Activation of ataxia telangiectasia-mutated DNA damage checkpoint signal transduction elicited by herpes simplex virus infection

Eukaryotic cells are equipped with machinery to monitor and repair damaged DNA. Herpes simplex virus (HSV) DNA replication occurs at discrete sites in nuclei, the replication compartment, where viral replication proteins cluster and synthesize a large amount of viral DNA. In the present study, HSV infection was found to elicit a cellular DNA damage response, with activation of the ataxia-telangiectasia-mutated (ATM) signal transduction pathway, as observed by autophosphorylation of ATM and phosphorylation of multiple downstream targets including Nbs1, Chk2, and p53, while infection with a UV-inactivated virus or with a replication-defective virus did not. Activated ATM and the DNA damage sensor MRN complex composed of Mre11, Rad50, and Nbs1 were recruited and retained at sites of viral DNA replication, probably recognizing newly synthesized viral DNAs as abnormal DNA structures. These events were not observed in ATM-deficient cells, indicating ATM dependence. In Nbs1-deficient cells, HSV infection induced an ATM DNA damage response that was delayed, suggesting a functional MRN complex requirement for efficient ATM activation. However, ATM silencing had no effect on viral replication in 293T cells. Our data open up an interesting question of how the virus is able to complete its replication, although host cells activate ATM checkpoint signaling in response to the HSV infection.     

DNA Damage Signaling Is Induced in the Absence of Epstein—Barr Virus (EBV) Lytic DNA Replication and in Response to Expression of ZEBRA

Epstein Barr virus (EBV), like other oncogenic viruses, modulates the activity of cellular DNA damage responses (DDR) during its life cycle. Our aim was to characterize the role of early lytic proteins and viral lytic DNA replication in activation of DNA damage signaling during the EBV lytic cycle. Our data challenge the prevalent hypothesis that activation of DDR pathways during the EBV lytic cycle occurs solely in response to large amounts of exogenous double stranded DNA products generated during lytic viral DNA replication. In immunofluorescence or immunoblot assays, DDR activation markers, specifically phosphorylated ATM (pATM), H2AX (γH2AX), or 53BP1 (p53BP1), were induced in the presence or absence of viral DNA amplification or replication compartments during the EBV lytic cycle. In assays with an ATM inhibitor and DNA damaging reagents in Burkitt lymphoma cell lines, γH2AX induction was necessary for optimal expression of early EBV genes, but not sufficient for lytic reactivation. Studies in lytically reactivated EBV-positive cells in which early EBV proteins, BGLF4, BGLF5, or BALF2, were not expressed showed that these proteins were not necessary for DDR activation during the EBV lytic cycle. Expression of ZEBRA, a viral protein that is necessary for EBV entry into the lytic phase, induced pATM foci and γH2AX independent of other EBV gene products. ZEBRA mutants deficient in DNA binding, Z(R183E) and Z(S186E), did not induce foci of pATM. ZEBRA co-localized with HP1β, a heterochromatin associated protein involved in DNA damage signaling. We propose a model of DDR activation during the EBV lytic cycle in which ZEBRA induces ATM kinase phosphorylation, in a DNA binding dependent manner, to modulate gene expression. ATM and H2AX phosphorylation induced prior to EBV replication may be critical for creating a microenvironment of viral and cellular gene expression that enables lytic cycle progression.     

Parvovirus minute virus of mice induces a DNA damage response that facilitates viral replication

