Induction of apoptosis in purified animal and plant nuclei by Xenopus egg extracts

We have developed a cell-free system that can trigger the nuclei purified from mouse liver and suspensioncultured carrot cells to undergo apoptosis as defined by the formation of apoptotic bodies and nucleosomal DNA fragments.The effects of different divalent cations and cycloheximide on DNA cleavage in this system were assessed.The fact that nuclei of plant cells can be induced to undergo apoptosis in a cell-free animal system suggests that animals and plants share a common signal transduction pathway triggering in the initiation stage of apoptosis.     

Importin α/β mediates nuclear import of individual SUMO E1 subunits and of the holo-enzyme

SUMOylation, reversible attachment of small ubiquitin-related modifier (SUMO), serves to regulate hundreds of proteins. Consistent with predominantly nuclear targets, enzymes required for attachment and removal of SUMO are highly enriched in this compartment. This is true also for the first enzyme of the SUMOylation cascade, the SUMO E1 enzyme heterodimer, Aos1/Uba2 (SAE1/SAE2). This essential enzyme serves to activate SUMO and to transfer it to the E2-conjugating enzyme Ubc9. Although the last 40 amino acids in yeast Uba2 have been implicated in its nuclear localization, little was known about the import pathways of Aos1, Uba2, and/or of the assembled E1 heterodimer. Here we show that the mammalian E1 subunits can be imported separately, identify nuclear localization signals (NLSs) in Aos1 and in Uba2, and demonstrate that their import is mediated by importin α/β in vitro and in intact cells. Once assembled into a stable heterodimer, the E1 enzyme can still be efficiently imported by importin α/β, due to the Uba2 NLS that is still accessible. These pathways may serve distinct purposes: import of nascent subunits prior to assembly and reimport of stable E1 enzyme complex after mitosis.     

Interaction between Rad9–Hus1–Rad1 and TopBP1 activates ATR–ATRIP and promotes TopBP1 recruitment to sites of UV-damage

The checkpoint clamp Rad9–Hus1–Rad1 (9–1–1) interacts with TopBP1 via two casein kinase 2 (CK2)-phosphorylation sites, Ser-341 and Ser-387 in Rad9. While this interaction is known to be important for the activation of ATR-Chk1 pathway, how the interaction contributes to their accumulation at sites of DNA damage remains controversial. Here, we have studied the contribution of the 9–1–1/TopBP1 interaction to the assembly and activation of checkpoint proteins at damaged DNA. UV-irradiation enhanced association of Rad9 with chromatin and its localization to sites of DNA damage without a direct interaction with TopBP1. TopBP1, as well as RPA and Rad17 facilitated Rad9 recruitment to DNA damage sites. Similar to Rad9, TopBP1 also localized to sites of UV-induced DNA damage. The DNA damage-induced TopBP1 redistribution was delayed in cells expressing a TopBP1 binding-deficient Rad9 mutant. Pharmacological inhibition of ATR recapitulated the delayed accumulation of TopBP1 in the cells, suggesting that ATR activation will induce more efficient accumulation of TopBP1. Taken together, TopBP1 and Rad9 can be independently recruited to damaged DNA. Once recruited, a direct interaction of 9–1–1/TopBP1 occurs and induces ATR activation leading to further TopBP1 accumulation and amplification of the checkpoint signal. Thus, we propose a new positive feedback mechanism that is necessary for successful formation of the damage-sensing complex and DNA damage checkpoint signaling in human cells.     

DNA topoisomerase IIα controls replication origin cluster licensing and firing time in Xenopus egg extracts

Sperm chromatin incubated in Xenopus egg extracts undergoes origin licensing and nuclear assembly before DNA replication. We found that depletion of DNA topoisomerase IIα (topo IIα), the sole topo II isozyme of eggs and its inhibition by ICRF-193, which clamps topo IIα around DNA have opposite effects on these processes. ICRF-193 slowed down replication origin cluster activation and fork progression in a checkpoint-independent manner, without altering replicon size. In contrast, topo IIα depletion accelerated origin cluster activation, and topo IIα add-back negated overinitiation. Therefore, topo IIα is not required for DNA replication, but topo IIα clamps slow replication, probably by forming roadblocks. ICRF-193 had no effect on DNA synthesis when added after nuclear assembly, confirming that topo IIα activity is dispensable for replication and revealing that topo IIα clamps formed on replicating DNA do not block replication, presumably because topo IIα acts behind and not in front of forks. Topo IIα depletion increased, and topo IIα addition reduced, chromatin loading of MCM2-7 replicative helicase, whereas ICRF-193 did not affect MCM2-7 loading. Therefore, topo IIα restrains MCM2-7 loading in an ICRF-193-resistant manner during origin licensing, suggesting a model for establishing the sequential firing of origin clusters.     

