Mre11 Nuclease Activity and Ctp1 Regulate Chk1 Activation by Rad3ATR and Tel1ATM Checkpoint Kinases at Double-Strand Breaks

Rad3, the Schizosaccharomyces pombe ortholog of human ATR and Saccharomyces cerevisiae Mec1, activates the checkpoint kinase Chk1 in response to DNA double-strand breaks (DSBs). Rad3^ATR/Mec1 associates with replication protein A (RPA), which binds single-stranded DNA overhangs formed by DSB resection. In humans and both yeasts, DSBs are initially detected and processed by the Mre11-Rad50-Nbs1^Xrs2 (MRN) nucleolytic protein complex in association with the Tel1^ATM checkpoint kinase and the Ctp1^CtIP/Sae2 DNA-end processing factor; however, in budding yeast, neither Mre11 nuclease activity or Sae2 are required for Mec1 signaling at irreparable DSBs. Here, we investigate the relationship between DNA end processing and the DSB checkpoint response in fission yeast, and we report that Mre11 nuclease activity and Ctp1 are critical for efficient Rad3-to-Chk1 signaling. Moreover, deleting Ctp1 reveals a Tel1-to-Chk1 signaling pathway that bypasses Rad3. This pathway requires Mre11 nuclease activity, the Rad9-Hus1-Rad1 (9-1-1) checkpoint clamp complex, and Crb2 checkpoint mediator. Ctp1 negatively regulates this pathway by controlling MRN residency at DSBs. A Tel1-to-Chk1 checkpoint pathway acting at unresected DSBs provides a mechanism for coupling Chk1 activation to the initial detection of DSBs and suggests that ATM may activate Chk1 by both direct and indirect mechanisms in mammalian cells.DNA double-strand breaks (DSBs), formed by clastogens or from endogenous damage, trigger multiple cellular responses that are critical for maintaining genome integrity. Of particular importance is the cell cycle checkpoint that restrains the onset of mitosis while DSB repair is under way. Chk1 is the critical effector of this checkpoint in the fission yeast Schizosaccharomyces pombe and mammalian cells, whereas the budding yeast Saccharomyces cerevisiae uses both Chk1 and Rad53 (orthologous to human Chk2 and fission yeast Cds1) to delay anaphase entry and mitotic exit. These kinases are regulated by ATM (ataxia-telangiectasia mutated) and ATR (ATM and Rad3-related) checkpoint kinases (5). Curiously, the regulatory connections between ATM/ATR and Chk1/Chk2 orthologs are not strictly conserved between species (Fig. ​(Fig.1A).1A). In mammals, ATM activates Chk2 while ATR activates Chk1. In S. cerevisiae and S. pombe, ATR orthologs (Mec1 and Rad3, respectively) activate Chk2 orthologs and Chk1, while Tel1 (ATM ortholog) is primarily involved in telomere maintenance (14, 38, 40).Open in a separate windowFIG. 1.Deletion of Ctp1 restores the DNA damage checkpoint in rad3Δ cells. (A) Regulatory connections between ATM/ATR and Chk1/Chk2 orthologs in mammals, S. cerevisiae, and S. pombe. ATM phosphorylates Chk2 and ATR phosphorylates Chk1. CtIP mediates an ATM-to-ATR switch through DNA end resection in mammals (44, 53). ATM promotes Chk1 activation by stimulating CtIP-dependent resection through an unknown mechanism. In S. cerevisiae, Mec1 phosphorylates both Rad53 and Chk1. Deleting Sae2 uncovers a Tel1-to-Rad53 signaling pathway and enhances Rad53 activation (47). In S. pombe, Cds1 and Chk1 activation is dependent on Rad3. (B) Chk1 phosphorylation peaks in wild-type (wt) (top panel) and ctp1Δ cells (bottom panel) 30 min after exposure to 90 Gy of IR in log-phase cultures. Chk1 phosphorylation in ctp1Δ cells prior to IR exposure likely arises from an inability to repair spontaneous DNA damage (23). Immunoblots were probed for the HA epitope-tagged Chk1 or Cdc2 as a loading control. (C) Chk1 phosphorylation is reduced at least 2-fold in ctp1Δ cells relative to the wild type. Quantification of blots from panel B expressed as a ratio of phospho-Chk1 (upper band) versus nonphospho-Chk1 (lower band) was performed. The phospho-Chk1 signal in untreated ctp1Δ cells was subtracted from the IR-treated samples to more accurately measure the IR-induced phosphorylation. (D) The ctp1Δ mutation restores Chk1 phosphorylation in rad3Δ cells. Cells were harvested immediately after mock or 90-Gy IR treatment and blotted for HA epitope tag. Ponceau staining shows equal loading. (E) Quantitation of Chk1 phosphorylation. Error bars represent the standard errors from three independent experiments. (F) The checkpoint arrest is restored in ctp1Δ rad3Δ cells. Cells synchronized in G₂ by elutriation were mock treated or exposed to 100 Gy of IR. Cell cycle progression was tracked by microscopic observation.The functions of ATM and ATR orthologs are intimately tied to the detection