Infection by DNA viruses can elicit DNA damage responses (DDRs) in host cells. In some cases the DDR presents a block to viral replication that must be overcome, and in other cases the infecting agent exploits the DDR to facilitate replication. We find that low multiplicity infection with the autonomous parvovirus minute virus of mice (MVM) results in the activation of a DDR, characterized by the phosphorylation of H2AX, Nbs1, RPA32, Chk2 and p53. These proteins are recruited to MVM replication centers, where they co-localize with the main viral replication protein, NS1. The response is seen in both human and murine cell lines following infection with either the MVMp or MVMi strains. Replication of the virus is required for DNA damage signaling. Damage response proteins, including the ATM kinase, accumulate in viral-induced replication centers. Using mutant cell lines and specific kinase inhibitors, we show that ATM is the main transducer of the signaling events in the normal murine host. ATM inhibitors restrict MVM replication and ameliorate virus-induced cell cycle arrest, suggesting that DNA damage signaling facilitates virus replication, perhaps in part by promoting cell cycle arrest. Thus it appears that MVM exploits the cellular DNA damage response machinery early in infection to enhance its replication in host cells.     

Induction and utilization of an ATM signaling pathway by polyomavirus

Progression from G(1) to S is essential for polyomavirus DNA replication and depends on the interaction of large T with the retinoblastoma gene product pRb. This virus-induced replication pathway is accompanied by p53 activation resembling a DNA damage response (12). We sought to determine whether this pathway depends in part on activation of the ATM (ataxia telangiectasia mutated) kinase and whether the virus gains advantages from this pathway beyond that of entry into S. We show that polyomavirus infection activates the S- and G(2)-phase checkpoints in primary as well as established mouse cells. Infected cells undergo a prolonged S phase compared to uninfected serum-stimulated cells and show no evidence of a G(2)-->M transition before lytic death ensues. Infection is accompanied by increases in ATM activity in vitro and in the level of ATM-S1981-P in vivo. The incubation of infected cells with caffeine, a known ATM inhibitor, did not block entry into S but reduced the rate of viral compared to cellular DNA synthesis. Importantly, caffeine lowered the yields of viral DNA an average of 3- to 6-fold and those of infectious virus by as much as 10-fold. Virus yields were 10-fold lower in ATM (-/-) p53(-/-) than in ATM(+/+) p53(-/-) mouse embryo fibroblasts, indicating a p53-independent role of ATM in productive infection. Replacement of the normal SMC1 (structural maintenance of chromosomes, or cohesin) protein, a critical ATM substrate in the DNA repair pathway, with its phosphorylation mutant SMC1(S957AS966A) also lowered virus yields by roughly 90%. We suggest that polyomavirus activates and utilizes a component(s) of an ATM pathway of DNA repair to prolong S phase and aid its own replication.     

Degradation of Phosphorylated p53 by Viral Protein-ECS E3 Ligase Complex

p53-signaling is modulated by viruses to establish a host cellular environment advantageous for their propagation. The Epstein-Barr virus (EBV) lytic program induces phosphorylation of p53, which prevents interaction with MDM2. Here, we show that induction of EBV lytic program leads to degradation of p53 via an ubiquitin-proteasome pathway independent of MDM2. The BZLF1 protein directly functions as an adaptor component of the ECS (Elongin B/C-Cul2/5-SOCS-box protein) ubiquitin ligase complex targeting p53 for degradation. Intringuingly, C-terminal phosphorylation of p53 resulting from activated DNA damage response by viral lytic replication enhances its binding to BZLF1 protein. Purified BZLF1 protein-associated ECS could be shown to catalyze ubiquitination of phospho-mimetic p53 more efficiently than the wild-type in vitro. The compensation of p53 at middle and late stages of the lytic infection inhibits viral DNA replication and production during lytic infection, suggesting that the degradation of p53 is required for efficient viral propagation. Taken together, these findings demonstrate a role for the BZLF1 protein-associated ECS ligase complex in regulation of p53 phosphorylated by activated DNA damage signaling during viral lytic infection.     

