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Smc5/6, like cohesin and condensin, is a structural maintenance of chromosomes complex crucial for genome stability. Unlike cohesin and condensin, Smc5/6 carries an essential Nse2 subunit with SUMO E3 ligase activity. While screening for new DNA replication checkpoint mutants in fission yeast, we have identified two previously uncharacterized mutants in Smc5/6. Characterization of the mutants and a series of previously reported Smc5/6 mutants uncovered that sumoylation of the RecQ helicase Rqh1 by Nse2 facilitates the checkpoint signaling at the replication fork. We found that mutations that eliminate the sumoylation sites or the helicase activity of Rqh1 compromised the checkpoint signaling similar to a nse2 mutant lacking the ligase activity. Surprisingly, introducing a sumoylation site mutation to a helicase-inactive rqh1 mutant promoted cell survival under stress. These findings, together with other genetic data, support a mechanism that sumoylation of Rqh1 by Smc5/6-Nse2 recruits Rqh1 or modulates its helicase activity at the fork to facilitate the checkpoint signaling. Since the Smc5/6 complex, Rqh1, and the replication checkpoint are conserved in eukaryotes, a similar checkpoint mechanism may be operating in human cells.

Introduction Patients with high-grade serous ovarian cancer (HGSOC) have a poor prognosis, and current chemotherapy regimens for treating advanced disease are far from satisfactory. Prexasertib (LY2606368) is a novel checkpoint kinase inhibitor (CHK) under investigation for the treatment of HGSOC. Data from a recent phase II trial showed promising efficacy and safety results for treating wild-type BRCA HGSOC. Areas covered This article reviews the available data on the pharmacokinetics, pharmacodynamics, clinical efficacy, and safety of prexasertib in the treatment of HGSOC. Expert opinion Until now, prexasertib demonstrated clinical activity in phase I and II clinical trial for treating wild-type BRCA HGSOC, whereas its promising efficacy as monotherapy and combined with olaparib in BRCA-mutated HGSOC has been preliminary evidenced only in phase I studies. Compared to other drugs of the same class, prexasertib showed a better tolerability profile, causing moderate hematological toxicity. Further studies are needed to confirm efficacy and safety profiles of prexasertib in combined regimens. New early clinical trials may investigate prexasertib administered with programmed cell death ligand 1 (PD-L1) and PI3 K inhibitors due to the preclinical evidence of a synergic action.

Breast cancer (BC) is the most frequent malignancy in both pre- and postmenopausal women. However, it is exceedingly rare in very young patients, and especially in adolescents. Herein, we report a case of an 18-year-old female diagnosed with invasive BC. The proband had been found to be negative for BC in close family members. A common BC genetic screening test for the Polish population did not detect any known founder mutations in the BRCA1 gene. Further evaluation identified a p.Ile157Thr (I157T) mutation in the CHEK2 gene, a p.Ala1991Val (A1991V) variant of unknown significance in the BRCA2 gene, p.Lys751Gln (K751Q) variant in the XPD (ERCC2) gene, and a homozygous p.Glu1008Ter (E1008*) mutation in the NOD2 gene. No other mutation had been found by next generation sequencing in major BC high-risk susceptibility genes BRCA1, BRCA2, as well as 92 other genes. To date, all these found alterations have been considered as low to moderate risk factors in the general population and moderate risk factors in younger women (<35 years of age). There are no previous articles relating low and moderate risk gene mutations to very young onset (below 20 years) BC with a fatal outcome. In our patient, a possible cumulative or synergistic risk effect for these 4 alterations, and a mutation in the NOD2 gene in particular, of which both presumably healthy parents were found to be carriers, is suggested.

The essential yeast kinases Mec1 and Rad53, or human ATR and Chk1, are crucial for checkpoint responses to exogenous genotoxic agents, but why they are also required for DNA replication in unperturbed cells remains poorly understood. Here we report that even in the absence of DNA-damaging agents, the rad53-4AQ mutant, lacking the N-terminal Mec1 phosphorylation site cluster, is synthetic lethal with a deletion of the RAD9 DNA damage checkpoint adaptor. This phenotype is caused by an inability of rad53-4AQ to activate the downstream kinase Dun1, which then leads to reduced basal deoxynucleoside triphosphate (dNTP) levels, spontaneous replication fork stalling, and constitutive activation of and dependence on S phase DNA damage checkpoints. Surprisingly, the kinase-deficient rad53-K227A mutant does not share these phenotypes but is rendered inviable by additional phosphosite mutations that prevent its binding to Dun1. The results demonstrate that ultralow Rad53 catalytic activity is sufficient for normal replication of undamaged chromosomes as long as it is targeted toward activation of the effector kinase Dun1. Our findings indicate that the essential S phase function of Rad53 is comprised by the combination of its role in regulating basal dNTP levels and its compensatory kinase function if dNTP levels are perturbed.

The central histone H3/H4 chaperone Asf1 comprises a highly conserved globular core and a divergent C-terminal tail. While the function and structure of the Asf1 core are well known, the function of the tail is less well understood. Here, we have explored the role of the yeast (yAsf1) and human (hAsf1a and hAsf1b) Asf1 tails in Saccharomyces cerevisiae. We show, using a photoreactive, unnatural amino acid, that Asf1 tail residue 210 cross-links to histone H3 in vivo and, further, that loss of C-terminal tail residues 211 to 279 weakens yAsf1-histone binding affinity in vitro nearly 200-fold. Via several yAsf1 C-terminal truncations and yeast-human chimeric proteins, we found that truncations at residue 210 increase transcriptional silencing and that the hAsf1a tail partially substitutes for full-length yAsf1 with respect to silencing but that full-length hAsf1b is a better overall substitute for full-length yAsf1. In addition, we show that the C-terminal tail of Asf1 is phosphorylated at T270 in yeast. Loss of this phosphorylation site does not prevent coimmunoprecipitation of yAsf1 and Rad53 from yeast extracts, whereas amino acid residue substitutions at the Asf1-histone H3/H4 interface do. Finally, we show that residue substitutions in yAsf1 near the CAF-1/HIRA interface also influence yAsf1's function in silencing.

