DNA damage-induced replication arrest in Xenopus egg extracts

Chromosomal replication is sensitive to the presence of DNA-damaging alkylating agents, such as methyl methanesulfonate (MMS). MMS is known to inhibit replication though activation of the DNA damage checkpoint and through checkpoint-independent slowing of replication fork progression. Using Xenopus egg extracts, we now report an additional pathway that is stimulated by MMS-induced damage. We show that, upon incubation in egg extracts, MMS-treated DNA activates a diffusible inhibitor that blocks, in trans, chromosomal replication. The downstream effect of the inhibitor is a failure to recruit proliferating cell nuclear antigen, but not DNA polymerase alpha, to the nascent replication fork. Thus, alkylation damage activates an inhibitor that intercepts the replication pathway at a point between the polymerase alpha and proliferating cell nuclear antigen execution steps. We also show that activation of the inhibitor does not require the DNA damage checkpoint; rather, stimulation of the pathway described here results in checkpoint activation. These data describe a novel replication arrest pathway, and they also provide an example of how subpathways within the DNA damage response network are integrated to promote efficient cell cycle arrest in response to damaged DNA.     

Human rabaptin-5 is selectively cleaved by caspase-3 during apoptosis

We have previously shown that Xenopus rabaptin-5 is cleaved in apoptotic extracts, with a concomitant reduction in the ability of these extracts to support endosomal membrane fusion (Cosulich, S. C., Horiuchi, H., Zerial, M., Clarke, P. R., and Woodman, P. G. (1997) EMBO J. 16, 6182-6191). In this report we demonstrate that caspase-dependent cleavage is a conserved feature of rabaptin-5. Human rabaptin-5 is cleaved at two sites (HSLD(379) and DESD(438)) in apoptotic HeLa extracts. Cleavage is effected by caspase-3, since it is prevented when caspase-3 activity is either inhibited by Ac-DEVD-CHO or removed by immunodepletion. Moreover, an identical pattern of cleavage is observed using recombinant caspase-3. The action of caspase-3 is highly selective; neither caspase-2 nor caspase-7 are able to cleave recombinant or cytosolic rabaptin-5. Caspase-dependent cleavage of rabaptin-5 generates two physically separated coiled coil-forming domains, the C-terminal of which retains the ability to bind the Rab5 exchange factor rabex-5.     

Protein disulfide isomerase is cleaved by caspase-3 and -7 during apoptosis

Apoptotic signals are typically accompanied by activation of aspartate-specific cysteine proteases called caspases, and caspase-3 and -7 play crucial roles in the execution of apoptosis. Previously, using the proteomic approach, protein disulfide isomerase (PDI) was found to be a candidate substrate of caspase-7. This abundant 55 kDa protein introduces disulfide bonds into proteins (via its oxidase activity) and catalyzes the rearrangement of incorrect disulfide bonds (via its isomerase activity). PDI is abundant in the ER but is also found in non-ER locations. In this study we demonstrated that PDI is cleaved by caspase-3 and -7 in vitro. In addition, in vivo experiment showed that it is cleaved during etoposide-induced apoptosis in HL-60 cells. Subcellular fractionation showed that PDI was also present in the cytosol. Furthermore, only cytosolic PDI was clearly digested by caspase-3 and -7. It was also confirmed by confocal image analysis that PDI and caspase-7 partially co-localize in both resting and apoptotic MCF-7 cells. Overexpression of cytosolic PDI (ER retention sequence deleted) inhibited cell death after an apoptotic stimulus. These data indicate that cytosolic PDI is a substrate of caspase-3 and -7, and that it has an anti-apoptotic action.     

HS 1-associated protein X-1 is cleaved by caspase-3 during apoptosis

Caspase-3 (CASP3) plays a key role in apoptosis. In this study, HAX-1 was identified as a new substrate of CASP3 during apoptosis. HAX-1 was cleaved by CASP3 during etoposide-(ETO) induced apoptosis, and this event was inhibited by a CASP3-specific inhibitor. The cleavage site of HAX-1, at Asp(127), was located using N-terminal amino acid sequencing of in vitro cleavage products of recombinant HAX-1. Overexpression of HAX-1 inhibited ETO-induced apoptotic cell death. It also inhibited CASP3 activity. Together, these results suggest that HAX-1, a substrate of CASP3, inhibits the apoptotic process by inhibiting CASP3 activity.     

