Arsenic-induced Mre11 phosphorylation is cell cycle-dependent and defective in NBS cells

Cancer-prone diseases ataxia-telangiectasia (AT), Nijmegen breakage syndrome (NBS) and ataxia-telangiectasia-like disorder (ATLD) are defective in the repair of DNA double-stranded break (DSB). On the other hand, arsenic (As) has been reported to cause DSB and to be involved in the occurrence of skin, lung and bladder cancers. To dissect the repair mechanism of As-induced DSB, wild type, AT and NBS cells were treated with sodium arsenite to study the complex formation and post-translational modification of Rad50/NBS1/Mre11 repair proteins. Our results showed that Mre11 went through cell cycle-dependent phosphorylation upon sodium arsenite treatment and this post-translational modification required NBS1 but not ATM. Defective As-induced Mre11 phosphorylation was rescued by reconstitution with full length NBS1 in NBS cells. Although As-induced Mre11 phosphorylation was not required for Rad50/NBS1/Mre11 complex formation, it might be required for the formation of Rad50/NBS1/Mre11 nuclear foci upon DNA damage.     

The Mre11/Rad50/Nbs1 complex limits adeno-associated virus transduction and replication

Adeno-associated virus (AAV) is a parvovirus with a small single-stranded DNA genome that relies on cellular replication machinery together with functions supplied by coinfecting helper viruses. The impact of host factors on AAV infection is not well understood. We explored the connection between AAV helper functions supplied by adenovirus and cellular DNA repair proteins. The adenoviral E1b55K/E4orf6 proteins induce degradation of the cellular Mre11 repair complex (MRN) to promote productive adenovirus infection. These viral proteins also augment recombinant AAV transduction and provide crucial helper functions for wild-type AAV replication. Here, we show that MRN poses a barrier to AAV and that the helper function provided by E1b55K/E4orf6 involves MRN degradation. Using a fluorescent method to visualize the viral genome, we show an effect at the viral DNA level. MRN components accumulate at AAV replication centers and recognize the viral inverted terminal repeats. Together, our data suggest that AAV is targeted by MRN and has evolved to exploit adenoviral proteins that degrade these cellular factors.     

Effects of adeno-associated virus DNA hairpin structure on recombination

Hairpin DNA ends are evolutionarily conserved intermediates in DNA recombination. The hairpin structures present on the ends of the adeno-associated virus (AAV) genome are substrates for recombination that give rise to persistent circular and concatemeric DNA episomes through intramolecular and intermolecular recombination, respectively. We have developed circularization-dependent and orientation-specific self-complementary AAV (scAAV) vectors as a reporter system to examine recombination events involving distinct hairpin structures, i.e., closed versus open hairpins. The results suggest that intramolecular recombination (circularization) is far more efficient than intermolecular recombination (concatemerization). Among all possible combinations of terminal repeats (TRs) involved in intermolecular recombination, the closed-closed TR structures are twice as efficient as the open-open TR substrates for recombination. In addition, both intramolecular recombination and intermolecular recombination exhibit the common dependency on specific DNA polymerases and topoisomerases. The circularization-dependent and orientation-specific scAAV vectors can serve as an efficient and controlled system for the delivery of DNA structures that mimic mammalian recombination intermediates and should be useful in assaying recombination in different experimental settings as well as elucidating the molecular mechanism of recombinant AAV genome persistence.     

Intracellular viral processing, not single-stranded DNA accumulation, is crucial for recombinant adeno-associated virus transduction

Adeno-associated virus (AAV) is a unique gene transfer vector which takes approximately 4 to 6 weeks to reach its expression plateau. The mechanism for this slow-rise expression profile was proposed to be inefficient second-strand DNA synthesis from the input single-stranded (ss) DNA viral genome. In order to clarify the status of ss AAV genomes, we generated AAV vectors labeled with bromodeoxyuridine (BrdU), a nucleotide analog that can be incorporated into the AAV genome and packaged into infectious virions. Since BrdU-DNA can be detected only by an anti-BrdU antibody when DNA is in an ss form, not in a double-stranded (ds) form, ss AAV genomes with BrdU can be readily tracked in situ. Although ss AAV DNA was abundant by Southern blot analysis, free ss AAV genomes were not detectable after AAV transduction by this new detection method. Further Southern blot analysis of viral DNA and virions revealed that ss AAV DNA was protected within virions. Extracted cellular fractions demonstrated that viral particles in host cells remained infectious. In addition, a significant amount of AAV genomes was degraded after AAV transduction. Therefore, we conclude that the amount of free ss DNA is not abundant during AAV transduction. AAV transduction is limited by the steps that affect AAV ss DNA release (i.e., uncoating) before second-strand DNA synthesis can occur. AAV ss DNA released from viral uncoating is either converted into ds DNA efficiently or degraded by cellular DNA repair mechanisms as damaged DNA. This study elucidates a mechanism that can be exploited to develop new strategies to improve AAV vector transduction efficiency.     