Recent studies have highlighted an apparently paradoxical link between self-renewal and senescence triggered by DNA damage in certain cell types. In addition, the finding that TP53 can suppress senescence has caused a re-evaluation of its functional role in regulating these outcomes. To investigate these phenomena and their relationship to pluripotency and senescence, we examined the response of the TP53-competent embryonal carcinoma (EC) cell line PA-1 to etoposide-induced DNA damage. Nuclear POU5F1/OCT4A and P21CIP1 were upregulated in the same cells following etoposide-induced G 2M arrest. However, while accumulating in the karyosol, the amount of OCT4A was reduced in the chromatin fraction. Phosphorylated CHK2 and RAD51/γH2AX-positive nuclear foci, overexpression of AURORA B kinase and moderate macroautophagy were evident. Upon release from G 2M arrest, cells with repaired DNA entered mitoses, while the cells with persisting DNA damage remained at this checkpoint or underwent mitotic slippage and gradually senesced. Reduction of TP53 using sh- or si-RNA prevented the upregulation of OCT4A and P21CIP1 and increased DNA damage. Subsequently, mitoses, micronucleation and senescence were all enhanced after TP53 reduction with senescence confirmed by upregulation of CDKN2A/P16INK4A and increased sa-β-galactosidase positivity. Those mitoses enhanced by TP53 silencing were shown to be multicentrosomal and multi-polar, containing fragmented and highly deranged chromosomes, indicating a loss of genome integrity. Together, these data suggest that TP53-dependent coupling of self-renewal and senescence pathways through the DNA damage checkpoint provides a mechanism for how embryonal stem cell-like EC cells safeguard DNA integrity, genome stability and ultimately the fidelity of self-renewal.

The Mec1 and Rad53 protein kinases are essential for budding yeast cell viability and are also required to activate the S-phase checkpoint, which supports DNA replication under stress conditions. Whether these two functions are related to each other remains to be determined, and the nature of the replication stress-dependent lethality of mec1 and rad53 mutants is still unclear. We show here that a decrease in cyclin-dependent kinase 1 (Cdk1) activity alleviates the lethal effects of mec1 and rad53 mutations both in the absence and in the presence of replication stress, indicating that the execution of a certain Cdk1-mediated event(s) is detrimental in the absence of Mec1 and Rad53. This lethality involves Cdk1 functions in both G(1) and mitosis. In fact, delaying either the G(1)/S transition or spindle elongation in mec1 and rad53 mutants allows their survival both after exposure to hydroxyurea and under unperturbed conditions. Altogether, our studies indicate that inappropriate entry into S phase and segregation of incompletely replicated chromosomes contribute to cell death when the S-phase checkpoint is not functional. Moreover, these findings suggest that the essential function of Mec1 and Rad53 is not necessarily separated from the function of these kinases in supporting DNA synthesis under stress conditions.

Hydroxyurea (HU) treatment activates the intra-S phase checkpoint proteins Cds1 and Mrc1 to prevent replication fork collapse. We found that prolonged DNA synthesis occurs in cds1Δ and mrc1Δ checkpoint mutants in the presence of HU and continues after release. This is coincident with increased DNA damage measured by phosphorylated histone H2A in whole cells during release. High-resolution live-cell imaging shows that mutants first accumulate extensive replication protein A (RPA) foci, followed by increased Rad52. Both DNA synthesis and RPA accumulation require the MCM helicase. We propose that a replication fork "collapse point" in HU-treated cells describes the point at which accumulated DNA damage and instability at individual forks prevent further replication. After this point, cds1Δ and mrc1Δ forks cannot complete genome replication. These observations establish replication fork collapse as a dynamic process that continues after release from HU block.

The circadian clock is a global regulatory mechanism that confers daily rhythmicity on many biochemical and physiological functions, including DNA excision repair in mammalian organisms. Here, we investigated the effect of the circadian clock on the major DNA damage response pathways by using mouse cell lines mutated in genes encoding proteins in the positive (Bmal1, CLOCK) or negative (Cry 1/2, Per 1/2) arms of the transcription-translation feedback loop that generates the circadian clock. We find that cells mutated in these genes are indistinguishable from wild-type in their response to UV, ionizing radiation and mitomycin C. We conclude that either the majority of DNA damage response reactions are not controlled by the circadian clock or that, even if such a control exists at the organism level, it is supplanted by homeostatic control mechanisms at the cellular level in tissue culture. We suggest that caution must be exercised in extrapolating from experiments in tissue culture to whole animals with respect to the effect of the circadian clock on cellular response to DNA damaging agents.

