Naked Replication Forks Break apRPArt

Abstract

Stalled replication forks occasionally collapse, leading to potentially catastrophic DNA double-strand breaks. Now, Toledo et al. (2013) reveal that fork breakage occurs when the pool of the single-strand DNA-binding protein RPA becomes exhausted. This study has important implications for the origin and treatment of cancers with high levels of replicative stress.

During DNA synthesis, the progression of replication forks is challenged by sequences that are difficult to replicate or by DNA lesions, resulting in the arrest of replication forks. The conditions that cause replication fork stalling, collectively referred to as “replication stress,” include DNA base or topoisomerase adducts, limiting nucleotide concentrations, intra-strand crosslinks, and deregulated expression of oncogenes. Although replication stress is known to be a major source of genome instability in precancerous lesions (Halazonetis et al., 2008), the series of events leading from replication fork stalling to genomic rearrangements are largely unknown. Toledo et al. (2013) now take an important step forward by explaining how DNA breaks arise at sites of replication stress.

More than a decade ago, it was observed that drugs that perturb replication fork progression lead to a catastrophic collapse of forks in budding yeast mutants lacking the checkpoint kinases Mec1/Rad53 (Lopes et al., 2001; Tercero and Diffley, 2001) and in mouse cells lacking the Mec1 homolog ATR (Brown and Baltimore, 2000). These observations suggest that a conserved signaling cascade mediated by the DNA damage checkpoint prevents irreversible breakdown of stalled replication forks.

How checkpoints are activated during S phase is an area of intense interest. Drugs that perturb replication progression, such as the alkylating agent MMS or the ribonucleotide reductase inhibitor HU, cause checkpoint activation by un-coupling the stalled polymerase from the helicase that unwinds the DNA in front of the lesion (Byun et al., 2005). This uncou pling leads to a vulnerable stretch of single-strand DNA (ssDNA) that is rapidly coated by the ssDNA protein RPA. RPA then loads the ATR kinase on the damaged region, which initiates the checkpoint-signaling cascade. The best-characterized ATR substrate Chk1 transmits the DNA damage signal to the rest of the nucleus. Chk1 targets cell-cycle transitions that prevent damaged cells from entering mitosis and slows down the rate of DNA synthesis by inhibiting new DNA replication origins from firing. Similar to ATR inhibition, compromised Chk1 activity results in impaired checkpoints and fragmented chromosomes, further strengthening the notion that cell-cycle checkpoints somehow prevent uncontrolled fork collapse.

It remains mysterious how such checkpoint mechanisms that act away from the stalled replication fork maintain local fork stability. One possibility is that checkpoint signaling also impacts locally on replisome components, which would contribute directly to fork stabilization. Another possibility is that ATR signaling regulates recombinational repair, which is necessary to restart already collapsed forks. In the current issue, Toledo and colleagues demonstrate an alternative mechanism for replication fork stability that unifies the local and global activities of ATR.

By an elegant use of high-throughput microscopy, the authors reveal that excessive RPA loading at ssDNA regions away from the originally stalled fork is coupled to DNA break formation at all active forks. Absence of ATR activity leads to an unscheduled firing of dormant replication origins. When cells are simultaneously treated with a source of replication stress, Toledo et al. observe that the number of stalled forks exceeds the pool of RPA, and ssDNA becomes simultaneously exposed at all forks. By yet unknown mechanisms, exposed ssDNA is cleaved, leading to the formation of DNA breaks (Figure 1). Consistent with the idea that RPA is limiting for fork breakage, depletion of RPA lowers the threshold of replication stress necessary for DNA break formation and, conversely, increased levels of RPA delays fork collapse. As noted by the authors, such increased replication fidelity may be beneficial for cancer cells, and indeed recent studies demonstrate that supraphysiological levels of Chk1 facilitate oncogene-mediated transformation by protecting cells from replication stress (López-Contreras et al., 2012).

Figure 1. DNA Breaks Arising at Stalled Replication Forks.

The figure classifies the different kinds of chromosome breaks that are induced by replication stress, on the basis of: the cell-cycle stage in which they occur (WHEN); the cause of the replication stress (WHY); the enzymes that produce the cleavage (HOW); and the signal that triggers the process (SIGNAL). The work of Toledo et al. demonstrates global breakage at every active fork when ssDNA is exposed through the exhaustion of the RPA pool. A more restricted activation of this process could initiate complex genomic rearrangements such as chromothripsis. To what extent the pathway revealed here contributes to DNA breaks that arise at specific fragile loci such as common fragile sites (CFS) or early replicating fragile sites (ERFS) remains to be determined.

This higher reliance of cancer cells on pathways that suppress replication stress may explain their sensitivity to ATR and Chk1 inhibitors, which are currently being explored for cancer chemotherapy. One prediction from this study is that therapeutic strategies that increase the generation of ssDNA tracks could synergize with compounds that lead to unscheduled origin firing. For example, conditions that increase end resection of DNA breaks, an essential intermediary step in homologous recombination, might be particularly cytotoxic in combination with inhibition of ATR activity due to the sequestering of RPA.

One of the most surprising findings from this manuscript is that, once naked ssDNA starts to accumulate, DNA breaks appear suddenly and simultaneously at every replication fork. This is consistent with the observed shattering of chromosomes that accumulate in cells lacking ATR or Chk1. How unshielded ssDNA leads to DSB formation remains unclear, but one possibility is that ssDNA becomes vulnerable to nucleases in the absence of RPA. The study by Toledo et al. may also provide a mechanistic framework for understanding the phenomenon of chromothripsis, a single catastrophic event leading to massive localized chromosomal rearrangements in cancer cells. These cancer-driving aberrations could originate from isolated genomic regions undergoing replication in RPA-devoid locations such as micronuclei.

It is important to note that RPA exhaustion is not the only mechanism that promotes replication fork instability. Replication stress has also been shown to preferentially target late replicating genomic regions referred to as common fragile sites (CFS) and early replicating fragile sites (ERFS), which are detected as individual breaks or gaps in metaphase chromosomes (Barlow et al., 2013; Casper et al., 2002). In the case of CFS, structure-specific nucleases cleave replication forks that have entered mitosis without completing replication (Mankouri et al., 2013), whereas replication fork collapse at ERFS is thought to be a result of collisions between the replication and transcription machinery during early S phase (Barlow et al., 2013). Thus, when RPA is limiting, all forks collapse, but milder conditions of RS can cause individual forks to be destabilized (Figure 1).

The sudden and massive phenomenon described here is reminiscent of phase transitions observed throughout nature. The most familiar example is the one that occurs in water: the abrupt, discontinuous transition from a liquid to a gas or a solid, induced by a subtle environmental change. An ever-so-slight shift in temperature or pressure can induce an astonishing transition from one state of matter to another entity that bears little resemblance to the first. Now it seems that a similar all-or-nothing transition might occur within cells suffering from replication stress, whereby every nonharmful replication fork is suddenly converted into a toxic DNA knife. A better understanding of this phenomenon might provide us with sharper weapons to fight the war on cancer.