Unperturbed Cells Research Articles

ChAM, a novel motif that mediates PALB2 intrinsic chromatin binding and facilitates DNA repair

The partner and localizer of breast cancer 2 susceptibility protein (PALB2) is crucial for the repair of DNA damage by homologous recombination. Here, we report that chromatin-association motif (ChAM), an evolutionarily conserved motif in PALB2, is necessary and sufficient to mediate its chromatin association in both unperturbed and damaged cells. ChAM is distinct from the previously described PALB2 DNA-binding regions. Deletion of ChAM decreases PALB2 and Rad51 accumulation at DNA damage sites and confers cellular hypersensitivity to the genotoxic drug mitomycin C. These results suggest that PALB2 chromatin association via ChAM facilitates PALB2 function in the cellular resistance to DNA damage.

Checkpoint kinase 1 (Chk1)-short is a splice variant and endogenous inhibitor of Chk1 that regulates cell cycle and DNA damage checkpoints

Checkpoint kinase 1 (Chk1) is a key regulator of checkpoint signaling in both the unperturbed cell cycle and DNA damage response. Under these conditions, Chk1 becomes active to prevent premature CDK1 activation and mitotic entry until DNA is properly replicated or repaired. It is unclear how Chk1 activity is controlled in the unperturbed cell cycle. During DNA damage, Chk1 is activated by ataxia telangiectasia and Rad3 related (ATR)-mediated phosphorylation; however, it is not entirely clear how this phosphorylation results in Chk1 activation. Here we report an N-terminally truncated alternative splice variant of Chk1, Chk1-S. Importantly, we show that Chk1-S is an endogenous repressor and regulator of Chk1. In the unperturbed cell cycle, Chk1-S interacts with and antagonizes Chk1 to promote the S-to-G2/M phase transition. During DNA damage, Chk1 is phosphorylated, which disrupts the Chk1-Chk1-S interaction, resulting in free, active Chk1 to arrest the cell cycle and facilitate DNA repair. Higher levels of Chk1-S are expressed, along with Chk1, in fetal and cancer tissues than in normal tissues. However, forced overexpression of Chk1-S in cultured cells and tumor xenografts induces premature mitotic entry, mitotic catastrophe, and reduction of tumor growth. The identification of Chk1-S as a unique splice variant and key regulator of Chk1 provides insights into cell cycle regulation and DNA damage response.

Interaction of LHBs with C53 promotes hepatocyte mitotic entry: A novel mechanism for HBV-induced hepatocellular carcinoma

The pre-S mutant LHBs, especially the pre-S2 type, is believed to be crucial in HBV-associated hepatocellular carcinogenesis. However, the mechanism of HBV-induced hepatocellular carcinoma is not fully understood. To identify the mechanism, pre-S2 LHBs-interacting proteins were studied, by performing a yeast two-hybrid screen of a human liver cDNA library. Screening of the library resulted in the isolation of several positive clones. Sequencing of the positive clones identified the full-length cDNA of the C53 gene. After identification of the interaction, the roles of LHBs on Cdk1, Chk1 activation and mitotic entry were studied. Screening of the library resulted in the isolation of several positive clones, that encoded the full-length cDNA of the C53 gene. We found that C53 interacts with pre-S2 LHBs both in vitro and invivo, but not with LHBs or other mutants. The binding of pre-S2 LHBs with C53 causes increased Cdk1 activation and mitotic entry, and the function of Chk1 is partially inhibited by the binding of pre-S2 LHBs with C53. Taken together, our results strongly suggest that the binding of pre-S2 LHBs with C53 is a novel negative regulator of the checkpoint response. By counteracting C53, pre-S2 LHBs promotes Cdk1 activation and mitotic entry in unperturbed cell cycle progression and delays the function of Chk1, which may be a novel potential mechanism for HBV-induced hepatocellular carcinoma (HCC).

