ATR-mediated DNA Damage Checkpoint Research Articles

Bringing It All Together: Coupling Excision Repair to the DNA Damage Checkpoint.

Nucleotide excision repair and the ATR-mediated DNA damage checkpoint are two critical cellular responses to the genotoxic stress induced by ultraviolet (UV) light and are important for cancer prevention. In vivo genetic data indicate that these global responses are coupled. Aziz Sancar et al. developed an in vitro coupled repair-checkpoint system to analyze the basic steps of these DNA damage stress responses in a biochemically defined system. The minimum set of factors essential for repair-checkpoint coupling include damaged DNA, the excision repair factors (XPA, XPC, XPF-ERCC1, XPG, TFIIH, RPA), the 5'-3' exonuclease EXO1, and the damage checkpoint proteins ATR-ATRIP and TopBP1. This coupled repair-checkpoint system was used to demonstrate that the ~30 nucleotide single-stranded DNA (ssDNA) gap generated by nucleotide excision repair is enlarged by EXO1 and bound by RPA to generate the signal that activates ATR.

WWOX modulates the ATR-mediated DNA damage checkpoint response.

For many decades genomic instability is considered one of the hallmarks of cancer. Role of the tumor suppressor WWOX (WW domain-containing oxidoreductase) in DNA damage response upon double strand breaks has been recently revealed. Here we demonstrate unforeseen functions for WWOX upon DNA single strand breaks (SSBs) checkpoint activation. We found that WWOX levels are induced following SSBs and accumulate in the nucleus. WWOX deficiency is associated with reduced activation of ataxia telangiectasia and Rad3-related protein (ATR) checkpoint proteins and increased chromosomal breaks. At the molecular level, we show that upon SSBs WWOX is modified at lysine 274 by ubiquitination mediated by the ubiquitin E3 ligase ITCH and interacts with ataxia telangiectasia-mutated (ATM). Interestingly, ATM inhibition was associated with reduced activation of ATR checkpoint proteins suggesting that WWOX manipulation of ATR checkpoint proteins is ATM-dependent. Taken together, the present findings indicate that WWOX plays a key role in ATR checkpoint activation, while its absence might facilitate genomic instability.

Functions and regulation of the multitasking FANCM family of DNA motor proteins.

Members of the conserved FANCM family of DNA motor proteins play key roles in genome maintenance processes. FANCM supports genome duplication and repair under different circumstances and also functions in the ATR-mediated DNA damage checkpoint. Some of these roles are shared among lower eukaryotic family members. Human FANCM has been linked to Fanconi anemia, a syndrome characterized by cancer predisposition, developmental disorder, and bone marrow failure. Recent studies on human FANCM and its orthologs from other organisms have provided insights into their biological functions, regulation, and collaboration with other genome maintenance factors. This review summarizes the progress made, with the goal of providing an integrated view of the functions and regulation of these enzymes in humans and model organisms and how they advance our understanding of genome maintenance processes.

Coupling of Human DNA Excision Repair and the ATR‐mediated DNA Damage Checkpoint

DNA repair and DNA damage checkpoints work in concert to help maintain genomic integrity. In vivo data suggest that these two global responses to DNA damage are coupled. It has been proposed that the single-stranded DNA gap generated by nucleotide excision repair is the signal that activates the ATR-mediated DNA damage checkpoint response and that the signal is enhanced by gap enlargement by EXO1. Here we have used purified core nucleotide excision repair factors (RPA, XPA, XPC, TFIIH, XPG, and XPF-ERCC1), core DNA damage checkpoint proteins (ATR-ATRIP, TopBP1, RPA), and DNA damaged by a UV-mimetic agent to analyze the basic steps of DNA damage checkpoint response in a biochemically defined system. We find that checkpoint signaling as measured by phosphorylation of target proteins by the ATR kinase requires enlargement of the excision gap generated by the excision repair system by the 5′ to 3′ exonuclease activity of EXO1. We conclude that, in addition to damaged DNA, RPA, XPA, XPC, TFIIH, XPG, XPF-ERCC1, ATR-ATRIP, TopBP1, and EXO1 constitute the minimum essential set of factors for ATR-mediated DNA damage checkpoint response.

