PrefaceGenome maintenance is a constant problem in all cells and a coordinated response to DNA damage is required to maintain cellular viability and prevent disease. The ATR and ATM protein kinases are master regulators of the DNA damage response, signaling to control cell cycle transitions, DNA replication, DNA repair, and apoptosis. Recent studies have provided insights into the mechanisms controlling ATR activation, helped to explain the overlapping but non-redundant activities of ATR and ATM in DNA damage signaling, and clarified the critical functions of ATR in maintaining genome integrity. IntroductionAll cells have elaborate mechanisms to maintain their genomes. DNA can be damaged during replication, by reactive metabolic byproducts as well as environmental mutagens. Responding to and repairing DNA damage is critical for cell viability and disease prevention.The DNA damage response (DDR) is a signal transduction pathway that coordinates cell cycle transitions, DNA replication, DNA repair and apoptosis. The major regulators of the DDR are the phosphoinositide 3-kinase related protein kinases (PIKKs), including ataxia-telangiectasia mutated (ATM) and ATM and Rad3 related (ATR). ATM and ATR share many biochemical and functional similarities. Both are large kinases with significant sequence homology and a strong preference to phosphorylate serine or threonine residues that are followed by glutamine. Both target an overlapping set of substrates that promote cell cycle arrest and DNA repair. However, ATR is essential for the viability of replicating human and mouse cells, whereas ATM is not 1-3 . ATM functions in response to rare occurrences of double strand breaks. By contrast, ATR is activated during every S-phase to regulate the firing of replication origins, the repair of damaged replication forks and to prevent the premature onset of mitosis 4, 5 (Fig. 1).Mutations in ATM predispose carriers to cancer and are found in approximately 0.5-1% of the population 6, 7 . People with mutations in both alleles of ATM suffer from the neurodegenerative and cancer predisposition disorder ataxia-telangiectasia 8 . Mutations in ATR are rare and probably only compatible with viability when heterozygous or hypomorphic. While the only clear link between ATR gene mutation and disease is in a few patients with the rare Seckel syndrome (characterized by growth retardation and microcephaly) 9 , disruptions in the ATR pathway do cause genomic instability, and ATR is activated by most cancer chemotherapies. Furthermore, ATR signaling is a promising target for cancer drug development 10, 11 . This review will focus on ATR signaling in the DNA damage response, and compare and contrast it with the more specialized role of ATM. NIH Public Access Mechanisms of ATR ActivationThe broad functions and physiological importance of ATR derive in large part from recognizing the signals that lead to its activation versus that of ATM. Thus, we will address the mechanism of activation in some detail. Recognizing DNA damageAlthough ATR is activ...

Replication stress is a complex phenomenon which has serious implications for genome stability, cell survival, and human disease. Generation of aberrant replication fork structures containing single-stranded DNA activates the replication stress response, primarily mediated by the kinase ATM- and Rad3-related (ATR). ATR and its downstream effectors stabilize and help to restart stalled replication forks, avoiding the generation of DNA damage and genome instability. Understanding these pathways may be key to diagnosis and treatment of human diseases caused by defective responses to replication stress.

The p53 tumor suppressor protein is activated and phosphorylated on serine-15 in response to various DNA damaging agents. The gene product mutated in ataxia telangiectasia, ATM, acts upstream of p53 in a signal transduction pathway initiated by ionizing radiation. Immunoprecipitated ATM had intrinsic protein kinase activity and phosphorylated p53 on serine-15 in a manganese-dependent manner. Ionizing radiation, but not ultraviolet radiation, rapidly enhanced this p53-directed kinase activity of endogenous ATM. These observations, along with the fact that phosphorylation of p53 on serine-15 in response to ionizing radiation is reduced in ataxia telangiectasia cells, suggest that ATM is a protein kinase that phosphorylates p53 in vivo.