Chloroethylnitrosureas (CNUs) are powerful DNA-reactive alkylating agents used in cancer therapy. Here, we analyzed cyto- and genotoxicity of nimustine (ACNU), a representative of CNUs, in synchronized cells and in cells deficient in repair proteins involved in homologous recombination (HR) or nonhomologous end-joining (NHEJ). We show that HR mutants are extremely sensitive to ACNU, as measured by colony formation, induction of apoptosis and chromosomal aberrations. The NHEJ mutants differed in their sensitivity, with Ku80 mutants being moderately sensitive and DNA-PKcs mutated cells being resistant. HR mutated cells displayed a sustained high level of γH2AX foci, which co-stained with Rad51 and 53BP1, indicating DNA double-strand breaks (DSB) to be formed. Using synchronized cells, we analyzed whether DSB formation after ACNU treatment was replication-dependent. We show that γH2AX foci were not induced in G(1) but increased significantly in S phase and remained at a high level in G(2), where a fraction of cells became arrested and underwent, with a delay of > 12 h, cell death by apoptosis and necrosis. Rad51, ATM, MDC-1 and RPA-2 foci were also formed and shown to co-localize with γH2AX foci induced in S phase, indicating that the DNA damage response was activated. All effects observed were abrogated by MGMT, which repairs O(6)-chloroethylguanine that is converted into DNA cross-links. We deduce that the major genotoxic and killing lesion induced by CNUs are O(6)-chloroethylguanine-triggered cross-links, which give rise to DSBs in the treatment cell cycle, and that HR, but not NHEJ, is the major route of protection against this group of anticancer drugs. Base excision repair had no significant impact on ACNU-induced cytotoxicity.

Protein phosphatase 2A (PP2A) bearing the B'γ (=B'α/B56γ1/PR61γ) subunit is recruited to dephosphorylation targets by cyclin G. We demonstrate here that cyclin G-associated kinase (GAK), a component of the GAK/B'γ/cyclin G complex, directly phosphorylates the B'γ-Thr104 residue and regulates PP2A activity. Indeed, an anti-B'γ-pT104 antibody detected immunofluorescence signals at the chromosome and centrosome during mitosis; these signals were reduced by siRNA-mediated GAK knockdown. After DNA damage by γ-irradiation, the chromosome signals formed foci that colocalized with a DNA double-strand break (DSB) marker H2AX-pS139 (γH2AX) and CHK2-pT68. Moreover, B'γ-pT104 enhanced PP2A holoenzyme assembly and PP2A activity, as shown by the results of an in vitro phosphatase assay. These results suggest a novel role for GAK as a regulator of dephosphorylation events under the control of the PP2A B'γ subunit.

von-Hippel Lindau protein (pVHL) suppresses tumorigenesis in the kidney, in part through regulation of hypoxia-inducible factor alpha (HIF alpha). However, HIF has been proposed to be necessary but insufficient for renal tumorigenesis. p53 was implicated as a transcription factor that is regulated by pVHL, but the molecular mechanism by which pVHL regulates p53 on DNA damage is unknown. We demonstrated that checkpoint kinase-2 (Chk2) binds to the beta-domain of pVHL and phosphorylates Ser 111 on DNA damage. Notably, this modification enhances pVHL-mediated transactivation of p53 by recruiting p300 and Tip60 to the chromatin of p53 target gene. Further, the naturally occurring pVHL mutants pVHL-S111R and pVHL-S111C showed diminished binding to coactivators, ultimately retarding p53-mediated growth arrest and apoptosis. In this study, we determined the molecular mechanism by which pVHL transactivates p53 on DNA damage and demonstrated that p53-related pVHL subtype mutants regulate tumorigenecity in VHL diseases.

The S phase checkpoint response slows down replication in the presence of replication stress such that replication can resume normally once conditions are favorable. Both proper activation and deactivation of the checkpoint are crucial for genome stability. However, the mechanisms of checkpoint deactivation have been largely unknown. Here, we show that two highly conserved Saccharomyces cerevisiae ATP-dependent chromatin remodeling factors, Isw2 and Ino80, function to attenuate and deactivate S phase checkpoint activity. Genetic interactions revealed that these chromatin remodeling factors and the Rad53 phosphatases function in parallel in the DNA replication stress response. Following a transient replication stress, an isw2 nhp10 double mutant displays stronger and prolonged checkpoint activation without experiencing increased replication fork troubles. Isw2 and Ino80 are both enriched at stalled replication forks and physically and specifically interact with a single-stranded DNA binding protein, replication protein A (RPA). Based on these results, we propose that Isw2 and Ino80 are targeted to stalled replication forks via RPA and directly control the amplitude of S phase checkpoint activity and the subsequent deactivation process.

Myc is a transcription factor frequently found deregulated in human cancer. The Myc-mediated cellular transformation process is associated with fast proliferative cells and inherent genomic instability, giving rise to malignant, invasive neoplasms with poor prognosis for survival. Transcription-independent functions of Myc include stimulation of replication. Excessive Myc expression stimulates a replication-associated DNA damage response that signals via the phosphoinositide-3-kinase (PI3K)-related protein kinases (PIKKs) ATM and ATR. These, in turn, activate the DNA damage transducers Chk1 and Chk2. Here, we show that Myc can stimulate Chek2 transcript indirectly in vitro as well as in B cells of λ-Myc transgenic mice or in the intestine of Apc (Min) mice. However, Chk2 is dispensable for Myc's ability to transform cells in vitro and for the survival of established lymphoma cells from λ-Myc transgenic mice. Chk2 deficiency induces polyploidy and slow growth, but the cells are viable and protected against DNA damage. Furthermore, inhibition of both Chk1/Chk2 with AZD7762 induces cell death and significantly delays disease progression of transplanted lymphoma cells in vivo. DNA damage recruits PARP family members to sites of DNA breaks that, in turn, facilitate the induction of DNA repair. Strikingly, combining Chk2 and PARP inhibition elicits a synergistic lethal response in the context of Myc overexpression. Our data indicates that only certain types of chemotherapy would give rise to a synergistic lethal response in combination with specific Chk2 inhibitors, which will be important if Chk2 inhibitors enter the clinic.