and nucleolytic processing of DSBs. ATM^Tel1 localizes at DSBs by interacting with Mre11-Rad50-Nbs1^Xrs2 (MRN) protein complex, which directly binds DNA ends (12, 20, 24, 50, 52). The MRN complex is essential for ATM^Tel1 function in all species. The Mre11 subunit of MRN complex has DNase activities that are critical for radioresistance in S. pombe and mice but not in budding yeast (3, 19, 22, 50). In fission yeast, MRN complex also recruits Ctp1 DNA end-processing factor to DSBs (25, 49). Ctp1 is structurally and functionally related to CtIP in mammals and Sae2 in budding yeast, the latter of which has nuclease activity in vitro (21, 23, 43). Ctp1 and CtIP are essential for survival of ionizing radiation and other clastogens (23, 43, 54), whereas sae2Δ mutants are not radiosensitive except at very high doses of ionizing radiation (IR), although both Ctp1 and Sae2 are required for repair of meiotic DSBs formed by a Spo11/Rec12-dependent mechanism (17, 23, 36). Genetic and biochemical studies indicate that Sae2/Ctp1/CtIP collaborate with MRN complex to initiate the 5′-to-3′ resection of DSBs (7, 23, 28, 43, 53, 55), which leads to the generation of 3′ single-strand overhangs (SSOs) that are critical for DSB repair by homologous recombination (HR). Replication protein A (RPA) binding to SSOs is essential for HR repair of DSBs, but it is also important for recruiting ATR^Rad3/Mec1, which interacts with RPA through its regulatory subunit ATRIP (Rad26 in fission yeast, Ddc2 in budding yeast) (5, 56). Subsequent phosphorylation of Chk1 by ATR also requires the Rad9-Hus1-Rad1 (9-1-1) checkpoint clamp, which is loaded at the single-strand/double-strand DNA junctions (26, 48, 57), the ATR activating protein TopBP1 (Cut5 in fission yeast), and a checkpoint mediator protein such as Crb2 in fission yeast (34, 41, 48).In this mechanism of DNA damage checkpoint signaling, DNA end resection is critical for ATR (Rad3/Mec1) activation, and therefore resection defective mutants should be unable to mount a fully active checkpoint response (44). However, Rad53 activation is not diminished in budding yeast sae2Δ mutants that suffer an irreparable DSB by expressing HO endonuclease. In fact, there is a defect in turning off the checkpoint signal (6). A similar effect is observed in S. cerevisiae strains expressing the mre11-H125N nuclease-defective form of Mre11. Moreover, overexpression of SAE2 strongly inhibits Rad53 activation (6). The reasons for these phenotypes are unknown, since neither Sae2 nor Mre11 nuclease activity are required for DSB resection or radioresistance. However, deleting Sae2 delays resection while at the same time enhancing a cryptic Tel1-to-Rad53 checkpoint pathway (6, 47). These effects correlate with delayed disassembly of Mre11 foci at DSBs in sae2Δ cells, suggesting that Sae2 may negatively regulate checkpoint signaling by modulating Mre11 association at damaged DNA (1, 6, 24). Enhancement of a Tel1-to-Rad53 checkpoint pathway by eliminating Sae2 suggests that the signaling pathways between ATM/ATR and Chk1/Chk2 checkpoint kinases are not hard wired but are adaptable to changes in DNA end processing (47). However, as yet there is no evidence that ATM^Tel1 can activate Chk1 in any organism.Since SAE2 deletion or overexpression has unexpected effects on Rad53 activation in budding yeast, we decided to explore the relationship between Ctp1 and Chk1 activation in fission yeast. Here, we show that Chk1 activation is substantially diminished in ctp1Δ cells exposed to ionizing radiation. These data are consistent with studies showing that CtIP is required for efficient Chk1 activation in mammalian cells treated with camptothecin (CPT), a topoisomerase I poison that causes replication fork collapse (43, 53). We also investigate the role of Mre11 nuclease activity and find that while ablating Mre11 nuclease activity enhances Rad53 activation in budding yeast, the equivalent Mre11 mutation in fission yeast severely impairs Chk1 activation by ionizing radiation. Furthermore, we find that deleting Ctp1 reveals a previously unknown Tel1-to-Chk1 signaling pathway in S. pombe, a finding analogous to the enhancement of a Tel1-to-Rad53 checkpoint pathway by eliminating Sae2 in S. cerevisiae (47). This Tel1-to-Chk1 pathway also requires Mre11 nuclease activity. These data establish that Tel1^ATM can activate Chk1 independently of Rad3^ATR, which has implications for studies linking ATM to Chk1 activation in mammalian cells (16, 31). Characterization of this pathway allows us to propose a more detailed model of how Chk1 is activated in response to DSBs.     

