ATR: a master conductor of cellular responses to DNA replication stress

The integrity of the genome is constantly challenged by intrinsic and extrinsic genotoxic stresses that damage DNA. The cellular responses to DNA damage are orchestrated by DNA damage signaling pathways, also known as DNA damage checkpoints. These signaling pathways play crucial roles in detecting DNA damage, regulating DNA repair and coordinating DNA repair with other cellular processes. In vertebrates, the ATM- and Rad3-related (ATR) kinase plays a key role in the response to a broad spectrum of DNA damage and DNA replication stress. Here, we will discuss the recent findings on how ATR is activated by DNA damage and how it protects the genome against interference with DNA replication.     

SV40 DNA replication intermediates: Analysis of drugs which target mammalian DNA replication

The simian virus 40 chromosome, a model for the mammalian replicon, is a uniquely powerful system for the study of drugs and treatments which target enzymes of the mammalian replication apparatus. High resolution gel electrophoretic analysis of normal and aberrant viral replication intermediates can be used effectively to understand the molecular events of replication failure. These events include breakage of replication forks, aberrant topoisomerase action, failure to separate daughter chromosomes, protein-DNA crosslinking, single and double strand DNA breakage, alterations in topology and inactivation of replication intermediates. The SV40 replication system can also be used to study the recombinational events which often follow drug-induced replication failure.     

Ku70/80 modulates ATM and ATR signaling pathways in response to DNA double strand breaks

Double strand break (DSB) recognition is the first step in the DSB damage response and involves activation of ataxia telangiectasia-mutated (ATM) and phosphorylation of targets such as p53 to trigger cell cycle arrest, DNA repair, or apoptosis. It was reported that activation of ATM- and Rad3-related (ATR) kinase by DSBs also occurs in an ATM-dependent manner. On the other hand, Ku70/80 is known to participate at a later time point in the DSB response, recruiting DNA-PKcs to facilitate non-homologous end joining. Because Ku70/80 has a high affinity for broken DNA ends and is abundant in nuclei, we examined their possible involvement in other aspects of the DSB damage response, particularly in modulating the activity of ATM and other phosphatidylinositol (PI) 3-related kinases during DSB recognition. We thus analyzed p53(Ser18) phosphorylation in irradiated Ku-deficient cells and observed persistent phosphorylation in these cells relative to wild type cells. ATM or ATR inhibition revealed that this phosphorylation is mainly mediated by ATM-dependent ATR activity at 2 h post-ionizing radiation in wild type cells, whereas in Ku-deficient cells, this occurs mainly through direct ATM activity, with a secondary contribution from ATR via a novel ATM-independent mechanism. Using ATM/Ku70 double-null cell lines, which we generated, we confirmed that ATM-independent ATR activity contributed to persistent phosphorylation of p53(Ser18) in Ku-deficient cells at 12 h post-ionizing radiation. In summary, we discovered a novel role for Ku70/80 in modulating ATM-dependent ATR activation during DSB damage response and demonstrated that these proteins confer a protective effect against ATM-independent ATR activation at later stages of the DSB damage response.     

RIF1: A novel regulatory factor for DNA replication and DNA damage response signaling

DNA double strand breaks (DSBs) are highly toxic to the cells and accumulation of DSBs results in several detrimental effects in various cellular processes which can lead to neurological, immunological and developmental disorders. Failure of the repair of DSBs spurs mutagenesis and is a driver of tumorigenesis, thus underscoring the importance of the accurate repair of DSBs. Two major canonical DSB repair pathways are the non-homologous end joining (NHEJ) and homologous recombination (HR) pathways. 53BP1 and BRCA1 are the key mediator proteins which coordinate with other components of the DNA repair machinery in the NHEJ and HR pathways respectively, and their exclusive recruitment to DNA breaks/ends potentially decides the choice of repair by either NHEJ or HR. Recently, Rap1 interacting factor 1 has been identified as an important component of the DNA repair pathway which acts downstream of the ATM/53BP1 to inhibit the 5′–3′ end resection of broken DNA ends, in-turn facilitating NHEJ repair and inhibiting homology directed repair. Rif1 is conserved from yeast to humans but its function has evolved from telomere length regulation in yeast to the maintenance of genome integrity in mammalian cells. Recently its role in the maintenance of genomic integrity has been expanded to include the regulation of chromatin structure, replication timing and intra-S phase checkpoint. We present a summary of these important findings highlighting the various aspects of Rif1 functions and discuss the key implications for genomic integrity.     