The deregulation of the DNA damage response (DDR) can contribute to leukemogenesis and favor the progression from myelodysplastic syndrome (MDS) to acute myeloid leukemia (AML). Since hypomethylating agent, notably azacitidine, constitute an efficient therapy for patients with high-risk MDS, we assessed whether such compounds can activate the DDR in malignant blasts. While azacitidine and decitabine had moderate effects on apoptosis and cell cycle progression, both agents induced profound changes in the expression and functionality of DDR-related proteins. Decitabine, and to a lesser degree azacitidine, induced the activation of checkpoint kinases Chk-1 and Chk-2 and the phosphorylation of the DDR-sensor H2AX. In addition, hypomethylating agents were found to cause the dephosphorylation of the transcriptional regulator forkhead box O3, best known as FOXO3A, whose phosphorylation has been related to poor prognosis in AML. The dephoasphorylation of FOXO3A induced by azacitidine or decitabine in malignant blasts was accompanied by the translocation of FOXO3A from the cytoplasm to the nucleus. Upon stimulation with azacitidine, MDS/AML-derived, azacitidine-sensitive SKM-1S cells upregulated FOXO3A and the pro-apoptotic FOXO3A targets BIM and PUMA, and this effect was attenuated or abolished in azacitidine-resistant SMK-1R cells. Altogether, our results suggest that the reactivation of FOXO3A may contribute to the effects of hypomethylating agents in malignant blasts.

Genomic instability is thought to be critical for the development of cancer. Among its causes microsatellite instability (MIN) and chromosomal instability (CIN) have attracted the most attention. Cell cycle checkpoints and DNA repair mechanisms are the first line of defense against DNA damage. Among the most dangerous DNA lesions are double-strand breaks. The response to DNA double strand breaks is regulated mainly by the serine/threonine kinases ATM and Chk2 and their downstream target the tumor suppressor p53, which in turn stimulates the expression of growth-inhibitory genes like p21 or pro-apoptotic genes like Bax. The balance between these gene products determines the fate of a cell. EAPP is a nuclear phosphoprotein that is frequently upregulated in human tumors. We have recently shown that EAPP levels are critical for cellular homeostasis. DNA damage elevates EAPP levels and its overexpression results in G1 arrest and impairs apoptosis in a p21-dependent manner. EAPP binds to the p21 promoter, stimulates its activity and seems to be essential for transcription initiation. In the present work we show that EAPP also regulates the phosphorylation status and thus the activity of Chk2. EAPP binding seems to trigger the dephosphorylation of P-Chk2 resulting in its inactivation. A newly described function of Chk2 in mitosis that secures genomic integrity might also be affected by EAPP overexpression. This might explain the abundance of EAPP in aneuploid tumor cells.

Ionizing radiation induces DNA Double-Strand Breaks (DSBs) which activate the ATM/CHEK2/p53 pathway leading to cell cycle arrest and apoptosis through transcription of genes including CDKN1A (p21) and BBC3 (PUMA). This pathway prevents genomic instability and tumorigenesis as demonstrated in heritable syndromes [e.g. Ataxia Telangiectasia (AT); Li-Fraumeni syndrome (LFS)]. Here, a simple assay based on gene expression in peripheral blood to measure accurately ATM/CHEK2/p53 pathway activity is described. The expression of p21, Puma and Sesn2 was determined in blood from mice with different gene copy numbers of Atm, Trp53 (p53), Chek2 or Arf and in human blood and mitogen stimulated T-lymphocyte (MSTL) cultures from AT, AT carriers, LFS patients, and controls, both before and after ex vivo ionizing irradiation. Mouse Atm/Chek2/p53 activity was highly dependent on the copy number of each gene except Arf. In human MSTL, an AT case, AT carriers and LFS patients showed responses distinct from healthy donors. The relationship between gene copy number and transcriptional induction upon radiation was linear for p21 and Puma and correlated well with cancer incidence in p53 variant mice. This reliable blood test provides an assay to determine ATM/CHEK2/p53 pathway activity and demonstrates the feasibility of assessing the activity of this essential cancer protection pathway in simple assays. These findings may have implications for the individualized prediction of cancer susceptibility.

When DNA replication is challenged, cells activate a DNA-synthesis checkpoint blocking cell cycle progression until they are able to overcome the replication defects. In fission yeast, Cds1 is the effector kinase of this checkpoint, inhibiting M phase entry through inactivation of the phosphatase Cdc25, stabilizing stalled replication forks to prevent deleterious DNA structures and triggering transcriptional activation of S-phase genes. The MBF complex controls the transcription of genes required for the S phase and Yox1, a homeodomain-containing protein, binds and represses MBF-dependent transcription at the end of S phase in a cell cycle-regulated manner. Interestingly, when the DNA synthesis checkpoint is activated, Yox1 is phosphorylated by Cds1 resulting in the abrogation of its binding to MBF. As a consequence, MBF-dependent transcription is maintained active until cells are able to overcome the replication challenge. Thus, Yox1 couples normal cell cycle regulation and the DNA synthesis checkpoint in a single transcriptional complex.

