Activation of the Prereplication Complex Is Blocked by Mimosine through Reactive Oxygen Species-activated Ataxia Telangiectasia Mutated (ATM) Protein without DNA Damage

Mimosine is an effective cell synchronization reagent used for arresting cells in late G₁ phase. However, the mechanism underlying mimosine-induced G₁ cell cycle arrest remains unclear. Using highly synchronous cell populations, we show here that mimosine blocks S phase entry through ATM activation. HeLa S3 cells are exposed to thymidine for 15 h, released for 9 h by washing out the thymidine, and subsequently treated with 1 mm mimosine for a further 15 h (thymidine → mimosine). In contrast to thymidine-induced S phase arrest, mimosine treatment synchronizes >90% of cells at the G₁-S phase boundary by inhibiting the transition of the prereplication complex to the preinitiation complex. Mimosine treatment activates ataxia telangiectasia mutated (ATM)/ataxia telangiectasia and Rad3-related (ATR)-mediated checkpoint signaling without inducing DNA damage. Inhibition of ATM activity is found to induce mimosine-arrested cells to enter S phase. In addition, ATM activation by mimosine treatment is mediated by reactive oxygen species (ROS). These results suggest that, upon mimosine treatment, ATM blocks S phase entry in response to ROS, which prevents replication fork stalling-induced DNA damage.     

Thymine DNA Glycosylase Is a CRL4Cdt2 Substrate

The E3 ubiquitin ligase CRL4^Cdt2 targets proteins for destruction in S phase and after DNA damage by coupling ubiquitylation to DNA-bound proliferating cell nuclear antigen (PCNA). Coupling to PCNA involves a PCNA-interacting peptide (PIP) degron motif in the substrate that recruits CRL4^Cdt2 while binding to PCNA. In vertebrates, CRL4^Cdt2 promotes degradation of proteins whose presence in S phase is deleterious, including Cdt1, Set8, and p21. Here, we show that CRL4^Cdt2 targets thymine DNA glycosylase (TDG), a base excision repair enzyme that is involved in DNA demethylation. TDG contains a conserved and nearly perfect match to the PIP degron consensus. TDG is ubiquitylated and destroyed in a PCNA-, Cdt2-, and PIP degron-dependent manner during DNA repair in Xenopus egg extract. The protein can also be destroyed during DNA replication in this system. During Xenopus development, TDG first accumulates during gastrulation, and its expression is down-regulated by CRL4^Cdt2. Our results expand the group of vertebrate CRL4^Cdt2 substrates to include a bona fide DNA repair enzyme.     

Proliferating cell nuclear antigen-dependent rapid recruitment of Cdt1 and CRL4Cdt2 at DNA-damaged sites after UV irradiation in HeLa cells

The licensing factor Cdt1 is degraded by CRL4(Cdt2) ubiquitin ligase dependent on proliferating cell nuclear antigen (PCNA) during S phase and when DNA damage is induced in G(1) phase. Association of both Cdt2 and PCNA with chromatin was observed in S phase and after UV irradiation. Here we used a micropore UV irradiation assay to examine Cdt2 accumulation at cyclobutane pyrimidine dimer-containing DNA-damaged sites in the process of Cdt1 degradation in HeLa cells. Cdt2, present in the nucleus throughout the cell cycle, accumulated rapidly at damaged DNA sites during G(1) phase. The recruitment of Cdt2 is dependent on prior PCNA chromatin binding because Cdt2 association was prevented when PCNA was silenced. Cdt1 was also recruited to damaged sites soon after UV irradiation through its PIP-box. As Cdt1 was degraded, the Cdt2 signal at damaged sites was reduced, but PCNA, cyclobutane pyrimidine dimer, and XPA (xeroderma pigmentosum, complementation group A) signals remained at the same levels. These findings suggest that Cdt1 degradation following UV irradiation occurs rapidly at damaged sites due to PCNA chromatin loading and the recruitment of Cdt1 and CRL4(Cdt2), before DNA damage repair is completed.     

Analysis of mutations that dissociate G(2) and essential S phase functions of human ataxia telangiectasia-mutated and Rad3-related (ATR) protein kinase

ATR (ataxia telangiectasia-mutated and Rad3-related) contains 16 conserved candidate autophosphorylation sites that match its preferred S/TQ consensus. To determine whether any is functionally important, we mutated the 16 candidate residues to alanine in a single cDNA to create a 16A-ATR mutant. The 16A-ATR mutant maintains kinase and G(2) checkpoint activities. However, it fails to rescue the essential function of ATR in maintaining cell viability and fails to promote replication recovery from a transient exposure to replication stress. Further analysis identified T1566A/T1578A/T1589A (3A-ATR) as critical mutations causing this separation of function activity. Secondary structure predictions indicate that these residues occur in a region between ATR HEAT repeats 31R and 32R that aligns with regions of ATM and DNA-PK containing regulatory autophosphorylation sites. Although this region is important for ATR function, the 3A-ATR residues do not appear to be sites of autophosphorylation. Nevertheless, our analysis identifies an important regulatory region of ATR that is shared among the PI3K-related protein kinase family. Furthermore, our data indicate that the essential function of ATR for cell viability is linked to its function in promoting proper replication in the context of replication stress and is independent of G(2) checkpoint activity.     