Polypyrimidine tract-binding proteins are cleaved by caspase-3 during apoptosis

The polypyrimidine tract-binding protein (PTB), an RNA-binding protein, is required for efficient translation of some mRNAs containing internal ribosomal entry sites (IRESs). Here we provide evidence that the addition of apoptosis-inducing agents to cells results in the cleavage of PTB isoforms 1, 2, and 4 by caspase-3. This cleavage of PTB separated the N-terminal region, containing NLS-RRM1, from the C-terminal region, containing RRM2-3-4. Our data indicate that there are three noncanonical caspase-3 target sites in PTBs, namely Ile-Val-Pro-Asp(7)Ile, Leu-Tyr-Thr-Asp(139)Ser, and Ala-Ala-Val-Asp(172)Ala. The C-terminal PTB fragments localized to the cytoplasm, as opposed to the nucleus where most intact PTBs are found. Moreover, these C-terminal PTB fragments inhibited translation of polioviral mRNA, which contains an IRES element requiring PTB for its activation. This suggests that translation of some IRES-containing mRNAs is regulated by proteolytic cleavage of PTB during apoptosis.     

Crk is required for apoptosis in Xenopus egg extracts.

Apoptosis is essential for the development and homeostasis of multicellular organisms. Recently, a cell-free extract prepared from Xenopus eggs was shown to recapitulate intracellular apoptotic pathways in vitro. While many stimuli have been shown to trigger apoptosis in a variety of cell types, the intracellular signaling pathways involved in apoptosis remain largely unknown. Here we show that addition of a recombinant protein containing the phosphotyrosine binding (SH2) domain from the adaptor protein crk, but not those derived from a panel of other signaling proteins, can prevent apoptosis in the Xenopus egg extract system. Furthermore, immunodepletion of endogenous crk protein from the egg extracts, or addition of anti-crk antisera to these extracts, prevents apoptosis. The ability to undergo apoptosis can be restored to these extracts by addition of recombinant crk protein. These results directly demonstrate that crk participates in apoptotic signaling.     

Inositol 1,4,5-trisphosphate receptor type 1 is a substrate for caspase-3 and is cleaved during apoptosis in a caspase-3-dependent manner

The inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R), an IP(3)-gated Ca(2+) channel located on intracellular Ca(2+) stores, modulates intracellular Ca(2+) signaling. During apoptosis of the human T-cell line, Jurkat cells, as induced by staurosporine or Fas ligation, IP(3)R type 1 (IP(3)R1) was found to be cleaved. IP(3)R1 degradation during apoptosis was inhibited by pretreatment of Jurkat cells with the caspase-3 (-like protease) inhibitor, Ac-DEVD-CHO, and the caspases inhibitor, z-VAD-CH(2)DCB but not by the caspase-1 (-like protease) inhibitor, Ac-YVAD-CHO, suggesting that IP(3)R1 was cleaved by a caspase-3 (-like) protease. The recombinant caspase-3 cleaved IP(3)R1 in vitro to produce a fragmentation pattern consistent with that seen in Jurkat cells undergoing apoptosis. N-terminal amino acid sequencing revealed that the major cleavage site is (1888)DEVD*(1892)R (mouse IP(3)R1), which involves consensus sequence for caspase-3 cleavage (DEVD). To determine whether IP(3)R1 is cleaved by caspase-3 or is proteolyzed in its absence by other caspases, we examined the cleavage of IP(3)R1 during apoptosis in the MCF-7 breast carcinoma cell line, which has genetically lost caspase-3. Tumor necrosis factor-alpha- or staurosporine-induced apoptosis in caspase-3-deficient MCF-7 cells failed to demonstrate cleavage of IP(3)R1. In contrast, MCF-7/Casp-3 cells stably expressing caspase-3 showed IP(3)R1 degradation upon apoptotic stimuli. Therefore IP(3)R1 is a newly identified caspase-3 substrate, and caspase-3 is essential for the cleavage of IP(3)R1 during apoptosis. This cleavage resulted in a decrease in the channel activity as IP(3)R1 was digested, indicating that caspase-3 inactivates IP(3)R1 channel functions.     