Mitotic Exit Control: A Space and Time Odyssey

The mitotic exit network (MEN), a protein kinase cascade under the switch-like control of the small GTPase Tem1, triggers exit from mitosis in budding yeast. Now it emerges that signals from both Tem1 and the yeast Polo kinase Cdc5 converge onto the MEN kinase Cdc15 to accurately restrict MEN activation to late mitosis.

Safeguarding genome integrity: the checkpoint kinases ATR, CHK1 and WEE1 restrain CDK activity during normal DNA replication

Mechanisms that preserve genome integrity are highly important during the normal life cycle of human cells. Loss of genome protective mechanisms can lead to the development of diseases such as cancer. Checkpoint kinases function in the cellular surveillance pathways that help cells to cope with DNA damage. Importantly, the checkpoint kinases ATR, CHK1 and WEE1 are not only activated in response to exogenous DNA damaging agents, but are active during normal S phase progression. Here, we review recent evidence that these checkpoint kinases are critical to avoid deleterious DNA breakage during DNA replication in normal, unperturbed cell cycle. Possible mechanisms how loss of these checkpoint kinases may cause DNA damage in S phase are discussed. We propose that the majority of DNA damage is induced as a consequence of deregulated CDK activity that forces unscheduled initiation of DNA replication. This could generate structures that are cleaved by DNA endonucleases leading to the formation of DNA double-strand breaks. Finally, we discuss how these S phase effects may impact on our understanding of cancer development following disruption of these checkpoint kinases, as well as on the potential of these kinases as targets for cancer treatment.

Cdc15 integrates Tem1 GTPase-mediated spatial signals with Polo kinase-mediated temporal cues to activate mitotic exit

In budding yeast, a Ras-like GTPase signaling cascade known as the mitotic exit network (MEN) promotes exit from mitosis. To ensure the accurate execution of mitosis, MEN activity is coordinated with other cellular events and restricted to anaphase. The MEN GTPase Tem1 has been assumed to be the central switch in MEN regulation. We show here that during an unperturbed cell cycle, restricting MEN activity to anaphase can occur in a Tem1 GTPase-independent manner. We found that the anaphase-specific activation of the MEN in the absence of Tem1 is controlled by the Polo kinase Cdc5. We further show that both Tem1 and Cdc5 are required to recruit the MEN kinase Cdc15 to spindle pole bodies, which is both necessary and sufficient to induce MEN signaling. Thus, Cdc15 functions as a coincidence detector of two essential cell cycle oscillators: the Polo kinase Cdc5 synthesis/degradation cycle and the Tem1 G-protein cycle. The Cdc15-dependent integration of these temporal (Cdc5 and Tem1 activity) and spatial (Tem1 activity) signals ensures that exit from mitosis occurs only after proper genome partitioning.

Imaging Specific Protein Labels on Eukaryotic Cells in Liquid with Scanning Transmission Electron Microscopy

Understanding the structure and dynamics of the protein complexes that underlie cellular function is a central scientific challenge. Biochemical techniques used to identify such complexes would be enhanced by the imaging of specific molecular positions in the context of intact cells, with protein-scale resolution (on the order of a few nanometers). Currently, though, nanometer resolution can only be achieved at the cost of less-direct imaging of the unperturbed cell. Cellular ultrastructure is traditionally studied by transmission electron microscopy (TEM), which yields nanometer resolution on embedded and stained sections, or cryo sections. These cellular samples are neither intact nor in their native liquid state. Light microscopy is used to image protein distributions in fluorescently labeled cells in liquid to investigate cellular function, but even recent improvements in resolution by nanoscopy techniques are still insufficient to resolve the individual constituents of protein complexes. Thus, development of techniques capable of high-resolution imaging in native cellular states would contribute significantly to our understanding of cellular function at the molecular level. The development of liquid compartments that include electron-transparent silicon nitride membrane windows has led to the introduction of a novel concept to achieve nanometer resolution on tagged proteins in cells.

