Safeguarding against unfavorable changes in the genome during cell division is vital. Although packaging DNA in chromatin may protect it, genes need to be transcribed, and chromosomes need to be duplicated and segregated. Thus, even for normal DNA metabolism, DNA is precariously unwound, nicked, copied, broken, and recombined. The cell also produces reactive metabolites and oxidation products that damage DNA. Finally, exogenous sources of DNA damage like radiation and other genotoxins are prevalent in the environment. This combined assault on DNA yields tens of thousands of DNA lesions per day in every human cell.
In response, the cell mounts an evolutionarily conserved DNA damage response (DDR) that coordinates cell cycle progression, DNA repair, DNA replication, DNA transcription, and even cell death to promote genome maintenance. Mutation or deletion of many DDR genes results in lethality, cancer susceptibility syndromes, neurodegenerative disorders, and premature aging syndromes. Therefore, genome maintenance via the DDR is essential to prevent disease.
At the apex of the DDR are three related protein kinases, Ataxia-telangiectasia Mutated (ATM), ATM and Rad3-related (ATR), and DNA-dependent protein kinase (DNA-PK). These kinases belong to the phosphoinositide-3-like kinase kinase (PIKK) family and share similar domain architecture and several modes of regulation [1
]. While DNA double-strand breaks (DSBs) activate ATM and DNA-PK, many types of DNA damage activate ATR, including DSBs, base adducts, and crosslinks [4
]. Once activated, these kinases preferentially phosphorylate serines and threonines followed by a glutamine (S/TQ) in hundreds of protein substrates. In some cases, substrates contain multiple phosphorylated S/TQs within a small region of the primary sequence
S/TQ cluster domains [5
The most common signal for ATR activation likely involves replication stress
interference of replication fork progression caused by DNA damage, lack of sufficient deoxynucleotides, and even difficult to replicate DNA sequences. In response to replication stress, ATR regulates replisome stability, origin firing, and prevents premature mitotic entry [4
]. ATR, unlike ATM and DNA-PK, is an essential gene in replicating cells [7
]. This probably is due to ATR activation by replication stress in every S phase and perhaps regulation of specific aspects of DNA replication such as origin firing or nucleotide production. Failure to resolve stalled replication forks results in unreplicated DNA, single-stranded DNA replication intermediates, and DSBs. Single-stranded DNA (ssDNA) and DSBs are highly recombinogenic, and aberrant recombination of these structures yields chromatid- and chromosome-type errors [10
Homozygous loss of function mutations in ATR
are not compatible with mammalian cell viability [7
]. However, hypomorphic mutations in ATR
that cause reduced ATR function are found in a few patients with the rare Seckel Syndrome, which is characterized by microcephaly and growth retardation [12
]. In certain genetic backgrounds ATR is a haploinsufficient tumor suppressor [13
], and ATR
mutations in microsatellite instability tumors are associated with reduced overall survival and disease-free survival [15
]. Oncogene-induced replication stress activates the ATR pathway explaining high levels of DDR activation in many neoplasias [17
]. Therefore, the ATR pathway and the DDR potentially constitute a barrier to cancer [20
]. Bypass of this barrier may be a double-edged sword for the cancer cell, as cancer cells may exhibit an increased dependency on the ATR pathway, analogous to oncogene addiction, to continue to replicate in the presence of oncogene-induced replication stress. Many current therapies are DNA damaging agents that increase the signaling burden on the ATR pathway, and ATR pathway proteins may be good drug targets in cancers containing elevated levels of replicative stress.
Canonical ATR signaling
Replication stress often results in the generation of excess ssDNA through uncoupling of enzymatic activities at the replication fork. For example, many DNA lesions stall the DNA polymerase but not the replicative helicase [21
]. While lesions, such as interstrand crosslinks, block both the polymerase and helicase, enzymatic remodeling of the blocked replication fork by helicases and nucleases also creates ssDNA. Similarly, nuclease-mediated resection of DNA DSBs produces ssDNA [22
]. ssDNA is bound by the ssDNA binding protein Replication Protein A (RPA). In the canonical ATR signaling pathway, RPA-ssDNA is the ligand that recruits ATR and other ATR signaling components to sites of replication stress. Once RPA-ssDNA appears, ATR signaling canonically requires: 1. Recruitment of ATR via its obligate partner ATR Interacting Protein (ATRIP) to RPA-ssDNA; 2. Independent recruitment of a checkpoint clamp, containing RAD9-HUS1-RAD1 proteins, and the ATR activator Topoisomerase Binding Protein 1 (TOPBP1) to RPA-ssDNA; 3. Activation of ATR by TOPBP1; and 4 Phosphorylation of ATR substrates (). Cases where ATR activation occurs independently of steps 1-4 and RPA-ssDNA are defined as non-canonical for the purposes of this review. For a more detailed description of the canonical pathway we refer the readers to a recent review [4
]. Here we will review new advances in understanding each of these steps in ATR signaling, emphasizing novel ATR regulatory factors, ATR targets, and non-canonical pathways of ATR regulation.
Canonical ATR signaling pathway and functions of newly identified regulators.