The Mre11 complex is required for ATM activation and the G2/M checkpoint

The maintenance of genome integrity requires a rapid and specific response to many types of DNA damage. The conserved and related PI3-like protein kinases, ataxia-telangiectasia mutated (ATM) and ATM-Rad3-related (ATR), orchestrate signal transduction pathways in response to genomic insults, such as DNA double-strand breaks (DSBs). It is unclear which proteins recognize DSBs and activate these pathways, but the Mre11/Rad50/NBS1 complex has been suggested to act as a damage sensor. Here we show that infection with an adenovirus lacking the E4 region also induces a cellular DNA damage response, with activation of ATM and ATR. Wild-type virus blocks this signaling through degradation of the Mre11 complex by the viral E1b55K/E4orf6 proteins. Using these viral proteins, we show that the Mre11 complex is required for both ATM activation and the ATM-dependent G(2)/M checkpoint in response to DSBs. These results demonstrate that the Mre11 complex can function as a damage sensor upstream of ATM/ATR signaling in mammalian cells.     

Virus Infection Induces NF-kappaB-dependent interchromosomal associations mediating monoallelic IFN-beta gene expression

The Mre11/Rad50/NBS1 (MRN) complex maintains genomic stability by bridging DNA ends and initiating DNA damage signaling through activation of the ATM kinase. Mre11 possesses DNA nuclease activities that are highly conserved in evolution but play unknown roles in mammals. To define the functions of Mre11, we engineered targeted mouse alleles that either abrogate nuclease activities or inactivate the entire MRN complex. Mre11 nuclease deficiency causes a striking array of phenotypes indistinguishable from the absence of MRN, including early embryonic lethality and dramatic genomic instability. We identify a crucial role for the nuclease activities in homology-directed double-strand-break repair and a contributing role in activating the ATR kinase. However, the nuclease activities are not required to activate ATM after DNA damage or telomere deprotection. Therefore, nucleolytic processing by Mre11 is an essential function of fundamental importance in DNA repair, distinct from MRN control of ATM signaling.     

Human cytomegalovirus disrupts both ataxia telangiectasia mutated protein (ATM)- and ATM-Rad3-related kinase-mediated DNA damage responses during lytic infection

Many viruses (herpes simplex virus type 1, polyomavirus, and human immunodeficiency virus type 1) require the activation of ataxia telangiectasia mutated protein (ATM) and/or Mre11 for a fully permissive infection. However, the longer life cycle of human cytomegalovirus (HCMV) may require more specific interactions with the DNA repair machinery to maximize viral replication. A prototypical damage response to the double-stranded ends of the incoming linear viral DNA was not observed in fibroblasts at early times postinfection (p.i.). Apparently, a constant low level of phosphorylated ATM was enough to phosphorylate its downstream targets, p53 and Nbs1. p53 was the only cellular protein observed to relocate at early times, forming foci in infected cell nuclei between 3.5 and 5.5 h p.i. Approximately half of these foci localized with input viral DNA, and all localized with viral UL112/113 prereplication site foci. No other DNA repair proteins localized with the virus or prereplication foci in the first 24 h p.i. When viral replication began in earnest, between 24 and 48 h p.i., there were large increases in steady-state levels and phosphorylation of many proteins involved in the damage response, presumably triggered by ATM-Rad3-related kinase activation. However, a sieving process occurred in which only certain proteins were specifically sequestered into viral replication centers and others were particularly excluded. In contrast to other viruses, activation of a damage response is neither necessary nor detrimental to infection, as neither ATM nor Mre11 was required for full virus replication and production. Thus, by preventing simultaneous relocalization of all the necessary repair components to the replication centers, HCMV subverts full activation and completion of both double-stranded break and S-phase checkpoints that should arrest all replication within the cell and likely lead to apoptosis.     