Fluorescence Correlation Spectroscopy of Intact Nuclear Pore Complexes

No methods proposed thus far have the sensitivity to measure the transport of single molecules through single nuclear pore complexes (NPCs) in intact cells. Here we demonstrate that fluorescence correlation spectroscopy (FCS) combined with real-time tracking of the center of mass of single NPCs in live, unperturbed cells allows us to detect the transport of single molecules in a reference system of a pore with high temporal (millisecond) and spatial (limited by diffraction) resolution. We find that the transport of the classical receptor karyopherin-β1 (Kapβ1) is regulated so as to produce a peculiar distribution of characteristic times at the NPC. This regulation, which is spatially restricted to the pore, depends on the properties and metabolic energy of Kapβ1. As such, this method provides a powerful tool for studying nucleocytoplasmic shuttling at the nanometer scale under physiological conditions.

Single-Agent Inhibition of Chk1 Is Antiproliferative in Human Cancer Cell Lines In Vitro and Inhibits Tumor Xenograft Growth In Vivo

Chk1 is a serine/threonine kinase that plays several important roles in the cellular response to genotoxic stress. Since many current standard-of-care therapies for human cancer directly damage DNA or inhibit DNA synthesis, there is interest in using small molecule inhibitors of Chk1 to potentiate their clinical activity. Additionally, Chk1 is known to be critically involved in cell cycle progression of unperturbed cells. Therefore, it is plausible that treatment with a Chkl inhibitor alone could also be an efficacious cancer therapy. Here we report that Chk1-A, a potent and highly selective small molecule inhibitor of Chk1, is antiproliferative as a single agent in a variety of human cancer cell lines in vitro. The inhibition of proliferation is associated with collapse of DNA replication and apoptosis. Rapid decreases in inhibitory phosphorylation of CDKs and a concomitant increase in CDK kinase activity and chromatin loading of Cdc45 suggest that the antiproliferative and proapoptotic activity of Chk1-A is at least in part due to deregulation of DNA synthesis. We extend these in vitro studies by demonstrating that Chk1-A inhibits the growth of tumor xenografts in vivo in a treatment regimen that is well tolerated. Together, these results suggest that single-agent inhibition of Chk1 may be an effective treatment strategy for selected human malignancies.

The cortical protein Lte1 promotes mitotic exit by inhibiting the spindle position checkpoint kinase Kin4

The spindle position checkpoint (SPOC) is an essential surveillance mechanism that allows mitotic exit only when the spindle is correctly oriented along the cell axis. Key SPOC components are the kinase Kin4 and the Bub2-Bfa1 GAP complex that inhibit the mitotic exit-promoting GTPase Tem1. During an unperturbed cell cycle, Kin4 associates with the mother spindle pole body (mSPB), whereas Bub2-Bfa1 is at the daughter SPB (dSPB). When the spindle is mispositioned, Bub2-Bfa1 and Kin4 bind to both SPBs, which enables Kin4 to phosphorylate Bfa1 and thereby block mitotic exit. Here, we show that the daughter cell protein Lte1 physically interacts with Kin4 and inhibits Kin4 kinase activity. Specifically, Lte1 binds to catalytically active Kin4 and promotes Kin4 hyperphosphorylation, which restricts Kin4 binding to the mSPB. This Lte1-mediated exclusion of Kin4 from the dSPB is essential for proper mitotic exit of cells with a correctly aligned spindle. Therefore, Lte1 promotes mitotic exit by inhibiting Kin4 activity at the dSPB.

Cancer Cells Cyclically Lose and Regain Drug-Resistant Highly Tumorigenic Features Characteristic of a Cancer Stem-like Phenotype