The Circadian Clock Controls Sunburn Apoptosis and Erythema in Mouse Skin

Epidemiological studies of humans and experimental studies with mouse models suggest that sunburn resulting from exposure to excessive UV light and damage to DNA confers an increased risk for melanoma and non-melanoma skin cancer. Previous reports have shown that both nucleotide excision repair, which is the sole pathway in humans for removing UV photoproducts, and DNA replication, are regulated by the circadian clock in mouse skin. Furthermore, the timing of UV exposure during the circadian cycle has been shown to affect skin carcinogenesis in mice. Because sunburn and skin cancer are causally related, we investigated UV-induced sunburn apoptosis and erythema in mouse skin as a function of circadian time. Interestingly, we observed that sunburn apoptosis, inflammatory cytokine induction, and erythema were maximal following an acute early morning exposure to UV and minimal following an afternoon exposure. Early morning exposure to UV also produced maximal activation of Atr-mediated DNA damage checkpoint signaling including activation of the tumor suppressor p53, which is known to control the process of sunburn apoptosis. To our knowledge these data provide the first evidence that the circadian clock plays an important role in the erythemal response in UV-irradiated skin. The early morning is when DNA repair is at a minimum, thus the acute responses likely are associated with unrepaired DNA damage. The prior report that mice are more susceptible to skin cancer induction following chronic irradiation in the AM, when p53 levels are maximally induced, is discussed in terms of the mutational inactivation of p53 during chronic irradiation.

E3 Ubiquitin Ligase HOIP Attenuates Apoptotic Cell Death Induced by Cisplatin

The genotoxin cisplatin is commonly used in chemotherapy to treat solid tumors, yet our understanding of the mechanism underlying the drug response is limited. In a focused siRNA screen, using an siRNA library targeting genes involved in ubiquitin and ubiquitin-like signaling, we identified the E3 ubiquitin ligase HOIP as a key regulator of cisplatin-induced genotoxicity. HOIP forms, with SHARPIN and HOIL-1L, the linear ubiquitin assembly complex (LUBAC). We show that cells deficient in the HOIP ligase complex exhibit hypersensitivity to cisplatin. This is due to a dramatic increase in caspase-8/caspase-3-mediated apoptosis that is strictly dependent on ATM-, but not ATR-mediated DNA damage checkpoint activation. Moreover, basal and cisplatin-induced activity of the stress response kinase JNK is enhanced in HOIP-depleted cells and, conversely, JNK inhibition can increase cellular resistance to cisplatin and reverse the apoptotic hyperactivation in HOIP-depleted cells. Furthermore, we show that HOIP depletion sensitizes cancer cells, derived from carcinomas of various origins, through an enhanced apoptotic cell death response. We also provide evidence that ovarian cancer cells classified as cisplatin-resistant can regain sensitivity following HOIP downregulation. Cumulatively, our study identifies a HOIP-regulated antiapoptotic signaling pathway, and we envisage HOIP as a potential target for the development of combinatorial chemotherapies to potentiate the efficacy of platinum-based anticancer drugs.

Coupling of Human DNA Excision Repair and the DNA Damage Checkpoint in a Defined in Vitro System

DNA repair and DNA damage checkpoints work in concert to help maintain genomic integrity. In vivo data suggest that these two global responses to DNA damage are coupled. It has been proposed that the canonical 30 nucleotide single-stranded DNA gap generated by nucleotide excision repair is the signal that activates the ATR-mediated DNA damage checkpoint response and that the signal is enhanced by gap enlargement by EXO1 (exonuclease 1) 5' to 3' exonuclease activity. Here we have used purified core nucleotide excision repair factors (RPA, XPA, XPC, TFIIH, XPG, and XPF-ERCC1), core DNA damage checkpoint proteins (ATR-ATRIP, TopBP1, RPA), and DNA damaged by a UV-mimetic agent to analyze the basic steps of DNA damage checkpoint response in a biochemically defined system. We find that checkpoint signaling as measured by phosphorylation of target proteins by the ATR kinase requires enlargement of the excision gap generated by the excision repair system by the 5' to 3' exonuclease activity of EXO1. We conclude that, in addition to damaged DNA, RPA, XPA, XPC, TFIIH, XPG, XPF-ERCC1, ATR-ATRIP, TopBP1, and EXO1 constitute the minimum essential set of factors for ATR-mediated DNA damage checkpoint response.