Dyrk2-associated EDD-DDB1-VprBP E3 Ligase Inhibits Telomerase by TERT Degradation

Telomerase maintains the telomere, a specialized chromosomal end structure that is essential for genomic stability and cell immortalization. Telomerase is not active in most somatic cells, but its reactivation is one of the hallmarks of cancer. In this study, we found that dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 2 (Dyrk2) negatively regulates telomerase activity. Dyrk2 phosphorylates TERT protein, a catalytic subunit of telomerase. Phosphorylated TERT is then associated with the EDD-DDB1-VprBP E3 ligase complex for subsequent ubiquitin-mediated TERT protein degradation. During the cell cycle, Dyrk2 interacts with TERT at the G2/M phase and induces degradation. In contrast, depletion of endogenous Dyrk2 disrupts the cell cycle-dependent regulation of TERT and elicits the constitutive activation of telomerase. Similarly, a Dyrk2 nonsense mutation identified in breast cancer compromises ubiquitination-mediated TERT protein degradation. Our findings suggest the novel molecular mechanism of kinase-associated telomerase regulation.     

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.     

Interplay between the DNA Damage Proteins MDC1 and ATM in the Regulation of the Spindle Assembly Checkpoint

To avoid genomic instability, cells have developed surveillance mechanisms such as the spindle assembly checkpoint (SAC) and the DNA damage response. ATM and MDC1 are central players of the cellular response to DNA double-strand breaks. Here, we identify a new role for these proteins in the regulation of mitotic progression and in SAC activation. MDC1 localizes at mitotic kinetochores following SAC activation in an ATM-dependent manner. ATM phosphorylates histone H2AX at mitotic kinetochores, and this phosphorylation is required for MDC1 localization at kinetochores. ATM and MDC1 are needed for kinetochore localization of the inhibitory mitotic checkpoint complex components, Mad2 and Cdc20, and for the maintenance of the mitotic checkpoint complex integrity. This probably relies on the interaction of MDC1 with the MCC. In this work, we have established that ATM and MDC1 maintain genomic stability not only by controlling the DNA damage response, but also by regulating SAC activation, providing an important link between these two essential biological processes.     

A Dbf4 mutant contributes to bypassing the Rad53-mediated block of origins of replication in response to genotoxic stress

An intra-S phase checkpoint slows the rate of DNA replication in response to DNA damage and replication fork blocks in eukaryotic cells. In the budding yeast Saccharomyces cerevisiae, such down-regulation is achieved through the Rad53 kinase-dependent block of origins of replication. We have identified the Rad53 phosphorylation sites on Dbf4, the activator subunit of the essential S phase Dbf4-dependent kinase, and generated a non-phosphorylatable Dbf4 mutant (dbf4(7A)). We show here that dbf4(7A) is a bona fide intra-S phase checkpoint bypass allele that contributes to abrogating the Rad53 block of origin firing in response to genotoxic stress.     

Thr-1989 phosphorylation is a marker of active ataxia telangiectasia-mutated and Rad3-related (ATR) kinase

The DNA damage response kinases ataxia telangiectasia-mutated (ATM), DNA-dependent protein kinase (DNA-PK), and ataxia telangiectasia-mutated and Rad3-related (ATR) signal through multiple pathways to promote genome maintenance. These related kinases share similar methods of regulation, including recruitment to specific nucleic acid structures and association with protein activators. ATM and DNA-PK also are regulated via phosphorylation, which provides a convenient biomarker for their activity. Whether phosphorylation regulates ATR is unknown. Here we identify ATR Thr-1989 as a DNA damage-regulated phosphorylation site. Selective inhibition of ATR prevents Thr-1989 phosphorylation, and phosphorylation requires ATR activation. Cells engineered to express only a non-phosphorylatable T1989A mutant exhibit a modest ATR functional defect. Our results suggest that, like ATM and DNA-PK, phosphorylation regulates ATR, and phospho-peptide specific antibodies to Thr-1989 provide a proximal marker of ATR activation.     