Drug resistance and brisk tumor initiation have traditionally been viewed as preexisting phenotypes present in small subpopulations of neoplastic cells sometimes termed cancer stem cells. However, recent work in cancer cell lines has shown that drug-resistant tumor-initiating features can emerge de novo within fractionated subpopulations of cells initially lacking these phenotypes. In the present study, we asked whether such phenotypic plasticity exists broadly in unperturbed cancer cell lines and tumor xenografts growing spontaneously without interventions such as drug selection or fractionation into subpopulations used in prior studies. To address this question, we used side population (SP) analysis combined with fluorescence labeling to identify a drug-resistant highly tumorigenic subpopulation and to track and analyze its interaction with the larger phenotypically negative population over time. Remarkably, we observed that SP size fluctuated in a cyclical manner: first contracting via differentiation into the non-SP (NSP) and then reexpanding via simultaneous direct conversion of numerous NSP cells back to the SP phenotype both in culture and in tumor xenografts. These findings show for the first time that adaptive, cancer-promoting traits such as drug resistance and brisk tumor initiation arise not only as solitary events under selective pressures but also as highly orchestrated transitions occurring concurrently in large numbers of cells even without specifically induced drug selection, ectopic gene expression, or fractionation into subpopulations. This high level of coordinated phenotypic plasticity bears consideration when using cancer cell lines as experimental models and may have significant implications for therapeutic efforts targeting cancer stem cells, which are marked by a drug-resistant tumor-initiating phenotype.

Budding yeast Dma1 and Dma2 participate in regulation of Swe1 levels and localization

Timely down-regulation of the evolutionarily conserved protein kinase Swe1 plays an important role in cell cycle control, as Swe1 can block nuclear division through inhibitory phosphorylation of the catalytic subunit of cyclin-dependent kinase. In particular, Swe1 degradation is important for budding yeast cell survival in case of DNA replication stress, whereas it is inhibited by the morphogenesis checkpoint in response to alterations in actin cytoskeleton or septin structure. We show that the lack of the Dma1 and Dma2 ubiquitin ligases, which moderately affects Swe1 localization and degradation during an unperturbed cell cycle with no apparent phenotypic effects, is toxic for cells that are partially defective in Swe1 down-regulation. Moreover, Swe1 is stabilized, restrained at the bud neck, and hyperphosphorylated in dma1Δ dma2Δ cells subjected to DNA replication stress, indicating that the mechanism stabilizing Swe1 under these conditions is different from the one triggered by the morphogenesis checkpoint. Finally, the Dma proteins are required for proper Swe1 ubiquitylation. Taken together, the data highlight a previously unknown role of these proteins in the complex regulation of Swe1 and suggest that they might contribute to control, directly or indirectly, Swe1 ubiquitylation.

Ixr1 Is Required for the Expression of the Ribonucleotide Reductase Rnr1 and Maintenance of dNTP Pools

The Saccharomyces cerevisiae Dun1 protein kinase is a downstream target of the conserved Mec1-Rad53 checkpoint pathway. Dun1 regulates dNTP pools during an unperturbed cell cycle and after DNA damage by modulating the activity of ribonucleotide reductase (RNR) by multiple mechanisms, including phosphorylation of RNR inhibitors Sml1 and Dif1. Dun1 also activates DNA-damage-inducible genes by inhibiting the Crt1 transcriptional repressor. Among the genes repressed by Crt1 are three out of four RNR genes: RNR2, RNR3, and RNR4. The fourth RNR gene, RNR1, is also DNA damage-inducible, but is not controlled by Crt1. It has been shown that the deletion of DUN1 is synthetic lethal with the deletion of IXR1, encoding an HMG-box-containing DNA binding protein, but the reason for this lethality is not known. Here we demonstrate that the dun1 ixr1 synthetic lethality is caused by an inadequate RNR activity. The deletion of IXR1 results in decreased dNTP levels due to a reduced RNR1 expression. The ixr1 single mutants compensate for the reduced Rnr1 levels by the Mec1-Rad53-Dun1-Crt1–dependent elevation of Rnr3 and Rnr4 levels and downregulation of Sml1 levels, explaining why DUN1 is indispensible in ixr1 mutants. The dun1 ixr1 synthetic lethality is rescued by an artificial elevation of the dNTP pools. We show that Ixr1 is phosphorylated at several residues and that Ser366, a residue important for the interaction of HMG boxes with DNA, is required for Ixr1 phosphorylation. Ixr1 interacts with DNA at multiple loci, including the RNR1 promoter. Ixr1 levels are decreased in Rad53-deficient cells, which are known to have excessive histone levels. A reduction of the histone gene dosage in the rad53 mutant restores Ixr1 levels. Our results demonstrate that Ixr1, but not Dun1, is required for the proper RNR1 expression both during an unperturbed cell cycle and after DNA damage.