Modulation of ATR-mediated DNA damage checkpoint response by cryptochrome 1

Mammalian cryptochromes (Crys) are essential circadian clock factors implicated in diverse clock-independent physiological functions, including DNA damage responses. Here we show that Cry1 modulates the ATR-mediated DNA damage checkpoint (DDC) response by interacting with Timeless (Tim) in a time-of-day-dependent manner. The DDC capacity in response to UV irradiation showed a circadian rhythm. Interestingly, clock-deficient Cry1 and Cry2 double knockout (CryDKO) cells retained substantial DDC capacity compared with clock-proficient wild-type cells, although the Cry1-modulated oscillation of the DDC capacity was abolished in CryDKO cells. We found temporal interaction of Cry1 and Tim in the nucleus. When Cry1 was expressed in the nucleus, it was critical for circadian ATR activity. We regenerated rhythmic DDC responses by ectopically expressing Cry1 in CryDKO cells. In addition, we also investigated the DDC capacity in the liver of mice that were intraperitoneally injected with cisplatin at different circadian times (CT). When mice were injected at CT20, about 2-fold higher expression of phosphorylated minichromosome maintenance protein 2 (p-MCM2) was detected compared with mice injected at CT08, which consequently affected the removal rate of cisplatin-DNA adducts from genomic DNA. Taken together, our data demonstrate the intimate interaction between the circadian clock and the DDC system during genotoxic stress in clock-ticking cells.

Differential Effect of Schisandrin B Stereoisomers on ATR-Mediated DNA Damage Checkpoint Signaling

We have previously reported that schisandrin B (SchB) is a specific inhibitor of ATR (ataxia telangiectasia and Rad-3-related) protein kinase. Since SchB consists of a mixture of its diastereomers gomisin N (GN) and γ-schisandrin (γ-Sch), the inhibitory action of SchB might result from a stereospecific interaction between one of the stereoisomers of SchB and ATR. Therefore, we investigated the effect of GN and γ-Sch on UV (UVC at 254 nm)-induced activation of DNA damage checkpoint signaling in A549 cells. UV-induced cell death (25 – 75 J/m2) was amplified by the presence of the diastereomers, especially GN. At the same time, GN, but not γ-Sch, inhibited the phosphorylation of checkpoint proteins such as p53, structural maintenance of chromosomes 1, and checkpoint kinase 1 in UV-irradiated cells. Moreover, GN inhibited the G2/M checkpoint during UV-induced DNA damage. The in vitro kinase activity of immunoaffinity-purified ATR was dose-dependently inhibited by GN (IC50: 7.28 μM) but not by γ-Sch. These results indicate that GN is the active component of SchB and suggest that GN inhibits the DNA damage checkpoint signaling by stereospecifically interacting with ATR.

Inhibition of ATR-Dependent Signaling by Protoapigenone and Its Derivative Sensitizes Cancer Cells to Interstrand Cross-link–Generating Agents In Vitro and In Vivo

DNA damage caused during cancer treatment can rapidly activate the ataxia telangiectasia-mutated (ATM) and ATM and Rad3-related (ATR)-dependent phosphorylation of Chk2 and Chk1 kinases, which are hallmarks of the DNA damage response (DDR). Pharmacologic inhibition of ATR causes a synthetic lethal effect on ATM- or p53-defective cancers, suggesting that such inhibition is an effective way to improve the sensitivity of cancers to DNA-damaging agents. Here, both the natural compound protoapigenone (WYC02) and its synthetic derivative WYC0209 exhibited cytotoxic effects on various cancer cell lines. WYC02 causes chromosomal aberration in the mitotic spreads of Chinese hamster ovary cells. Interestingly, cancer cells did not exhibit typical DDR markers upon exposure to WYC02 and WYC0209 (WYCs). Further investigation into the molecular mechanisms of WYCs function revealed that they have a potential ability to inhibit DDR, particularly on activation of Chk1 and Fanconi anemia group D2 protein (FANCD2), but not Chk2. In this way, WYCs inhibited ATR-mediated DNA damage checkpoint and repair. Furthermore, when combined with the DNA cross-linking agent cisplatin, treatment with WYCs resulted in increased tumor sensitivity to interstrand cross-link-generating agents both in vitro and in vivo. Our results therefore especially implicate WYCs in enhancing tumor chemosensitivity when the ATR checkpoint is constitutively active in states of oncogene-driven replicative stress or tolerance to DNA-interfering agents.