Ataxia Telangiectasia-mutated- and Rad3-related Protein Regulates the DNA Damage-induced G2/M Checkpoint through the Aurora A Cofactor Bora Protein

Polo-like kinase1 (Plk1) activation is inhibited in response to DNA damage, and this inhibition contributes to the activation of the G₂/M checkpoint, although the molecular mechanism by which Plk1 is inhibited is not clear. Here we report that the DNA damage signaling pathway inhibits Plk1 activity through Bora. Following UV irradiation, ataxia telangiectasia-mutated- and Rad3-related protein phosphorylates Bora at Thr-501. The phosphorylated Thr-501 is subsequently recognized by the E3 ubiquitin ligase SCF-β-TRCP, which targets Bora for degradation. The degradation of Bora compromises Plk1 activation and contributes to DNA damage-induced G₂ arrest. These findings shed new light on Plk1 regulation by the DNA damage response pathway.     

The PSO4 Protein Complex Associates with Replication Protein A (RPA) and Modulates the Activation of Ataxia Telangiectasia-mutated and Rad3-related (ATR)

The PSO4 core complex is composed of PSO4/PRP19/SNEV, CDC5L, PLRG1, and BCAS2/SPF27. Besides its well defined functions in pre-mRNA splicing, the PSO4 complex has been shown recently to participate in the DNA damage response. However, the specific role for the PSO4 complex in the DNA damage response pathways is still not clear. Here we show that both the BCAS2 and PSO4 subunits of the PSO4 complex directly interact and colocalize with replication protein A (RPA). Depletion of BCAS2 or PSO4 impairs the recruitment of ATR-interacting protein (ATRIP) to DNA damage sites and compromises CHK1 activation and RPA2 phosphorylation. Moreover, we demonstrate that both the RPA1-binding ability of BCAS2 and the E3 ligase activity of PSO4 are required for efficient accumulation of ATRIP at DNA damage sites and the subsequent CHK1 activation and RPA2 phosphorylation. Our results suggest that the PSO4 complex functionally interacts with RPA and plays an important role in the DNA damage response.     

Nuclear BAG6-UBL4A-GET4 Complex Mediates DNA Damage Signaling and Cell Death

BCL2-associated athanogene 6 (BAG6) is a member of the BAG protein family, which is implicated in diverse cellular processes including apoptosis, co-chaperone, and DNA damage response (DDR). Recently, it has been shown that BAG6 forms a stable complex with UBL4A and GET4 and functions in membrane protein targeting and protein quality control. The BAG6 sequence contains a canonical nuclear localization signal and is localized predominantly in the nucleus. However, GET4 and UBL4A are found mainly in cytoplasm. Whether GET4 and UBL4A are also involved in DDR in the context of the BAG6 complex remains unknown. Here, we provide evidence that nuclear BAG6-UBL4A-GET4 complex mediates DDR signaling and damage-induced cell death. BAG6 appears to be the central component for the process, as depletion of BAG6 leads to the loss of both UBL4A and GET4 proteins and resistance to cell killing by DNA-damaging agents. In addition, nuclear localization of BAG6 and phosphorylation of BAG6 by ATM/ATR are also required for cell killing. UBL4A and GET4 translocate to the nucleus upon DNA damage and appear to play redundant roles in cell killing, as depletion of either one has no effect but co-depletion leads to resistance. All three components of the BAG6 complex are required for optimal DDR signaling, as BAG6, and to a lesser extent, GET4 and UBL4A, regulate the recruitment of BRCA1 to sites of DNA damage. Together our results suggest that the nuclear BAG6 complex is an effector in DNA damage response pathway and its phosphorylation and nuclear localization are important determinants for its function.     

Ubiquitin E3 Ligase CRL4CDT2/DCAF2 as a Potential Chemotherapeutic Target for Ovarian Surface Epithelial Cancer

Cullin-RING ubiquitin ligases (CRLs) are the largest family of E3 ligases and require cullin neddylation for their activation. The NEDD8-activating enzyme inhibitor MLN4924 reportedly blocked cullin neddylation and inactivated CRLs, which resulted in apoptosis induction and tumor suppression. However, CRL roles in ovarian cancer cell survival and the ovarian tumor repressing effects of MLN4924 are unknown. We show here that CRL4 components are highly expressed in human epithelial ovarian cancer tissues. MLN4924-induced DNA damage, cell cycle arrest, and apoptosis in ovarian cancer cells in a time- and dose-dependent manner. In addition, MLN4924 sensitized ovarian cancer cells to other chemotherapeutic drug treatments. Depletion of CRL4 components Roc1/2, Cul4a, and DDB1 had inhibitory effects on ovarian cancer cells similar to MLN4924 treatment, which suggested that CRL4 inhibition contributed to the chemotherapeutic effect of MLN4924 in ovarian cancers. We also investigated for key CRL4 substrate adaptors required for ovarian cancer cells. Depleting Vprbp/Dcaf1 did not significantly affect ovarian cancer cell growth, even though it was expressed by ovarian cancer tissues. However, depleting Cdt2/Dcaf2 mimicked the pharmacological effects of MLN4924 and caused the accumulation of its substrate, CDT1, both in vitro and in vivo. MLN4924-induced DNA damage and apoptosis were partially rescued by Cdt1 depletion, suggesting that CRL4^CDT2 repression and CDT1 accumulation were key biochemical events contributing to the genotoxic effects of MLN4924 in ovarian cancer cells. Taken together, these results indicate that CRL4^CDT2 is a potential drug target in ovarian cancers and that MLN4924 may be an effective anticancer agent for targeted ovarian cancer therapy.     

