ATRIP binding to replication protein A-single-stranded DNA promotes ATR-ATRIP localization but is dispensable for Chk1 phosphorylation

ATR associates with the regulatory protein ATRIP that has been proposed to localize ATR to sites of DNA damage through an interaction with single-stranded DNA (ssDNA) coated with replication protein A (RPA). We tested this hypothesis and found that ATRIP is required for ATR accumulation at intranuclear foci induced by DNA damage. A domain at the N terminus of ATRIP is necessary and sufficient for interaction with RPA-ssDNA. Deletion of the ssDNA-RPA interaction domain of ATRIP greatly diminished accumulation of ATRIP into foci. However, the ATRIP-RPA-ssDNA interaction is not sufficient for ATRIP recognition of DNA damage. A splice variant of ATRIP that cannot bind to ATR revealed that ATR association is also essential for proper ATRIP localization. Furthermore, the ATRIP-RPA-ssDNA interaction is not absolutely essential for ATR activation because ATR phosphorylates Chk1 in cells expressing only a mutant of ATRIP that does not bind to RPA-ssDNA. These data suggest that binding to RPA-ssDNA is not the essential function of ATRIP in ATR-dependent checkpoint signaling and ATR has an important function in properly localizing the ATR-ATRIP complex.     

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.     

A novel protein activity mediates DNA binding of an ATR-ATRIP complex

The function of the ATR (ataxia-telangiectasia mutated and Rad3-related)-ATRIP (ATR-interacting protein) protein kinase complex is central to the cellular response to replication stress and DNA damage. In order to better understand the function of this complex, we have studied its interaction with DNA. We find that both ATR and ATRIP associate with chromatin in vivo, and they exist as a large molecular weight complex that can bind single-stranded (ss)DNA cellulose in vitro. Although replication protein A (RPA) is sufficient for the recruitment of ATRIP to ssDNA, we show that a distinct ATR-ATRIP complex is able to bind to DNA with lower affinity in the absence of RPA. In this latter complex, we show that neither ATR nor ATRIP are able to bind DNA individually, nor do they bind DNA in a cooperative manner. However, the addition of HeLa nuclear extract is able to reconstitute the DNA binding of both ATR and ATRIP, suggesting the requirement for an additional protein activity. We also show that ATR is necessary for ATRIP to bind DNA in this low affinity mode and to form a large DNA binding complex. These observations suggest that there are at least two in vitro ATR-ATRIP DNA binding complexes, one which binds DNA with high affinity in an RPA-dependent manner and a second, which binds DNA with lower affinity in an RPA-independent manner but which requires an as of yet unidentified protein.     

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.     

Dimerization of the ATRIP protein through the coiled-coil motif and its implication to the maintenance of stalled replication forks

ATR (ATM and Rad3-related), a PI kinase-related kinase (PIKK), has been implicated in the DNA structure checkpoint in mammalian cells. ATR associates with its partner protein ATRIP to form a functional complex in the nucleus. In this study, we investigated the role of the ATRIP coiled-coil domain in ATR-mediated processes. The coiled-coil domain of human ATRIP contributes to self-dimerization in vivo, which is important for the stable translocation of the ATR-ATRIP complex to nuclear foci that are formed after exposure to genotoxic stress. The expression of dimerization-defective ATRIP diminishes the maintenance of replication forks during treatment with replication inhibitors. By contrast, it does not compromise the G2/M checkpoint after IR-induced DNA damage. These results show that there are two critical functions of ATR-ATRIP after the exposure to genotoxic stress: maintenance of the integrity of replication machinery and execution of cell cycle arrest, which are separable and are achieved via distinct mechanisms. The former function may involve the concentrated localization of ATR to damaged sites for which the ATRIP coiled-coil motif is critical.     

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.     

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.     

Role of the C terminus of Mec1 checkpoint kinase in its localization to sites of DNA damage

The large protein kinases, ataxia-telangiectasia mutated (ATM) and ATM-Rad3-related (ATR), coordinate the cellular response to DNA damage. In budding yeast, ATR homologue Mec1 plays a central role in DNA damage signaling. Mec1 interacts physically with Ddc2 and functions in the form of the Mec1-Ddc2 complex. To identify proteins interacting with the Mec1-Ddc2 complex, we performed a modified two-hybrid screen and isolated RFA1 and RFA2, genes that encode subunits of replication protein A (RPA). Using the two-hybrid system, we found that the extreme C-terminal region of Mec1 is critical for RPA binding. The C-terminal substitution mutation does not affect the Mec1-Ddc2 complex formation, but it does impair the interaction of Mec1 and Ddc2 with RPA as well as their association with DNA lesions. The C-terminal mutation also decreases Mec1 kinase activity. However, the Mec1 kinase-defect by itself does not perturb Mec1 association with sites of DNA damage. We also found that Mec1 and Ddc2 associate with sites of DNA damage in an interdependent manner. Our findings support the model in which Mec1 and Ddc2 localize to sites of DNA damage by interacting with RPA in the form of the Mec1-Ddc2 complex.     