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.     

Adeno-Associated Virus Replication Induces a DNA Damage Response Coordinated by DNA-Dependent Protein Kinase

The parvovirus adeno-associated virus (AAV) contains a small single-stranded DNA genome with inverted terminal repeats that form hairpin structures. In order to propagate, AAV relies on the cellular replication machinery together with functions supplied by coinfecting helper viruses such as adenovirus (Ad). Here, we examined the host cell response to AAV replication in the context of Ad or Ad helper proteins. We show that AAV and Ad coinfection activates a DNA damage response (DDR) that is distinct from that seen during Ad or AAV infection alone. The DDR was also triggered when AAV replicated in the presence of minimal Ad helper proteins. We detected autophosphorylation of the kinases ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and signaling to downstream targets SMC1, Chk1, Chk2, H2AX, and XRCC4 and multiple sites on RPA32. The Mre11 complex was not required for activation of the DDR to AAV infection. Additionally, we found that DNA-PKcs was the primary mediator of damage signaling in response to AAV replication. Immunofluorescence revealed that some activated damage proteins were found in a pan-nuclear pattern (phosphorylated ATM, SMC1, and H2AX), while others such as DNA-PK components (DNA-PKcs, Ku70, and Ku86) and RPA32 accumulated at AAV replication centers. Although expression of the large viral Rep proteins contributed to some damage signaling, we observed that the full response required replication of the AAV genome. Our results demonstrate that AAV replication in the presence of Ad helper functions elicits a unique damage response controlled by DNA-PK.Replication of viral genomes produces a large amount of extrachromosomal DNA that may be recognized by the cellular DNA damage machinery. This is often accompanied by activation of DNA damage response (DDR) signaling pathways and recruitment of cellular repair proteins to sites of viral replication. Viruses therefore provide good model systems to study the recognition and response to DNA damage (reviewed in reference 48). The Mre11/Rad50/Nbs1 (MRN) complex functions as a sensor of chromosomal DNA double-strand breaks (DSBs) and is involved in activation of damage signaling (reviewed in reference 41). The MRN complex also localizes to DNA DSBs and is found at viral replication compartments during infection with a number of DNA viruses (6, 40, 47, 70, 75, 77, 87, 93). The phosphatidylinositol 3-kinase-like kinases (PIKKs) ataxia telangiectasia mutated (ATM), ATM and Rad3-related kinase (ATR), and the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) are involved in the signal transduction cascades activated by DNA damage (reviewed in references 43, 51, and 71). These kinases respond to distinct types of damage and regulate DSB repair during different phases of the cell cycle (5), either through nonhomologous end-joining (NHEJ) or homologous recombination pathways (reviewed in references 63, 81, and 86). The DNA-PK holoenzyme is composed of DNA-PKcs and two regulatory subunits, the Ku70 and Ku86 heterodimer. DNA-PK functions with XRCC4/DNA ligase IV to repair breaks during NHEJ, and works with Artemis to process DNA hairpin structures during VDJ recombination and during a subset of DNA DSB events (46, 50, 86). While the kinase activity of DNA-PKcs leads to phosphorylation of a large number of substrates in vitro as well as autophosphorylation of specific residues (reviewed in references 16 and 85), it is currently unclear how DNA-PKcs contributes to signaling in cells upon different types of damage.The adeno-associated virus (AAV) genome consists of a molecule of single-stranded DNA with inverted terminal repeats (ITRs) at both ends that form double-hairpin structures due to their palindromic sequences (reviewed in reference 52). The ITRs are important for replication and packaging of the viral genome and for integration into the host genome. Four viral Rep proteins (Rep78, Rep68, Rep52, and Rep40) are also required for replication and packaging of the AAV genome into virions assembled from the Cap proteins. Although the Rep and Cap genes are replaced in recombinant AAV vectors (rAAV) that retain only the ITRs flanking the gene of interest, these vectors can be replicated by providing Rep in trans (reviewed in reference 7). Productive AAV infection requires helper functions supplied by adenovirus (Ad) or other viruses such as herpes simplex virus (HSV) (reviewed in reference 27), together with components of the host cell DNA replication machinery (54, 55, 58). In the presence of helper viruses or minimal helper proteins from Ad or HSV, AAV replicates in the nucleus at centers where the viral DNA and Rep proteins accumulate (35, 76, 84, 89). Cellular and viral proteins involved in AAV replication, including replication protein A (RPA), Ad DNA-binding protein (DBP), and HSV ICP8, localize with Rep proteins at these viral centers (29, 33, 76).A number of published reports suggest associations between AAV and the cellular DNA damage machinery. For example, transduction