Distinct roles for DNA-PK,ATM and ATR in RPA phosphorylation and checkpoint activation in response to replication stress

DNA damage encountered by DNA replication forks poses risks of genome destabilization, a precursor to carcinogenesis. Damage checkpoint systems cause cell cycle arrest, promote repair and induce programed cell death when damage is severe. Checkpoints are critical parts of the DNA damage response network that act to suppress cancer. DNA damage and perturbation of replication machinery causes replication stress, characterized by accumulation of single-stranded DNA bound by replication protein A (RPA), which triggers activation of ataxia telangiectasia and Rad3 related (ATR) and phosphorylation of the RPA32, subunit of RPA, leading to Chk1 activation and arrest. DNA-dependent protein kinase catalytic subunit (DNA-PKcs) [a kinase related to ataxia telangiectasia mutated (ATM) and ATR] has well characterized roles in DNA double-strand break repair, but poorly understood roles in replication stress-induced RPA phosphorylation. We show that DNA-PKcs mutant cells fail to arrest replication following stress, and mutations in RPA32 phosphorylation sites targeted by DNA-PKcs increase the proportion of cells in mitosis, impair ATR signaling to Chk1 and confer a G2/M arrest defect. Inhibition of ATR and DNA-PK (but not ATM), mimic the defects observed in cells expressing mutant RPA32. Cells expressing mutant RPA32 or DNA-PKcs show sustained H2AX phosphorylation in response to replication stress that persists in cells entering mitosis, indicating inappropriate mitotic entry with unrepaired damage.     

Recruitment of the cell cycle checkpoint kinase ATR to chromatin during S-phase

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.     

RPA70 depletion induces hSSB1/2-INTS3 complex to initiate ATR signaling

The primary eukaryotic single-stranded DNA-binding protein, Replication protein A (RPA), binds to single-stranded DNA at the sites of DNA damage and recruits the apical checkpoint kinase, ATR via its partner protein, ATRIP. It has been demonstrated that absence of RPA incapacitates the ATR-mediated checkpoint response. We report that in the absence of RPA, human single-stranded DNA-binding protein 1 (hSSB1) and its partner protein INTS3 form sub-nuclear foci, associate with the ATR-ATRIP complex and recruit it to the sites of genomic stress. The ATRIP foci formed after RPA depletion are abrogated in the absence of INTS3, establishing that hSSB-INTS3 complex recruits the ATR-ATRIP checkpoint complex to the sites of genomic stress. Depletion of homologs hSSB1/2 and INTS3 in RPA-deficient cells attenuates Chk1 phosphorylation, indicating that the cells are debilitated in responding to stress. We have identified that TopBP1 and the Rad9-Rad1-Hus1 complex are essential for the alternate mode of ATR activation. In summation, we report that the single-stranded DNA-binding protein complex, hSSB1/2-INTS3 can recruit the checkpoint complex to initiate ATR signaling.     

The basic cleft of RPA70N binds multiple checkpoint proteins, including RAD9, to regulate ATR signaling

ATR kinase activation requires the recruitment of the ATR-ATRIP and RAD9-HUS1-RAD1 (9-1-1) checkpoint complexes to sites of DNA damage or replication stress. Replication protein A (RPA) bound to single-stranded DNA is at least part of the molecular recognition element that recruits these checkpoint complexes. We have found that the basic cleft of the RPA70 N-terminal oligonucleotide-oligosaccharide fold (OB-fold) domain is a key determinant of checkpoint activation. This protein-protein interaction surface is able to bind several checkpoint proteins, including ATRIP, RAD9, and MRE11. RAD9 binding to RPA is mediated by an acidic peptide within the C-terminal RAD9 tail that has sequence similarity to the primary RPA-binding surface in the checkpoint recruitment domain (CRD) of ATRIP. Mutation of the RAD9 CRD impairs its localization to sites of DNA damage or replication stress without perturbing its ability to form the 9-1-1 complex or bind the ATR activator TopBP1. Disruption of the RAD9-RPA interaction also impairs ATR signaling to CHK1 and causes hypersensitivity to both DNA damage and replication stress. Thus, the basic cleft of the RPA70 N-terminal OB-fold domain binds multiple checkpoint proteins, including RAD9, to promote ATR signaling.     

