DNA-damage response pathways triggered by viral replication

Many viruses, with distinct replication strategies, activate DNA-damage response pathways, including the lentivirus human immunodeficiency virus (HIV) and the DNA viruses Epstein-Barr virus (EBV), herpes simplex virus 1, adenovirus and SV40. DNA-damage response pathways involving DNA-dependent protein kinase, ataxia-telengiectasia mutated (ATM) and 'ataxia-telengiectasia and Rad3-related' (ATR) have all been implicated. This review focuses on the effects of HIV and EBV replication on DNA repair pathways. It has been suggested that activation of cellular DNA repair and recombination enzymes is beneficial for viral replication, as illustrated by the ability of suppressors of the ATM and ATR family to inhibit HIV replication. However, activation of DNA-damage response pathways can also promote apoptosis. Viruses can tailor the cellular response by suppressing downstream signalling from DNA-damage sensors, as exemplified by EBV. New small-molecule inhibitors of the DNA-damage response pathways could therefore be of value to treat viral infections.     

ATR signalling: more than meeting at the fork

Preservation of genome integrity via the DNA-damage response is critical to prevent disease. ATR (ataxia telangiectasia mutated- and Rad3-related) is essential for life and functions as a master regulator of the DNA-damage response, especially during DNA replication. ATR controls and co-ordinates DNA replication origin firing, replication fork stability, cell cycle checkpoints and DNA repair. Since its identification 15 years ago, a model of ATR activation and signalling has emerged that involves localization to sites of DNA damage and activation through protein-protein interactions. Recent research has added an increasingly detailed understanding of the canonical ATR pathway, and an appreciation that the canonical model does not fully capture the complexity of ATR regulation. In the present article, we review the ATR signalling process, focusing on mechanistic findings garnered from the identification of new ATR-interacting proteins and substrates. We discuss how to incorporate these new insights into a model of ATR regulation and point out the significant gaps in our understanding of this essential genome-maintenance pathway.     

ATM- and cell cycle-dependent regulation of ATR in response to DNA double-strand breaks

It is generally thought that the DNA-damage checkpoint kinases, ataxia-telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR), work independently of one another. Here, we show that ATM and the nuclease activity of meiotic recombination 11 (Mre11) are required for the processing of DNA double-strand breaks (DSBs) to generate the replication protein A (RPA)-coated ssDNA that is needed for ATR recruitment and the subsequent phosphorylation and activation of Chk1. Moreover, we show that efficient ATM-dependent ATR activation in response to DSBs is restricted to the S and G2 cell cycle phases and requires CDK kinase activity. Thus, in response to DSBs, ATR activation is regulated by ATM in a cell-cycle dependent manner.     

BRCA1 is required for hMLH1 stabilization following doxorubicin-induced DNA damage

Human DNA mismatch repair (MMR) is involved in the removal of DNA base mismatches that arise either during DNA replication or are caused by DNA damage. In this study, we show that the activation of the MMR component hMLH1 in response to doxorubicin (DOX) treatment requires the presence of BRCA1 and that this phenomenon is mediated by an ATM/ATR dependent phosphorylation of the hMLH1 Ser-406 residue. BRCA1 is an oncosuppressor protein with a central role in the DNA damage response and it is a critical component of the ATM/ATR mediated checkpoint signaling. Starting from a previous finding in which we demonstrated that hMLH1 is able to bind to BRCA1, in this study we asked whether BRCA1 might be the bridge for ATM/ATR dependent phosphorylation of the hMLH1 molecular partner. We found that: (i) the negative modulation of BRCA1 expression is able to produce a remarkable reversal of hMLH1 stabilization, (ii) BRCA1 is required for post-translational modification produced by DOX treatment on hMLH1 which is, in turn, attributed to the ATM/ATR activity, (iii) the serine 406 phosphorylatable residue is critical for hMLH1 activation by ATM/ATR via BRCA1. Taken together, our data lend support to the hypothesis suggesting an important role of this oncosuppressor as a scaffold or bridging protein in DNA-damage response signaling via downstream phosphorylation of the ATM/ATR substrate hMLH1.     