Targeted disruption of NBS1 reveals its roles in mouse development and DNA repair

Nijmegen breakage syndrome (NBS) is an autosomal recessive hereditary disease that shares some common defects with ataxia-telangiectasia. The gene product mutated in NBS, named NBS1, is a component of the Mre11 complex that is involved in DNA strand-break repair. To elucidate the physiological roles of NBS1, we disrupted the N-terminal exons of the NBS1 gene in mice. NBS1(m/m) mice are viable, growth retarded and hypersensitive to ionizing radiation (IR). NBS1(m/m) mice exhibit multiple lymphoid developmental defects, and rapidly develop thymic lymphoma. In addition, female NBS1(m/m) mice are sterile due to oogenesis failure. NBS1(m/m) cells are impaired in cellular responses to IR and defective in cellular proliferation. Most systematic and cellular defects identified in NBS1(m/m) mice recapitulate those in NBS patients, and are essentially identical to those observed in Atm(-/-) mice. In contrast to Atm(-/-) mice, spermatogenesis is normal in NBS1(m/m) mice, indicating that distinct roles of ATM have differential requirement for NBS1 activity. Thus, NBS1 and ATM have overlapping and distinct functions in animal development and DNA repair.     

A murine model of Nijmegen breakage syndrome

Nijmegen breakage syndrome (NBS) is a rare autosomal recessive disorder characterized by microcephaly, immunodeficiency, and predisposition to hematopoietic malignancy. The clinical and cellular phenotypes of NBS substantially overlap those of ataxia-telangiectasia (A-T). NBS is caused by mutation of the NBS1 gene, which encodes a member of the Mre11 complex, a trimeric protein complex also containing Mre11 and Rad50. Several lines of evidence indicate that the ataxia-telangiectasia mutated (ATM) kinase and the Mre11 complex functionally interact. Both NBS and A-T cells exhibit ionizing radiation (IR) sensitivity and defects in the intra S phase checkpoint, resulting in radioresistant DNA synthesis (RDS)-the failure to suppress DNA replication origin firing after IR exposure. NBS1 is phosphorylated by ATM in response to IR, and this event is required for activation of the intra S phase checkpoint (the RDS checkpoint). We derived a murine model of NBS, the Nbs1(DeltaB/DeltaB) mouse. Nbs1(DeltaB/DeltaB) cells are phenotypically identical to those established from NBS patients. The Nbs1(DeltaB) allele was synthetically lethal with ATM deficiency. We propose that the ATM-Mre11 complex DNA damage response pathway is essential and that ATM or the Mre11 complex serves as a nexus to additional components of the pathway.     

Aspergillus nidulans uvsBATR and scaANBS1 genes show genetic interactions during recovery from replication stress and DNA damage

The ATM/ATR kinases and the Mre11 (Mre11-Rad50-Nbs1) protein complex are central players in the cellular DNA damage response. Here we characterize possible interactions between Aspergillus nidulans uvsB(ATR) and the Mre11 complex (scaA(NBS1)). We demonstrate that there is an epistatic relationship between uvsB(ATR), the homolog of the ATR/MEC1 gene, and scaA(NBS1), the homolog of the NBS1/XRS2 gene, for both repair and checkpoint functions and that correct ScaA(NBS1) expression during recovery from replication stress depends on uvsB(ATR). In addition, we also show that the formation of UvsC foci during recovery from replication stress is dependent on both uvsB(ATR) and scaA(NBS1) function. Furthermore, ScaA(NBS1) is also dependent on uvsB(ATR) for nuclear focus formation upon the induction of DNA double-strand breaks by phleomycin. Our results highlight the extensive genetic interactions between UvsB and the Mre11 complex that are required for S-phase progression and recovery from DNA damage.     

MRE11/RAD50/NBS1: complex activities

The MRE11/RAD50/NBS1 complex (Mre11 complex) is a central player in most aspects of the cellular response to DNA double-strand breaks, including homologous recombination, non-homologous end joining, telomere maintenance and DNA damage checkpoint activation. Several of these findings are explained by the unusual enzymatic activities and macromolecular structure of the Mre11 complex. The Mre11 complex possesses an ATP-stimulated nuclease to process heterogeneous DNA ends and long coiled-coil tails to link DNA ends and/or sister chromatids. However, the mechanistic role of the Mre11 complex in checkpoint activation has been unclear until recently. New data suggest that the Mre11 complex can directly activate the ATM checkpoint kinase at DNA breaks. These findings, together with newly determined functional interactions, identify the Mre11 complex as an architectural and mechanistic keystone of cellular response events emerging from DNA breaks.     

