DNA Damage Checkpoint Recovery Research Articles

The ATM-E6AP-MASTL axis mediates DNA damage checkpoint recovery

Checkpoint activation after DNA damage causes a transient cell cycle arrest by suppressing cyclin-dependent kinases (CDKs). However, it remains largely elusive how cell cycle recovery is initiated after DNA damage. In this study, we discovered the upregulated protein level of MASTL kinase hours after DNA damage. MASTL promotes cell cycle progression by preventing PP2A/B55-catalyzed dephosphorylation of CDK substrates. DNA damage-induced MASTL upregulation was caused by decreased protein degradation, and was unique among mitotic kinases. We identified E6AP as the E3 ubiquitin ligase that mediated MASTL degradation. MASTL degradation was inhibited upon DNA damage as a result of the dissociation of E6AP from MASTL. E6AP depletion reduced DNA damage signaling, and promoted cell cycle recovery from the DNA damage checkpoint, in a MASTL-dependent manner. Furthermore, we found that E6AP was phosphorylated at Ser-218 by ATM after DNA damage and that this phosphorylation was required for its dissociation from MASTL, the stabilization of MASTL, and the timely recovery of cell cycle progression. Together, our data revealed that ATM/ATR-dependent signaling, while activating the DNA damage checkpoint, also initiates cell cycle recovery from the arrest. Consequently, this results in a timer-like mechanism that ensures the transient nature of the DNA damage checkpoint.

The ATM-E6AP-MASTL axis mediates DNA damage checkpoint recovery.

Checkpoint activation after DNA damage causes a transient cell cycle arrest by suppressing cyclin-dependent kinases (CDKs). However, it remains largely elusive how cell cycle recovery is initiated after DNA damage. In this study, we discovered the upregulated protein level of MASTL kinase hours after DNA damage. MASTL promotes cell cycle progression by preventing PP2A/B55-catalyzed dephosphorylation of CDK substrates. DNA damage-induced MASTL upregulation was caused by decreased protein degradation, and was unique among mitotic kinases. We identified E6AP as the E3 ubiquitin ligase that mediated MASTL degradation. MASTL degradation was inhibited upon DNA damage as a result of the dissociation of E6AP from MASTL. E6AP depletion reduced DNA damage signaling, and promoted cell cycle recovery from the DNA damage checkpoint, in a MASTL-dependent manner. Furthermore, we found that E6AP was phosphorylated at Ser-218 by ATM after DNA damage and that this phosphorylation was required for its dissociation from MASTL, the stabilization of MASTL, and the timely recovery of cell cycle progression. Together, our data revealed that ATM/ATR-dependent signaling, while activating the DNA damage checkpoint, also initiates cell cycle recovery from the arrest. Consequently, this results in a timer-like mechanism that ensures the transient nature of the DNA damage checkpoint.

The Role of Polo-Like Kinase 1 in Regulating the Forkhead Box Family Transcription Factors.

Polo-like kinase 1 (PLK1) is a serine/threonine kinase with more than 600 phosphorylation substrates through which it regulates many biological processes, including mitosis, apoptosis, metabolism, RNA processing, vesicle transport, and G2 DNA-damage checkpoint recovery, among others. Among the many PLK1 targets are members of the FOX family of transcription factors (FOX TFs), including FOXM1, FOXO1, FOXO3, and FOXK1. FOXM1 and FOXK1 have critical oncogenic roles in cancer through their antagonism of apoptotic signals and their promotion of cell proliferation, metastasis, angiogenesis, and therapeutic resistance. In contrast, FOXO1 and FOXO3 have been identified to have broad functions in maintaining cellular homeostasis. In this review, we discuss PLK1-mediated regulation of FOX TFs, highlighting the effects of PLK1 on the activity and stability of these proteins. In addition, we review the prognostic and clinical significance of these proteins in human cancers and, more importantly, the different approaches that have been used to disrupt PLK1 and FOX TF-mediated signaling networks. Furthermore, we discuss the therapeutic potential of targeting PLK1-regulated FOX TFs in human cancers.

