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.

SUMMARYOncogene activation has been shown to generate replication-born DNA damage, also known as replicative stress (RS). Notably, the ATR kinase -and not ATM-is the primary responder to RS. One limitation for the study of ATR is the lack of potent inhibitors. We here describe a cell-based screening strategy that has allowed us to identify compounds with ATR inhibitory activity in the nanomolar range. Pharmacological inhibition of ATR generates RS, leading to chromosomal breakage in the presence of conditions that stall replication forks. Moreover, ATR inhibition is particularly toxic for p53 deficient cells, this toxicity being exacerbated by RS-generating conditions such as the overexpression of cyclin E. Importantly, one of the compounds is NVP-BEZ235, a dual PI3K/mTOR inhibitor that is currently being tested for cancer chemotherapy, but which we now show is also very potent against ATM, ATR and DNA-PKcs.

Our paper identified nuclear proteins likely harboring disordered low-complexity sequences via precipitation by b-isox microcrystals. In Table S2, we ranked 580 nuclear proteins isolated in this manner and indicated that they were ordered according to the density of spectral counts. It has come to our attention that the proteins in this table are ordered by the relative density of [G/S]Y[G/S] triplet repeats rather than by spectral counts. This error affects the following sentence in the text of the Results section: ''Among the 580 mammalian proteins selectively precipitated by b-isox microcrystals, TAF15 registered the second highest number of spectral counts, and the largest subunit of RNA polymerase II registered the third highest number of spectral counts (Table S2).'' This is because the named positions had been based on ranking by triplet repeat density. We now provide with the article online the correctly ordered Table S2 (by spectral counts instead of triplet repeat density), and the affected sentence has now been changed to indicate the ranking positions of these proteins when ordered by spectral counts, such that TAF15 is 23 rd on the list and the largest subunit of RNA polymerase is 46 th. All proteins on the list are well above the false discovery rate, and the fact that both TAF15 and the largest subunit of RNA polymerase II are close to the very top of the list means that these adjustments do not alter any results or conclusions presented in the paper. We note that Table S3, which presents the yeast nuclear proteins precipitated by b-isox microcrystals, was correctly ordered by density of spectral counts as indicated. We wish to thank David Trudgian, a computational scientist in our Mass Sepctrometry Shared Resource Core, for pointing out the inconsistency in the organization and annotation of the original Table S2.

Oncogene-induced replicative stress activates an Atr- and Chk1-dependent response, which has been proposed to be widespread in tumors. We explored whether the presence of replicative stress could be exploited for the selective elimination of cancer cells. To this end, we evaluated the impact of targeting the replicative stress-response on cancer development. In mice (Mus musculus), the reduced levels of Atr found on a mouse model of the Atr-Seckel syndrome completely prevented the development of Myc-induced lymphomas or pancreatic tumors, both of which showed abundant levels of replicative stress. Moreover, Chk1 inhibitors were highly effective in killing Myc-driven lymphomas. By contrast, pancreatic adenocarcinomas initiated by K-Ras(G12V) showed no detectable evidence of replicative stress and were nonresponsive to this therapy. Besides its impact on cancer, Myc overexpression aggravated the phenotypes of Atr-Seckel mice, revealing that oncogenes can modulate the severity of replicative stress-associated diseases.