by rAAV vectors is increased by genotoxic agents and DNA damaging treatments (1, 62, 91) although the mechanisms involved remain unclear. Additionally, the ATM kinase negatively regulates rAAV transduction (64, 92), and we have shown that the MRN complex poses a barrier to both rAAV transduction and wild-type AAV replication (11, 67). UV-inactivated AAV particles also appear to activate a DDR involving ATM and ATR kinases that perturbs cell cycle progression (39, 60, 88). It has been suggested that this response is provoked by the AAV ITRs (60) and that UV-treated particles mimic stalled replication forks in infected cells (39). In addition to AAV genome components, the viral Rep proteins have been observed to exhibit cytotoxicity and induce S-phase arrest (3, 65).The role of cellular repair proteins in AAV genome processing has also been explored by examining the molecular fate of rAAV vectors, which are converted into circular and concatemeric forms that persist episomally (18, 19, 66). Proteins shown to regulate circularization in cell culture include ATM and the MRN complex (14, 64), while in vivo experiments using mouse models have implicated ATM and DNA-PK in this process (14, 20, 72). Additionally, DNA-PKcs and Artemis have recently been shown to cleave the ITR hairpins of rAAV vectors in vivo in a tissue-dependent manner (36). Despite these studies, it is not clear how damage response factors function together and how they impact AAV transduction and replication in human cells.In this study we examined the cellular response to AAV replication in the context of Ad infection or helper proteins. We show that coinfection with AAV and Ad activates a DDR that is distinct from that seen during infection with Ad alone. The ATM and DNA-PKcs damage kinases are activated and signal to downstream substrates, but the response does not require the MRN complex and is primarily mediated by DNA-PKcs. Although expression of the large Rep proteins induced some DDR events, full signaling appeared to require AAV replication and was accompanied by accumulation of DNA-PK at viral replication compartments. Our results demonstrate that AAV replication induces a unique DNA damage signal transduction response and provides a model system for studying DNA-PK.     

Homologous Recombinational Repair Factors Are Recruited and Loaded onto the Viral DNA Genome in Epstein-Barr Virus Replication Compartments

Homologous recombination is an important biological process that facilitates genome rearrangement and repair of DNA double-strand breaks (DSBs). The induction of Epstein-Barr virus (EBV) lytic replication induces ataxia telangiectasia-mutated (ATM)-dependent DNA damage checkpoint signaling, leading to the clustering of phosphorylated ATM and Mre11/Rad50/Nbs1 (MRN) complexes to sites of viral genome synthesis in nuclei. Here we report that homologous recombinational repair (HRR) factors such as replication protein A (RPA), Rad51, and Rad52 as well as MRN complexes are recruited and loaded onto the newly synthesized viral genome in replication compartments. The 32-kDa subunit of RPA is extensively phosphorylated at sites in accordance with those with ATM. The hyperphosphorylation of RPA32 causes a change in RPA conformation, resulting in a switch from the catalysis of DNA replication to the participation in DNA repair. The levels of Rad51 and phosphorylated RPA were found to increase with the progression of viral productive replication, while that of Rad52 proved constant. Furthermore, biochemical fractionation revealed increases in levels of DNA-bound forms of these HRRs. Bromodeoxyuridine-labeled chromatin immunoprecipitation and PCR analyses confirmed the loading of RPA, Rad 51, Rad52, and Mre11 onto newly synthesized viral DNA, and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling analysis demonstrated DSBs in the EBV replication compartments. HRR factors might be recruited to repair DSBs on the viral genome in viral replication compartments. RNA interference knockdown of RPA32 and Rad51 prevented viral DNA synthesis remarkably, suggesting that homologous recombination and/or repair of viral DNA genome might occur, coupled with DNA replication to facilitate viral genome synthesis.Replication protein A (RPA), the eukaryotic single-stranded DNA (ssDNA)-binding protein, is a heterotrimeric complex composed of three tightly associated subunits of 70, 32, and 14 kDa (referred as to RPA70, RPA32, and RPA14, respectively) that is essential for DNA replication, recombination, and all major types of DNA repair (4). RPA participates in such diverse pathways through its ability to interact with DNA and numerous proteins involved in its processing. During DNA replication, RPA associates with ssDNA at forks and facilitates nascent-strand DNA synthesis by replicative DNA polymerases localized at replication foci during S phase. Under DNA-damaging conditions, RPA binds to ssDNA at damaged sites and interacts with repair and recombination components to process double-strand DNA breaks (DSBs) and other lesions (6, 14, 21, 32, 38, 41).RPA undergoes both DNA damage-independent and -dependent phosphorylation on the N-terminal 