Skp2 E3 ligase integrates ATM activation and homologous recombination repair by ubiquitinating NBS1

The Mre11/Rad50/NBS1 (MRN) complex is thought to be a critical sensor that detects damaged DNA and recruits ATM to DNA foci for activation. However, it remains to be established how the MRN complex regulates ATM recruitment to the DNA foci during DNA double-strand breaks (DSBs). Here we show that Skp2 E3 ligase is a key component for the MRN complex-mediated ATM activation in response to DSBs. Skp2 interacts with NBS1 and triggers K63-linked ubiquitination of NBS1 upon DSBs, which is critical for the interaction of NBS1 with ATM, thereby facilitating ATM recruitment to the DNA foci for activation. Finally, we show that Skp2 deficiency exhibits a defect in homologous recombination (HR) repair, thereby increasing IR sensitivity. Our results provide molecular insights into how Skp2 and the MRN complex coordinate to activate ATM, and identify Skp2-mediatetd NBS1 ubiquitination as a vital event for ATM activation in response to DNA damage.     

The Mre11 complex and ATM: collaborating to navigate S phase

Recently, findings regarding a group of cancer predisposition and chromosome instability syndromes, Nijmegen breakage syndrome (NBS), the ataxia-telangiectasia-like disorder (A-TLD) and ataxia telangiectasia have shed light on the unexpected role of recombinational DNA repair proteins in DNA-damage-dependent cell-cycle regulation. Mutations in the Mre11 complex cause A-TLD and NBS. In addition, functions of the Mre11 complex have been biochemically linked to ATM, the large protein kinase that is defective in ataxia-telangiectasia cells by the observation that Nbs1 is a bona fide substrate of the ATM kinase.     

Widespread Phosphorylation of Histone H2AX by Species C Adenovirus Infection Requires Viral DNA Replication