Atm is dispensable for p53 apoptosis and tumor suppression triggered by cell cycle dysfunction

Both p53 and ATM are checkpoint regulators with roles in genetic stabilization and cancer susceptibility. ATM appears to function in the same DNA damage checkpoint pathway as p53. However, ATM's role in p53-dependent apoptosis and tumor suppression in response to cell cycle dysregulation is unknown. In this study, we tested the role of murine ataxia telangiectasia protein (Atm) in a transgenic mouse brain tumor model in which p53-mediated apoptosis results in tumor suppression. These p53-mediated activities are induced by tissue-specific inactivation of pRb family proteins by a truncated simian virus 40 large T antigen in brain epithelium. We show that p53-dependent apoptosis, transactivation, and tumor suppression are unaffected by Atm deficiency, suggesting that signaling in the DNA damage pathway is distinct from that in the oncogene-induced pathway. In addition, we show that Atm deficiency has no overall effect on tumor growth and progression in this model.     

Viral transport of DNA damage that mimics a stalled replication fork

Adeno-associated virus type 2 (AAV2) infection incites cells to arrest with 4N DNA content or die if the p53 pathway is defective. This arrest depends on AAV2 DNA, which is single stranded with inverted terminal repeats that serve as primers during viral DNA replication. Here, we show that AAV2 DNA triggers damage signaling that resembles the response to an aberrant cellular DNA replication fork. UV treatment of AAV2 enhances the G2 arrest by generating intrastrand DNA cross-links which persist in infected cells, disrupting viral DNA replication and maintaining the viral DNA in the single-stranded form. In cells, such DNA accumulates into nuclear foci with a signaling apparatus that involves DNA polymerase delta, ATR, TopBP1, RPA, and the Rad9/Rad1/Hus1 complex but not ATM or NBS1. Focus formation and damage signaling strictly depend on ATR and Chk1 functions. Activation of the Chk1 effector kinase leads to the virus-induced G2 arrest. AAV2 provides a novel way to study the cellular response to abnormal DNA replication without damaging cellular DNA. By using the AAV2 system, we show that in human cells activation of phosphorylation of Chk1 depends on TopBP1 and that it is a prerequisite for the appearance of DNA damage foci.     

Gamma-herpesvirus kinase actively initiates a DNA damage response by inducing phosphorylation of H2AX to foster viral replication

DNA virus infection can elicit the DNA damage response in host cells, including ATM kinase activation and H2AX phosphorylation. This is considered to be the host cell response to replicating viral DNA. In contrast, we show that during infection of macrophages murine gamma-herpesvirus 68 (gammaHV68) actively induces H2AX phosphorylation by expressing a viral kinase (orf36). GammaHV68-encoded orf36 kinase and its EBV homolog, BGLF4, induce H2AX phosphorylation independently of other viral genes. The process requires the kinase domain of Orf36 and is enhanced by ATM. Orf36 is important for gammaHV68 replication in infected animals, and orf36, H2AX, and ATM are all critical for efficient gammaHV68 replication in primary macrophages. Thus, activation of proximal components of the DNA damage signaling response is an active viral kinase-driven strategy required for efficient gamma-herpesvirus replication.     

A Novel Cell Cycle Inhibitor Stalls Replication Forks and Activates S Phase Checkpoint

DNA replication checkpoint is activated in response to replication stresses. It maintains the integrity of stalled replication forks and prevents premature segregation of largely unreplicated chromosomes. In budding yeast, Mec1 and Rad53 kinases (homologous to mammalian ATM/ATR and Chk2 kinases, respectively) are the main effectors of this checkpoint control. Using a yeast based screen, we have identified acompound (named here ENA) which inhibits DNA replication and activatesMec1/Rad53 checkpoint. A brief exposure to this compound stops fork progression at or near replication origin and renders the forks incompetent to resume replication despite the presence of a functional checkpoint. ENA also inhibits DNA synthesis in mammalian cells leading to the activation of ATM/ATR pathway and the induction of apoptosis in a p53 independent manner. Interestingly, ENA acts as an effective antiproliferative agent against a subset of cancer cell lines and as an anti-tumor agent against human xenografts in mice. Thus, ENA is a potent cell cycle inhibitor with conceivable therapeutic potential.     