Systems biology approach reveals a link between mTORC1 and G2/M DNA damage checkpoint recovery

Checkpoint recovery, the process that checkpoint-arrested cells with normal DNA repair capacity resume cell cycle progression, is essential for genome stability. However, the signaling network of the process has not been clearly defined. Here, we combine functional proteomics, mathematical modeling, and molecular biology to identify mTORC1, the nutrient signaling integrator, as the determinant for G2/M checkpoint recovery. Inhibition of the mTORC1 pathway delays mitotic entry after DNA damage through KDM4B-mediated regulation of CCNB1 and PLK1 transcription. Cells with hyper-mTORC1 activity caused by TSC2 depletion exhibit accelerated G2/M checkpoint recovery. Those Tsc2-null cells are sensitive to WEE1 inhibition in vitro and in vivo by driving unscheduled mitotic entry and inducing mitotic catastrophe. These results reveal that mTORC1 functions as a mediator between nutrition availability sensing and cell fate determination after DNA damage, suggesting that checkpoint inhibitors may be used to treat mTORC1-hyperactivated tumors such as those associated with tuberous sclerosis complex.

Discovery of a dual protease mechanism that promotes DNA damage checkpoint recovery.

The DNA damage response is a signaling pathway found throughout biology. In many bacteria the DNA damage checkpoint is enforced by inducing expression of a small, membrane bound inhibitor that delays cell division providing time to repair damaged chromosomes. How cells promote checkpoint recovery after sensing successful repair is unknown. By using a high-throughput, forward genetic screen, we identified two unrelated proteases, YlbL and CtpA, that promote DNA damage checkpoint recovery in Bacillus subtilis. Deletion of both proteases leads to accumulation of the checkpoint protein YneA. We show that DNA damage sensitivity and increased cell elongation in protease mutants depends on yneA. Further, expression of YneA in protease mutants was sufficient to inhibit cell proliferation. Finally, we show that both proteases interact with YneA and that one of the two proteases, CtpA, directly cleaves YneA in vitro. With these results, we report the mechanism for DNA damage checkpoint recovery in bacteria that use membrane bound cell division inhibitors.

Dihydrocoumarin, an HDAC Inhibitor, Increases DNA Damage Sensitivity by Inhibiting Rad52.

Effective DNA repair enables cancer cells to survive DNA damage induced by chemotherapeutic or radiotherapeutic treatments. Therefore, inhibiting DNA repair pathways is a promising therapeutic strategy for increasing the efficacy of such treatments. In this study, we found that dihydrocoumarin (DHC), a flavoring agent, causes deficiencies in double-stand break (DSB) repair and prolonged DNA damage checkpoint recovery in yeast. Following DNA damage, Rad52 recombinase was revealed to be inhibited by DHC, which results in deficiencies in DSB repair and prolonged DNA damage checkpoint recovery. The deletion of RPD3, a class I histone deacetylase (HDAC), was found to mimic DHC-induced suppression of Rad52 expression, suggesting that the HDAC inhibitor activity of DHC is critical to DSB repair and DNA damage sensitivity. Overall, our findings delineate the regulatory mechanisms of DHC in DSB repair and suggest that it might potentially be used as an inhibitor of the DNA repair pathway in human cells.

Delineation of the role of chromatin assembly and the Rtt101Mms1 E3 ubiquitin ligase in DNA damage checkpoint recovery in budding yeast

The DNA damage checkpoint is activated in response to DNA double-strand breaks (DSBs). We had previously shown that chromatin assembly mediated by the histone chaperone Asf1 triggers inactivation of the DNA damage checkpoint in yeast after DSB repair, also called checkpoint recovery. Here we show that chromatin assembly factor 1 (CAF-1) also contributes to chromatin reassembly after DSB repair, explaining its role in checkpoint recovery. Towards understanding how chromatin assembly promotes checkpoint recovery, we find persistent presence of the damage sensors Ddc1 and Ddc2 after DSB repair in asf1 mutants. The genes encoding the E3 ubiquitin ligase complex Rtt101Mms1 are epistatic to ASF1 for survival following induction of a DSB, and Rtt101Mms1 are required for checkpoint recovery after DSB repair but not for chromatin assembly. By contrast, the Mms22 substrate adaptor that is degraded by Rtt101Mms1 is required for DSB repair per se. Deletion of MMS22 blocks loading of Rad51 at the DSB, while deletion of ASF1 or RTT101 leads to persistent Rad51 loading. We propose that checkpoint recovery is promoted by Rtt101Mms1-mediated ubiquitylation of Mms22 in order to halt Mms22-dependent loading of Rad51 onto double-stranded DNA after DSB repair, in concert with the chromatin assembly-mediated displacement of Rad51 and checkpoint sensors from the site of repair.