A Novel Non-canonical Forkhead-associated (FHA) Domain-binding Interface Mediates the Interaction between Rad53 and Dbf4 Proteins

Forkhead-associated (FHA) and BRCA1 C-terminal (BRCT) domains are overrepresented in DNA damage and replication stress response proteins. They function primarily as phosphoepitope recognition modules but can also mediate non-canonical interactions. The latter are rare, and only a few have been studied at a molecular level. We have identified a crucial non-canonical interaction between the N-terminal FHA1 domain of the checkpoint effector kinase Rad53 and the BRCT domain of the regulatory subunit of the Dbf4-dependent kinase that is critical to suppress late origin firing and to stabilize stalled forks during replication stress. The Rad53-Dbf4 interaction is phosphorylation-independent and involves a novel non-canonical interface on the FHA1 domain. Mutations within this surface result in hypersensitivity to genotoxic stress. Importantly, this surface is not conserved in the FHA2 domain of Rad53, suggesting that the FHA domains of Rad53 gain specificity by engaging additional interaction interfaces beyond their phosphoepitope-binding site. In general, our results point to FHA domains functioning as complex logic gates rather than mere phosphoepitope-targeting modules.     

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 γ-H2AX) and γ-H2AX focus formation after UV treatment. Consequently, depletion of MTA1 results in a defect in the G₂-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.     

Repair-specific functions of replication protein A

Replication protein A (RPA), the major eukaryotic single-strand DNA (ssDNA)-binding protein, is essential for replication, repair, recombination, and checkpoint activation. Defects in RPA-associated cellular activities lead to genomic instability, a major factor in the pathogenesis of cancer and other diseases. ssDNA binding activity is primarily mediated by two domains in the 70-kDa subunit of the RPA complex. These ssDNA interactions are mediated by a combination of polar residues and four conserved aromatic residues. Mutation of the aromatic residues causes a modest decrease in binding to long (30-nucleotide) ssDNA fragments but results in checkpoint activation and cell cycle arrest in cells. We have used a combination of biochemical analysis and knockdown replacement studies in cells to determine the contribution of these aromatic residues to RPA function. Cells containing the aromatic residue mutants were able to progress normally through S-phase but were defective in DNA repair. Biochemical characterization revealed that mutation of the aromatic residues severely decreased binding to short ssDNA fragments less than 20 nucleotides long. These data indicate that altered binding of RPA to short ssDNA intermediates causes a defect in DNA repair but not in DNA replication. These studies show that cells require different RPA functions in DNA replication and DNA repair.     

The unstructured C-terminal tail of yeast Dpb11 (human TopBP1) protein is dispensable for DNA replication and the S phase checkpoint but required for the G2/M checkpoint

Budding yeast Dpb11 (human TopBP1, fission yeast Cut5) is an essential protein required for replisome assembly and for the DNA damage checkpoint. Previous studies with the temperature-sensitive dpb11-1 allele, truncated at amino acid 583 of the 764-amino acid protein, have suggested the model that Dpb11 couples DNA replication to the replication checkpoint. However, the dpb11-1 allele shows distinct replication defects even at permissive temperatures. Here, we determine that the 1-600-amino acid domain of DPB11 is both required and sufficient for full replication function of Dpb11 but that this domain is defective for activation of the principal checkpoint kinase Mec1 (human ataxia telangiectasia and Rad3-related) in vitro and in vivo. Remarkably, mutants of DPB11 that leave its replication function intact but abrogate its ability to activate Mec1 are proficient for the replication checkpoint, but they are compromised for the G(2)/M DNA damage checkpoint. These data suggest that replication checkpoint defects may result indirectly from defects in replisome assembly. Two conserved aromatic amino acids in the C terminus of Dpb11 are critical for Mec1 activation in vitro and for the G(2)/M checkpoint in yeast. Together with aromatic motifs identified previously in the Ddc1 subunit of 9-1-1, another activator of Mec1 kinase, they define a consensus structure for Mec1 activation.