Distinct roles of ATR and DNA‐PKcs in triggering DNA damage responses in ATM‐deficient cells

The cellular response to DNA double‐strand breaks involves direct activation of ataxia telangiectasia mutated (ATM) and indirect activation of ataxia telangiectasia and Rad3 related (ATR) in an ATM/Mre11/cell‐cycle‐dependent manner. Here, we report that the crucial checkpoint signalling proteins—p53, structural maintainance of chromosomes 1 (SMC1), p53 binding protein 1 (53BP1), checkpoint kinase (Chk)1 and Chk2—are phosphorylated rapidly by ATR in an ATM/Mre11/cell‐cycle‐independent manner, albeit at low levels. We observed the sequential recruitment of replication protein A (RPA) and ATR to the sites of DNA damage in ATM‐deficient cells, which provides a mechanistic basis for the observed phosphorylations. The recruitment of ATR and consequent phosphorylations do not require Mre11 but are dependent on Exo1. We show that these low levels of phosphorylation are biologically important, as ATM‐deficient cells enforce an early G2/M checkpoint that is ATR‐dependent. ATR is also essential for the late G2 accumulation that is peculiar to irradiated ATM‐deficient cells. Interestingly, phosphorylation of KRAB associated protein 1 (KAP‐1), a protein involved in chromatin remodelling, is mediated by DNA‐dependent protein kinase catalytic subunit (DNA‐PKcs) in a spatio‐temporal manner in addition to ATM. We posit that ATM substrates involved in cell‐cycle checkpoint signalling can be minimally phosphorylated independently by ATR, while a small subset of proteins involved in chromatin remodelling are phosphorylated by DNA‐PKcs in addition to ATM.     

ATR-dependent phosphorylation and activation of ATM in response to UV treatment or replication fork stalling

The phosphatidyl inositol 3-kinase-like kinases (PIKKs), ataxia-telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR) regulate parallel damage response signalling pathways. ATM is reported to be activated by DNA double-strand breaks (DSBs), whereas ATR is recruited to single-stranded regions of DNA. Although the two pathways were considered to function independently, recent studies have demonstrated that ATM functions upstream of ATR following exposure to ionising radiation (IR) in S/G2. Here, we show that ATM phosphorylation at Ser1981, a characterised autophosphorylation site, is ATR-dependent and ATM-independent following replication fork stalling or UV treatment. In contrast to IR-induced ATM-S1981 phosphorylation, UV-induced ATM-S1981 phosphorylation does not require the Nbs1 C-terminus or Mre11. ATR-dependent phosphorylation of ATM activates ATM phosphorylation of Chk2, which has an overlapping function with Chk1 in regulating G2/M checkpoint arrest. Our findings provide insight into the interplay between the PIKK damage response pathways.     

ATR and Chk1 Suppress a Caspase-3–Dependent Apoptotic Response Following DNA Replication Stress

The related PIK-like kinases Ataxia-Telangiectasia Mutated (ATM) and ATM- and Rad3-related (ATR) play major roles in the regulation of cellular responses to DNA damage or replication stress. The pro-apoptotic role of ATM and p53 in response to ionizing radiation (IR) has been widely investigated. Much less is known about the control of apoptosis following DNA replication stress. Recent work indicates that Chk1, the downstream phosphorylation target of ATR, protects cells from apoptosis induced by DNA replication inhibitors as well as IR. The aim of the work reported here was to determine the roles of ATM- and ATR-protein kinase cascades in the control of apoptosis following replication stress and the relationship between Chk1-suppressed apoptotic pathways responding to replication stress or IR. ATM and ATR/Chk1 signalling pathways were manipulated using siRNA-mediated depletions or specific inhibitors in two tumour cell lines or fibroblasts derived from patients with inherited mutations. We show that depletion of ATM or its downstream phosphorylation targets, NBS1 and BID, has relatively little effect on apoptosis induced by DNA replication inhibitors, while ATR or Chk1 depletion strongly enhances cell death induced by such agents in all cells tested. Furthermore, early events occurring after the disruption of DNA replication (accumulation of RPA foci and RPA34 hyperphosphorylation) in ATR- or Chk1-depleted cells committed to apoptosis are not detected in ATM-depleted cells. Unlike the Chk1-suppressed pathway responding to IR, the replication stress-triggered apoptotic pathway did not require ATM and is characterized by activation of caspase 3 in both p53-proficient and -deficient cells. Taken together, our results show that the ATR-Chk1 signalling pathway plays a major role in the regulation of death in response to DNA replication stress and that the Chk1-suppressed pathway protecting cells from replication stress is clearly distinguishable from that protecting cells from IR.     