The CDK1 inhibitory kinase MYT1 in DNA damage checkpoint recovery

Inhibition of cyclin-dependent kinase 1 (CDK1) by phosphorylation is a key regulatory mechanism for both the unperturbed cell cycle and the DNA damage checkpoint. Although both WEE1 and MYT1 can phosphorylate CDK1, little is known about the contribution of MYT1. We found that in contrast to WEE1, MYT1 was not important for the normal cell cycle or checkpoint activation. Time-lapse microscopy indicated that MYT1 did, however, have a rate-determining role during checkpoint recovery. Depletion of MYT1 induced precocious mitotic entry when the checkpoint was abrogated with inhibitors of either CHK1 or WEE1, indicating that MYT1 contributes to checkpoint recovery independently of WEE1. The acceleration of checkpoint recovery in MYT1-depleted cells was due to a lowering of threshold for CDK1 activation. The kinase activity of MYT1 was high during checkpoint activation and reduced during checkpoint recovery. Importantly, although depletion of MYT1 alone did not affect long-term cell growth, it potentiated with DNA damage to inhibit cell growth in clonogenic survival and tumor xenograft models. These results reveal the functions of MYT1 in checkpoint recovery and highlight the potential of MYT1 as a target for anti-cancer therapies.

Greatwall and Polo-like Kinase 1 Coordinate to Promote Checkpoint Recovery

Checkpoint recovery upon completion of DNA repair allows the cell to return to normal cell cycle progression and is thus a crucial process that determines cell fate after DNA damage. We previously studied this process in Xenopus egg extracts and established Greatwall (Gwl) as an important regulator. Here we show that preactivated Gwl kinase can promote checkpoint recovery independently of cyclin-dependent kinase 1 (Cdk1) or Plx1 (Xenopus polo-like kinase 1), whereas depletion of Gwl from extracts exhibits no synergy with that of Plx1 in delaying checkpoint recovery, suggesting a distinct but related relationship between Gwl and Plx1. In further revealing their functional relationship, we found mutual dependence for activation of Gwl and Plx1 during checkpoint recovery, as well as their direct association. We characterized the protein association in detail and recapitulated it in vitro with purified proteins, which suggests direct interaction. Interestingly, Gwl interaction with Plx1 and its phosphorylation by Plx1 both increase at the stage of checkpoint recovery. More importantly, Plx1-mediated phosphorylation renders Gwl more efficient in promoting checkpoint recovery, suggesting a functional involvement of such regulation in the recovery process. Finally, we report an indirect regulatory mechanism involving Aurora A that may account for Gwl-dependent regulation of Plx1 during checkpoint recovery. Our results thus reveal novel mechanisms underlying the involvement of Gwl in checkpoint recovery, in particular, its functional relationship with Plx1, a well characterized regulator of checkpoint recovery. Coordinated interplays between Plx1 and Gwl are required for reactivation of these kinases from the G(2)/M DNA damage checkpoint and efficient checkpoint recovery.

Abstract 1999: ATR- and ATM-regulated H3K56 acetylation mediates recovery from UV-induced cell-cycle checkpoint arrest