33 residues of RPA32. Unstressed cell cycle-dependent phosphorylation occurs during the G₁/S-phase transition and in M phase, primarily at the conserved cyclin-CDK phosphorylation sites of Ser-23 and Ser-29 in the N terminus of the RPA32 subunit (13, 15). In contrast, stress-induced hyperphosphorylation of RPA is much more extensive. Nine potential phosphorylation sites within the N-terminal domain of RPA32, Ser-4, Ser-8, Ser-11/Ser-12/Ser-13, Thr-21, Ser-23, Ser-29, and Ser-33, in response to DNA-damaging agents, have been suggested (33, 54). Although this region of RPA32 is not required for the ssDNA-binding activity of RPA (5, 22), a phosphorylation-induced subtle conformation change in RPA, resulting from altered intersubunit interactions, regulates the interaction of RPA with both interacting proteins and DNA (30). The hyperphosphorylated form of RPA32 is unable to localize to replication centers in normal cells, while binding to DNA damage foci is unaffected (46). Therefore, RPA phosphorylation following damage is thought to both prevent RPA from catalyzing DNA replication and potentially serve as a marker to recruit repair factors to sites of DNA damage. RPA localizes to nuclear foci where DNA repair is occurring after DNA damage and is essential for multiple DNA repair pathways, participating in damage recognition, excision, and resynthesis reactions (4, 56).Mammalian cells can repair DSBs by homologous recombination (HR) or by nonhomologous end joining. HR is an accurate repair process, the first step of which is the resection of the 5′ ends of the DSB to generate 3′ ssDNA overhangs. This reaction is carried out by the Mre11/Rad50/Nbs1 (MRN) complex, which not only functions as a damage sensor upstream of ataxia telangiectasia-mutated (ATM)/ATM-Rad3-related (ATR) activation but also plays a role in DSB repair (4). RPA and members of the RAD52 epistasis group of gene products, such as Rad51, Rad52, and Rad54, bind to the resulting 3′ ssDNA strands and form a helical, nucleoprotein filament that facilitates the invasion of a damaged DNA strand into the homologous double-stranded DNA partner. The human Rad51 protein is a structural and functional homolog of the Escherichia coli RecA protein, which promotes homologous pairing and strand transfer reactions in vitro. Both Rad51 and Rad52 bind specifically to the terminal regions of tailed duplex DNA, the substrate thought to initiate recombination in vivo. Furthermore, nucleoprotein filaments of Rad51, formed on tailed DNA, catalyze strand invasion of homologous duplex DNA in a reaction that is stimulated by Rad52 and RPA (3).Epstein-Barr virus (EBV) is a human herpesvirus that infects B lymphocytes, inducing their continuous proliferation. In B-lymphoblastoid cell lines, there is no production of virus particles, which is termed latent infection (52). Reactivation from latency is characterized by the expression of lytic genes, and one of the first detectable changes is the expression of the BZLF1 immediate-early gene product, which trans-activates viral promoters (16), leading to an ordered cascade of viral early and late gene expression. This lytic EBV DNA replication occurs in discrete sites in nuclei, called replication compartments, in which seven viral replication proteins are assembled (44). The viral genome is amplified several hundredfold by the viral replication machinery and is thought to generate highly branched replication intermediates through HR coupled with viral DNA replication (48). With the progression of lytic replication, the replication compartments become larger and appeared to fuse to form large globular structures that eventually filled the nucleus at late stages of infection (8, 45).We previously isolated latently EBV-infected Tet-BZLF1/B95-8 cells in which the exogenous BZLF1 protein is conditionally expressed under the control of a tetracycline-regulated promoter, leading to a highly efficient induction of lytic replication (28). Using this system, we have demonstrated that the induction of the EBV lytic program results in the inhibition of replication of cellular DNA in spite of the replication of viral DNA (28) and elicits a cellular DNA damage response, with the activation of the ATM-Chk2-p53 DNA damage transduction pathway (29). The DNA damage sensor MRN complex and phosphorylated ATM are recruited and retained in viral replication compartments (29).Here we report that RPA32 is extensively phosphorylated after EBV lytic replication is induced, with the phosphorylation sites in accordance with those for ATM. Phosphorylated RPA, Rad51, and Rad52, which are involved in HR repair (HRR), are recruited and retained in viral replication compartments as well as the MRN complex. Furthermore, DSBs could be demonstrated to occur during viral genome synthesis in the EBV replication compartments. HRR factors might be recruited to repair DSBs on the viral genome in viral replication compartments. RNA interference (RNAi) knockdown of RPA32 and Rad51 prevented viral DNA synthesis remarkably, suggesting that HR and/or repair of viral DNA genome might occur, coupled with DNA replication, to facilitate viral genome synthesis.     

