The Replication Checkpoint Prevents Two Types of Fork Collapse without Regulating Replisome Stability.

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.

ATR phosphorylates SMARCAL1 to prevent replication fork collapse.

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 single-stranded 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.

Analysis of protein dynamics at active, stalled, and collapsed replication forks.

Successful DNA replication and packaging of newly synthesized DNA into chromatin are essential to maintain genome integrity. Defects in the DNA template challenge genetic and epigenetic inheritance. Unfortunately, tracking DNA damage responses (DDRs), histone deposition, and chromatin maturation at replication forks is difficult in mammalian cells. Here we describe a technology called iPOND (isolation of proteins on nascent DNA) to analyze proteins at active and damaged replication forks at high resolution. Using this methodology, we define the timing of histone deposition and chromatin maturation. Class 1 histone deacetylases are enriched at replisomes and remove predeposition marks on histone H4. Chromatin maturation continues even when decoupled from replisome movement. Furthermore, fork stalling causes changes in the recruitment and phosphorylation of proteins at the damaged fork. Checkpoint kinases catalyze H2AX phosphorylation, which spreads from the stalled fork to include a large chromatin domain even prior to fork collapse and double-strand break formation. Finally, we demonstrate a switch in the DDR at persistently stalled forks that includes MRE11-dependent RAD51 assembly. These data reveal a dynamic recruitment of proteins and post-translational modifications at damaged forks and surrounding chromatin. Furthermore, our studies establish iPOND as a useful methodology to study DNA replication and chromatin maturation.

Substrate-selective repair and restart of replication forks by DNA translocases.

Stalled replication forks are sources of genetic instability. Multiple fork-remodeling enzymes are recruited to stalled forks, but how they work to promote fork restart is poorly understood. By combining ensemble biochemical assays and single-molecule studies with magnetic tweezers, we show that SMARCAL1 branch migration and DNA-annealing activities are directed by the single-stranded DNA-binding protein RPA to selectively regress stalled replication forks caused by blockage to the leading-strand polymerase and to restore normal replication forks with a lagging-strand gap. We unveil the molecular mechanisms by which RPA enforces SMARCAL1 substrate preference. E. coli RecG acts similarly to SMARCAL1 in the presence of E. coli SSB, whereas the highly related human protein ZRANB3 has different substrate preferences. Our findings identify the important substrates of SMARCAL1 in fork repair, suggest that RecG and SMARCAL1 are functional orthologs, and provide a comprehensive model of fork repair by these DNA translocases.

Monitoring the spatiotemporal dynamics of proteins at replication forks and in assembled chromatin using isolation of proteins on nascent DNA.

Understanding the processes of DNA replication, chromatin assembly and maturation, and the replication stress response requires the ability to monitor protein dynamics at active and damaged replication forks. Detecting protein accumulation at replication forks or damaged sites has primarily relied on immunofluorescence imaging, which is limited in resolution and antibody sensitivity. Here we describe a procedure to isolate proteins on nascent DNA (iPOND) that permits a high-resolution spatiotemporal analysis of proteins at replication forks or on chromatin following DNA replication in cultured cells. iPOND relies on labeling of nascent DNA with the nucleoside analog 5-ethynyl-2'-deoxyuridine (EdU). Biotin conjugation to EdU-labeled DNA using click chemistry facilitates a single-step streptavidin purification of proteins bound to the nascent DNA. iPOND permits an interrogation of any cellular process linked to DNA synthesis using a 3- to 4-d protocol.