ATR/ATM targets are phosphorylated by ATR in response to hypoxia and ATM in response to reoxygenation

The ATR kinase phosphorylates both p53 and Chk1 in response to extreme hypoxia (oxygen concentrations of less than 0.02%). In contrast to ATR, loss of ATM does not affect the phosphorylation of these or other targets in response to hypoxia. However, hypoxia within tumors is often transient and is inevitably followed by reoxygenation. We hypothesized that ATR activity is induced under hypoxic conditions because of growth arrest and ATM activity increases in response to the oxidative stress of reoxygenation. Using the comet assay to detect DNA damage, we find that reoxygenation induced significant amounts of DNA damage. Two ATR/ATM targets, p53 serine 15 and histone H2AX, were both phosphorylated in response to hypoxia in an ATR-dependent manner. These phosphorylations were then maintained in response to reoxygenation-induced DNA damage in an ATM-dependent manner. The reoxygenation-induced p53 serine 15 phosphorylation was inhibited by the addition of N-acetyl-l-cysteine (NAC), indicating that free radical-induced DNA damage was mediated by reactive oxygen species. Taken together these data implicate both ATR and ATM as critical roles in the response of hypoxia and reperfusion in solid tumors.     

Regulation of DNA replication by the S-phase DNA damage checkpoint

Cells slow replication in response to DNA damage. This slowing was the first DNA damage checkpoint response discovered and its study led to the discovery of the central checkpoint kinase, Ataxia Telangiectasia Mutated (ATM). Nonetheless, the manner by which the S-phase DNA damage checkpoint slows replication is still unclear. The checkpoint could slow bulk replication by inhibiting replication origin firing or slowing replication fork progression, and both mechanisms appear to be used. However, assays in various systems using different DNA damaging agents have produced conflicting results as to the relative importance of the two mechanisms. Furthermore, although progress has been made in elucidating the mechanism of origin regulation in vertebrates, the mechanism by which forks are slowed remains unknown. We review both past and present efforts towards determining how cells slow replication in response to damage and try to resolve apparent conflicts and discrepancies within the field. We propose that inhibition of origin firing is a global checkpoint mechanism that reduces overall DNA synthesis whenever the checkpoint is activated, whereas slowing of fork progression reflects a local checkpoint mechanism that only affects replisomes as they encounter DNA damage and therefore only affects overall replication rates in cases of high lesion density.     

Step-wise progression of mammalian 10-kb DNA replication intermediates to mature chromatin

In mammalian DNA synthesis the primary replication intermediates are joined to larger intermediates. After the joining process is complete one can detect a distinct stage called the post-elongation stage. Furthermore a 10-kb DNA1 population is detected before the post-elongation stage whereas a 10-kb DNA2 population is part of this stage DNA. When cells are treated with 3-aminobenzamide, an inhibitor of poly(ADP-ribose) synthetase, an altered post-elongation-stage DNA was detected, which does not contain 10-kb fragments. The step(s) affected by 3-aminobenzamide prevents the appearance of 10-kb DNA in the post-elongation stage. The drug effect is reversible with the appearance of 10-kb DNA in the post-elongation stage when the cells are washed free of drug. Hence there is a step-wise progression from 10-kb DNA, via the post-elongation stage, to mature chromatin.     

Rapidly labeled intermediates in DNA replication in higher plants.

In detached Vicia embryos, the incorporation of [3-H] thymidine into DNA starts at about 25 h after the beginning of imbibition and reaches maximum at about 33 h. The DNA synthesized during the first replication phase was extracted. Alkaline sucrose density-gradient analyses of the DNA indicated the occurrence of several short pieces of rapidly labeled DNA having sedimentation values of approx. 10 S and 14 S, after a pulse for 5 to 10 min. Prolonged labeling and chase incubation led to a shift of the shorter fragments to longer ones of 19 S and 22 S or more, thus indicating the nature of intermediates during DNA replication of these short fragments.     

