The DNA damage response kinase ataxia telangiectasia and Rad3-related (ATR) coordinates much of the cellular response to replication stress. The exact mechanisms by which ATR regulates DNA synthesis in conditions of replication stress are largely unknown, but this activity is critical for the viability and proliferation of cancer cells, making ATR a potential therapeutic target. Here we use selective ATR inhibitors to demonstrate that acute inhibition of ATR kinase activity yields rapid cell lethality, disrupts the timing of replication initiation, slows replication elongation, and induces fork collapse. We define the mechanism of this fork collapse, which includes SLX4-dependent cleavage yielding double-strand breaks and CtIP-dependent resection generating excess singlestranded template and nascent DNA strands. Our data suggest that the DNA substrates of these nucleases are generated at least in part by the SMARCAL1 DNA translocase. Properly regulated SMARCAL1 promotes stalled fork repair and restart; however, unregulated SMARCAL1 contributes to fork collapse when ATR is inactivated in both mammalian and Xenopus systems. ATR phosphorylates SMARCAL1 on S652, thereby limiting its fork regression activities and preventing aberrant fork processing. Thus, phosphorylation of SMARCAL1 is one mechanism by which ATR prevents fork collapse, promotes the completion of DNA replication, and maintains genome integrity.

Summary The ATR replication checkpoint ensures that stalled forks remain stable when replisome movement is impeded. Using an improved iPOND protocol combined with SILAC mass spectrometry, we characterized human replisome dynamics in response to fork stalling. Our data provide a quantitative picture of the replisome and replication stress response proteomes in 32 experimental conditions. Importantly, rather than stabilize the replisome, the checkpoint prevents two distinct types of fork collapse. Unsupervised hierarchical clustering of protein abundance on nascent DNA is sufficient to identify protein complexes and place newly identified replisome-associated proteins into functional pathways. As an example, we demonstrate that ZNF644 complexes with the G9a/GLP methyltransferase at replication forks and is needed to prevent replication-associated DNA damage. Our data reveal how the replication checkpoint preserves genome integrity, provide insights into the mechanism of action of ATR inhibitors, and will be a useful resource for replication, DNA repair, and chromatin investigators.

The ATR (ATM and Rad3-related) kinase and its regulatory partner ATRIP (ATR-interacting protein) coordinate checkpoint responses to DNA damage and replication stress. TopBP1 functions as a general activator of ATR. However, the mechanism by which TopBP1 activates ATR is unknown. Here, we show that ATRIP contains a TopBP1-interacting region that is necessary for the association of TopBP1 and ATR, for TopBP1-mediated activation of ATR, and for cells to survive and recover DNA synthesis following replication stress. We demonstrate that this region is functionally conserved in the Saccharomyces cerevisiae ATRIP ortholog Ddc2, suggesting a conserved mechanism of regulation. In addition, we identify a domain of ATR that is critical for its activation by TopBP1. Mutations of the ATR PRD (PIKK [phosphoinositide 3-kinase related kinase] Regulatory Domain) do not affect the basal kinase activity of ATR but prevent its activation. Cellular complementation experiments demonstrate that TopBP1-mediated ATR activation is required for checkpoint signaling and cellular viability. The PRDs of ATM and mTOR (mammalian target of rapamycin) were shown previously to regulate the activities of these kinases, and our data indicate that the DNA-PKcs (DNA-dependent protein kinase catalytic subunit) PRD is important for DNA-PKcs regulation. Therefore, divergent amino acid sequences within the PRD and a unique protein partner allow each of these PIK kinases to respond to distinct cellular events.[Keywords: ATR; ATRIP; TopBP1; checkpoint; PIKK; Ddc2] Supplemental material is available at http://www.genesdev.org.

Mutations in SMARCAL1 (HARP) cause Schimke immunoosseous dysplasia (SIOD). The mechanistic basis for this disease is unknown. Using functional genomic screens, we identified SMARCAL1 as a genome maintenance protein. Silencing and overexpression of SMARCAL1 leads to activation of the DNA damage response during S phase in the absence of any genotoxic agent. SMARCAL1 contains a Replication protein A (RPA)-binding motif similar to that found in the replication stress response protein TIPIN (Timeless-Interacting Protein), which is both necessary and sufficient to target SMARCAL1 to stalled replication forks. RPA binding is critical for the cellular function of SMARCAL1; however, it is not necessary for the annealing helicase activity of SMARCAL1 in vitro. An SIOD-associated SMARCAL1 mutant fails to prevent replication-associated DNA damage from accumulating in cells in which endogenous SMARCAL1 is silenced. Ataxia-telangiectasia mutated (ATM), ATM and Rad3-related (ATR), and DNA-dependent protein kinase (DNA-PK) phosphorylate SMARCAL1 in response to replication stress. Loss of SMARCAL1 activity causes increased RPA loading onto chromatin and persistent RPA phosphorylation after a transient exposure to replication stress. Furthermore, SMARCAL1-deficient cells are hypersensitive to replication stress agents. Thus, SMARCAL1 is a replication stress response protein, and the pleiotropic phenotypes of SIOD are at least partly due to defects in genome maintenance during DNA replication.[Keywords: SMARCAL1; HARP; replication; DNA damage response; RPA; checkpoint] Supplemental material is available at http://www.genesdev.org.

The minichromosome maintenance (MCM) 2-7 helicase complex functions to initiate and elongate replication forks. Cell cycle checkpoint signaling pathways regulate DNA replication to maintain genomic stability. We describe four lines of evidence that ATM/ATR-dependent (ataxia-telangiectasia-mutated/ATM- and Rad3-related) checkpoint pathways are directly linked to three members of the MCM complex. First, ATM phosphorylates MCM3 on S535 in response to ionizing radiation. Second, ATR phosphorylates MCM2 on S108 in response to multiple forms of DNA damage and stalling of replication forks. Third, ATR-interacting protein (ATRIP)-ATR interacts with MCM7. Fourth, reducing the amount of MCM7 in cells disrupts checkpoint signaling and causes an intra-S-phase checkpoint defect. Thus, the MCM complex is a platform for multiple DNA damage-dependent regulatory signals that control DNA replication.