Adenovirus infection activates cellular DNA damage response and repair pathways. Viral proteins that are synthesized before viral DNA replication prevent recognition of viral genomes as a substrate for DNA repair by targeting members of the sensor complex composed of Mre11/Rad50/NBS1 for degradation and relocalization, as well as targeting the effector protein DNA ligase IV. Despite inactivation of these cellular sensor and effector proteins, infection results in high levels of histone 2AX phosphorylation, or γH2AX. Although phosphorylated H2AX is a characteristic marker of double-stranded DNA breaks, this modification was widely distributed throughout the nucleus of infected cells and was coincident with the bulk of cellular DNA. H2AX phosphorylation occurred after the onset of viral DNA replication and after the degradation of Mre11. Experiments with inhibitors of the serine-threonine kinases ataxia telangiectasia mutated (ATM), AT- and Rad3-related (ATR), and DNA protein kinase (DNA-PK), the kinases responsible for H2AX phosphorylation, indicate that H2AX may be phosphorylated by ATR during a wild-type adenovirus infection, with some contribution from ATM and DNA-PK. Viral DNA replication appears to be the stimulus for this phosphorylation event, since infection with a nonreplicating virus did not elicit phosphorylation of H2AX. Infected cells also responded to high levels of input viral DNA by localized phosphorylation of H2AX. These results are consistent with a model in which adenovirus-infected cells sense and respond to both incoming viral DNA and viral DNA replication.Cellular DNA damage response pathways protect and preserve the integrity of the genome. These pathways, which are activated in response to various forms of DNA damage, involve a number of proteins that participate in both DNA repair and cell cycle progression (62). The serine-threonine kinases ataxia telangiectasia mutated (ATM), AT- and Rad3-related (ATR), and DNA protein kinase (DNA-PK) are activated in response to distinct types of damage. The ATM pathway is activated primarily by double-stranded DNA breaks (4, 30). DNA-PK acts in conjunction with the DNA ligase IV/XRCC4 complex to mediate the ligation of double-stranded breaks through nonhomologous end joining (34). The ATR pathway can be activated in response to a wide range of genotoxic stresses, such as base or nucleotide excision, double-stranded breaks, or single-stranded breaks. Activation of ATR is generally thought to occur via the recognition of single-stranded tracks of DNA (63). Each of these pathways leads to the phosphorylation and activation of a number of cellular proteins such as the variant histone H2AX, checkpoint kinases 1 and 2 (Chk1 and Chk2), and Nijmegen break syndrome protein 1 (NBS1), among others (62). Signals transmitted by a cascade of phosphorylation events result in cell cycle arrest and the accumulation of repair protein complexes at sites of DNA damage.Upon recognition of a double-stranded DNA break by the cell, H2AX is phosphorylated on an extended C-terminal tail at serine 139 by the phosphatidylinositol 3-kinase (PI3K)-related kinases ATM, ATR, and DNA-PK (9, 41, 44, 58). Considered one of the earliest indications of a double-stranded DNA break, phosphorylated H2AX (γH2AX) acts as a scaffolding protein to which a number of DNA repair factors can dock to facilitate repair of the damaged DNA (36, 42, 53). Areas of phosphorylated H2AX, termed γH2AX foci, are enriched for proteins involved in both homologous recombination and nonhomologous end joining, such as NBS1, BRCA1 (42), and Mdc1 (24, 50).Although adenovirus is able to activate both ATM and ATR pathways (11), adenoviral proteins limit the extent and consequences of signaling through these pathways. The E1B-55K and E4orf6 proteins form an E3 ubiquitin ligase with the cellular proteins Cullin-5, elongins B and C, and Rbx1 (28, 43). This complex targets key cellular proteins involved in cellular response to DNA damage, including p53 (28, 43), Mre11 (51), and DNA ligase IV (3). The E4orf3 gene product targets cellular proteins central to both the cellular DNA damage response and the antiviral response. The E4orf3 protein of species C adenoviruses alters the localization of Mre11/Rad50/NBS1 (MRN) complex members within the nucleus to prevent association with centers of viral DNA replication and to ensure efficient viral DNA replication (17, 18, 52). In addition, these three viral early proteins direct members of the MRN complex (2, 35) and the single-stranded DNA-binding protein 2 (20) to cytoplasmic aggresomes, where these sequestered proteins are effectively inactivated. These viral activities, along with the inactivation of DNA-PK by E4orf3 and E4orf6 gene products (7), appear to prevent recognition of viral genomes by the MRN complex and prevent ligation of these genomes through nonhomologous end joining. In cells infected with a virus with E4 deleted, Mre11 physically binds to viral DNA in an NBS1-dependent manner and may prevent efficient genome replication (37). The overlapping means by which adenovirus disables the MRN complex and prevents DNA damage repair serves to illustrate the importance of this activity for a productive adenovirus infection. However, despite having DNA damage signaling and DNA repair pathways dismantled, adenovirus-infected cells exhibit some characteristic changes associated with DNA damage signaling events, such as the phosphorylation of H2AX (6, 15). Thus, it appears that adenovirus effectively inhibits DNA repair activity but may not fully suppress the early events of DNA damage signaling.The focus of the present study was to elucidate the activation of DNA damage signaling pathways revealed by phosphorylation of the variant histone H2AX during wild-type adenovirus infection and to determine what stage of the virus life cycle leads to this activation. We demonstrate that infected cells respond to viral genome replication with high levels of H2AX phosphorylation throughout the cell nucleus. This phosphorylation event is not localized to viral replication centers and does not appear to be concurrent with cellular double-stranded DNA breaks; rather, H2AX phosphorylation occurs coincident with the bulk of cellular chromatin. H2AX phosphorylation follows viral DNA replication and reaches peak levels after the degradation of the Mre11. In addition, we observed that infected cells can respond to both the presence of incoming viral genomes and genome replication by initiating H2AX phosphorylation.     

In vitro excision of adeno-associated virus DNA from recombinant plasmids: isolation of an enzyme fraction from HeLa cells that cleaves DNA at poly(G) sequences.