ATMIN defines an NBS1-independent pathway of ATM signalling

The checkpoint kinase ATM (ataxia telangiectasia mutated) transduces genomic stress signals to halt cell cycle progression and promote DNA repair in response to DNA damage. Here, we report the characterisation of an essential cofactor for ATM, ATMIN (ATM INteracting protein). ATMIN interacts with ATM through a C-terminal motif, which is also present in Nijmegen breakage syndrome (NBS)1. ATMIN and ATM co-localised in response to ATM activation by chloroquine and hypotonic stress, but not after induction of double-strand breaks by ionising radiation (IR). ATM/ATMIN complex disruption by IR was attenuated in cells with impaired NBS1 function, suggesting competition of NBS1 and ATMIN for ATM binding. ATMIN protein levels were reduced in ataxia telangiectasia cells and ATM protein levels were low in primary murine fibroblasts lacking ATMIN, indicating reciprocal stabilisation. Whereas phosphorylation of Smc1, Chk2 and p53 was normal after IR in ATMIN-deficient cells, basal ATM activity and ATM activation by hypotonic stress and inhibition of DNA replication was impaired. Thus, ATMIN defines a novel NBS1-independent pathway of ATM signalling.     

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.     

E1B55K-Deleted Adenovirus (ONYX-015) Overrides G1/S and G2/M Checkpoints and Causes Mitotic Catastrophe and Endoreduplication in p53-Proficient Normal Cells

In order to take advantage of cell replication machinery, viruses have evolved complex strategies to override cell cycle checkpoints and force host cells into S phase. To do so, virus products must interfere not only with the basal cell cycle regulators, such as pRb or Mad2, but also with the main surveillance pathways such as those controlled by p53 and ATM. Recently, a number of defective viruses has been produced which, lacking the latter ability, are incapable of replicating in normal cells but should be able to grow and finally lyse those cells that, such as the tumor cells, have lost their surveillance mechanisms. A prototype of these oncolytic viruses is the E1B55K-defective Adenovirus ONYX-015, which was predicted to selectively replicate and kill p53-deficient cancer cells. We found that, despite wt p53 and notwithstanding the activation of the checkpoint regulators p53, ATM, and Mad2, ONYX-015 actively replicated in HUVEC cells. Furthermore, ONYX-015 replication induced a specific phenotype, which is distinct from that of the E4-deleted adenovirus dlE4 Ad5, although both viruses express the main regulatory region E1A. This phenotype includes overriding of the G1/S and G2/M checkpoints, over-expression of MAD2 and retardation of mitosis and accumulation of polyploid cells, suggesting the occurrence of alterations at the mitotic-spindle checkpoint and impairment of the post-mitotic checkpoint. Our data suggest that viral E1A and E4 region products can override all host cell-checkpoint response even at the presence of a full activation of the ATM/p53 pathway. Furthermore, the E4 region alone seems to act independently of the E1B55K virus product in impairing the ATM-dependent, p53-independent G2/M checkpoint since dlE4 Ad5-infected cells arrested in G2 while ONYX-015-infected cells did enter mitosis.     

Inactivation of ATM/ATR DNA Damage Checkpoint Promotes Androgen Induced Chromosomal Instability in Prostate Epithelial Cells

The ATM/ATR DNA damage checkpoint functions in the maintenance of genetic stability and some missense variants of the ATM gene have been shown to confer a moderate increased risk of prostate cancer. However, whether inactivation of this checkpoint contributes directly to prostate specific cancer predisposition is still unknown. Here, we show that exposure of non-malignant prostate epithelial cells (HPr-1AR) to androgen led to activation of the ATM/ATR DNA damage response and induction of cellular senescence. Notably, knockdown of the ATM gene expression in HPr-1AR cells can promote androgen-induced TMPRSS2: ERG rearrangement, a prostate-specific chromosome translocation frequently found in prostate cancer cells. Intriguingly, unlike the non-malignant prostate epithelial cells, the ATM/ATR DNA damage checkpoint appears to be defective in prostate cancer cells, since androgen treatment only induced a partial activation of the DNA damage response. This mechanism appears to preserve androgen induced autophosphorylation of ATM and phosphorylation of H2AX, lesion processing and repair pathway yet restrain ATM/CHK1/CHK2 and p53 signaling pathway. Our findings demonstrate that ATM/ATR inactivation is a crucial step in promoting androgen-induced genomic instability and prostate carcinogenesis.     

