ATM and ATR are two related kinases essential for signalling DNA damage. Although ATM is thought to be the principle kinase responsible for signalling ionising radiation (IR)-induced DNA damage, ATR also contributes to signalling this form of genotoxic stress. However, the molecular basis of differential ATM and ATR activation in response to IR remains unclear. Here, we report that ATR is recruited to sites of IR-induced DNA damage significantly later than activation of ATM. We show that ATR is recruited to IR-induced nuclear foci in G 1 and S phase of the cell cycle, supporting a role for ATR in detecting DNA damage outside of S phase. In addition, we report that recruitment of ATR to sites of IRinduced DNA damage is concomitant with appearance of large tracts of single-stranded DNA (ssDNA) and that this event is dependent on ATM and components of the Mre11/Rad50/Nbs1 (MRN) protein complex.

The ataxia telangiectasia-mutated (ATM) and Rad3-related kinase (ATR) is a central component of the cell cycle checkpoint machinery required to induce cell cycle arrest in response to DNA damage. Accumulating evidence suggests a role for ATR in signaling DNA damage during S-phase. Here we show that ATR is recruited to nuclear foci induced by replication fork stalling in a manner that is dependent on the single stranded binding protein replication protein A (RPA). ATR associates with chromatin in asynchronous cell cultures, and we use a variety of approaches to examine the association of ATR with chromatin in the absence of agents that cause genotoxic stress. Under our experimental conditions, ATR exhibits a decreased affinity for chromatin in quiescent cells and cells synchronized at mitosis but an increased affinity for chromatin as cells re-enter the cell cycle. Using centrifugal elutriation to obtain cells enriched at various stages of the cell cycle, we show that ATR associates with chromatin in a cell cycle-dependent manner, specifically during S-phase. Cell cycle association of ATR with chromatin mirrors that of RPA in addition to claspin, a cell cycle checkpoint protein previously shown to be a component of the replication machinery. Furthermore, association of ATR with chromatin occurs in the absence of detectable DNA damage and cell cycle checkpoint activation. These data are consistent with a model whereby ATR is recruited to chromatin during the unperturbed cell cycle and points to a role of ATR in monitoring genome integrity during normal S-phase progression.DNA damage activates a cascade of phosphorylation events that affect a variety of cellular processes such as DNA repair, transcription, apoptotic cell death, and cell cycle progression. Cell cycle checkpoints are signal transduction pathways that result in cell cycle arrest in response to DNA damage to allow repair of DNA prior to duplication of DNA during S-phase, or segregation of the genome at mitosis (1). Cell cycle checkpoints are critical for the maintenance of genome integrity, and this is reflected in the observations that dysfunction of these pathways can lead to a variety of clinical symptoms, including an increased risk of cancer (2). Recently, much progress has been made in our understanding of the downstream effector proteins, activated in response to DNA damage, that lead to cell cycle arrest (3); however, the mechanisms by which DNA damage is detected and signaled remain elusive.S-phase is a period of the cell cycle when the genome is particularly susceptible to gaining mutations. Replication stress can occur during S-phase when replication forks encounter un-repaired DNA lesions, or when DNA polymerase activity is inhibited by depletion of intracellular nucleotide levels or exposure of cells to chemical agents such as aphidicolin. Replication stress can result in stalling of replication forks that under certain circumstances induces replication fork collapse and DNA damage that is potentially lethal to the cell (4). Monitoring bot...