Abstract DNA damage results in chromatin remodeling to allow prompt access and efficient loading of repair and checkpoint proteins at damage sites to initiate cell cycle arrest and successfully complete DNA repair. Subsequently, chromatin structure is restored and cells begin to recover from checkpoint arrest. This ‘access-repair-restore’ model of DNA repair entails a coordinated functional interaction among chromatin remodelers, histone chaperones and a variety of histone modifications. Recent studies have implicated a role for H3K56 acetylation in the DNA damage response and checkpoint recovery. In this study, we determined the role of H3K56 acetylation in response to UV-induced DNA damage. We show that H3K56 is deacetylated within 15 minutes of UV-irradiation of normal human fibroblasts (NHFs), and acetylation is promptly recovered with full restoration at 48 hours. In yeast, acetylated H3K56 is deposited at the sites of DNA damage in an ASF1-dependent manner and is involved in checkpoint recovery. Therefore, we tested whether the checkpoint kinases, ATR and ATM, regulate the acetylation of H3K56. Interestingly, H3K56 acetylation was significantly lower in Seckel cells compared to NHFs, indicating a role of ATR in regulating H3K56 acetylation even in the absence of DNA damage. On the other hand, in AT and ATM-depleted HeLa cells, the post-repair recovery of H3K56 acetylation was defective. These data indicate a contributory role of ATR and ATM in regulating H3K56 acetylation. Next, we assessed the role of histone chaperone ASF1A in deacetylation/acetylation of H3K56. shRNA-mediated knockdown of ASF1A did not show a discernable defect in the deacetylation of H3K56 in response to UV-induced DNA damage, but the restoration of H3K56 acetylation was impaired. Thus, ASF1A appears to be essential for the post-repair recovery of H3K56 acetylation. To further confirm the role of ASF1A-mediated H3K56 acetylation in checkpoint recovery, we determined the level of H2AX phosphorylation at various times following UV-irradiation in ASF1A-deficient and H3K56R mutant cells, which are defective in H3K56 acetylation. Both cell types were shown to retain phosphorylated H2AX for a longer time at damage sites as compared to the control wild-type cells, indicating the persistence of checkpoint arrest in the absence of H3K56 acetylation. Similarly, the phosphorylated state of checkpoint kinases, Chk1 and Chk2, was maintained for longer times in the absence of ASF1A and in H3K56R mutant cells. FACS analysis of cell cycle progression after UV-irradiation of both ASF1A-deficient and H3K56R mutant cells revealed a slower recovery from the checkpoint arrest. Taken together, our results demonstrate an important function of H3K56 acetylation in the recovery from DNA damage-induced cell cycle checkpoint arrest. (This work was supported by NIH grants ES2388, ES12991 and CA93413) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1999. doi:10.1158/1538-7445.AM2011-1999

Deregulated Ras signaling compromises DNA damage checkpoint recovery in S. cerevisiae

The DNA damage checkpoint maintains genome stability by arresting the cell cycle and promoting DNA repair under genotoxic stress. Cells must downregulate the checkpoint signaling pathways in order to resume cell division after completing DNA repair. While the mechanisms of checkpoint activation have been well-characterized, the process of checkpoint recovery, and the signals regulating it, has only recently been investigated. We have identified a new role for the Ras signaling pathway as a regulator of DNA damage checkpoint recovery. Here we report that in budding yeast, deletion of the IRA1 and IRA2 genes encoding negative regulators of Ras prevents cellular recovery from a DNA damage induced arrest. The checkpoint kinase Rad53 is dephosphorylated in an IRA-deficient strain, indicating that recovery failure is not caused by constitutive checkpoint pathway activation. The ira1D ira2D recovery defect requires the checkpoint kinase Chk1 and the cAMP-dependent protein kinase (PKA) catalytic subunit Tpk2. Furthermore, PKA phosphorylation sites on the anaphase promoting complex specificity factor Cdc20 are required for the recovery defect, indicating a link between the recovery defect and PKA regulation of mitosis. This work identifies a new signaling pathway that can regulate DNA damage checkpoint recovery, and implicates the Ras signaling pathway as an important regulator of mitotic events.

Human Papillomavirus 16 E7 Oncoprotein Attenuates DNA Damage Checkpoint Control by Increasing the Proteolytic Turnover of Claspin