Regulation of DNA replication by homopurine/homopyrimidine sequences

The simple repeating homopurine/homopyrimidine sequences dispersed throughout many eukaryotic genomes are known to form triple helical structures comprising three-stranded and single-stranded DNA. Several lines of evidence suggest that these structures influence DNA replication in cells. Homopurine/homopyrimidine sequences cloned into simian virus 40 (SV40) or SV40 origin-containing plasmids caused a reduced rate of DNA synthesis due to the pausing of replication forks. More prominent arrests were observed in in vitro experiments using single-stranded and double-stranded DNA with triplex-forming sequences. Nucleotides unable to form triplexes when present in the template DNA or when incorporated into the nascent strand prevented termination. Similarly, mutations destroying the triplex potential did not cause arrest while compensatory mutations restoring triplex potential restored it. These and other observations from a number of laboratories indicating that homopurine/homopyrimidine sequences act as arrest signals in vitro and as pause sites in vivo during replication fork movement suggest that these naturally occurring sequences play a regulatory role in DNA replication and gene amplification.     

ATR and ATM regulate the timing of DNA replication origin firing

Timing of DNA replication initiation is dependent on S-phase-promoting kinase (SPK) activity at discrete origins and the simultaneous function of many replicons. DNA damage prevents origin firing through the ATM- and ATR-dependent inhibition of Cdk2 and Cdc7 SPKs. Here, we establish that modulation of ATM- and ATR-signalling pathways controls origin firing in the absence of DNA damage. Inhibition of ATM and ATR with caffeine or specific neutralizing antibodies, or upregulation of Cdk2 or Cdc7, promoted rapid and synchronous origin firing; conversely, inhibition of Cdc25A slowed DNA replication. Cdk2 was in equilibrium between active and inactive states, and the concentration of replication protein A (RPA)-bound single-stranded DNA (ssDNA) correlated with Chk1 activation and inhibition of origin firing. Furthermore, ATM was transiently activated during ongoing replication. We propose that ATR and ATM regulate SPK activity through a feedback mechanism originating at active replicons. Our observations establish that ATM- and ATR-signalling pathways operate during an unperturbed cell cycle to regulate initiation and progression of DNA synthesis, and are therefore poised to halt replication in the presence of DNA damage.     

Inhibition of ATR protein kinase activity by schisandrin B in DNA damage response

ATM and ATR protein kinases play a crucial role in cellular DNA damage responses. The inhibition of ATM and ATR can lead to the abolition of the function of cell cycle checkpoints. In this regard, it is expected that checkpoint inhibitors can serve as sensitizing agents for anti-cancer chemo/radiotherapy. Although several ATM inhibitors have been reported, there are no ATR-specific inhibitors currently available. Here, we report the inhibitory effect of schisandrin B (SchB), an active ingredient of Fructus schisandrae, on ATR activity in DNA damage response. SchB treatment significantly decreased the viability of A549 adenocarcinoma cells after UV exposure. Importantly, SchB treatment inhibited both the phosphorylation levels of ATM and ATR substrates, as well as the activity of the G2/M checkpoint in UV-exposed cells. The protein kinase activity of immunoaffinity-purified ATR was dose-dependently decreased by SchB in vitro (IC₅₀: 7.25 μM), but the inhibitory effect was not observed in ATM, Chk1, PI3K, DNA-PK, and mTOR. The extent of UV-induced phosphorylation of p53 and Chk1 was markedly reduced by SchB in ATM-deficient but not siATR-treated cells. Taken together, our demonstration of the ability of SchB to inhibit ATR protein kinase activity following DNA damage in cells has clinical implications in anti-cancer therapy.     

Regulation of DNA replication during development

As development unfolds, DNA replication is not only coordinated with cell proliferation, but is regulated uniquely in specific cell types and organs. This differential regulation of DNA synthesis requires crosstalk between DNA replication and differentiation. This dynamic aspect of DNA replication is highlighted by the finding that the distribution of replication origins varies between differentiated cell types and changes with differentiation. Moreover, differential DNA replication in some cell types can lead to increases or decreases in gene copy number along chromosomes. This review highlights the recent advances and technologies that have provided us with new insights into the developmental regulation of DNA replication.