ATR Prohibits Replication Catastrophe by Preventing Global Exhaustion of RPA

Abstract

ATR, activated by replication stress, protects replication forks locally and suppresses origin firing globally. Here, we show that these functions of ATR are mechanistically coupled. Although initially stable, stalled forks in ATR-deficient cells undergo nucleus-wide breakage after unscheduled origin firing generates an excess of single-stranded DNA that exhausts the nuclear pool of RPA. Partial reduction of RPA accelerated fork breakage, and forced elevation of RPA was sufficient to delay such "replication catastrophe" even in the absence of ATR activity. Conversely, unscheduled origin firing induced breakage of stalled forks even in cells with active ATR. Thus, ATR-mediated suppression of dormant origins shields active forks against irreversible breakage via preventing exhaustion of nuclear RPA. This study elucidates how replicating genomes avoid destabilizing DNA damage. Because cancer cells commonly feature intrinsically high replication stress, this study also provides a molecular rationale for their hypersensitivity to ATR inhibitors.

LY2606368 Causes Replication Catastrophe and Antitumor Effects through CHK1-Dependent Mechanisms

“…Toledo and colleagues recently reported that the ATR/CHK1 signaling axis maintains the integrity of stalled replication forks in part by regulating the available pool of RPA to coat exposed ssDNA (13). Under conditions of replication stress, inhibition of ATR and CHK1 resulted in depletion of the nuclear RPA pool, breakage of stalled forks, and fragmentation of chromosomes.…”

“…Another hallmark of replication catastrophe caused by loss of ATR/CHK1 origin control is depletion of the pool of RPA protein available to protect ssDNA at replication forks (13). This is due to the excess of open replication forks with exposed ssDNA and can be measured by high-content image analysis of the coaccumulation of RPA2 protein and phosphorylated H2AX (S139) on the chromatin of each cell.…”

“…In a cascade effect, loss of CHK1 activity may also lead to fork instability and collapse. A recent study using inhibitors of ATR and CHK1 reported that the abundance of ssDNA during replication stress exhausts the available pool of RPA increasing the likelihood that unprotected ssDNA will be cleaved by endonucleases (13). Stalled forks are believed to be repaired primarily, but not exclusively, by homologous recombination (14,15).…”

“…Ultimately, the cell enters mitosis with fragmented chromosomes resulting in cell death. Cell death caused by failure of the ATR/ CHK1 axis during replication stress has been described as replication catastrophe (13).…”

Replication stress and cancer: It takes two to tango

“…In their absence, stalled forks persist and additional backup origins are fired, leading to an exhaustion of dNTP pools and a further accumulation of non-progressive forks. When the amount of ssDNA exceeds the pool of available RPA, and by yet not fully understood mechanisms, forks collapse leading to the generation of DNA double strand breaks (DSB) [7]. The lack of checkpoint activation in these cells allows them to progress to mitosis with unreplicated chromosomes leading to death by mitotic catastrophe.…”