Abstract 2971: The modulation checkpoint signaling by natural products during the DNA damage response

Abstract ATM (ataxia telangiectasia mutated) and ATR (AT and Rad3 related) protein kinases play a crucial role in cellular DNA damage responses. The inhibition of these kinases and/or related signaling molecules lead to an abolishment of one such signal pathways termed as checkpoints. It is expected that the discovery of checkpoint modulator such as natural products will be an effective assistance of anti-cancer therapy with lower side effect. In the present study, we demonstrated the inhibitory effect of Squalene (SQ), flavonoids derived from Persimmon leaves (PL) and Schisandrin-B (SchB) on ATM/ATR-dependent checkpoint signaling pathway. The cell viability was decreased by SQ, PL or SchB treated A549 adenocarcinoma after gamma-irradiation (IR) or UV exposure. In each SQ, PL and SchB treated cells, G2/M checkpoint activity was abolished in DNA damaged cells. In vitro kinase assay revealed that PL and SchB inhibits ATM and ATR, respectively. SQ, on the other hand, showed no effect on both kinases. Interestingly, SQ increased Wip1 protein expression in unperturbed cells, and suppressed ATM activation in IR treated cells. Consistent with potential inhibition of ATM by SQ or PL, IR-induced phosphorylations of ATM effectors such as p53 (S15) and Chk1 (S317) were remarkably inhibited by the treatment of SQ or PL. SchB inhibited the specific phosphorylation of these effectors by ATR. Moreover, Xenograft and allograft studies with natural products were demonstrated that the tumor growth of both cancer cells in vivo were obviously inhibited by SQ, PL or SchB treatment. These results suggest that some natural products such as SQ, PL and SchB might be applicable to the sensitization of anti-cancer therapies such as radio- /chemotherapy. 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 2971. doi:10.1158/1538-7445.AM2011-2971

Abstract 4194: Constitutive DNA-damage signaling promotes cancer cell proliferation through Chk1-CIP2A pathway independent of ATM-ATR

Abstract DNA damage is a hallmark of malignantly transformed cells. Accordingly, overexpression of the DNA damage activated kinases has been observed in different human cancers. However, it is unclear whether constitutive DNA damage present in unperturbed cancer cells promotes tumourigenesis. Here we show that increased activity of DNA damage sensitive kinase Chk1 promotes proliferation of several cancer cell types by stimulating expression of CIP2A protein. Either chemical or genetic inhibition of Chk1, and not of ATM-ATR, results in potent inhibition of CIP2A protein expression levels. Importantly, Chk1 siRNA-elicited inhibition of cell proliferation and clonogenic growth is rescued by overexpression of CIP2A from a heterologous promoter. Moreover, in clinical (human) tumor samples there is a significant association between CIP2A and Chk1 expression in both ovarian and gastric cancers. Intriguingly, meta-analysis of seventeen published genome wide studies on different types of cancers unveils a striking similarity in the gene expression patterns of both CIP2A and Chk1. Amongst these, 12/17 studies show overexpression (top 10%) of both these proteins. Additionally, similarly to CIP2A, increased Chk1 expression is shown to correlate with tumor progression in human cancer. In summary, these results identify a novel function for DNA damage kinase Chk1 in regulation of the human oncoprotein CIP2A. In general, these results provide an unprecented molecular mechanism by which constitutive DNA damage present in cancer cells promotes tumourigenesis. Additionally, as CIP2A is not significantly expressed in most of the normal human tissues, it is proposed that targeting of CIP2A might abrogate the Chk1-mediated support of proliferation without concerns related to anticancer therapy directly targeting Chk1. 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 4194. doi:10.1158/1538-7445.AM2011-4194