ATR and Chk1 Suppress a Caspase-3–Dependent Apoptotic Response Following DNA Replication Stress

The related PIK-like kinases Ataxia-Telangiectasia Mutated (ATM) and ATM- and Rad3-related (ATR) play major roles in the regulation of cellular responses to DNA damage or replication stress. The pro-apoptotic role of ATM and p53 in response to ionizing radiation (IR) has been widely investigated. Much less is known about the control of apoptosis following DNA replication stress. Recent work indicates that Chk1, the downstream phosphorylation target of ATR, protects cells from apoptosis induced by DNA replication inhibitors as well as IR. The aim of the work reported here was to determine the roles of ATM- and ATR-protein kinase cascades in the control of apoptosis following replication stress and the relationship between Chk1-suppressed apoptotic pathways responding to replication stress or IR. ATM and ATR/Chk1 signalling pathways were manipulated using siRNA-mediated depletions or specific inhibitors in two tumour cell lines or fibroblasts derived from patients with inherited mutations. We show that depletion of ATM or its downstream phosphorylation targets, NBS1 and BID, has relatively little effect on apoptosis induced by DNA replication inhibitors, while ATR or Chk1 depletion strongly enhances cell death induced by such agents in all cells tested. Furthermore, early events occurring after the disruption of DNA replication (accumulation of RPA foci and RPA34 hyperphosphorylation) in ATR- or Chk1-depleted cells committed to apoptosis are not detected in ATM-depleted cells. Unlike the Chk1-suppressed pathway responding to IR, the replication stress-triggered apoptotic pathway did not require ATM and is characterized by activation of caspase 3 in both p53-proficient and -deficient cells. Taken together, our results show that the ATR-Chk1 signalling pathway plays a major role in the regulation of death in response to DNA replication stress and that the Chk1-suppressed pathway protecting cells from replication stress is clearly distinguishable from that protecting cells from IR.     

Hop1 and the meiotic DNA-damage response

During the DNA-damage response, adaptor proteins mediate signaling between the PI3K-like sensor kinases, ATM and ATR, and serine/threonine effector kinases. Carballo et al. (2008) now show that the chromosomal protein Hop1 mediates PI3K-like kinase signaling during the repair of DNA double-strand breaks (DSBs) in meiosis.     

Homeostatic regulation of meiotic DSB formation by ATM/ATR

Ataxia–telangiectasia mutated (ATM) and RAD3-related (ATR) are widely known as being central players in the mitotic DNA damage response (DDR), mounting responses to DNA double-strand breaks (DSBs) and single-stranded DNA (ssDNA) respectively. The DDR signalling cascade couples cell cycle control to damage-sensing and repair processes in order to prevent untimely cell cycle progression while damage still persists [1]. Both ATM/ATR are, however, also emerging as essential factors in the process of meiosis; a specialised cell cycle programme responsible for the formation of haploid gametes via two sequential nuclear divisions. Central to achieving accurate meiotic chromosome segregation is the introduction of numerous DSBs spread across the genome by the evolutionarily conserved enzyme, Spo11. This review seeks to explore and address how cells utilise ATM/ATR pathways to regulate Spo11-DSB formation, establish DSB homeostasis and ensure meiosis is completed unperturbed.     

