Regulation of Rad17 Protein Turnover Unveils an Impact of Rad17-APC Cascade in Breast Carcinogenesis and Treatment

Aberrant regulation of DNA damage checkpoint function leads to genome instability that in turn can predispose cellular tissues to become cancerous. Previous works from us and others demonstrated the role of Rad17 in either activation or termination of DNA damage checkpoint function. In the current study, we have revealed the unexpected accumulation of Rad17 in various types of breast cancer cell lines as well as human breast cancer tissues. We observed that Rad17 protein turnover rate in breast epithelial cells is much faster than in breast cancer cells, where the turnover of Rad17 is regulated by the Cdh1/APC pathway. We further observed that Rad17-mediated checkpoint function is modulated by proteolysis. Stabilization of Rad17 disrupts cellular response to chemotherapeutic drug-induced DNA damage and enhances cellular transformation. In addition, manipulation of Rad17 by RNA interference or stabilization of Rad17 significantly sensitize breast cancer cell to various chemotherapeutic drugs. Our present results indicate the manipulation of Rad17 proteolysis could be a valuable approach to sensitize breast cancer cell to the chemotherapeutic treatment despite of the critical role in governing DNA damage response and cellular recovery from genotoxic stress.     

The Cdc14B-Cdh1-Plk1 axis controls the G2 DNA-damage-response checkpoint

In response to DNA damage in G2, mammalian cells must avoid entry into mitosis and instead initiate DNA repair. Here, we show that, in response to genotoxic stress in G2, the phosphatase Cdc14B translocates from the nucleolus to the nucleoplasm and induces the activation of the ubiquitin ligase APC/C(Cdh1), with the consequent degradation of Plk1, a prominent mitotic kinase. This process induces the stabilization of Claspin, an activator of the DNA-damage checkpoint, and Wee1, an inhibitor of cell-cycle progression, and allows an efficient G2 checkpoint. As a by-product of APC/C(Cdh1) reactivation in DNA-damaged G2 cells, Claspin, which we show to be an APC/C(Cdh1) substrate in G1, is targeted for degradation. However, this process is counteracted by the deubiquitylating enzyme Usp28 to permit Claspin-mediated activation of Chk1 in response to DNA damage. These findings define a novel pathway that is crucial for the G2 DNA-damage-response checkpoint.     

The ubiquitous role of ubiquitin in the DNA damage response

Protein ubiquitylation has emerged as an important regulatory mechanism that impacts almost every aspect of the DNA damage response. In this review, we discuss how DNA repair and checkpoint pathways utilize the diversity offered by the ubiquitin conjugation system to modulate the response to genotoxic lesions in space and time. In particular, we will highlight recent work done on the regulation of DNA double-strand breaks signalling and repair by the RNF8/RNF168 E3 ubiquitin ligases, the Fanconi anemia pathway and the role of protein degradation in the enforcement and termination of checkpoint signalling. We also discuss the various functions of deubiquitylating enzymes in these processes along with potential avenues for exploiting the ubiquitin conjugation/deconjugation system for therapeutic purposes.     

DNA repair protein Rad55 is a terminal substrate of the DNA damage checkpoints

Checkpoints, which are integral to the cellular response to DNA damage, coordinate transient cell cycle arrest and the induced expression of DNA repair genes after genotoxic stress. DNA repair ensures cellular survival and genomic stability, utilizing a multipathway network. Here we report evidence that the two systems, DNA damage checkpoint control and DNA repair, are directly connected by demonstrating that the Rad55 double-strand break repair protein of the recombinational repair pathway is a terminal substrate of DNA damage and replication block checkpoints. Rad55p was specifically phosphorylated in response to DNA damage induced by the alkylating agent methyl methanesulfonate, dependent on an active DNA damage checkpoint. Rad55p modification was also observed after gamma ray and UV radiation. The rapid time course of phosphorylation and the recombination defects identified in checkpoint-deficient cells are consistent with a role of the DNA damage checkpoint in activating recombinational repair. Rad55p phosphorylation possibly affects the balance between different competing DNA repair pathways.     