Abstract 4458: Chk1 inhibition as a novel therapeutic strategy for treating triple negative breast and ovarian cancers

Abstract The serine-threonine kinase Chk1 plays a critical role in cell cycle checkpoints induced in response to genotoxic stress and protecting tumour cells with a defective G1 checkpoint from DNA damage induced by cytotoxic chemotherapeutic drugs. Chk1 inhibitors are currently being pursued in the clinic as a therapeutic strategy to potentiate the anti-tumour efficacy of cytotoxic chemotherapeutic drugs without increasing their systemic toxicity. Several genetic studies have suggested that Chk1 may be critical to ensure accurate DNA replication and division during an unperturbed cell cycle and therefore Chk1 inhibitors, administered as single agents, may be beneficial anti-cancer treatments. Triple-negative breast cancer is characterised by the lack of expression of estrogen and progesterone receptors as well as Her2, along with a gene expression profile and phenotypic characteristics consistent with basal-like breast cancer. Such cancers initially respond well to cytotoxic chemotherapy and PARP inhibitors though the overall prognosis for patients with these tumour types is poor. We therefore hypothesised that triple-negative breast cancer may exhibit sensitivity to single-agent Chk1 inhibition. V158411 is a potent, selective inhibitor of the Chk1 and Chk2 kinases and potently inhibited the proliferation of triple-negative breast (average GI50 0.17µM) compared to ER-positive breast (2.3µM), lung (6.3µM) and colon (2.5µM) cancer cells. In addition, two out of three ovarian cell lines exhibited high sensitivity to growth inhibition by V158411 (SKOV-3 0.06µM and A2780 0.39µM). This sensitivity was independent of p53 mutational status, did not correlate with sensitivity to DNA damaging agents and could not be reversed in ER-positive breast cancer cells with 4-hydroxytamoxifen. Chk1 inhibition reduced triple-negative breast and ovarian cancer cell viability, activated H2AX phosphorylation and induced caspase-3/7 dependent apoptosis at doses that correlated with the anti-proliferative effects. V158411 did not induce a consistent cell cycle arrest or cells to undergo a premature mitosis. V158411 still potentiated the cytotoxicity of cisplatin and gemcitabine in p53 deficient cell lines. In an attempt to understand the sensitivity of certain cancer cell types and identify biomarkers to select sensitive patient populations, tumour cell lysates were profiled for proteins involved in the cell cycle, DNA damage response and apoptosis. Sensitivity to V158411 correlated with increased expression of pChk1(S296) and reduced expression of Mcl-1. This study suggests that triple-negative breast and ovarian cancers rely on Chk1 to complete an unperturbed cell cycle and that inhibiting Chk1 results in increased DNA damage and cell death in these tumour types. V158411, administered as a single agent or in combination with chemotherapy could be a novel, effective strategy in treating these cancer types. 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 4458. doi:10.1158/1538-7445.AM2011-4458

Abstract LB-188: Chk1 suppresses bypass of mitosis and tetraploidization in p53-deficient cells

Abstract Many checkpoint-deficient cancer cells are unable to maintain a numerically stable chromosome complement. The Chk1 kinase has a well-established role in the DNA damage checkpoint responses and is also essential for viability; however little is known about its role throughout the unperturbed cell cycle. Here, we rigorously examine the role of Chk1 in the maintenance of cell ploidy and mitotic progression. Using recombinant adeno-associated virus-based gene targeting techniques, we disrupted one allele of CHK1 in human cancer cell lines that are proficient or deficient for the tumor suppressor p53. We assessed ploidy by fluorescence in situ hybridization and metaphase chromosome analysis, and examined cell cycle progression by live cell time-lapse fluorescent microscopy, DNA labeling, flow cytometry and biochemical analysis. Loss of one CHK1 allele caused an elevated frequency of mitotic bypass in p53-deficient cells. CHK1-haploinsufficient, p53-deficient cells frequently underwent sequential rounds of DNA synthesis without an intervening mitosis, which caused whole genome endoreduplication and tetraploidization. CHK1 haploinsufficient cells exhibited reduced Cdk1 activity, which has been shown to lead to the bypass of mitosis. This mitotic bypass phenotype could be suppressed by targeted reversion of a p53 mutation or by exogenous Cdk1 expression. Interestingly, the rate of mitotic bypass was independent of upstream signaling by ATR to Chk1 and was not associated with DNA damage. These findings demonstrate a previously unrecognized role for Chk1 in promoting the initiation of mitosis during the cancer cell cycle and thereby maintaining ploidy. 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 LB-188. doi:10.1158/1538-7445.AM2011-LB-188