Ataxia telangiectasia mutated- and Rad3-related kinase drives both the early and the late DNA-damage response to the monofunctional antitumour alkylator S23906

Numerous anticancer agents and environmental mutagens target DNA. Although all such compounds interfere with the progression of the replication fork and inhibit DNA synthesis, there are marked differences in the DNA-damage response pathways they trigger, and the relative impact of the proximal or the distal signal transducers on cell survival is mainly lesion-specific. Accordingly, checkpoint kinase inhibitors in current clinical development show synergistic activity with some DNA-targeting agents, but not with others. In the present study, we characterize the DNA-damage response to the antitumour acronycine derivative S23906, which forms monofunctional adducts with guanine residues in the minor groove of DNA. S23906 exposure is accompanied by specific recruitment of RPA (replication protein A) at replication sites and rapid Chk1 activation. In contrast, neither MRN (Mre11-Rad50-Nbs1) nor ATM (ataxia-telangiectasia mutated), contributes to the initial response to S23906. Interestingly, genetic attenuation of ATR (ATM- and Ras3-related) activity inhibits not only the early phosphorylation of histone H2AX and Chk1, but also interferes with the late phosphorylation of Chk2. Moreover, loss of ATR function or pharmacological inhibition of the checkpoint kinases by AZD7762 is accompanied by abrogation of the S-phase arrest and increased sensitivity towards S23906. These findings identify ATR as a central co-ordinator of the DNA-damage response to S23906, and provide a mechanistic rationale for combinations of S23906 and similar agents with checkpoint abrogators.     

Functional interaction between FOXO3a and ATM regulates DNA damage response

The maintenance of genomic stability in cells is relentlessly challenged by environmental stresses that induce DNA breaks, which activate the DNA-damage pathway mediated by ataxia-telangiectasia mutated (ATM) and its downstream mediators to control damage-induced cell-cycle checkpoints and DNA repair. Here, we show that FOXO3a interacts with ATM to promote phosphorylation of ATM at Ser 1981 and prompting its downstream mediators to form nuclear foci in response to DNA damage. Silencing FOXO3a in cells abrogates the formation of ATM-pS1981 and phospho-histone H2AX foci after DNA damage. Increasing FOXO3a in cells promotes ATM-regulated signalling, the intra-S-phase or G2-M cell-cycle checkpoints, and the repair of damaged DNA, whereas cells lacking FOXO3a did not trigger the DNA-repair mechanism after DNA damage. The carboxy-terminal domain of FOXO3a binds to the FAT domain of ATM, thereby contributing to the activation of ATM. These results suggest that ATM may be regulated directly by FOXO3a in the DNA-damage response.     

SQ/TQ cluster domains: concentrated ATM/ATR kinase phosphorylation site regions in DNA-damage-response proteins

ATM/ATR-like protein kinases play central roles in the maintenance of genome stability and phosphorylate numerous substrates in response to DNA damage, preferentially on SQ or TQ motifs. ATM/ATR substrates often contain several closely spaced SQ/TQ motifs in regions that have been termed SQ/TQ cluster domains (SCDs). SCDs are now considered a structural hallmark of DNA-damage-response proteins. Mutational analyses of a number of SCD-containing proteins indicate that multisite phosphorylation of SQ/TQ motifs is required for normal DNA-damage responses, most commonly by mediating protein-protein interactions in the formation of DNA-damage-induced complexes. SCD sequences are highly diverse and these domains may be largely unfolded in their native state rather than adopting a common three-dimensional fold. Structural disorder of SCDs could be advantageous for efficient phosphorylation by ATM/ATR kinases and also enable them to be molded into distinct conformations to facilitate flexible interactions with multiple binding partners.     

A proteomic analysis of ataxia telangiectasia-mutated (ATM)/ATM-Rad3-related (ATR) substrates identifies the ubiquitin-proteasome system as a regulator for DNA damage checkpoints

ATM (ataxia telangiectasia-mutated) and ATR (ATM-Rad3-related) are proximal checkpoint kinases that regulate DNA damage response (DDR). Identification and characterization of ATM/ATR substrates hold the keys for the understanding of DDR. Few techniques are available to identify protein kinase substrates. Here, we screened for potential ATM/ATR substrates using phospho-specific antibodies against known ATM/ATR substrates. We identified proteins cross-reacting to phospho-specific antibodies in response to DNA damage by mass spectrometry. We validated a subset of the candidate substrates to be phosphorylated in an ATM/ATR-dependent manner in vivo. Combining with a functional checkpoint screen, we identified proteins that belong to the ubiquitin-proteasome system (UPS) to be required in mammalian DNA damage checkpoint control, particularly the G(1) cell cycle checkpoint, thus revealing protein ubiquitylation as an important regulatory mechanism downstream of ATM/ATR activation for checkpoint control.     