Cell cycle checkpoints ensure that eukaryotic cells do not enter mitosis after ionizing irradiation (IR). The G(2)-arrest after IR is the result of activation of multiple signalling pathways, the contributions of which vary with time after irradiation. We have studied the time evolution of the IR-induced G(2)-arrest in human B-lymphocyte cancer cell lines, as well as the molecular mechanisms responsible for the arrest. Cells that were in G(2) phase at the time of irradiation experienced a transient arrest that blocked entry into mitosis at 0-2 hours after IR (0.5 or 4 Gy). Activation of ATM and CHEK2 occurred at the same time as this early arrest and was, like the arrest, abrogated by the ATM-inhibitor KU-55933. A late, permanent and ATM-independent arrest (≥6 hours after IR) of cells that were in G(2)/S/G(1) at the time of irradiation (4 Gy) was inactivated by caffeine. This late G(2)-arrest could not be explained by down-regulation of genes with functions in G(2)/mitosis (e.g. PLK1, CCNB1/2), since the down-regulation was transient and not accompanied by reduced protein levels. However, the persistent phosphorylation of CHEK1 after 4 Gy suggested a role for CHEK1 in the late arrest, consistent with the abrogation of the arrest in CHEK1-depleted cells. TP53 was not necessary for the late G(2)-arrest, but mediated an intermediate arrest (2-10 hours after IR) independently of ATM and CHEK1. In conclusion, the IR-induced arrest in G(2) is mediated by ATM immediately after irradiation, with TP53 for independent and transient back-up, while CHEK1 is necessary for the late arrest., (© 2011 Landes Bioscience)

Never-in-mitosis A related protein kinase 1 (Nek1) is involved early in a DNA damage sensing/repair pathway. We have previously shown that cells without functional Nek1 fail to activate the more distal kinases Chk1 and Chk2 and fail to arrest properly at G1/S or M-phase checkpoints in response to DNA damage. As a consequence, foci of damaged DNA in Nek1 null cells persist long after the instigating insult, and Nek1 null cells develop unstable chromosomes at a rate much higher than identically cultured wild type cells. Here we show that Nek1 functions independently of canonical DNA damage responses requiring the PI3 kinase-like proteins ATM and ATR. Chemical inhibitors of ATM/ATR or mutation of the genes that encode them fail to alter the kinase activity of Nek1 or its localization to nuclear foci of DNA damage. Moreover ATM and ATR activities, including the localization of the proteins to DNA damage sites and phosphorylation of early DNA damage response substrates, are intact in Nek1 -/- murine cells and in human cells with Nek1 expression silenced by siRNA. Our results demonstrate that Nek1 is important for proper checkpoint control and characterize for the first time a DNA damage response that does not directly involve one of the known upstream mediator kinases, ATM or ATR.

Checkpoints monitor the successful completion of cell cycle processes, such as DNA replication, and also regulate the expression of cell cycle-dependent genes that are required for responses. In the model yeast Schizosaccharomyces pombe G 1/S phase-specific gene expression is regulated by the MBF (also known as DSC1) transcription factor complex and is also activated by the mammalian ATM/ATR-related Rad3 DNA replication checkpoint. Here, we show that the Yox1 homeodomain transcription factor acts to co-ordinate the expression of MBF-regulated genes during the cell division cycle. Moreover, our data suggests that Yox1 is inactivated by the Rad3 DNA replication checkpoint via phosphorylation by the conserved Cds1 checkpoint kinase. Collectively, our data has implications for understanding the mechanisms underlying the coordination of cell cycle processes in eukaryotes.

Rad3, the Schizosaccharomyces pombe ortholog of human ATR and Saccharomyces cerevisiae Mec1, activates the checkpoint kinase Chk1 in response to DNA double-strand breaks (DSBs). Rad3(ATR/Mec1) associates with replication protein A (RPA), which binds single-stranded DNA overhangs formed by DSB resection. In humans and both yeasts, DSBs are initially detected and processed by the Mre11-Rad50-Nbs1(Xrs2) (MRN) nucleolytic protein complex in association with the Tel1(ATM) checkpoint kinase and the Ctp1(CtIP/Sae2) DNA-end processing factor; however, in budding yeast, neither Mre11 nuclease activity or Sae2 are required for Mec1 signaling at irreparable DSBs. Here, we investigate the relationship between DNA end processing and the DSB checkpoint response in fission yeast, and we report that Mre11 nuclease activity and Ctp1 are critical for efficient Rad3-to-Chk1 signaling. Moreover, deleting Ctp1 reveals a Tel1-to-Chk1 signaling pathway that bypasses Rad3. This pathway requires Mre11 nuclease activity, the Rad9-Hus1-Rad1 (9-1-1) checkpoint clamp complex, and Crb2 checkpoint mediator. Ctp1 negatively regulates this pathway by controlling MRN residency at DSBs. A Tel1-to-Chk1 checkpoint pathway acting at unresected DSBs provides a mechanism for coupling Chk1 activation to the initial detection of DSBs and suggests that ATM may activate Chk1 by both direct and indirect mechanisms in mammalian cells.

In this study, MLPA assay was performed for detection of large rearrangements of BRCA1 and BRCA2 genes in 16 familial, 29 early onset, 3 male breast cancer, and 2 bilateral breast/ovarian cancer high risk Turkish index cases. MLPA assay for all exons of both genes and for 1100delC variant of CHEK2 gene were performed. Analyses, revealed no large genomic rearrangements in both genes, and, no 1100del variant in CHEK2 gene. Our data which represents the first results for Turkish patients, suggest that, the frequency of BRCA1 and BRCA2 genes' large rearrangements is very low.