Herpes Simplex Virus Type 1 Single Strand DNA Binding Protein and Helicase/Primase Complex Disable Cellular ATR Signaling

Herpes Simplex Virus type 1 (HSV-1) has evolved to disable the cellular DNA damage response kinase, ATR. We have previously shown that HSV-1-infected cells are unable to phosphorylate the ATR substrate Chk1, even under conditions in which replication forks are stalled. Here we report that the HSV-1 single stranded DNA binding protein (ICP8), and the helicase/primase complex (UL8/UL5/UL52) form a nuclear complex in transfected cells that is necessary and sufficient to disable ATR signaling. This complex localizes to sites of DNA damage and colocalizes with ATR/ATRIP and RPA, but under these conditions, the Rad9-Rad1-Hus1 checkpoint clamp (9-1-1) do not. ATR is generally activated by substrates that contain ssDNA adjacent to dsDNA, and previous work from our laboratory has shown that ICP8 and helicase/primase also recognize this substrate. We suggest that these four viral proteins prevent ATR activation by binding to the DNA substrate and obstructing loading of the 9-1-1 checkpoint clamp. Exclusion of 9-1-1 prevents recruitment of TopBP1, the ATR kinase activator, and thus effectively disables ATR signaling. These data provide the first example of viral DNA replication proteins obscuring access to a DNA substrate that would normally trigger a DNA damage response and checkpoint signaling. This unusual mechanism used by HSV suggests that it may be possible to inhibit ATR signaling by preventing recruitment of the 9-1-1 clamp and TopBP1.     

DDK phosphorylates checkpoint clamp component Rad9 and promotes its release from damaged chromatin

When inappropriate DNA structures arise, they are sensed by DNA structure-dependent checkpoint pathways and subsequently repaired. Recruitment of checkpoint proteins to such structures precedes recruitment of proteins involved in DNA metabolism. Thus, checkpoints can regulate DNA metabolism. We show that fission yeast Rad9, a 9-1-1 heterotrimeric checkpoint-clamp component, is phosphorylated by Hsk1(Cdc7), the Schizosaccharomyces pombe?Dbf4-dependent kinase (DDK) homolog, in response to replication-induced DNA damage. Phosphorylation of Rad9 disrupts its interaction with replication protein A (RPA) and is dependent on 9-1-1 chromatin loading, the Rad9-associated protein Rad4/Cut5(TopBP1), and prior phosphorylation by Rad3(ATR). rad9 mutants defective in DDK phosphorylation show wild-type checkpoint responses but abnormal DNA repair protein foci and decreased viability after replication stress. We propose that Rad9 phosphorylation by DDK releases Rad9 from DNA damage sites to facilitate DNA repair.     

ATR and ATM-Dependent Movement of BLM Helicase during Replication Stress Ensures Optimal ATM Activation and 53BP1 Focus Formation

The BLM helicase, a deficiency in which markedly increases cancer incidence in humans, isrequired for optimal repair during DNA replication. We show that BLM rapidly moves fromPML nuclear bodies to damaged replication forks, returning to PML bodies several hours later,owing to activities of the DNA damage response kinases ATR and ATM, respectively.Immunofluorescence and cellular fractionation demonstrate that BLM partitions to different subcellularcompartments after replication stress. Unexpectedly, fibroblasts lacking BLM weredeficient in phospho-ATM (S-1981) and 53-binding protein-1 (53BP1), and these proteins failedto form foci following replication stress. Expression of a dominant p53 mutant or helicasedeficientBLM restored replication stress-induced 53BP1 foci, but only mutant p53 restoredoptimal ATM activation. Thus, optimal repair of damaged replication fork lesions likelyrequires both ATR and ATM, BLM recruits 53BP1 to these lesions independent of its helicaseactivity, and optimal activation of ATM requires both p53 and BLM helicase activities.

Supplemental material for this paper can be found at the following link:

http://www.landesbioscience.com/journals/cc/davalosCC3-12-sup.pdf     

RPA2 is a direct downstream target for ATR to regulate the S-phase checkpoint

Upon DNA damage, replication is inhibited by the S-phase checkpoint. ATR (ataxia telangiectasia mutated- and Rad3-related) is specifically involved in the inhibition of replicon initiation when cells are treated with DNA damage-inducing agents that stall replication forks, but the mechanism by which it acts to prevent replication is not yet fully understood. We observed that RPA2 is phosphorylated on chromatin in an ATR-dependent manner when replication forks are stalled. Mutation of the ATR-dependent phosphorylation sites in RPA2 leads to a defect in the down-regulation of DNA synthesis following treatment with UV radiation, although ATR activation is not affected. Threonine 21 and serine 33, two residues among several phosphorylation sites in the amino terminus of RPA2, are specifically required for the UV-induced, ATR-mediated inhibition of DNA replication. RPA2 mutant alleles containing phospho-mimetic mutations at ATR-dependent phosphorylation sites have an impaired ability to associate with replication centers, indicating that ATR phosphorylation of RPA2 directly affects the replication function of RPA. Our studies suggest that in response to UV-induced DNA damage, ATR rapidly phosphorylates RPA2, disrupting its association with replication centers in the S-phase and contributing to the inhibition of DNA replication.     