RSC functions as an early double-strand-break sensor in the cell's response to DNA damage

The detection of a DNA double-strand break (DSB) is necessary to initiate DSB repair. Several proteins, including the MRX/N complex, Tel1/ATM (ataxia telangiectasia mutated), and Mec1/ATR (ATM and Rad3 related), have been proposed as sensors of DNA damage, yet how they recognize the breaks is poorly understood. DSBs occur in the context of chromatin, implicating factors capable of altering local and/or global chromatin structure in the cellular response to DNA damage, including DSB sensing. Emerging evidence indicates that ATP-dependent chromatin-remodeling complexes function in DNA repair. Here we describe an important and novel early role for the RSC ATP-dependent chromatin remodeler linked to DSB sensing in the cell's DNA-damage response. RSC is required for full levels of H2A phosphorylation because it facilitates the recruitment of Tel1/ATM and Mec1/ATR to the break site. Consistent with these results, we also show that Rsc2 is needed for efficient activation of the Rad53-dependent checkpoint, as well as for Cohesin's association with the break site. Finally, Rsc2 is needed for the DNA-damage-induced changes in nucleosome structure surrounding the DSB site. Together, these new findings functionally link RSC to DSB sensing, highlighting the importance of ATP-dependent chromatin-remodeling factors in the cell's early response to DNA damage.     

Drosophila ATM and ATR have distinct activities in the regulation of meiotic DNA damage and repair

Ataxia telangiectasia-mutated (ATM) and ataxia telangiectasia-related (ATR) kinases are conserved regulators of cellular responses to double strand breaks (DSBs). During meiosis, however, the functions of these kinases in DSB repair and the deoxyribonucleic acid (DNA) damage checkpoint are unclear. In this paper, we show that ATM and ATR have unique roles in the repair of meiotic DSBs in Drosophila melanogaster. ATR mutant analysis indicated that it is required for checkpoint activity, whereas ATM may not be. Both kinases phosphorylate H2AV (γ-H2AV), and, using this as a reporter for ATM/ATR activity, we found that the DSB repair response is surprisingly dynamic at the site of DNA damage. γ-H2AV is continuously exchanged, requiring new phosphorylation at the break site until repair is completed. However, most surprising is that the number of γ-H2AV foci is dramatically increased in the absence of ATM, but not ATR, suggesting that the number of DSBs is increased. Thus, we conclude that ATM is primarily required for the meiotic DSB repair response, which includes functions in DNA damage repair and negative feedback control over the level of programmed DSBs during meiosis.     

Comparison of hypoxia-induced replication arrest with hydroxyurea and aphidicolin-induced arrest

Severe levels of hypoxia (oxygen concentrations of less that 0.02%) have been shown to induce a rapid S-phase arrest. The mechanism behind hypoxia-induced S-phase arrest is unclear, we show here that it was not mediated by a shortage of nucleosides and was not dependent on p53, p21 or Hif 1alpha status. The drugs aphidicolin and hydroxyurea both induce rapid replication arrest and have been used throughout the literature to study the ATR-mediated response to stalled replication. We have shown previously that hypoxia induces ATR-dependent phosphorylation of p53, Chk1 and histone H2AX. Using comet-assays to detect DNA-damage we found that both aphidicolin and hydroxyurea induced significant levels of DNA-damage while hypoxia did not. Here we show that like aphidicolin and hydroxyurea, hypoxia induces phosphorylation of Nbs1 at serine 343 and Rad17 serine 645. Hypoxia-dependent phosphorylation of Nbs1 and Rad17 was ATM-independent and therefore likely to be a result of the ATR kinase activity. In contrast, p53 was phosphorylated differentially in response to the three treatments considered here. p53 was phosphorylated at serine 15 in response to all three treatments but was only phosphorylated at serine 20 in response to the drug treatments. We propose that treatment with either aphidicolin or hydroxyurea leads to not only replication arrest but also DNA-damage and therefore both ATM and ATR-mediated signaling. In contrast replication arrest induced by severe hypoxia is sensed exclusively through ATR, with ATM only having a role to play after re-oxygenation.     