Entry into and precise progression through the cell cycle depends on the sequential expression and activation of cyclin dependent kinases (CDK). In accord, CDK dysregulation is a hallmark of many cancers. The function of Cdk2 is still an enigma as in vitro studies revealed that it is required for S phase-entry, whereas in vivo studies showed that Cdk2 is not an essential gene. Moreover, unlike other Cdks, or its cyclin E regulator, Cdk2-overexpressing tumors were reported only in one type of tumor. In this report we used budding yeast as a tool to explore Cdk2 function. We showed that hCdk2 promoted S phase in cells carrying a temperature-sensitive mutation in yCDK1, albeit, only when expressed at low or moderate levels. Overexpression of hCdk2 resulted in a defect in the G1 to S transition and a reduction in viability. The same phenotypes were observed in cells overexpressing its yeast functional homolog, Ime2, which is a meiosis-specific CDK-like kinase. A genetic interaction with the DNA damage checkpoint was demonstrated by showing an increased toxicity of hCdk2 and Ime2 in RAD53-deleted cells, and delayed Rad53 activation in response to MMS treatment in cells overexpressing hCdk2 or Ime2.

Mek1 is a Chk2/Rad53/Cds1-related protein kinase that is required for proper meiotic progression of Schizosaccharomyces pombe. However, the molecular mechanisms of Mek1 regulation and Mek1 phosphorylation targets are unclear. Here, we report that Mek1 is phosphorylated at serine-12 (S12), S14 and threonine-15 (T15) by Rad3 (ATR) and/or Tel1 (ATM) kinases that are activated by meiotic programmed double-strand breaks (DSBs). Mutations of these sites by alanine replacement caused abnormal meiotic progression and recombination rates. Phosphorylation of these sites triggers autophosphorylation of Mek1; indeed, alanine replacement mutations of Mek1-T318 and -T322 residues in the activation loop of Mek1 reduced Mek1 kinase activity and meiotic recombination rates. Substrates of Mek1 include Mus81-T275, Rdh54-T6 and Rdh54-T673. Mus81-T275 is known to regulate the Mus81 function in DNA cleavage, whereas Rdh54-T6A/T673A mutant cells showed abnormal meiotic recombination. Taken together, we conclude that the phosphorylation of Mek1 by Rad3 or Tel1, Mek1 autophosphorylation and Mus81 or Rdh54 phosphorylation by Mek1 regulate meiotic progression in S. pombe.

Studies in budding yeast suggest the protein kinase Rad53 plays novel roles in controlling initiation of DNA replication and in maintaining cellular histone levels, and these roles are independent of Rad53-mediated regulation of the checkpoint and of nucleotide levels. In order to elucidate the role of Rad53 in replication initiation, we isolated a novel allele of RAD53, rad53-rep, that separates the checkpoint function of RAD53 from the DNA replication function. rad53-rep mutants display a chromosome loss phenotype that is suppressed by increased origin dosage, providing further evidence that Rad53 plays a role in the initiation of DNA replication. Deletion of the major histone H3-H4 pair suppresses rad53-rep-cdc7-1 synthetic lethality, suggesting Rad53's functions in degradation of excess cellular histone and in replication initiation are related. Rad53-rep is active as a protein kinase yet fails to interact with origins of replication and like the rad53D mutant, the rad53-rep mutant accumulates excess soluble histones, and it is sensitive to histone dosage. In contrast, a checkpoint defective allele of RAD53 with mutations in both FHA domains, binds origins, and growth of a rad53-FHA mutant is unaffected by histone dosage. Based on these observations, we hypothesize that the origin binding and the histone degradation activities of Rad53 are central to its function in DNA replication and are independent of its checkpoint functions. We propose a model in which Rad53 acts as a "nucleosome buffer," interacting with origins of replication to prevent the binding of excess histones to origin DNA and to maintain proper chromatin configuration.

In fission yeast, replication fork arrest activates the replication checkpoint effector kinase Cds1(Chk2/Rad53) through the Rad3(ATR/Mec1)-Mrc1(Claspin) pathway. Hsk1, the Cdc7 homologue of fission yeast required for efficient initiation of DNA replication, is also required for Cds1 activation. Hsk1 kinase activity is required for induction and maintenance of Mrc1 hyperphosphorylation, which is induced by replication fork block and mediated by Rad3. Rad3 kinase activity does not change in an hsk1 temperature-sensitive mutant, and Hsk1 kinase activity is not affected by rad3 mutation. Hsk1 kinase vigorously phosphorylates Mrc1 in vitro, predominantly at non-SQ/TQ sites, but this phosphorylation does not seem to affect the Rad3 action on Mrc1. Interestingly, the replication stress-induced activation of Cds1 and hyperphosphorylation of Mrc1 is almost completely abrogated in an initiation-defective mutant of cdc45, but not in an mcm2 or polε mutant. The results suggest that Hsk1-mediated loading of Cdc45 onto replication origins may play important roles in replication stress-induced checkpoint.

DNA damage promotes the activation of a signal transduction cascade referred to as the DNA damage checkpoint. This pathway initiates with the Mec1/ATR kinase, which then phosphorylates the Rad53/Chk2 kinase. Mec1 phosphorylation of Rad53 is then thought to promote Rad53 autophosphorylation, ultimately leading to a fully active Rad53 molecule that can go on to phosphorylate substrates important for DNA damage resistance. In the absence of DNA repair, this checkpoint is eventually downregulated in a Cdc5-dependent process referred to as checkpoint adaptation. Recently, we showed that overexpression of Cdc5 leads to checkpoint inactivation and loss of the strong electrophoretic shift associated with Rad53 inactivation. Interestingly, this same overexpression did not strongly inhibit Rad53 autophosphorylation activity as measured by the in situ assay (ISA). The ISA involves incubating the re-natured Rad53 protein with γ ³²P labeled ATP after electrophoresis and western blotting. Using a newly identified Rad53 target, we show that despite strong ISA activity, Rad53 does not maintain phosphorylation of this substrate. We hypothesize that, during adaptation, Rad53 may be in a unique state in which it maintains some Mec1 phosphorylation, but does not have the auto-phosphorylations required for full activity towards exogenous substrates.