The anaphase-promoting complex or cyclosome supports cell survival in response to endoplasmic reticulum stress

The anaphase-promoting complex or cyclosome (APC/C) is a multi-subunit ubiquitin ligase that regulates exit from mitosis and G1 phase of the cell cycle. Although the regulation and function of APC/C(Cdh1) in the unperturbed cell cycle is well studied, little is known of its role in non-genotoxic stress responses. Here, we demonstrate the role of APC/C(Cdh1) (APC/C activated by Cdh1 protein) in cellular protection from endoplasmic reticulum (ER) stress. Activation of APC/C(Cdh1) under ER stress conditions is evidenced by Cdh1-dependent degradation of its substrates. Importantly, the activity of APC/C(Cdh1) maintains the ER stress checkpoint, as depletion of Cdh1 by RNAi impairs cell cycle arrest and accelerates cell death following ER stress. Our findings identify APC/C(Cdh1) as a regulator of cell cycle checkpoint and cell survival in response to proteotoxic insults.     

Cell cycle-dependent processing of DNA lesions controls localization of Rad9 to sites of genotoxic stress

The Rad9/Rad1/Hus1 complex functions to facilitate the ATR-mediated phosphorylation of several substrates that control the checkpoint arrest induced by DNA damage. Here we show that in response to genotoxic stress induced by different types of damaging agents, Rad9 rapidly relocalized to sites of single stranded DNA, as visualized by discrete nuclear foci that co-localize with RPA. UV light-induced Rad9 foci also colocalized with TopBP1 and γ-H2AX. Interestingly, Rad9 foci were predominately formed in G1 and S phase after UV light, while treatment of cells with ionizing radiation (IR) resulted in accumulation of Rad9 into foci in S and G2. Photobleaching experiments in living cells revealed that the Rad9 protein is highly mobile in undamaged cells. However, genotoxic stress induced the immobilization of a large proportion of the protein. The proportion of Rad9 immobilization was larger in S phase and the accumulation to sites of locally damaged areas induced by UV-laser irradiation was faster during DNA replication. Inactivation of nucleotide excision repair by knock down of XPA and XPC resulted in a decrease of G1 phase cells that displayed Rad9 foci in response to UV light, whereas IR-induced Rad9 foci were not affected. In contrast, downregulation of CtIP, which promotes DSB resection, abrogated the IR-induced Rad9 foci. These findings show that due to processing of DNA lesions into a common intermediate, which occurs in a cell cycle-dependent manner, Rad9 is able to respond to different types of genotoxic stress.     

Two distinct pathways for inhibiting pds1 ubiquitination in response to DNA damage

The presence of DNA damage activates a conserved cellular response known as the DNA damage checkpoint pathway. This pathway induces a cell cycle arrest that persists until the damage is repaired. Consequently, the failure to arrest in response to DNA damage is associated with genomic instability. In budding yeast, activation of the DNA damage checkpoint pathway leads to a mitotic cell cycle arrest. Following the detection of DNA damage, the checkpoint signal is transduced via the Mec1 kinase, which in turn activates two kinases, Rad53 and Chk1 that act in parallel pathways to bring about the cell cycle arrest. The downstream target of Rad53 is unknown. The target of Chk1 is Pds1, an inhibitor of anaphase initiation whose degradation is a prerequisite for mitotic progression. Pds1 degradation is dependent on its ubiquitination by the anaphase-promoting complex/cyclosome ubiquitin ligase, acting in conjunction with the Cdc20 protein (APC/CCdc20). Previous studies showed that the Rad53 and Chk1 pathways independently lead to Pds1 stabilization but the mechanism for this was unknown. In the present study we show that both the Chk1 and the Rad53 pathways inhibit the APC/CCdc20-dependent ubiquitination of Pds1 but they affect different steps of the process: the Rad53 pathway inhibits the Pds1-Cdc20 interaction whereas Chk1-dependent phosphorylation of Pds1 inhibits the ubiquitination reaction itself. Finally, we show that once the DNA damage is repaired, Pds1 dephosphorylation is involved in the recovery from the checkpoint induced cell cycle arrest.     