ATR kinase activation in G1 phase facilitates the repair of ionizing radiation-induced DNA damage

The kinase ATR is activated by RPA-coated single-stranded DNA generated at aberrant replicative structures and resected double strand breaks. While many hundred candidate ATR substrates have been identified, the essential role of ATR in the replicative stress response has impeded the study of ATR kinase-dependent signalling. Using recently developed selective drugs, we show that ATR inhibition has a significantly more potent effect than ATM inhibition on ionizing radiation (IR)-mediated cell killing. Transient ATR inhibition for a short interval after IR has long-term consequences that include an accumulation of RPA foci and a total abrogation of Chk1 S345 phosphorylation. We show that ATR kinase activity in G1 phase cells is important for survival after IR and that ATR colocalizes with RPA in the absence of detectable RPA S4/8 phosphorylation. Our data reveal that, unexpectedly, ATR kinase inhibitors may be more potent cellular radiosensitizers than ATM kinase inhibitors, and that this is associated with a novel role for ATR in G1 phase cells.     

Single-stranded oligonucleotide-mediated gene repair in mammalian cells has a mechanism distinct from homologous recombination repair

Single-stranded DNA oligonucleotide (SSO)-mediated gene repair has great potentials for gene therapy and functional genomic studies. However, its underlying mechanism remains unclear. Previous studies from other groups have suggested that DNA damage response via the ATM/ATR pathway may be involved in this process. In this study, we measured the effect of two ATM/ATR inhibitors caffeine and pentoxifylline on the correction efficiency in SSO-mediated gene repair. We also checked their effect on double-stranded break (DSB)-induced homologous recombination repair (HRR) as a control, which is well known to be dependent on the ATM/ATR pathway. We found these inhibitors could completely inhibit DSB-induced HRR, but could only partially inhibit SSO-mediated process, indicating SSO-mediated gene repair is not dependent on the ATM/ATR pathway. Furthermore, we found that thymidine treatment promotes SSO-mediated gene repair, but inhibits DSB-induced HRR. Collectively, our results demonstrate that SSO-mediated and DSB-induced gene repairs have distinct mechanisms.     

Nek1 kinase functions in DNA damage response and checkpoint control through a pathway independent of ATM and ATR

Never-in-mitosis A related protein kinase 1 (Nek1) is involved early in a DNA damage sensing/repair pathway. We have previously shown that cells without functional Nek1 fail to activate the more distal kinases Chk1 and Chk2 and fail to arrest properly at G₁/S or M-phase checkpoints in response to DNA damage. As a consequence, foci of damaged DNA in Nek1 null cells persist long after the instigating insult, and Nek1 null cells develop unstable chromosomes at a rate much higher than identically cultured wild-type cells. Here we show that Nek1 functions independently of canonical DNA damage responses requiring the PI3 kinase-like proteins ATM and ATR. Chemical inhibitors of ATM/ATR or mutation of the genes that encode them fail to alter the kinase activity of Nek1 or its localization to nuclear foci of DNA damage. Moreover ATM and ATR activities, including the localization of the proteins to DNA damage sites and phosphorylation of early DNA damage response substrates, are intact in Nek1^−/− murine cells and in human cells with Nek1 expression silenced by siRNA. Our results demonstrate that Nek1 is important for proper checkpoint control and characterize for the first time a DNA damage response that does not directly involve one of the known upstream mediator kinases, ATM or ATR.Key words: checkpoint control, DNA damage response, Nek1, ATM, ATR