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

Abstract

The ATR-CHK1 axis stabilizes stalled replication forks and prevents their collapse into DNA double-strand breaks (DSBs). Here, we show that fork collapse in Atr-deleted cells is mediated through the combined effects the sumo targeted E3-ubiquitin ligase RNF4 and activation of the AURKA-PLK1 pathway. As indicated previously, Atrdeleted cells exhibited a decreased ability to restart DNA replication following fork stalling in comparison with control cells. However, suppression of RNF4, AURKA, or PLK1 returned the reinitiation of replication in Atrdeleted cells to near wild-type levels. In RNF4-depleted cells, this rescue directly correlated with the persistence of sumoylation of chromatin-bound factors. Notably, RNF4 repression substantially suppressed the accumulation of DSBs in ATR-deficient cells, and this decrease in breaks was enhanced by concomitant inhibition of PLK1. DSBs resulting from ATR inhibition were also observed to be dependent on the endonuclease scaffold protein SLX4, suggesting that RNF4 and PLK1 either help activate the SLX4 complex or make DNA replication fork structures accessible for subsequent SLX4-dependent cleavage. Thus, replication fork collapse following ATR inhibition is a multistep process that disrupts replisome function and permits cleavage of the replication fork.

The SLX4 Complex Is a SUMO E3 Ligase that Impacts on Replication Stress Outcome and Genome Stability

“…The SUMO E3 Ligase Activity of SLX4 Mediates Toxicity in Response to Global Replication Stress Recent studies reported that SLX4 contributes to DSB formation in response to global replication stress (Couch et al, 2013;Fugger et al, 2013;Ragland et al, 2013). Our findings suggested that these effects might correlate with the SIM-and BTB-dependent cytotoxicity induced by high overexpression levels of SLX4 ( Figure 5B).…”

“…Remarkably, this SLX4-mediated toxicity was abolished when the SIMs or the BTB were mutated ( Figures 6A and S6B), suggesting that SLX4-dependent SUMO E3 ligase activity is toxic in cells facing global replication fork stalling. Low doses of HU combined with ATR checkpoint kinase inhibition (ATRi) result in cell death associated with replication fork collapse, DSB induction, and extensive ssDNA generation at the fork (Couch et al, 2013;Ragland et al, 2013). Depletion of SLX4 also attenuated the synthetic lethality of the double treatment HU+ATRi in the parental FIT 0 cells ( Figure 6B).…”

Regulation of recombination at yeast nuclear pores controls repair and triplet repeat stability

“…RNF4 has also been implicated in the response to DNA damage and facilitates damage-induced HR in human cells (Galanty et al 2012;Yin et al 2012;Ragland et al 2013). Moreover, DSBs in heterochromatin have been shown to move out of heterochromatin for repair in Drosophila and human cells (Chiolo et al 2011;Jakob et al 2011), and DSB repair pathway choice is also influenced by nuclear position in human cells, with end-joining more prevalent at the nuclear lamina, and HR more prevalent at nuclear pores (Lemaitre et al 2014).…”

Preventing replication fork collapse to maintain genome integrity

“…Evidence for checkpoint-dependent replisome stabilization comes from both indirect and direct experimental approaches. These data include decreases in the abundance of some replisome components at stalled forks in checkpoint-deficient yeast as measured by ChIP [17, 49–51], loss of early PCNA staining patterns in human and fission yeast cells [43, 52], and decreases in the amount of some replisome proteins bound to chromatin in both Xenopus in vitro replication systems and human cells [45, 53, 54]. …”