Replication protein A and gamma-H2AX foci assembly is triggered by cellular response to DNA double-strand breaks

Human replication protein A (RPA p34), a crucial component of diverse DNA excision repair pathways, is implicated in DNA double-strand break (DSB) repair. To evaluate its role in DSB repair, the intranuclear dynamics of RPA was investigated after DNA damage and replication blockage in human cells. Using two different agents [ionizing radiation (IR) and hydroxyurea (HU)] to generate DSBs, we found that RPA relocated into distinct nuclear foci and colocalized with a well-known DSB binding factor, gamma-H2AX, at the sites of DNA damage in a time-dependent manner. Colocalization of RPA and gamma-H2AX foci peaked at 2 h after IR treatment and subsequently declined with increasing postrecovery times. The time course of RPA and gamma-H2AX foci association correlated well with the DSB repair activity detected by a neutral comet assay. A phosphatidylinositol-3 (PI-3) kinase inhibitor, wortmannin, completely abolished both RPA and gamma-H2AX foci formation triggered by IR. Additionally, radiosensitive ataxia telangiectasia (AT) cells harboring mutations in ATM gene product were found to be deficient in RPA and gamma-H2AX colocalization after IR. Transfection of AT cells with ATM cDNA fully restored the association of RPA foci with gamma-H2AX illustrating the requirement of ATM gene product for this process. The exact coincidence of RPA and gamma-H2AX in response to HU specifically in S-phase cells supports their role in DNA replication checkpoint control. Depletion of RPA by small interfering RNA (SiRNA) substantially elevated the frequencies of IR-induced micronuclei (MN) and apoptosis in human cells suggestive of a role for RPA in DSB repair. We propose that RPA in association with gamma-H2AX contributes to both DNA damage checkpoint control and repair in response to strand breaks and stalled replication forks in human cells.     

RING finger and WD repeat domain 3 (RFWD3) associates with replication protein A (RPA) and facilitates RPA-mediated DNA damage response

DNA damage response is crucial for maintaining genomic integrity and preventing cancer by coordinating the activation of checkpoints and the repair of damaged DNA. Central to DNA damage response are the two checkpoint kinases ATM and ATR that phosphorylate a wide range of substrates. RING finger and WD repeat domain 3 (RFWD3) was initially identified as a substrate of ATM/ATR from a proteomic screen. Subsequent studies showed that RFWD3 is an E3 ubiquitin ligase that ubiquitinates p53 in vitro and positively regulates p53 levels in response to DNA damage. We report here that RFWD3 associates with replication protein A (RPA), a single-stranded DNA-binding protein that plays essential roles in DNA replication, recombination, and repair. Binding of RPA to single-stranded DNA (ssDNA), which is generated by DNA damage and repair, is essential for the recruitment of DNA repair factors to damaged sites and the activation of checkpoint signaling. We show that RFWD3 is physically associated with RPA and rapidly localizes to sites of DNA damage in a RPA-dependent manner. In vitro experiments suggest that the C terminus of RFWD3, which encompass the coiled-coil domain and the WD40 domain, is necessary for binding to RPA. Furthermore, DNA damage-induced phosphorylation of RPA and RFWD3 is dependent upon each other. Consequently, loss of RFWD3 results in the persistent foci of DNA damage marker γH2AX and the repair protein Rad51 in damaged cells. These findings suggest that RFWD3 is recruited to sites of DNA damage and facilitates RPA-mediated DNA damage signaling and repair.     

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     

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.     

Quaternary structure of ATR and effects of ATRIP and replication protein A on its DNA binding and kinase activities

ATR is an essential protein that functions as a damage sensor and a proximal kinase in the DNA damage checkpoint response in mammalian cells. It is a member of the phosphoinositide 3-kinase-like kinase (PIKK) family, which includes ATM, ATR, and DNA-dependent protein kinase. Recently, it was found that ATM is an oligomeric protein that is converted to an active monomeric form by phosphorylation in trans upon DNA damage, and this raised the possibility that other members of the PIKK family may be regulated in a similar manner. Here we show that ATR is a monomeric protein associated with a smaller protein called ATRIP with moderate affinity. The ATR protein by itself or in the form of the ATR-ATRIP heterodimer binds to naked or replication protein A (RPA)-covered DNAs with comparable affinities. However, the phosphorylation of RPA by ATR is dependent on single-stranded DNA and is stimulated by ATRIP. These findings suggest that the regulation and mechanism of action of ATR are fundamentally different from those of the other PIKK proteins.     

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.     