Importin β3 mediates the nuclear import of human ribosomal protein L7 through its interaction with the multifaceted basic clusters of L7

We show that importin β3 is essential for the nuclear import of L7. The import is mediated via the multifaceted basic amino acid clusters present in the NH₂-region of L7, and is RanGTP-dependent. Using a (EGFP)₃ reporter system and a FRAP assay, the role the individual clusters play as a functional NLS has been characterized, and each cluster was found to exhibit a different rate of real time nuclear uptake. We assume that having such a multiple NLS may provide L7 with preferential nuclear uptake.

Structured summary

MINT-7992735: Importin beta-3 (uniprotkb:O00410) binds (MI:0407) to L7 (uniprotkb:P18124) by biophysical (MI:0013)MINT-7992687: L7 (uniprotkb:P18124) binds (MI:0407) to Importin beta-3 (uniprotkb:O00410) by filter binding (MI:0049)MINT-7992699: L7 (uniprotkb:P18124) physically interacts (MI:0915) with Importin beta-3 (uniprotkb:O00410) by affinity chromatography technology (MI:0004)MINT-7992718: L7 (uniprotkb:P18124) physically interacts (MI:0915) with RAN (uniprotkb:P62826) by competition binding (MI:0405)MINT-7992671: L7 (uniprotkb:P18124) physically interacts (MI:0915) with Importin beta-3 (uniprotkb:O00410) by pull down (MI:0096)     

Nuclear assembly of purified Crythecodinium cohnii chromosomes in cell—free extracts of Xenopus laevis eggs

Incubation of dinoflagellate Crythecodinium cohnii chromosomes in cytoplasmic extracts of unfertilized Xenopus laevis eggs resulted in chromosomes decondensation and recondensation,nuclear envelope assembly,and nuclear reconstitution.Dinoflagellate Crythecodinium cohnii is a kind of primitive eukaryote which possesses numerous permanently condensed chromosomes and discontinuous double-layered nuclear membrane throughout the cell cycle.The assembled nuclei,being surrounded by a continuous double membrane containing nuclear pores and the uniformly dispersed chromatin fibers are morphologically distinguishable from that of Dinoflagellate Crythecodinium cohnii.However,incubation of dinoflagellate Cyrthecodinium cohnii chromosomes in the extracts from dinoflagellate Crythecodinium cohnii cells does not induce nuclear reconstitution.     

ATR and Chk1 Suppress a Caspase-3–Dependent Apoptotic Response Following DNA Replication Stress

The related PIK-like kinases Ataxia-Telangiectasia Mutated (ATM) and ATM- and Rad3-related (ATR) play major roles in the regulation of cellular responses to DNA damage or replication stress. The pro-apoptotic role of ATM and p53 in response to ionizing radiation (IR) has been widely investigated. Much less is known about the control of apoptosis following DNA replication stress. Recent work indicates that Chk1, the downstream phosphorylation target of ATR, protects cells from apoptosis induced by DNA replication inhibitors as well as IR. The aim of the work reported here was to determine the roles of ATM- and ATR-protein kinase cascades in the control of apoptosis following replication stress and the relationship between Chk1-suppressed apoptotic pathways responding to replication stress or IR. ATM and ATR/Chk1 signalling pathways were manipulated using siRNA-mediated depletions or specific inhibitors in two tumour cell lines or fibroblasts derived from patients with inherited mutations. We show that depletion of ATM or its downstream phosphorylation targets, NBS1 and BID, has relatively little effect on apoptosis induced by DNA replication inhibitors, while ATR or Chk1 depletion strongly enhances cell death induced by such agents in all cells tested. Furthermore, early events occurring after the disruption of DNA replication (accumulation of RPA foci and RPA34 hyperphosphorylation) in ATR- or Chk1-depleted cells committed to apoptosis are not detected in ATM-depleted cells. Unlike the Chk1-suppressed pathway responding to IR, the replication stress-triggered apoptotic pathway did not require ATM and is characterized by activation of caspase 3 in both p53-proficient and -deficient cells. Taken together, our results show that the ATR-Chk1 signalling pathway plays a major role in the regulation of death in response to DNA replication stress and that the Chk1-suppressed pathway protecting cells from replication stress is clearly distinguishable from that protecting cells from IR.     

