et al. 2011; Plechanovova et al. 2011). In S. cerevisiae, polysumoylation of replisome components is stimulated after fork stalling and occurs initially in a Mec1-independent manner (Branzei et al. 2006; Cremona et al. 2012), although loss of Mec1 activity increases sumoylation in the absence of fork stalling (Cremona et al. 2012). Thus, the known targeting of RNF4 to polySUMO2/3-conjugated proteins suggests that RNF4 is localized to replisome components after fork stalling and participates in replication fork collapse when ATR/Mec1 is absent. Although these advances imply that PLK1 and RNF4 may regulate replication fork collapse in ATR-deficient cells, this hypothesis has not been tested. Here, we show that suppression of either the AURKA–PLK1 pathway or RNF4 permits replication restart following collapse in Atr-deleted cells. Replication recovery in Atr-deleted cells is not associated with dormant origin firing but instead takes place at formerly stalled replication forks. Notably, combined suppression of RNF4 or the AURKA–PLK1 pathway almost completely prevents replication fork collapse into DSBs, with suppression of RNF4 alone having a more pronounced rescuing effect than inhibition of PLK1. These data indicate that replication fork collapse in mammalian cells is an active and cell-dominant signaling process that is necessary for the cleavage of replication forks into DSBs.