DNA-PK phosphorylation of RPA32 Ser4/Ser8 regulates replication stress checkpoint activation,fork restart,homologous recombination and mitotic catastrophe

Genotoxins and other factors cause replication stress that activate the DNA damage response (DDR), comprising checkpoint and repair systems. The DDR suppresses cancer by promoting genome stability, and it regulates tumor resistance to chemo- and radiotherapy. Three members of the phosphatidylinositol 3-kinase-related kinase (PIKK) family, ATM, ATR, and DNA-PK, are important DDR proteins. A key PIKK target is replication protein A (RPA), which binds single-stranded DNA and functions in DNA replication, DNA repair, and checkpoint signaling. An early response to replication stress is ATR activation, which occurs when RPA accumulates on ssDNA. Activated ATR phosphorylates many targets, including the RPA32 subunit of RPA, leading to Chk1 activation and replication arrest. DNA-PK also phosphorylates RPA32 in response to replication stress, and we demonstrate that cells with DNA-PK defects, or lacking RPA32 Ser4/Ser8 targeted by DNA-PK, confer similar phenotypes, including defective replication checkpoint arrest, hyper-recombination, premature replication fork restart, failure to block late origin firing, and increased mitotic catastrophe. We present evidence that hyper-recombination in these mutants is ATM-dependent, but the other defects are ATM-independent. These results indicate that DNA-PK and ATR signaling through RPA32 plays a critical role in promoting genome stability and cell survival in response to replication stress.     

How Cells Activate ATR

ATR is a critical upstream regulator of checkpoint responses to incompletely replicated and damaged DNA. However, it had not been understood how the kinase activity of ATR is switched on during checkpoint responses. TopBP1 and its homologs are necessary for both DNA replication and checkpoint control. A recent report from this laboratory demonstrated that TopBP1 functions as an activator of ATR. It had been known that TopBP1 accumulates at sites of replicative stress and DNA damage. Thus, interaction of ATR with a critical protein at stalled replication forks and sites of DNA damage triggers its activation. This finding helps to explain how aberrant DNA structures in the genome induce ATR-dependent signaling processes.     

Clamping the Mec1/ATR Checkpoint Kinase into Action

The yeast checkpoint protein kinase Mec1, the ortholog of human ATR, is the essential upstream regulator of the cell cycle checkpoint in response to DNA damage and to stalling of DNA replication forks. The activity of Mec1/ATR is not directly regulated by the DNA substrates that signal checkpoint activation. Rather the signal appears to be transduced to Mec1 by factors that interact with the signaling DNA substrates. One of these factors, the DNA damage checkpoint clamp Rad17-Mec3-Ddc1 (human 9-1-1) is loaded onto gapped DNA resulting from the partial repair of DNA damage, and the Ddc1 subunit of this complex activates Mec1. In vertebrate cells, the TopBP1 protein (Cut5 in S. pombe and Dpb11 in S. cervisiae) that is also required for establishment of the replication fork, functions during replication fork dysfunction to activate ATR. Both mechanisms of activation generally upregulate the kinase activity towards all downstream targets.     

Akt/PKB suppresses DNA damage processing and checkpoint activation in late G2

Using chemical genetics to reversibly inhibit Cdk1, we find that cells arrested in late G2 are unable to delay mitotic entry after irradiation. Late G2 cells detect DNA damage lesions and form γ-H2AX foci but fail to activate Chk1. This reflects a lack of DNA double-strand break processing because late G2 cells fail to recruit RPA (replication protein A), ATR (ataxia telangiectasia and Rad3 related), Rad51, or CtIP (C-terminal interacting protein) to sites of radiation-induced damage, events essential for both checkpoint activation and initiation of DNA repair by homologous recombination. Remarkably, inhibition of Akt/PKB (protein kinase B) restores DNA damage processing and Chk1 activation after irradiation in late G2. These data demonstrate a previously unrecognized role for Akt in cell cycle regulation of DNA repair and checkpoint activation. Because Akt/PKB is frequently activated in many tumor types, these findings have important implications for the evolution and therapy of such cancers.     

RPA mediates recombination repair during replication stress and is displaced from DNA by checkpoint signalling in human cells

The replication protein A (RPA) is involved in most, if not all, nuclear metabolism involving single-stranded DNA. Here, we show that RPA is involved in genome maintenance at stalled replication forks by the homologous recombination repair system in humans. Depletion of the RPA protein inhibited the formation of RAD51 nuclear foci after hydroxyurea-induced replication stalling leading to persistent unrepaired DNA double-strand breaks (DSBs). We demonstrate a direct role of RPA in homology directed recombination repair. We find that RPA is dispensable for checkpoint kinase 1 (Chk1) activation and that RPA directly binds RAD52 upon replication stress, suggesting a direct role in recombination repair. In addition we show that inhibition of Chk1 with UCN-01 decreases dissociation of RPA from the chromatin and inhibits association of RAD51 and RAD52 with DNA. Altogether, our data suggest a direct role of RPA in homologous recombination in assembly of the RAD51 and RAD52 proteins. Furthermore, our data suggest that replacement of RPA with the RAD51 and RAD52 proteins is affected by checkpoint signalling.