Nuclear factories for signalling and repairing DNA double strand breaks in living fission yeast

In mammalian and budding yeast cells treated with genotoxic agents, different proteins implicated in detecting, signalling or repairing DNA lesions form nuclear foci. We studied foci formed by proteins involved in these processes in living fission yeast cells, which is amenable to genetic and molecular analysis. Using fluorescent tags, we analysed subnuclear localisations of the DNA damage checkpoint protein Rad9, of the homologous recombination protein Rad22 and of PCNA, which are implicated in many aspects of DNA metabolism. After inducing double strand breaks (DSBs) with ionising radiations, Rad22, Rad9 and PCNA form a low number of nuclear foci. Rad9 recruitment to foci depends on the presence of Rad1, Hus1 and Rad17, but is independent of downstream checkpoint effectors and of homologous recombination proteins. Likewise, Rad22 and PCNA form foci despite inactive homologous recombination repair and impaired DNA damage checkpoint. Rad22 and Rad9 foci co-localise completely, whereas PCNA co-localises with Rad22 and Rad9 only partially. Foci do not disassemble in cells unable to repair DNA by homologous recombination. Thus, in fission yeast, DSBs are detected by the DNA damage checkpoint and are repaired by homologous recombination at a few spatially confined subnuclear compartments where Rad22, Rad9 and PCNA concentrate independently.     

Degradation of human RAP80 is cell cycle regulated by Cdc20 and Cdh1 ubiquitin ligases

Receptor-associated protein 80 (RAP80) is a component of the BRCA1-A complex that recruits BRCA1 to DNA damage sites in the DNA damage-induced ubiquitin signaling pathway. RAP80-depleted cells showed defective G(2)-M phase checkpoint control. In this study, we show that RAP80 protein levels fluctuate during the cell cycle. Its expression level peaked in the G(2) phase and declined during mitosis and progression into the G(1) phase. Also, RAP80 is polyubiquitinated and degraded by the anaphase-promoting complex (APC/C)(Cdc20) or (APC/C)(Cdh1). Consistent with this, knockdown of Cdc20 or Cdh1 expression by transfecting with small interfering RNAs blocked RAP80 degradation during mitosis or the G(1) phase, respectively. A conserved destruction box (D box) in RAP80 affected its stability and ubiquitination, which was dependent on APC/cyclosome(Cdc20) (C(Cdc20)) or APC/cyclosome(Cdh1) (C(Cdh1)). In addition, overexpression of RAP80 destruction box1 deletion mutant attenuated mitotic progression. Thus, APC/C(Cdc20) or APC/C(Cdh1) complexes regulate RAP80 stability during mitosis to the G(1) phase, and these events are critical for a novel function of RAP80 in mitotic progression.     

Cdc5L interacts with ATR and is required for the S‐phase cell‐cycle checkpoint

Cell division cycle 5‐like protein (Cdc5L) is a core component of the putative E3 ubiquitin ligase complex containing Prp19/Pso4, Plrg1 and Spf27. This complex has been shown to have a role in pre‐messenger RNA splicing from yeast to humans; however, more recent studies have described a function for this complex in the cellular response to DNA damage. Here, we show that Cdc5L interacts physically with the cell‐cycle checkpoint kinase ataxia‐telangiectasia and Rad3‐related (ATR). Depletion of Cdc5L by RNA‐mediated interference methods results in a defective S‐phase cell‐cycle checkpoint and cellular sensitivity in response to replication‐fork blocking agents. Furthermore, we show that Cdc5L is required for the activation of downstream effectors or mediators of ATR checkpoint function such as checkpoint kinase 1 (Chk1), cell cycle checkpoint protein Rad 17 (Rad17) and Fanconi anaemia complementation group D2 protein (FancD2). In addition, we have mapped the ATR‐binding region in Cdc5L and show that a deletion mutant that is unable to interact with ATR is defective in the rescue of the checkpoint deficiency in Cdc5L‐depleted cells. These findings show a new function for Cdc5L in the regulation of the ATR‐mediated cell‐cycle checkpoint in response to genotoxic agents.     