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.     

Proapoptotic Bid mediates the Atr-directed DNA damage response to replicative stress

Proapoptotic BH3 interacting domain death agonist (Bid), a BH3-only Bcl-2 family member, is situated at the interface between the DNA damage response and apoptosis, with roles in death receptor-induced apoptosis as well as cell cycle checkpoints following DNA damage.(1, 2, 3) In this study, we demonstrate that Bid functions at the level of the sensor complex in the Atm and Rad3-related (Atr)-directed DNA damage response. Bid is found with replication protein A (RPA) in nuclear foci and associates with the Atr/Atr-interacting protein (Atrip)/RPA complex following replicative stress. Furthermore, Bid-deficient cells show an impaired response to replicative stress manifest by reduced accumulation of Atr and Atrip on chromatin and at DNA damage foci, reduced recovery of DNA synthesis following replicative stress, and decreased checkpoint kinase 1 activation and RPA phosphorylation. These results establish a direct role for the BH3-only Bcl-2 family member, Bid, acting at the level of the damage sensor complex to amplify the Atr-directed cellular response to replicative DNA damage.     

ATR autophosphorylation as a molecular switch for checkpoint activation

The ataxia telangiectasia-mutated and Rad3-related (ATR) kinase is a master checkpoint regulator safeguarding the genome. Upon DNA damage, the ATR-ATRIP complex is recruited to sites of DNA damage by RPA-coated single-stranded DNA and activated by an elusive process. Here, we show that ATR is transformed into a hyperphosphorylated state after DNA damage, and that a single autophosphorylation event at Thr 1989 is crucial for ATR activation. Phosphorylation of Thr 1989 relies on RPA, ATRIP, and ATR kinase activity, but unexpectedly not on the ATR stimulator TopBP1. Recruitment of ATR-ATRIP to RPA-ssDNA leads to congregation of ATR-ATRIP complexes and promotes Thr 1989 phosphorylation in trans. Phosphorylated Thr 1989 is directly recognized by TopBP1 via the BRCT domains 7 and 8, enabling TopBP1 to engage ATR-ATRIP, to stimulate the ATR kinase, and to facilitate ATR substrate recognition. Thus, ATR autophosphorylation on RPA-ssDNA is a molecular switch to launch robust checkpoint response.     

Tipin-Replication Protein A Interaction Mediates Chk1 Phosphorylation by ATR in Response to Genotoxic Stress

Mammalian Timeless is a multifunctional protein that performs essential roles in the circadian clock, chromosome cohesion, DNA replication fork protection, and DNA replication/DNA damage checkpoint pathways. The human Timeless exists in a tight complex with a smaller protein called Tipin (Timeless-interacting protein). Here we investigated the mechanism by which the Timeless-Tipin complex functions as a mediator in the ATR-Chk1 DNA damage checkpoint pathway. We find that the Timeless-Tipin complex specifically mediates Chk1 phosphorylation by ATR in response to DNA damage and replication stress through interaction of Tipin with the 34-kDa subunit of replication protein A (RPA). The Tipin-RPA interaction stabilizes Timeless-Tipin and Tipin-Claspin complexes on RPA-coated ssDNA and in doing so promotes Claspin-mediated phosphorylation of Chk1 by ATR. Our results therefore indicate that RPA-covered ssDNA not only supports recruitment and activation of ATR but also, through Tipin and Claspin, it plays an important role in the action of ATR on its critical downstream target Chk1.     