The human papillomavirus (HPV) 16 E7 oncoprotein has been reported previously to stimulate DNA damage and to activate host cell DNA damage checkpoints. How HPV-16 E7 maintains proliferation despite activated DNA damage checkpoints is incompletely understood. Here, we provide evidence that cells expressing the HPV-16 E7 oncoprotein can enter mitosis in the presence of DNA damage. We show that this activity of HPV-16 E7 involves attenuation of DNA damage checkpoint control by accelerating the proteolytic turnover of claspin. Claspin mediates the activation of CHK1 by ATR in response to replication stress, and its degradation plays a critical role in DNA damage checkpoint recovery. Expression of a nondegradable mutant of claspin was shown to inhibit mitotic entry in HPV-16 E7-expressing cells. Multiple components of the SCF(beta-TrCP)-based claspin degradation machinery were found deregulated in the presence of HPV-16 E7, including cullin 1, beta-TrCP, Aurora A, and Polo-like kinase-1 (PLK1). In contrast, no difference in the expression level of the claspin deubiquitinating enzyme USP7 was detected. Levels of Aurora A and PLK1 as well as phosphorylated PLK1 at threonine 210, a prerequisite for DNA damage checkpoint recovery, remained detectable following replication stress in HPV-16 E7-expressing cells but not in control cells. In summary, our results suggest that the HPV-16 E7 oncoprotein alleviates DNA damage checkpoint responses and promotes mitotic entry by accelerating claspin degradation through a mechanism that involves deregulation of components of the SCF(beta-TrCP)-based claspin degradation machinery.

PP4R4/KIAA1622 Forms a Novel Stable Cytosolic Complex with Phosphoprotein Phosphatase 4

Protein serine/threonine phosphatase 4 (PP4c) is an essential polypeptide involved in critical cellular processes such as microtubule growth and organization, DNA damage checkpoint recovery, apoptosis, and tumor necrosis factor alpha signaling. Like other phosphatases of the PP2A family, PP4c interacts with regulatory proteins, which specify substrate targeting and intracellular localization. The identification of these regulatory proteins is, therefore, key to fully understanding the function of this enzyme class. Here, using a sensitive affinity purification/mass spectrometry approach, we identify a novel, stable cytosolic PP4c interacting partner, KIAA1622, which we have renamed PP4R4. PP4R4 displays weak sequence homology with the A (scaffolding) subunit of the PP2A holoenzyme and specifically associates with PP4c (and not with the related PP2Ac or PP6c phosphatases). The PP4c.PP4R4 interaction is disrupted by mutations analogous to those abrogating the association of PP2Ac with PP2A A subunit. However, unlike the PP2A A subunit, which plays a scaffolding role, PP4R4 does not bridge PP4c with previously characterized PP4 regulatory subunits. PP4c.PP4R4 complexes exhibit phosphatase activity toward a fluorogenic substrate and gammaH2AX, but this activity is lower than that associated with the PP4c.PP4R2.PP4R3 complex, which itself is less active than the free PP4c catalytic subunit. Our data demonstrate that PP4R4 forms a novel cytosolic complex with PP4c, independent from the complexes containing PP4R1, PP4R2.PP4R3, and alpha4, and that the regulatory subunits of PP4c have evolved different modes of interaction with the catalytic subunit.

Erratum: Corrigendum: A phosphatase complex that dephosphorylates γH2AX regulates DNA damage checkpoint recovery

Corrigendum: A phosphatase complex that dephosphorylates γH2AX regulates DNA damage checkpoint recovery

A phosphatase complex that dephosphorylates γH2AX regulates DNA damage checkpoint recovery

One of the earliest marks of a double-strand break (DSB) in eukaryotes is serine phosphorylation of the histone variant H2AX at the carboxy-terminal SQE motif to create gammaH2AX-containing nucleosomes. Budding-yeast histone H2A is phosphorylated in a similar manner by the checkpoint kinases Tel1 and Mec1 (ref. 2; orthologous to mammalian ATM and ATR, respectively) over a 50-kilobase region surrounding the DSB. This modification is important for recruiting numerous DSB-recognition and repair factors to the break site, including DNA damage checkpoint proteins, chromatin remodellers and cohesins. Multiple mechanisms for eliminating gammaH2AX as DNA repair completes are possible, including removal by histone exchange followed potentially by degradation, or, alternatively, dephosphorylation. Here we describe a three-protein complex (HTP-C, for histone H2A phosphatase complex) containing the phosphatase Pph3 that regulates the phosphorylation status of gammaH2AX in vivo and efficiently dephosphorylates gammaH2AX in vitro. gammaH2AX is lost from chromatin surrounding a DSB independently of the HTP-C, indicating that the phosphatase targets gammaH2AX after its displacement from DNA. The dephosphorylation of gammaH2AX by the HTP-C is necessary for efficient recovery from the DNA damage checkpoint.