A conserved histone deacetylase with a role in the regulation of cytokinesis in Schizosaccharomyces pombe

Background

The maintenance of genomic integrity is essential for cell viability. Complex signalling pathways (DNA integrity checkpoints) mediate the response to genotoxic stresses. Identifying new functions involved in the cellular response to DNA-damage is crucial. The Saccharomyces cerevisiae SLT2 gene encodes a member of the mitogen-activated protein kinase (MAPK) cascade whose main function is the maintenance of the cell wall integrity. However, different observations suggest that SLT2 may also have a role related to DNA metabolism.

Results

This work consisted in a comprehensive study to connect the Slt2 protein to genome integrity maintenance in response to genotoxic stresses. The slt2 mutant strain was hypersensitive to a variety of genotoxic treatments, including incubation with hydroxyurea (HU), methylmetanosulfonate (MMS), phleomycin or UV irradiation. Furthermore, Slt2 was activated by all these treatments, which suggests that Slt2 plays a central role in the cellular response to genotoxic stresses. Activation of Slt2 was not dependent on the DNA integrity checkpoint. For MMS and UV, Slt2 activation required progression through the cell cycle. In contrast, HU also activated Slt2 in nocodazol-arrested cells, which suggests that Slt2 may respond to dNTP pools alterations. However, neither the protein level of the distinct ribonucleotide reductase subunits nor the dNTP pools were affected in a slt2 mutant strain. An analysis of the checkpoint function revealed that Slt2 was not required for either cell cycle arrest or the activation of the Rad53 checkpoint kinase in response to DNA damage. However, slt2 mutant cells showed an elongated bud and partially impaired Swe1 degradation after replicative stress, indicating that Slt2 could contribute, in parallel with Rad53, to bud morphogenesis control after genotoxic stresses.

Conclusions

Slt2 is activated by several genotoxic treatments and is required to properly cope with DNA damage. Slt2 function is important for bud morphogenesis and optimal Swe1 degradation under replicative stress. The MAPK Slt2 appears as a new player in the cellular response to genotoxic stresses.     

The ubiquitin receptor Rad23: At the crossroads of nucleotide excision repair and proteasomal degradation

A protein that exemplifies the intimate link between the ubiquitin/proteasome system (UPS) and DNA repair is the yeast nucleotide excision repair (NER) protein Rad23 and its human orthologs hHR23A and hHR23B. Rad23, which was originally identified as an important factor involved in the recognition of DNA lesions, also plays a central role in targeting ubiquitylated proteins for proteasomal degradation, an activity that it shares with other ubiquitin receptors like Dsk2 and Ddi1. Although the finding that Rad23 serves as a ubiquitin receptor explains to a large extent its importance in proteasomal degradation, the precise mode of action of Rad23 in NER and the possible link with the UPS is less clear. In this review, we discuss our present knowledge on the functions of Rad23 in protein degradation and DNA repair and speculate on the importance of the dual roles of Rad23 for the cell's ability to cope with stress conditions.     

Interaction between human MCM7 and Rad17 proteins is required for replication checkpoint signaling

Human Rad17 (hRad17) is centrally involved in the activation of cell-cycle checkpoints by genotoxic agents or replication stress. Here we identify hMCM7, a core component of the DNA replication apparatus, as a novel hRad17-interacting protein. In HeLa cells, depletion of either hRad17 or hMCM7 with small-interfering RNA suppressed ultraviolet (UV) light- or aphidicolin-induced hChk1 phosphorylation, and abolished UV-induced S-phase checkpoint activation. Similar results were obtained after transfection of these cells with a fusion protein containing the hMCM7-binding region of hRad17. The hMCM7-depleted cells were also defective for the formation of ATR-containing nuclear foci after UV irradiation, suggesting that hMCM7 is required for stable recruitment of ATR to damaged DNA. These results demonstrate that hMCM7 plays a direct role in the transmission of DNA damage signals from active replication forks to the S-phase checkpoint machinery in human cells.     