RPA-coated single-stranded DNA promotes the ETAA1-dependent activation of ATR

Besides TopBP1, ETAA1 has been identified more recently as an activator of the ATR-ATRIP complex in human cells. We have examined the role of ETAA1 in the Xenopus egg-extract system, which has been instrumental in the study of ATR-ATRIP. Depletion of ETAA1 from egg extracts did not noticeably reduce the activation of ATR-ATRIP in response to replication stress, as monitored by the ATR-dependent phosphorylation of Chk1 and RPA. Moreover, lack of ETAA1 did not appear to affect DNA replication during an unperturbed S-phase. Significantly, we find that TopBP1 is considerably more abundant than ETAA1 in egg extracts. We proceeded to show that ETAA1 could support the activation of ATR-ATRIP in response to replication stress if we increased its concentration in egg extracts by adding extra full-length recombinant ETAA1. Thus, TopBP1 appears to be the predominant activator of ATR-ATRIP in response to replication stress in this system. We have also explored the biochemical mechanism by which ETAA1 activates ATR-ATRIP. We have developed an in vitro system in which full-length recombinant ETAA1 supports activation of ATR-ATRIP in the presence of defined components. We find that binding of ETAA1 to RPA associated with single-stranded DNA (ssDNA) greatly stimulates its ability to activate ATR-ATRIP. Thus, RPA-coated ssDNA serves as a direct positive effector in the ETAA1-mediated activation of ATR-ATRIP.     

Phosphorylation of replication protein A by S-phase checkpoint kinases

The major eukaryotic single-stranded DNA (ssDNA) binding protein, replication protein A (RPA), is a heterotrimer with subunits of 70, 32 and 14 kDa (RPA70, RPA32 and RPA14). RPA-coated ssDNA has been implicated as one of the triggers for intra-S-phase checkpoint activation. Phosphorylation of RPA occurs in cells with damaged DNA or stalled replication forks. Here we show that human RPA70 and RPA32 can be phosphorylated by purified S-phase checkpoint kinases, ATR and Chk1. While ATR phosphorylates the N-terminus of RPA70, Chk1 preferentially phosphorylates RPA's major ssDNA binding domain. Chk1 phosphorylated RPA70 shows reduced ssDNA binding activity, and binding of RPA to ssDNA blocks Chk1 phosphorylation, suggesting that Chk1 and ssDNA compete for RPA's major ssDNA binding domain. ssDNA stimulates RPA32 phosphorylation by ATR in a length dependent manner. Furthermore, 3'-, but not 5'-, recessed single strand/double strand DNA junctions produce an even stronger stimulatory effect on RPA32 phosphorylation by ATR. This stimulation occurs for both RNA and DNA recessed ends. RPA's DNA binding polarity and its interaction to 3'-primer-template junctions contribute to efficient RPA32 phosphorylation. Progression of DNA polymerase is able to block the accessibility of the 3'-recessed ends and prevent the stimulatory effects of primer-template junctions on RPA phosphorylation by ATR. We propose models for the role of RPA phosphorylation by Chk1 in S-phase checkpoint pathways, and the possible regulation of ATR activity by different nucleic acid structures.     

A role for E1B-AP5 in ATR signaling pathways during adenovirus infection

E1B-55K-associated protein 5 (E1B-AP5) is a cellular, heterogeneous nuclear ribonucleoprotein that is targeted by adenovirus (Ad) E1B-55K during infection. The function of E1B-AP5 during infection, however, remains largely unknown. Given the role of E1B-55K targets in the DNA damage response, we examined whether E1B-AP5 function was integral to these pathways. Here, we show a novel role for E1B-AP5 as a key regulator of ATR signaling pathways activated during Ad infection. E1B-AP5 is recruited to viral replication centers during infection, where it colocalizes with ATR-interacting protein (ATRIP) and the ATR substrate replication protein A 32 (RPA32). Indeed, E1B-AP5 associates with ATRIP and RPA complex component RPA70 in both uninfected and Ad-infected cells. Additionally, glutathione S-transferase pull-downs show that E1B-AP5 associates with RPA components RPA70 and RPA32 directly in vitro. E1B-AP5 is required for the ATR-dependent phosphorylation of RPA32 during infection and contributes to the Ad-induced phosphorylation of Smc1 and H2AX. In this regard, it is interesting that Ad5 and Ad12 differentially promote the phosphorylation of RPA32, Rad9, and Smc1 during infection such that Ad12 promotes a significant phosphorylation of RPA32 and Rad9, whereas Ad5 only weakly promotes RPA32 phosphorylation and does not induce Rad9 phosphorylation. These data suggest that Ad5 and Ad12 have evolved different strategies to regulate DNA damage signaling pathways during infection in order to promote viral replication. Taken together, our results define a role for E1B-AP5 in ATR signaling pathways activated during infection. This might have broader implications for the regulation of ATR activity during cellular DNA replication or in response to DNA damage.     