Physiology and pathology of nuclear phospholipase C β1

The existence and function of inositide signaling in the nucleus is well documented and we know that the existence of the inositide cycle inside the nucleus has a biological role. An autonomous lipid-dependent signaling system, independently regulated from its plasma membrane counterpart, acts in the nucleus and modulates cell cycle progression and differentiation.We and others focused on PLCβ1, which is the most extensively investigated PLC isoform in the nuclear compartment. PLCβ1 is a key player in the regulation of nuclear inositol lipid signaling, and, as discussed above, its function could also be involved in nuclear structure because it hydrolyses PtdIns(4,5)P2, a well accepted regulator of chromatin remodelling. The evidence, in a number of patients with myelodysplastic syndromes, that the mono-allelic deletion of PLCβ1 is associated with an increased risk of developing acute myeloid leukemia paves the way for an entirely new field of investigation. Indeed the genetic defect evidenced, in addition to being a useful prognostic tool, also suggests that altered expression of this enzyme could have a role in the pathogenesis of this disease, by causing an imbalance between proliferation and apoptosis. The epigenetics of PLCβ1 expression in MDS has been reviewed as well.     

Reduced ATR or Chk1 Expression Leads to Chromosome Instability and Chemosensitization of Mismatch Repair–deficient Colorectal Cancer Cells

Genomic instability in colorectal cancer is categorized into two distinct classes: chromosome instability (CIN) and microsatellite instability (MSI). MSI is the result of mutations in the mismatch repair (MMR) machinery, whereas CIN is often thought to be associated with a disruption in the APC gene. Clinical data has recently shown the presence of heterozygous mutations in ATR and Chk1 in human cancers that exhibit MSI, suggesting that those mutations may contribute to tumorigenesis. To determine whether reduced activity in the DNA damage checkpoint pathway would cooperate with MMR deficiency to induce CIN, we used siRNA strategies to partially decrease the expression of ATR or Chk1 in MMR-deficient colorectal cancer cells. The resultant cancer cells display a typical CIN phenotype, as characterized by an increase in the number of chromosomal abnormalities. Importantly, restoration of MMR proficiency completely inhibited induction of the CIN phenotype, indicating that the combination of partial checkpoint blockage and MMR deficiency is necessary to trigger CIN. Moreover, disruption of ATR and Chk1 in MMR-deficient cells enhanced the sensitivity to treatment with the commonly used colorectal chemotherapeutic compound, 5-fluorouracil. These results provide a basis for the development of a combination therapy for those cancer patients.     

Tim–Tipin dysfunction creates an indispensible reliance on the ATR–Chk1 pathway for continued DNA synthesis

The Tim (Timeless)–Tipin complex has been proposed to maintain genome stability by facilitating ATR-mediated Chk1 activation. However, as a replisome component, Tim–Tipin has also been suggested to couple DNA unwinding to synthesis, an activity expected to suppress single-stranded DNA (ssDNA) accumulation and limit ATR–Chk1 pathway engagement. We now demonstrate that Tim–Tipin depletion is sufficient to increase ssDNA accumulation at replication forks and stimulate ATR activity during otherwise unperturbed DNA replication. Notably, suppression of the ATR–Chk1 pathway in Tim–Tipin-deficient cells completely abrogates nucleotide incorporation in S phase, indicating that the ATR-dependent response to Tim–Tipin depletion is indispensible for continued DNA synthesis. Replication failure in ATR/Tim-deficient cells is strongly associated with synergistic increases in H2AX phosphorylation and DNA double-strand breaks, suggesting that ATR pathway activation preserves fork stability in instances of Tim–Tipin dysfunction. Together, these experiments indicate that the Tim–Tipin complex stabilizes replication forks both by preventing the accumulation of ssDNA upstream of ATR–Chk1 function and by facilitating phosphorylation of Chk1 by ATR.     