The mechanisms that control E2F-1 activity are complex. We previously showed that Chk1 and Chk2 are required for E2F1 stabilization and p73 target gene induction following DNA damage. To gain further insight into the processes regulating E2F1 protein stability, we focused our investigation on the mechanisms responsible for regulating E2F1 turnover. Here we show that E2F1 is a substrate of the anaphase promoting complex or cyclosome (APC/C), a ubiquitin ligase that plays an important role in cell cycle progression. Ectopic expression of the APC/C activators Cdh1 and Cdc20 reduced the levels of co-expressed E2F-1 protein. Co-expression of DP1 with E2F1 blocked APC/C-induced E2F1 degradation, suggesting that the E2F1/DP1 heterodimer is protected from APC/C regulation. Following Cdc20 knockdown, E2F1 levels increased and remained stable in extracts over a time course, indicating that APC/C(Cdc20) is a primary regulator of E2F1 stability in vivo. Moreover, cell synchronization experiments showed that siRNA directed against Cdc20 induced an accumulation of E2F1 protein in prometaphase cells. These data suggest that APC/C(Cdc20) specifically targets E2F1 for degradation in early mitosis and reveal a novel mechanism for limiting free E2F1 levels in cells, failure of which may compromise cell survival and/or homeostasis.

The cohesin network has an essential role in chromosome segregation, but also plays a role in DNA damage repair. Eco1 is an acetyltransferase that targets subunits of the cohesin complex and is involved in both the chromosome segregation and DNA damage repair roles of the network. Using budding yeast as a model system, we find that mutations in Eco1, including a genocopy of a human Roberts syndrome allele, do not cause gross defects in chromosome cohesion. We examined how mitotic and meiotic DNA damage repair is affected by mutations in Eco1. Strains containing mutations in Eco1 are sensitive to DNA damaging agents that cause double-strand breaks, such as X-rays and bleomycin. While meiotic crossing over is relatively unaffected in strains containing the Roberts mutation, reciprocal mitotic crossovers occur with extremely low frequency in this mutant background. Our results suggest that Eco1 promotes the reciprocal exchange of chromosome arms and maintenance of heterozygosity during mitosis.

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 ira1Δ ira2Δ 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.

The aim of the current study was to determine the signaling differences between gamma- and proton beam-irradiations. A549 lung adenocarcinoma cells were irradiated with 2 Gy proton beam or gamma-radiation. Proton beam was found to be more cytotoxic than gamma-radiation. Proton beam-irradiated cells showed phosphorylation of H2AX, ATM, Chk2, and p53. The mechanism of excessive cell killing in proton beam-irradiated cells was found to be upregulation of Bax and downregulation of Bcl-2. The noteworthy finding of this study is the biphasic activation of the sensor proteins, ATM, and DNA-PK and no activation of ATR by proton irradiation.

ATM-dependent initiation of the radiation-induced G(2)/M checkpoint arrest is well established. Recent results have shown that the majority of DNA double-strand breaks (DSBs) in G(2) phase are repaired by DNA nonhomologous end joining (NHEJ), while approximately 15% of DSBs are slowly repaired by homologous recombination. Here, we evaluate how the G(2)/M checkpoint is maintained in irradiated G(2) cells, in light of our current understanding of G(2) phase DSB repair. We show that ATM-dependent resection at a subset of DSBs leads to ATR-dependent Chk1 activation. ATR-Seckel syndrome cells, which fail to efficiently activate Chk1, and small interfering RNA (siRNA) Chk1-treated cells show premature mitotic entry. Thus, Chk1 significantly contributes to maintaining checkpoint arrest. Second, sustained ATM signaling to Chk2 contributes, particularly when NHEJ is impaired by XLF deficiency. We also show that cells lacking the mediator proteins 53BP1 and MDC1 initially arrest following radiation doses greater than 3 Gy but are subsequently released prematurely. Thus, 53BP1(-/-) and MDC1(-/-) cells manifest a checkpoint defect at high doses. This failure to maintain arrest is due to diminished Chk1 activation and a decreased ability to sustain ATM-Chk2 signaling. The combined repair and checkpoint defects conferred by 53BP1 and MDC1 deficiency act synergistically to enhance chromosome breakage.

Two major control systems regulate early stages of mitosis: activation of Cdk1 and anaphase control through assembly and disassembly of the mitotic spindle. In parallel to cell cycle progression, centrosomal duplication is regulated through proteins including Nek2. Recent studies suggest that centrosome-localized Chk1 forestalls premature activation of centrosomal Cdc25b and Cdk1 for mitotic entry, whereas Chk2 binds centrosomes and arrests mitosis only after activation by ATM and ATR in response to DNA damage. Here, we show that Chk2 centrosomal binding does not require DNA damage, but varies according to cell cycle progression. These and other data suggest a model in which binding of Chk2 to the centrosome at multiple cell cycle junctures controls co-localization of Chk2 with other cell cycle and centrosomal regulators., (© 2010 Landes Bioscience)