RNF8-dependent and RNF8-independent regulation of 53BP1 in response to DNA damage

The DNA damage surveillance network orchestrates cellular responses to DNA damage through the recruitment of DNA damage-signaling molecules to DNA damage sites and the concomitant activation of protein phosphorylation cascades controlled by the ATM (ataxia-telangiectasia-mutated) and ATR (ATM-Rad3-related) kinases. Activation of ATM/ATR triggers cell cycle checkpoint activation and adaptive responses to DNA damage. Recent studies suggest that protein ubiquitylation or degradation plays an important role in the DNA damage response. In this study, we examined the potential role of the proteasome in checkpoint activation and ATM/ATR signaling in response to UV light-induced DNA damage. HeLa cells treated with the proteasome inhibitor MG-132 showed delayed phosphorylation of ATM substrates in response to UV light. UV light-induced phosphorylation of 53BP1, as well as its recruitment to DNA damage foci, was strongly suppressed by proteasome inhibition, whereas the recruitment of upstream regulators of 53BP1, including MDC1 and H2AX, was unaffected. The ubiquitin-protein isopeptide ligase RNF8 was critical for 53BP1 focus targeting and phosphorylation in ionizing radiation-damaged cells, whereas UV light-induced 53BP1 phosphorylation and targeting exhibited partial dependence on RNF8 and the ubiquitin-conjugating enzyme UBC13. Suppression of RNF8 or UBC13 also led to subtle defects in UV light-induced G2/M checkpoint activation. These findings are consistent with a model in which RNF8 ubiquitylation pathways are essential for 53BP1 regulation in response to ionizing radiation, whereas RNF8-independent pathways contribute to 53BP1 targeting and phosphorylation in response to UV light and potentially other forms of DNA replication stress.     

DNA双链断裂损伤反应及它的医学意义

DNA损伤应激反应是维持基因组稳定性的基石.细胞在长期进化中形成了由损伤监视、周期调控、损伤修复、凋亡诱导等在内的自稳平衡机制.一方面,借助感应、识别并启动精细而复杂的修复机制修复损伤;另一方面,通过DNA损伤应激活化的细胞周期检查点机制,延迟或阻断细胞周期进程,为损伤修复提供时间,使细胞能安全进入新一轮细胞周期;损伤无法修复时则诱导细胞凋亡.DNA双链断裂(double strand breaks,DSBs)是真核基因组后果最严重的损伤类型之一,其修复不利,同肿瘤等人类疾病的发生发展密切相关.新进展揭示:DSBs损伤反应信号分子ATM-Chk2-p53、H2AX等的组成性活化,是肿瘤形成早期所激活的细胞内可诱导的抗癌屏障,其信号网络的精确、精细调控在基因组稳定性维持中发挥重要作用.此外,HIV病毒整合进入宿主细胞基因组的过程也依赖于宿主细胞中ATM介导的DSBs损伤反应信号转导;ATM特异性的小分子抑制剂在抗HIV感染中显示重要的功能意义.文中重点讨论调控DSBs损伤应激反应信号网络的主要研究进展,及其在肿瘤发生、发展及抗HIV感染中的新医学意义.     