APC/CCdh1-dependent proteolysis of USP1 regulates the response to UV-mediated DNA damage

Targeted protein destruction of critical cellular regulators during the G1 phase of the cell cycle is achieved by anaphase-promoting complex/cyclosome^Cdh1 (APC/C^Cdh1), a multisubunit E3 ubiquitin ligase. Cells lacking Cdh1 have been shown to accumulate deoxyribonucleic acid (DNA) damage, suggesting that it may play a previously unrecognized role in maintaining genomic stability. The ubiquitin-specific protease 1 (USP1) is a known critical regulator of DNA repair and genomic stability. In this paper, we report that USP1 was degraded in G1 via APC/C^Cdh1. USP1 levels were kept low in G1 to provide a permissive condition for inducing proliferating cell nuclear antigen (PCNA) monoubiquitination in response to ultraviolet (UV) damage before DNA replication. Importantly, expression of a USP1 mutant that cannot be degraded via APC/C^Cdh1 inhibited PCNA monoubiquitination during G1, likely compromising the recruitment of trans-lesion synthesis polymerase to UV repair sites. Thus, we propose a role for APC/C^Cdh1 in modulating the status of PCNA monoubiquitination and UV DNA repair before S phase entry.     

Rad17 phosphorylation is required for claspin recruitment and Chk1 activation in response to replication stress

The ATR-mediated checkpoint is not only critical for responding to genotoxic stress but also essential for cell proliferation. The RFC-related checkpoint protein Rad17, a phosphorylation substrate of ATR, is critical for ATR-mediated checkpoint signaling and cell survival. Here, we show that phosphorylation of Rad17 by ATR is important for genomic stability and restraint of S phase but is not essential for cell survival. The phosphomutant Rad17AA exhibits distinct defects in hydroxyurea- (HU) and ultraviolet- (UV) induced Chk1 activation, indicating that separate Rad17 functions are required differently in response to different types of replication interference. Although cells expressing Rad17AA can initiate Chk1 phosphorylation after HU treatment, they fail to sustain Chk1 phosphorylation after withdrawal of HU and are profoundly sensitive to HU. Importantly, we found that phosphorylated Rad17 interacts with Claspin and regulates its phosphorylation. These findings reveal a phosphorylation-dependent function of Rad17 in an ATR-Rad17-Claspin-Chk1-signaling cascade that responds to specific replication stress.     

KEN-box-dependent degradation of the Bub1 spindle checkpoint kinase by the anaphase-promoting complex/cyclosome

The spindle checkpoint is a cell cycle surveillance mechanism that ensures the fidelity of chromosome segregation during mitosis and meiosis. Bub1 is a protein serine-threonine kinase that plays multiple roles in chromosome segregation and the spindle checkpoint. In response to misaligned chromosomes, Bub1 directly inhibits the ubiquitin ligase activity of the anaphase-promoting complex or cyclosome (APC/C) by phosphorylating its activator Cdc20. The protein level and the kinase activity of Bub1 are regulated during the cell cycle; they peak in mitosis and are low in G1/S phase. Here we show that Bub1 is degraded during mitotic exit and that degradation of Bub1 is mediated by APC/C in complex with its activator Cdh1 (APC/C(Cdh1)). Overexpression of Cdh1 reduces the protein levels of ectopically expressed Bub1, whereas depletion of Cdh1 by RNA interference increases the level of the endogenous Bub1 protein. Bub1 is ubiquitinated by immunopurified APC/C(Cdh1) in vitro. We further identify two KEN-box motifs on Bub1 that are required for its degradation in vivo and ubiquitination in vitro. A Bub1 mutant protein with both KEN-boxes mutated is stable in cells but fails to elicit a cell cycle phenotype, indicating that degradation of Bub1 by APC/C(Cdh1) is not required for mitotic exit. Nevertheless, our study clearly demonstrates that Bub1, an APC/C inhibitor, is also an APC/C substrate. The antagonistic relationship between Bub1 and APC/C may help to prevent the premature accumulation of Bub1 during G1.