Claspin,a Chk1-regulatory protein,monitors DNA replication on chromatin independently of RPA,ATR, and Rad17

Claspin is required for the ATR-dependent activation of Chk1 in Xenopus egg extracts containing incompletely replicated DNA. We show here that Claspin associates with chromatin in a regulated manner during S phase. Binding of Claspin to chromatin depends on the pre-replication complex (pre-RC) and Cdc45 but not on replication protein A (RPA). These dependencies suggest that binding of Claspin occurs around the time of initial DNA unwinding at replication origins. By contrast, both ATR and Rad17 require RPA for association with DNA. Claspin, ATR, and Rad17 all bind to chromatin independently. These findings suggest that Claspin plays a role in monitoring DNA replication during S phase. Claspin, ATR, and Rad17 may collaborate in checkpoint regulation by detecting different aspects of a DNA replication fork.     

Novel checkpoint response to genotoxic stress mediated by nucleolin-replication protein a complex formation

Human replication protein A (RPA), the primary single-stranded DNA-binding protein, was previously found to be inhibited after heat shock by complex formation with nucleolin. Here we show that nucleolin-RPA complex formation is stimulated after genotoxic stresses such as treatment with camptothecin or exposure to ionizing radiation. Complex formation in vitro and in vivo requires a 63-residue glycine-arginine-rich (GAR) domain located at the extreme C terminus of nucleolin, with this domain sufficient to inhibit DNA replication in vitro. Fluorescence resonance energy transfer studies demonstrate that the nucleolin-RPA interaction after stress occurs both in the nucleoplasm and in the nucleolus. Expression of the GAR domain or a nucleolin mutant (TM) with a constitutive interaction with RPA is sufficient to inhibit entry into S phase. Increasing cellular RPA levels by overexpression of the RPA2 subunit minimizes the inhibitory effects of nucleolin GAR or TM expression on chromosomal DNA replication. The arrest is independent of p53 activation by ATM or ATR and does not involve heightened expression of p21. Our data reveal a novel cellular mechanism that represses genomic replication in response to genotoxic stress by inhibition of an essential DNA replication factor.     

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.     

ATR kinase activity regulates the intranuclear translocation of ATR and RPA following ionizing radiation

Upon damage of DNA in eukaryotic cells, several repair and checkpoint proteins undergo a dramatic intranuclear relocalization, translocating to nuclear foci thought to represent sites of DNA damage and repair. Examples of such proteins include the checkpoint kinase ATR (ATM and Rad3-related) as well as replication protein A (RPA), a single-stranded DNA binding protein required in DNA replication and repair. Here, we used a microscopy-based approach to investigate whether the damage-induced translocation of RPA is an active process regulated by ATR. Our data show that in undamaged cells, ATR and RPA are uniformly distributed in the nucleus or localized to promyelocytic leukemia protein (PML) nuclear bodies. In cells treated with ionizing radiation, both ATR and RPA translocate to punctate, abundant nuclear foci where they continue to colocalize. Surprisingly, an ATR mutant that lacks kinase activity fails to relocalize in response to DNA damage. Furthermore, this kinase-inactive mutant blocks the translocation of RPA in a cell cycle-dependent manner. These observations demonstrate that the kinase activity of ATR is essential for the irradiation-induced release of ATR and RPA from PML bodies and translocation of ATR and RPA to potential sites of DNA damage.     

Mislocalization of the MRN complex prevents ATR signaling during adenovirus infection

The protein kinases ataxia‐telangiectasia mutated (ATM) and ATM‐Rad3 related (ATR) are activated in response to DNA damage, genotoxic stress and virus infections. Here we show that during infection with wild‐type adenovirus, ATR and its cofactors RPA32, ATRIP and TopBP1 accumulate at viral replication centres, but there is minimal ATR activation. We show that the Mre11/Rad50/Nbs1 (MRN) complex is recruited to viral centres only during infection with adenoviruses lacking the early region E4 and ATR signaling is activated. This suggests a novel requirement for the MRN complex in ATR activation during virus infection, which is independent of Mre11 nuclease activity and recruitment of RPA/ATR/ATRIP/TopBP1. Unlike other damage scenarios, we found that ATM and ATR signaling are not dependent on each other during infection. We identify a region of the viral E4orf3 protein responsible for immobilization of the MRN complex and show that this prevents ATR signaling during adenovirus infection. We propose that immobilization of the MRN damage sensor by E4orf3 protein prevents recognition of viral genomes and blocks detrimental aspects of checkpoint signaling during virus infection.