Cell Cycle-Dependent Role of MRN at Dysfunctional Telomeres: ATM Signaling-Dependent Induction of Nonhomologous End Joining (NHEJ) in G1 and Resection-Mediated Inhibition of NHEJ in G2

Here, we address the role of the MRN (Mre11/Rad50/Nbs1) complex in the response to telomeres rendered dysfunctional by deletion of the shelterin component TRF2. Using conditional NBS1/TRF2 double-knockout MEFs, we show that MRN is required for ATM signaling in response to telomere dysfunction. This establishes that MRN is the only sensor for the ATM kinase and suggests that TRF2 might block ATM signaling by interfering with MRN binding to the telomere terminus, possibly by sequestering the telomere end in the t-loop structure. We also examined the role of the MRN/ATM pathway in nonhomologous end joining (NHEJ) of damaged telomeres. NBS1 deficiency abrogated the telomere fusions that occur in G₁, consistent with the requirement for ATM and its target 53BP1 in this setting. Interestingly, NBS1 and ATM, but not H2AX, repressed NHEJ at dysfunctional telomeres in G₂, specifically at telomeres generated by leading-strand DNA synthesis. Leading-strand telomere ends were not prone to fuse in the absence of either TRF2 or MRN/ATM, indicating redundancy in their protection. We propose that MRN represses NHEJ by promoting the generation of a 3′ overhang after completion of leading-strand DNA synthesis. TRF2 may ensure overhang formation by recruiting MRN (and other nucleases) to newly generated telomere ends. The activation of the MRN/ATM pathway by the dysfunctional telomeres is proposed to induce resection that protects the leading-strand ends from NHEJ when TRF2 is absent. Thus, the role of MRN at dysfunctional telomeres is multifaceted, involving both repression of NHEJ in G₂ through end resection and induction of NHEJ in G₁ through ATM-dependent signaling.Mammalian telomeres solve the end protection problem through their association with shelterin. The shelterin factor TRF2 (telomere repeat-binding factor 2) protects chromosome ends from inappropriate DNA repair events that threaten the integrity of the genome (reviewed in reference 32). When TRF2 is removed by Cre-mediated deletion from conditional knockout mouse embryo fibroblasts (TRF2^F/− MEFs), telomeres activate the ATM kinase pathway and are processed by the canonical nonhomologous end-joining (NHEJ) pathway to generate chromosome end-to-end fusions (10, 11).The repair of telomeres in TRF2-deficient cells is readily monitored in metaphase spreads. Over the course of four or five cell divisions, the majority of chromosome ends become fused, resulting in metaphase spreads displaying the typical pattern of long trains of joined chromosomes (10). The reproducible pace and the efficiency of telomere NHEJ have allowed the study of factors involved in its execution and regulation. In addition to depending on the NHEJ factors Ku70 and DNA ligase IV (10, 11), telomere fusions are facilitated by the ATM kinase (26). This aspect of telomere NHEJ is mediated through the ATM kinase target 53BP1. 53BP1 accumulates at telomeres in TRF2-depleted cells and stimulates chromatin mobility, thereby promoting the juxtaposition of distantly positioned chromosome ends prior to their fusion (18). Telomere NHEJ is also accelerated by the ATM phosphorylation target MDC1, which is required for the prolonged association of 53BP1 at sites of DNA damage (19).Although loss of TRF2 leads to telomere deprotection at all stages of the cell cycle, NHEJ of uncapped telomeres takes place primarily before their replication in G₁ (25). Postreplicative (G₂) telomere fusions can occur at a low frequency upon TRF2 deletion, but only when cyclin-dependent kinase activity is inhibited with roscovitine (25). The target of Cdk1 in this setting is not known.Here, we dissect the role of the MRN (Mre11/Rad50/Nbs1) complex and H2AX at telomeres rendered dysfunctional through deletion of TRF2. The highly conserved MRN complex has been proposed to function as the double-stranded break (DSB) sensor in the ATM pathway (reviewed in references 34 and 35). In support of this model, Mre11 interacts directly with DNA ends via two carboxy-terminal DNA binding domains (13, 14); the recruitment of MRN to sites of damage is independent of ATM signaling, as it occurs in the presence of the phosphoinositide-3-kinase-related protein kinase inhibitor caffeine (29, 44); in vitro analysis has demonstrated that MRN is required for activation of ATM by linear DNAs (27); a mutant form of Rad50 (Rad50S) can induce ATM signaling in the absence of DNA damage (31); and phosphorylation