Regulation of cellular and SV40 virus origins of replication by Chk1-dependent intrinsic and UVC radiation-induced checkpoints

DNA replication is inhibited by DNA damage through cis effects on replication fork progression and trans effects associated with checkpoints. In this study, we employed a combined pulse labeling and neutral-neutral two-dimensional gel-based approach to compare the effects of a DNA damaging agent frequently employed to invoke checkpoints, UVC radiation, on the replication of cellular and simian virus 40 (SV40) chromosomes in intact cells. UVC radiation induced similar inhibitory effects on the initiation and elongation phases of cellular and SV40 DNA replication. The initiation-inhibitory effects occurred independently of p53 and were abrogated by the ATM and ATR kinase inhibitor caffeine, or the Chk1 kinase inhibitor UCN-01. Inhibition of cellular origins was also abrogated by the expression of a dominant-negative Chk1 mutant. These results indicate that UVC induces a Chk1- and ATR or ATM-dependent checkpoint that targets both cellular and SV40 viral replication origins. Loss of Chk1 and ATR or ATM function also stimulated initiation of cellular and viral DNA replication in the absence of UVC radiation, revealing the existence of a novel intrinsic checkpoint that targets both cellular and SV40 viral origins of replication in the absence of DNA damage or stalled DNA replication forks. This checkpoint inhibits the replication in early S phase cells of a region of the repetitive rDNA locus that replicates in late S phase. The ability to detect these checkpoints using the well characterized SV40 model system should facilitate analysis of the molecular basis for these effects.     

Squalene Inhibits ATM-Dependent Signaling in γIR-Induced DNA Damage Response through Induction of Wip1 Phosphatase

Ataxia telangiectasia mutated (ATM) kinase plays a crucial role as a master controller in the cellular DNA damage response. Inhibition of ATM leads to inhibition of the checkpoint signaling pathway. Hence, addition of checkpoint inhibitors to anticancer therapies may be an effective targeting strategy. A recent study reported that Wip1, a protein phosphatase, de-phosphorylates serine 1981 of ATM during the DNA damage response. Squalene has been proposed to complement anticancer therapies such as chemotherapy and radiotherapy; however, there is little mechanistic information supporting this idea. Here, we report the inhibitory effect of squalene on ATM-dependent DNA damage signals. Squalene itself did not affect cell viability and the cell cycle of A549 cells, but it enhanced the cytotoxicity of gamma-irradiation (γIR). The in vitro kinase activity of ATM was not altered by squalene. However, squalene increased Wip1 expression in cells and suppressed ATM activation in γIR-treated cells. Consistent with the potential inhibition of ATM by squalene, IR-induced phosphorylation of ATM effectors such as p53 (Ser15) and Chk1 (Ser317) was inhibited by cell treatment with squalene. Thus, squalene inhibits the ATM-dependent signaling pathway following DNA damage through intracellular induction of Wip1 expression.     

Simian virus 40 large T antigen disrupts genome integrity and activates a DNA damage response via Bub1 binding

Simian virus 40 (SV40) large T antigen (LT) is a multifunctional protein that is important for viral replication and oncogenic transformation. Previously, infection of monkey or human cells with SV40 was shown to lead to the induction of DNA damage response signaling, which is required for efficient viral replication. However, it was not clear if LT is sufficient to induce the damage response and, if so, what the genetic requirements and functional consequences might be. Here, we show that the expression of LT alone, without a replication origin, can induce key DNA damage response markers including the accumulation of γ-H2AX and 53BP1 in nuclear foci. Other DNA damage-signaling components downstream of ATM/ATR kinases were induced, including chk1 and chk2. LT also bound the Claspin mediator protein, which normally facilitates the ATR activation of chk1 and monitors cellular replication origins. Stimulation of the damage response by LT depends mainly on binding to Bub1 rather than to the retinoblastoma protein. LT has long been known to stabilize p53 despite functionally inactivating it. We show that the activation of a DNA damage response by LT via Bub1 appears to play a major role in p53 stabilization by promoting the phosphorylation of p53 at Ser15. Accompanying the DNA damage response, LT induces tetraploidy, which is also dependent on Bub1 binding. Taken together, our data suggest that LT, via Bub1 binding, breaches genome integrity mechanisms, leading to DNA damage responses, p53 stabilization, and tetraploidy.