When circular recombinant plasmids containing adeno-associated virus (AAV) DNA sequences are transfected into human cells, the AAV provirus is rescued. Using these circular AAV plasmids as substrates, we isolated an enzyme fraction from HeLa cell nuclear extracts that excises intact AAV DNA in vitro from vector DNA and produces linear DNA products. The recognition signal for the enzyme is a polypurine-polypyrimidine sequence which is at least 9 residues long and rich in G.C base pairs. Such sequences are present in AAV recombinant plasmids as part of the first 15 base pairs of the AAV terminal repeat and in some cases as the result of cloning the AAV genome by G.C tailing. The isolated enzyme fraction does not have significant endonucleolytic activity on single-stranded or double-stranded DNA. Plasmid DNA that is transfected into tissue culture cells is cleaved in vivo to produce a pattern of DNA fragments similar to that seen with purified enzyme in vitro. The activity has been called endo R for rescue, and its behavior suggests that it may have a role in recombination of cellular chromosomes.     

A forward chemical genetic screen reveals an inhibitor of the Mre11-Rad50-Nbs1 complex

The MRN (Mre11-Rad50-Nbs1)-ATM (ataxia-telangiectasia mutated) pathway is essential for sensing and signaling from DNA double-strand breaks. The MRN complex acts as a DNA damage sensor, maintains genome stability during DNA replication, promotes homology-dependent DNA repair and activates ATM. MRN is essential for cell viability, which has limited functional studies of the complex. Small-molecule inhibitors of MRN could circumvent this experimental limitation and could also be used as cellular radio- and chemosensitization compounds. Using cell-free systems that recapitulate faithfully the MRN-ATM signaling pathway, we designed a forward chemical genetic screen to identify inhibitors of the pathway, and we isolated 6-(4-hydroxyphenyl)-2-thioxo-2,3-dihydro-4(1H)-pyrimidinone (mirin, 1) as an inhibitor of MRN. Mirin prevents MRN-dependent activation of ATM without affecting ATM protein kinase activity, and it inhibits Mre11-associated exonuclease activity. Consistent with its ability to target the MRN complex, mirin abolishes the G2/M checkpoint and homology-dependent repair in mammalian cells.     

Ataxia-telangiectasia-like disorder (ATLD)-its clinical presentation and molecular basis

Comparison of the clinical and cellular phenotypes of different genomic instability syndromes provides new insights into functional links in the complex network of the DNA damage response. A prominent example of this principle is provided by examination of three such disorders: ataxia-telangiectasia (A-T) caused by lack or inactivation of the ATM protein kinase, which mobilises the cellular response to double strand breaks in the DNA; ataxia-telangiectasia-like disease (ATLD), a result of deficiency of the human Mre11 protein; and the Nijmegen breakage syndrome (NBS), which represents defective Nbs1 protein. Mre11 and Nbs1 are members of the Mre11/Rad50/Nbs1 (MRN) protein complex. MRN and its individual components are involved in different responses to cellular damage induced by ionising radiation and radiomimetic chemicals, including complexing with chromatin and with other damage response proteins, formation of radiation-induced foci, and the induction of different cell cycle checkpoints. The phosphorylation of Nbs1 by ATM would indicate that ATM acts upstream of the MRN complex. Consistent with this were the suggestions that ATM could be activated in the absence of fully functional Nbs1 protein. In contrast, the regulation of some ATM target proteins, e.g. Smc1 requires the MRN complex as well as ATM. Nbs1 may, therefore, be both a substrate for ATM and a mediator of ATM function. Recent studies that indicate a requirement of the MRN complex for proper ATM activation suggest that the relationship between ATM and the MRN complex in the DNA damage response is yet to be fully determined. Despite the fact that both Mre11 and Nbs1 are part of the same MRN complex, deficiency in either protein in humans does not lead to the same clinical picture. This suggests that components of the complex may also act separately.     

PIKK-dependent phosphorylation of Mre11 induces MRN complex inactivation by disassembly from chromatin

The role of Mre11 phosphorylation in the cellular response to DNA double-strand breaks (DSBs) is not well understood. Here, we show that phosphorylation of Mre11 at SQ/TQ motifs by PIKKs (PI3 Kinase-related Kinases) induces MRN (Mre11–Rad50–Nbs1) complex dissociation from chromatin by reducing Mre11 affinity for DNA. Whereas phosphorylation of Mre11 at these residues is not required for DSB-induced ATM (Ataxia-Telangiectasia mutated) activation, abrogation of Mre11 dephosphorylation impairs ATM signaling. Our study provides a functional characterization of the DNA damage-induced Mre11 phosphorylation, and suggests that MRN inactivation participates in the down-regulation of damage signaling during checkpoint recovery following DSB repair.