Small Ubiquitin-related Modifier Ligase Activity of Mms21 Is Required for Maintenance of Chromosome Integrity during the Unperturbed Mitotic Cell Division Cycle in Saccharomyces cerevisiae

The SUMO ligase activity of Mms21/Nse2, a conserved member of the Smc5/6 complex, is required for resisting extrinsically induced genotoxic stress. We report that the Mms21 SUMO ligase activity is also required during the unchallenged mitotic cell cycle in Saccharomyces cerevisiae. SUMO ligase-defective cells were slow growing and spontaneously incurred DNA damage. These cells required caffeine-sensitive Mec1 kinase-dependent checkpoint signaling for survival even in the absence of extrinsically induced genotoxic stress. SUMO ligase-defective cells were sensitive to replication stress and displayed synthetic growth defects with DNA damage checkpoint-defective mutants such as mec1, rad9, and rad24. MMS21 SUMO ligase and mediator of replication checkpoint 1 gene (MRC1) were epistatic with respect to hydroxyurea-induced replication stress or methyl methanesulfonate-induced DNA damage sensitivity. Subjecting Mms21 SUMO ligase-deficient cells to transient replication stress resulted in enhancement of cell cycle progression defects such as mitotic delay and accumulation of hyperploid cells. Consistent with the spontaneous activation of the DNA damage checkpoint pathway observed in the Mms21-mediated sumoylation-deficient cells, enhanced frequency of chromosome breakage and loss was detected in these mutant cells. A mutation in the conserved cysteine 221 that is engaged in coordination of the zinc ion in Loop 2 of the Mms21 SPL-RING E3 ligase catalytic domain resulted in strong replication stress sensitivity and also conferred slow growth and Mec1 dependence to unchallenged mitotically dividing cells. Our findings establish Mms21-mediated sumoylation as a determinant of cell cycle progression and maintenance of chromosome integrity during the unperturbed mitotic cell division cycle in budding yeast.

Single-cell heterogeneity analysis provides rapid validation of biophysical parameters in living cells and quantifies heterogeneous sensitivity to interleukin-2, -7, and -15. (57.12)

Abstract The single-cell resolution of flow cytometry provides a quantitative measurement of the heterogeneity of protein expression within clonal populations of cells. As the biological significance of this variability is becoming more widely appreciated, there is a need for new tools and methodology to analyze and leverage the effects of endogenous variation. These tools can provide alternatives to genetic methods, such as siRNA knockdown or germline knockout, that are commonly used to manipulate protein expression. We introduce such a new tool, named ScatterSlice, that enables rapid quantification of the correlation between heterogeneity in cellular responses (measured by staining of phospho-proteins) and varied expression of signaling molecules. We demonstrate its application to the validation of biochemical models of cellular responses to IL-2, IL-7, and IL-15 in living, unperturbed cells. In addition, we demonstrate that heterogeneous distribution of receptor subunits within a population of lymphocytes results in gradations of sensitivity to each cytokine. Finally, using experimental measurements of error and uncertainty in combination with biochemical models, we demonstrate the robustness of this analysis to experimental noise, and consider both limits and extensions to its use.

The Smc5/6 complex and the difficulties cutting the ties of twin sisters

The Smc5/6 complex and the difficulties cutting the ties of twin sisters