of ATM targets in response to ionizing radiation is completely abrogated upon deletion of NBS1 from MEFs (17). These data and the striking similarities between syndromes caused by mutations in ATM, Nbs1, and Mre11 (ataxia telangiectasia, Nijmegen breakage syndrome, and ataxia telangiectasia-like disease, respectively) are consistent with a sensor function for MRN.MRN has also been implicated in several aspects of DNA repair. Potentially relevant to DNA repair events, Mre11 dimers can bridge and align the two DNA ends in vitro (49) and Rad50 may promote long-range tethering of sister chromatids (24, 50). In addition, a binding partner of the MRN complex, CtIP, has been implicated in end resection of DNA ends during homology-directed repair (39, 45). The role of MRN in NHEJ has been much less clear. MRX, the yeast orthologue of MRN, functions during NHEJ in Saccharomyces cerevisiae but not in Schizosaccharomyces pombe (28, 30). In mammalian cells, MRN is not recruited to I-SceI-induced DSBs in G₁, whereas Ku70 is, and MRN does not appear to be required for NHEJ-mediated repair of these DSBs (38, 54). On the other hand, MRN promotes class switch recombination (37) and has been implicated in accurate NHEJ repair during V(D)J recombination (22).The involvement of MRN in ATM signaling and DNA repair pathways has been intriguing from the perspective of telomere biology. While several of the attributes of MRN might be considered a threat to telomere integrity, MRN is known to associate with mammalian telomeres, most likely through an interaction with the TRF2 complex (48, 51, 57). MRN has been implicated in the generation of the telomeric overhang (12), the telomerase pathway (36, 52), the ALT pathway (55), and the protection of telomeres from stochastic deletion events (1). It has also been speculated that MRN may contribute to formation of the t-loop structure (16). t-loops, the lariats formed through the strand invasion of the telomere terminus into the duplex telomeric DNA (21), are thought to contribute to telomere protection by effectively shielding the chromosome end from DNA damage response factors that interact with DNA ends, including nucleases, and the Ku heterodimer (15).H2AX has been studied extensively in the context of chromosome-internal DSBs. When a DSB is formed, ATM acts near the lesion to phosphorylate a conserved carboxy-terminal serine of H2AX, a histone variant present throughout the genome (7). Phosphorylated H2AX (referred to as γ-H2AX) promotes the spreading of DNA damage factors over several megabases along the damaged chromatin and mediates the amplification of the DNA damage signal (43). The signal amplification is accomplished through a sequence of phospho-specific interactions among γ-H2AX, MDC1, NBS1, RNF8, and RNF168, which results in the additional binding of ATM and additional phosphorylation of H2AX in adjacent chromatin (reviewed in reference 33). The formation of these large domains of altered chromatin, referred to as irradiation-induced foci at DSBs and telomere dysfunction-induced foci (TIFs) at dysfunctional telomeres (44), promotes the binding of several factors implicated in DNA repair, including the BRCA1 A complex and 53BP1 (33).In agreement with a role for H2AX in DNA repair, H2AX-deficient cells exhibit elevated levels of irradiation-induced chromosome abnormalities (5, 9). In addition, H2AX-null B cells are prone to chromosome breaks and translocations in the immunoglobulin locus, indicative of impaired class switch recombination, a process that involves the repair of DSBs through the NHEJ pathway (9, 20). Since H2AX is dispensable for the activation of irradiation-induced checkpoints (8), these data argue that H2AX contributes directly to DNA repair. However, a different set of studies has concluded that H2AX is not required for NHEJ during V(D)J recombination (5, 9) but that it plays a role in homology-directed repair (53). In this study, we have further queried the contribution of H2AX to NHEJ in the context of dysfunctional telomeres.Our aim was to dissect the contribution of MRN and H2AX to DNA damage signaling and NHEJ-mediated repair in response to telomere dysfunction elicited by deletion of TRF2. Importantly, since ATM is the only kinase activated in this setting, deletion of TRF2 can illuminate the specific contribution of these factors in the absence of the confounding effects of ATR signaling (26). This approach revealed a dual role for MRN at telomeres, involving both its function as a sensor in the ATM pathway and its ability to protect telomeres from NHEJ under certain circumstances.     