Hypoxia-induced nuclear translocation of β-catenin in the healing process of frostbite

Frostbite occurs when the skin is exposed to localized low temperatures. The main causes of frostbite are thought to be direct cell injury due to freezing of cells and tissue ischemia due to abnormal blood circulation. However, the molecular mechanism of frostbite has not been elucidated. This study aims to explain the molecular dynamics of frostbite using a mouse frostbite model and keratinocyte cell culture. Comprehensive gene expression analysis performed on mouse skin samples revealed that β-catenin signaling is activated by frostbite. Immunohistochemistry showed nuclear translocation of β-catenin in the skin of frostbite model mice that was not observed in mice subjected to a mechanical skin damage model induced by tape stripping. Tissue hypoxia, as detected by pimonidazole staining, coexisted with nuclear expression of β-catenin. In keratinocyte cell cultures, nuclear translocation of β-catenin was induced by hypoxia, but not by low temperature. Hypoxia induced epithelial-mesenchymal transition - an important biological event in the healing process of skin - and in vitro wound-healing activity, both of which were suppressed by β-catenin inhibition. Our results suggest that during frostbite, impaired blood flow causes hypoxia, which in turn activates β-catenin that promotes keratinocyte motility and tissue repair.     

Separate Domains for Desensitization of GABA ρ1 and β2 Subunits Expressed in Xenopus Oocytes

Desensitization of ligand-gated receptor channels is an intrinsic feedback mechanism and prevents the receptor/channels from becoming overly activated thereby maintaining biological function of the nervous system. Desensitization also plays an important role in neuronal plasticity. By taking advantage of biophysical and pharmacological diversities of GABA β₂ subunits from the brain and ρ₁ subunits from the retina, structural determinants that confer agonist-induced desensitization were identified. A synthetic chimeric receptor/channel was created from the β₂ and ρ₁ subunits for this investigation. The chimera was constructed from the extracellular N-domain of the β₂ subunit, extending from the amino terminus to the beginning region of the M1 transmembrane segment, and from the C-domain of the ρ₁ subunit extending from the M1 transmembrane segment to the carboxyl terminus. The C-domain region included the M1 to M4 transmembrane regions and the large intracellular loop between the M3 and M4 transmembrane segments. Homo-oligomeric GABA β₂, ρ₁, and β₂/ρ₁ chimeric receptor/channels were individually expressed in Xenopus oocytes, and the desensitization characteristics attributable to each type of subunit were compared. Results from the present study reveal that motifs in the amino-terminal and carboxyl-terminal domains of the β₂ subunit conferred the agonist-induced desensitization; chloroform modulation was linked to specific phases of the GABA-activated current decay. Received: 2 April 1997/Revised: 27 March 1998     

Ddc1 checkpoint protein and DNA polymerase ɛ interact with nick-containing DNA repair intermediate in cell free extracts of Saccharomyces cerevisiae

To characterize proteins that interact with base excision/single-strand interruption repair DNA intermediates in cell free extracts of Saccharomyces cerevisiae, we used a combination of photoaffinity labeling with the protein identification by MALDI-TOF-MS peptide mapping. Photoreactive analogue of dCTP, namely exo-N-[4-(4-azido-2,3,5,6,-tetrafluorobenzylidenehydrazinocarbonyl)-butylcarbamoyl]-2'-deoxycytidine-5'-triphosphate, and [(32)P]-labeled DNA duplex containing one nucleotide gap were used to generate nick-containing DNA with a photoreactive dCMP residue at the 3'-margin of the nick. This photoreactive DNA derivative was incubated with the yeast cell extract and after UV irradiation a number of proteins were labeled. Two of the crosslinked proteins were identified as the catalytic subunit of DNA polymerase ? and Ddc1 checkpoint protein. Labeling of DNA polymerase ? catalytic subunit with the nick-containing DNA repair intermediate indicates that the DNA polymerase is involved in the DNA repair synthesis in yeast, at least at DNA single-strand interruptions. Crosslinking of Ddc1 to DNA nicks took place independently of the other components of checkpoint clamp, Mec3 and Rad17, suggesting that the protein alone is able to recognize DNA single-strand breaks. Indeed, purified GST-tagged Ddc1 protein was efficiently crosslinked to nick-containing DNA. The interaction of Ddc1 with DNA nicks may provide a link between the DNA damage checkpoint and DNA base excision/single-strand breaks repair pathways in yeast. In addition, we found that absence of Ddc1 protein greatly influences the overall pattern of other proteins crosslinked to DNA nick. We suggested that this last effect of Ddc1 is at least partially due to its capacity to prevent proteolytic degradation of the DNA-protein adducts.