While telomeres must provide mechanisms to prevent DNA repair and DNA damage checkpoint factors from fusing chromosome ends and causing permanent cell cycle arrest, these factors associate with functional telomeres and play critical roles in the maintenance of telomeres. Previous studies have established that Tel1 (ATM) and Rad3 (ATR) kinases play redundant but essential roles for telomere maintenance in fission yeast. In addition, the Rad9-Rad1-Hus1 (911) and Rad17-RFC complexes work downstream of Rad3 (ATR) in fission yeast telomere maintenance. Here, we investigated how 911, Rad17-RFC and another RFC-like complex Ctf18-RFC contribute to telomere maintenance in fission yeast cells lacking Tel1 and carrying a novel hypomorphic allele of rad3 (DBD-rad3), generated by the fusion between the DNA binding domain (DBD) of the fission yeast telomere capping protein Pot1 and Rad3. Our investigations have uncovered a surprising redundancy for Rad9 and Hus1 in allowing Rad1 to contribute to telomere maintenance in DBD-rad3 tel1 cells. In addition, we found that Rad17-RFC and Ctf18-RFC carry out redundant telomere maintenance functions in DBD-rad3 tel1 cells. Since checkpoint sensor proteins are highly conserved, genetic redundancies uncovered here may be relevant to telomere maintenance and detection of DNA damage in other eukaryotes.

In order to maintain genetic integrity, cells are equipped with cell cycle checkpoints that detect DNA damage, orchestrate repair, and if necessary, eliminate severely damaged cells by inducing apoptotic cell death. The mitotic machinery is now emerging as an important determinant of the cellular responses to DNA damage where it functions as both the downstream target and the upstream regulator of the G2/M checkpoint. Cell cycle kinases and the DNA damage checkpoint kinases appear to reciprocally control each other. Specifically, cell cycle kinases control the inactivation of DNA damage checkpoint signaling. Termination of a DNA damage response by mitotic kinases appears to be an evolutionary conserved mechanism that allows resumption of cell cycle progression. Here we review recent reports in which molecular mechanisms underlying checkpoint silencing at the G2/M transition are elucidated.

The decatenation G2 checkpoint is proposed to delay cellular progression from G2 into mitosis when intertwined daughter chromatids are insufficiently decatenated. Previous studies indicated that the ATM- and Rad3-related (ATR) checkpoint kinase, but not the ataxia telangiectasia-mutated (ATM) kinase, was required for decatenation G2 checkpoint function. Here, we show that the method used to quantify decatenation G2 checkpoint function can influence the identification of genetic requirements for the checkpoint. Normal human diploid fibroblast (NHDF) lines responded to the topoisomerase II (topo II) catalytic inhibitor ICRF-193 with a stringent G2 arrest and a reduction in the mitotic index. While siRNA-mediated depletion of ATR and CHEK1 increased the mitotic index in ICRF-193 treated NHDF lines, depletion of these proteins did not affect the mitotic entry rate, indicating that the decatenation G2 checkpoint was functional. These results suggest that ATR and CHEK1 are not required for the decatenation G2 checkpoint, but may influence mitotic exit after inhibition of topo II. A re-evaluation of ataxia telangiectasia (AT) cell lines using the mitotic entry assay indicated that ATM was required for the decatenation G2 checkpoint. Three NHDF cell lines responded to ICRF-193 with a mean 98% inhibition of the mitotic entry rate. Examination of the mitotic entry rates in AT fibroblasts upon treatment with ICRF-193 revealed a significantly attenuated decatenation G2 checkpoint response, with a mean 59% inhibition of the mitotic entry rate. In addition, a normal lymphoblastoid line exhibited a 95% inhibition of the mitotic entry rate after incubation with ICRF-193, whereas two AT lymphoblastoid lines displayed only 36% and 20% inhibition of the mitotic entry rate. Stable depletion of ATM in normal human fibroblasts with short hairpin RNA also attenuated decatenation G2 checkpoint function by an average of 40%. Western immunoblot analysis demonstrated that treatment with ICRF-193 induced ATM autophosphorylation and ATM-dependent phosphorylation of Ser15-p53 and Thr68 in Chk2, but no appreciable phosphorylation of Ser139-H2AX or Ser345-Chk1. The results suggest that inhibition of topo II induces ATM to phosphorylate selected targets that contribute to a G2 arrest independently of DNA damage.

The catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) plays a major role in the repair of DNA double-strand breaks (DSBs) by nonhomologous end joining (NHEJ). We have previously shown that DNA-PKcs is autophosphorylated in response to ionizing radiation (IR) and that dephosphorylation by a protein phosphatase 2A (PP2A)-like protein phosphatase (PP2A, PP4, or PP6) regulates the protein kinase activity of DNA-PKcs. Here we report that DNA-PKcs interacts with the catalytic subunits of PP6 (PP6c) and PP2A (PP2Ac), as well as with the PP6 regulatory subunits PP6R1, PP6R2, and PP6R3. Consistent with a role in the DNA damage response, silencing of PP6c by small interfering RNA (siRNA) induced sensitivity to IR and delayed release from the G(2)/M checkpoint. Furthermore, siRNA silencing of either PP6c or PP6R1 led to sustained phosphorylation of histone H2AX on serine 139 (gamma-H2AX) after IR. In contrast, silencing of PP6c did not affect the autophosphorylation of DNA-PKcs on serine 2056 or that of the ataxia-telangiectasia mutated (ATM) protein on serine 1981. We propose that a novel function of DNA-PKcs is to recruit PP6 to sites of DNA damage and that PP6 contributes to the dephosphorylation of gamma-H2AX, the dissolution of IR-induced foci, and release from the G(2)/M checkpoint in vivo.