One way to preserve a rare book is to lock it away from all potential sources of damage. Of course, an inaccessible book is also of little use, and the paper and ink will continue to degrade with age in any case. Like a book, the information stored in our DNA needs to be read, but it is also subject to continuous assault. In this review, we examine how the replication stress response that is controlled by the kinase ataxia telangiectasia and Rad3-related (ATR) senses and resolves threats to DNA integrity so the DNA remains available to read in all of our cells. We discuss the multiple data that have revealed an elegant yet increasingly complex mechanism of ATR activation. These involve a core set of components that recruit ATR to stressed replication forks, stimulate kinase activity and amplify ATR signaling. We focus on the activities of ATR in control of cell cycle checkpoints, origin firing and replication fork stability, and how proper regulation of these processes is crucial to ensure faithful duplication of a challenging genome.

The ATR-dependent DNA damage response pathway can respond to a diverse group of lesions as well as inhibitors of DNA replication. Using the Xenopus egg extract system, we show that lesions induced by UV irradiation and cis-platinum cause the functional uncoupling of MCM helicase and DNA polymerase activities, an event previously shown for aphidicolin. Inhibition of uncoupling during elongation with inhibitors of MCM7 or Cdc45, a putative helicase cofactor, results in abrogation of Chk1 phosphorylation, indicating that uncoupling is necessary for activation of the checkpoint. However, uncoupling is not sufficient for checkpoint activation, and DNA synthesis by Pol␣ is also required. Finally, using plasmids of varying size, we demonstrate that all of the unwound DNA generated at a stalled replication fork can contribute to the level of Chk1 phosphorylation, suggesting that uncoupling amplifies checkpoint signaling at each individual replication fork. Taken together, these observations indicate that functional uncoupling of MCM helicase and DNA polymerase activities occurs in response to multiple forms of DNA damage and that there is a general mechanism for generation of the checkpoint-activating signal following DNA damage.

Summary Conflicts between transcription and replication are a potent source of DNA damage. Co-transcriptional R-loops could aggravate such conflicts by creating an additional barrier to replication fork progression. Here, we use a defined episomal system to investigate how conflict orientation and R-loop formation influence genome stability in human cells. R-loops, but not normal transcription complexes, induce DNA breaks and orientation-specific DNA damage responses during conflicts with replication forks. Unexpectedly, the replisome acts as an orientation-dependent regulator of R-loop levels, reducing R-loops in the co-directional (CD) orientation but promoting their formation in the head-on (HO) orientation. Replication stress and deregulated origin firing increase the number of HO collisions leading to genome-destabilizing R-loops. Our findings connect DNA replication to R-loop homeostasis and suggest a mechanistic basis for genome instability resulting from deregulated DNA replication, observed in cancer and other disease states.

During transcription, the nascent RNA strand can base pair with its template DNA, displacing the non-template strand as ssDNA and forming a structure called an R-loop. R-loops are common across many domains of life and cause DNA damage in certain contexts. In this review, we summarize recent results implicating R-loops as important regulators of cellular processes such as transcription termination, gene regulation, and DNA repair. We also highlight recent work suggesting that R-loops can be problematic to cells as blocks to efficient transcription and replication that trigger the DNA damage response. Finally, we discuss how R-loops may contribute to cancer, neurodegeneration, and inflammatory diseases and compare the available next-generation sequencing-based approaches to map R-loops genome wide.

SUMMARY Signaling pathways that respond to DNA damage are essential for the maintenance of genome stability and are linked to many diseases, including cancer. Here, a genome-wide siRNA screen was employed to identify novel genes involved in genome stabilization by monitoring phosphorylation of the histone variant H2AX, an early mark of DNA damage. We identified hundreds of genes whose down-regulation led to elevated levels of H2AX phosphorylation (γH2AX) and revealed new links to cellular complexes and to genes with unclassified functions. We demonstrate a widespread role for mRNA processing factors in preventing DNA damage, which in some cases is caused by aberrant RNA-DNA structures. Furthermore, we connect increased γH2AX levels to the neurological disorder, Charcot-Marie-Tooth (CMT) syndrome, and we find a role for several CMT proteins in the DNA damage response. These data indicate that preservation of genome stability is mediated by a larger network of biological processes than previously appreciated.