Exhaustion of RPA Leads to Replication Catastrophe

When DNA replication forks stall, the dissociation of the replication fork helicase from DNA polymerase generates replication protein A (RPA)-coated single-stranded DNA (ssDNA), which leads to the activation of Ataxia Telangiectasia and Rad3-Related (ATR) kinase. ATR signaling prevents genomic instability by preventing collapse and breakage of stalled replication forks and suppressing firing of new replication origins throughout the nucleus, but it is unclear how these functions are related. After inhibiting ATR in cells with stalled replication forks, Toledo and colleagues unexpectedly observed a substantial lag between ATR inhibition and replication fork breakage, suggesting that an ATR-independent mechanism can protect replication forks for a limited time. Quantitative image-based cytometry analysis of immunofluorescently labeled single cells revealed that in the context of replication stress, ATR inhibition leads to DNA double-strand breaks specifically in cells where ssDNA generation exceeds the accumulation of chromatin-bound RPA. Together with the findings that RPA overexpression and suppression of replication origin activity each prevented replication fork breakage, this observation suggested that unscheduled origin firing and subsequent ssDNA generation caused by ATR loss exhausts the nuclear supply of RPA and promotes genome-wide replication fork breakage. The “replication catastrophe” caused by exhaustion of RPA irreversibly arrested cell-cycle progression and induced senescence due to irreparable DNA damage, with forced premature chromosome condensation revealing breaks in replicating loci within every chromosome. In addition to providing an explanation for how the local and global functions of ATR in preventing genomic instability are linked, these findings suggest that RPA exists in excess of the amount required for normal DNA replication to shield ssDNA and protect against replication fork breakage during replication stress. Because oncogenic transformation is associated with replication stress, these findings also predict that cancer cells might have a limited RPA pool and be sensitive to agents that induce replication catastrophe.

Toledo LI, Altmeyer M, Rask MB, Lukas C, Larsen DH, Povlsen LK, et al. ATR prohibits replication catastrophe by preventing global exhaustion of RPA. Cell 2013;155:1088–103.

• ©2014 American Association for Cancer Research.