Requirement of MTA1 in ATR-mediated DNA Damage Checkpoint Function

MTA1 (metastasis-associated protein 1), an integral component of the nucleosome remodeling and deacetylase complex, has recently been implicated in the ionizing radiation-induced DNA damage response. However, whether MTA1 also participates in the UV-induced DNA damage checkpoint pathway remains unknown. In response to UV radiation, ATR (ataxia teleangiectasia- and Rad3-related) is the major kinase activated that orchestrates cell cycle progression with DNA repair machinery by phosphorylating and activating a number of downstream substrates, such as Chk1 (checkpoint kinase 1) and H2AX (histone 2A variant X). Here, we report that UV radiation stabilizes MTA1 in an ATR-dependent manner and increases MTA1 binding to ATR. On the other hand, depletion of MTA1 compromises the ATR-mediated Chk1 activation following UV treatment, accompanied by a marked down-regulation of Chk1 and its interacting partner Claspin, an adaptor protein that is required for the phosphorylation and activation of Chk1 by ATR. Furthermore, MTA1 deficiency decreases the induction of phosphorylated H2AX (referred to as gamma-H2AX) and gamma-H2AX focus formation after UV treatment. Consequently, depletion of MTA1 results in a defect in the G(2)-M checkpoint and increases cellular sensitivity to UV-induced DNA damage. Thus, MTA1 is required for the activation of the ATR-Claspin-Chk1 and ATR-H2AX pathways following UV treatment, and the noted abrogation of the DNA damage checkpoint in the MTA1-depleted cells may be, at least in part, a consequence of dysregulation of the expression of these two pathways. These findings suggest that, in addition to its role in the repair of double strand breaks caused by ionizing radiation, MTA1 also participates in the UV-induced ATR-mediated DNA damage checkpoint pathway.

The human ATR-mediated DNA damage checkpoint in a reconstituted system

DNA damage checkpoints are signal transduction pathways that coordinate the cell cycle with other cellular responses to DNA damage in order to maintain genomic integrity. The ATR kinase is responsible for initiating the DNA damage checkpoint in response to UV-damaged DNA and incompletely replicated DNA. We have recently established an in vitro system that recapitulates most but not all features of the human ATR-mediated DNA damage checkpoint response. With this system we have shown that damaged DNA can be a direct signal for ATR activation, and that TopBP1 specifically stimulates ATR in the presence of damaged DNA under physiological ionic strength conditions. This system provides a powerful tool to gain insight into the molecular mechanism of the ATR pathway. Here we describe preparation of the checkpoint components and our specific kinase assay in more detail.

Role of Claspin in regulation of nucleotide excision repair factor DDB2

The replication checkpoint protein Claspin is important for maintenance of genomic stability and is required for cells to overcome genotoxic stress. Upon UV-induced DNA damage, Claspin is required for activation of the ATR-mediated DNA damage checkpoint response, leading to arrest of DNA replication and inhibition of cell cycle progression. Located at the DNA replication fork, Claspin is also suggested to monitor replication and sense damage. Our present studies in HeLa cells demonstrate associations between the Claspin/ATR-related DNA damage checkpoint response and the global genomic nucleotide excision repair pathway. siRNA-mediated knockdown of Claspin abolishes the UV-induced degradation of DDB2 and impairs the co-localization of DDB2 to DNA damage sites. Thus, the presence of Claspin is required for the total turnover of DNA damage binding protein DDB2, as well as for its functionality in DNA damage recognition. Claspin, however, seems not to be required for maintaining the cellular level of the NER factor XPC and its UV-induced post-translational modifications. Co-localization of XPC with DNA lesions is also intact in the absence of Claspin as is the repair of the UV-induced lesions CPD and 6-4PP. Claspin itself may be directly responsible for physical interaction between the two pathways since Claspin is able to associate with DDB1, DDB2 and XPC. Taken together, these findings reveal physical and functional interplay between Claspin and NER-related proteins and demonstrate crosstalk between the DNA damage checkpoint control and DNA damage repair pathways.

Telomerase-independent Regulation of ATR by Human Telomerase RNA

The human telomerase RNA (hTR), together with the telomerase reverse transcriptase, hTERT, constitute the core components of telomerase that is essential for telomere maintenance. While hTR is ubiquitously expressed, hTERT is normally restricted to germ cells and certain stem cells, but both are often deregulated during tumorigenesis. Here, we investigated the effects of changes in hTR cellular levels. Surprisingly, while inhibition of hTR expression triggers a rapid, telomerase-independent, growth arrest associated with p53 and CHK1 activation, its increased expression neutralizes activation of these pathways in response to genotoxic stress. These hTR effects are mediated through ATR and are sufficiently strong to impair ATR-mediated DNA-damage checkpoint responses. Furthermore, in response to low UV radiation, which activates ATR, endogenous hTR levels increase irrespective of telomerase status. Thus, we uncovered a novel, telomerase-independent, function of hTR that restrains ATR activity and participates in the recovery of cells from UV radiation.