Herpes Simplex Virus Type 1 Glycoprotein E Mediates Retrograde Spread from Epithelial Cells to Neurites

In animal models of infection, glycoprotein E (gE) is required for efficient herpes simplex virus type 1 (HSV-1) spread from the inoculation site to the cell bodies of innervating neurons (retrograde direction). Retrograde spread in vivo is a multistep process, in that HSV-1 first spreads between epithelial cells at the inoculation site, then infects neurites, and finally travels by retrograde axonal transport to the neuron cell body. To better understand the role of gE in retrograde spread, we used a compartmentalized neuron culture system, in which neurons were infected in the presence or absence of epithelial cells. We found that gE-deleted HSV-1 (NS-gEnull) retained retrograde axonal transport activity when added directly to neurites, in contrast to the retrograde spread defect of this virus in animals. To better mimic the in vivo milieu, we overlaid neurites with epithelial cells prior to infection. In this modified system, virus infects epithelial cells and then spreads to neurites, revealing a 100-fold retrograde spread defect for NS-gEnull. We measured the retrograde spread defect of NS-gEnull from a variety of epithelial cell lines and found that the magnitude of the spread defect from epithelial cells to neurons correlated with epithelial cell plaque size defect, indicating that gE plays a similar role in both types of spread. Therefore, gE-mediated spread between epithelial cells and neurites likely explains the retrograde spread defect of gE-deleted HSV-1 in vivo.Herpes simplex virus type 1 (HSV-1) is an alphaherpesvirus that characteristically infects skin and mucosal surfaces before spreading to sensory neurons, where it establishes a lifelong persistent infection. The virus periodically returns to the periphery via sensory axons and causes recurrent lesions as well as asymptomatic shedding. This life cycle requires viral transport along axons in two directions: toward the neuron cell body (retrograde direction) and away from the neuron cell body (anterograde direction).Many studies of alphaherpesvirus neuronal spread have focused on pseudorabies virus (PRV), a virus whose natural host is the pig. Three PRV proteins, glycoprotein E (gE), gI, and Us9, have been shown to mediate anterograde neuronal spread both in animal models of infection and in cultured neurons. However, these three proteins are dispensable for retrograde spread (3, 8, 11, 12, 31, 46). In contrast, numerous animal models of infection have shown that HSV-1 gE is required for retrograde spread from the inoculation site to the cell bodies of innervating neurons (4, 9, 44, 56). In the murine flank model, wild-type (WT) virus replicates in the skin and then infects sensory neurons and spreads in a retrograde direction to the dorsal root ganglia (DRG). In this model, gE-deleted HSV-1 replicates in the skin but is not detected in the DRG (9, 44). This phenotype differs from gE-deleted PRV, which is able to reach the DRG at WT levels (8). Thus, unlike PRV, gE-deleted HSV-1 viruses have a retrograde spread defect in vivo.HSV-1 gE is a 552-amino-acid type I membrane protein found in the virion membrane as well as in the trans-Golgi and plasma membranes of infected cells (1). gE forms a heterodimer with another viral glycoprotein, gI. The gE/gI complex is important for HSV-1 immune evasion through its Fc receptor activity. gE/gI binds to the Fc domain of antibodies directed against other viral proteins, sequestering these antibodies and blocking antibody effector functions (27, 32, 40). Additionally, gE/gI promotes spread between epithelial cells. Viruses lacking either gE or gI form characteristically small plaques in cell culture and small inoculation site lesions in mice (4, 9, 18, 40, 58). In animal models, gE and gI also mediate viral spread in both anterograde and retrograde directions (4, 19, 44, 56).In order to better understand the role of gE in HSV-1 retrograde neuronal spread, we employed a compartmentalized neuron culture system that has been used to study directional neuronal spread of PRV and West Nile virus (12, 14, 45). In the Campenot chamber system, neurites are contained in a compartment that is separate from their corresponding cell bodies. Therefore, spread in an exclusively retrograde direction can be measured by infecting neurites and detecting spread to neuron cell bodies.HSV-1 replication requires retrograde transport of incoming viral genomes to the nucleus. In neurites, fusion between viral and cellular membranes occurs at the plasma membrane (43, 48). Upon membrane fusion, the capsid and a subset of tegument proteins (the inner tegument) dissociate from glycoproteins and outer tegument proteins, which remain at the plasma membrane (28, 38). Unenveloped capsids and the associated inner tegument proteins are then transported in the retrograde direction to the nucleus (7, 48, 49).For both neurons and epithelial cells, retrograde transport is dependent upon microtubules, ATP, the retrograde microtubule motor dynein, and the dynein cofactor dynactin (22, 34, 49, 52). Several viral proteins interact with components of the dynein motor complex (23, 39, 60). However, none of these proteins suggest a completely satisfactory mechanism by which viral retrograde transport occurs, either because they are not components of the complex that is transported to the nucleus (UL34, UL9, VP11/12) or because capsids lacking that protein retain retrograde transport activity (VP26) (2, 17, 21, 28, 37). This implies that additional viral proteins are involved in retrograde trafficking.We sought to better characterize the role of gE in retrograde spread and found that gE is dispensable for retrograde axonal transport; however, it promotes HSV-1 spread from epithelial cells to neurites. This epithelial cell-to-neuron spread defect provides a plausible explanation for the retrograde spread defect of gE-deleted HSV-1 in animal models of infection.