Calcium oscillations in Xenopus egg cycling extracts

Cell cycle in various types of cells and in early embryos is often accompanied by transient changes in the concentration of free cytosolic calcium. In the present study, using fluorescent indicator fura-2, we demonstrate that Ca(2+) oscillates cyclically with an amplitude of about 100 nM and a period of mitotic cycle in cell-free Xenopus egg cycling extracts. It peaks in early metaphase just preceding mitotic reactivation of Cdc2 kinase and MAPK and reaches a minimum in interphase. The source of Ca(2+) in the extracts is a particulate fraction containing egg intracellular Ca(2+) stores, since the addition of a calcium-mobilizing second messenger, inositol 1,4,5-trisphosphate (IP3), induced a transient increase in Ca(2+). The inclusion of heparin, an IP3 receptor antagonist, or ultrafiltration of the extracts prevented Ca(2+)-releasing activity of IP3. The depletion of Ca(2+) in the extracts by the calcium chelator BAPTA resulted in the blockade of cell cycle at different stages, depending on the time of drug administration. The addition of BAPTA late in interphase blocked cell cycle at mitotic entry in prophase, whereas its application in anaphase or telophase blocked the extracts in early interphase. BAPTA administration in metaphase before transition to anaphase brought about a metaphase-like arrest in the cycling extracts. Inhibition of IP3-induced calcium release by heparin also arrested cell cycle progression in the cycling extracts.     

Progress in understanding DNA replication control

Completion of genome duplication during the S-phase of the cell cycle is crucial for the maintenance of genomic integrity. In eukaryotes, chromosomal DNA replication is accomplished by the activity of multiple origins of DNA replication scattered across the genome. Origin specification, selection and activity as well as the availability of replication factors and the regulation of DNA replication licensing, have unique and common features among eukaryotes. Although the initial studies on the semiconservative nature of chromosome duplication were carried out in the mid 1950s in Vicia faba, since then plant DNA replication studies have been scarce. However, they have received an unprecedented drive in the last decade after the completion of sequencing the Arabidopsis thaliana genome, and more recently of other plant genomes. In particular, the past year has witnessed major advances with the use of genomic approaches to study chromosomal replication timing, DNA replication origins and licensing control mechanisms. In this minireview article we discuss these recent discoveries in plants in the context of what is known at the genomic level in other eukaryotes. These studies constitute the basis for addressing in the future key questions about replication origin specification and function that will be of relevance not only for plants but also for the rest of multicellular organisms.     

Evidence that DNA replication is not regulated by ubiquitin-dependent proteolysis in Xenopus egg extract

The Xenopus early embryonic cell cycle consists of rapid oscillations between mitosis and DNA synthesis. We used ubiquitin (Ub)-dependent proteolysis inhibitors to determine whether Ub-mediated proteolysis regulates the initiation of DNA replication in Xenopus egg extract. Methylated Ub, a chemically modified Ub that cannot form chains, and S5a, a Ub chain-binding subunit of the 26S proteasome, were added to extract at concentrations known to inhibit cyclin B proteolysis and their effects on cell cycle progression and DNA replication were examined. DNA replication initiated concomitant with controls and proceeded in a semiconservative fashion in the presence of both methylated Ub and S5a. However, mitotic progression was halted, showing that the inhibitors were functional. We conclude that initiation of DNA replication is not regulated by Ub-dependent proteolysis in the early Xenopus cell cycle.     

Repair of psoralen interstrand cross-links in Xenopus laevis egg extracts is highly mutagenic

The recognition and removal of interstrand cross-links is perhaps the least understood of all repair pathways in eukaryotic cells. We have shown previously that uncoupling of cross-links occurs in mammalian cell extracts and have identified a number of factors that mediate this process. However, we have not observed complete repair of the substrate in this system. Here, we show that uncoupling of interstrand cross-links also occurs in Xenopus laevis egg extracts, and that the initial products of this reaction are identical to the products observed in mammalian cell extracts suggesting a common mechanism. However in contrast to mammalian cell extracts, we observe repair of the cross-linked substrate in the Xenopus extracts presumably by a translesion bypass mechanism that allows replication past the uncoupled monoadduct, and its likely subsequent removal by nucleotide excision repair. This repair process is shown to be highly mutagenic consistent with bypass synthesis.     

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.     

Budding yeast Cdc6p induces re-replication in fission yeast by inhibition of SCF(Pop)-mediated proteolysis

In fission yeast, overexpression of the replication initiator protein Cdc18p induces re-replication, a phenotype characterized by continuous DNA synthesis in the absence of cell division. In contrast, overexpression of Cdc6p, the budding yeast homolog of Cdc18p, does not cause re-replication in S. cerevisiae. However, we have found that Cdc6p has the ability to induce re-replication in fission yeast. Cdc6p cannot functionally replace Cdc18p, but instead interferes with the proteolysis of both Cdc18p and Rum1p, the inhibitor of the protein kinase Cdc2p. This activity of Cdc6p is entirely contained within a short N-terminal peptide, which forms a tight complex with Cdc2p and the F-box/WD-repeat protein Sud1p/Pop2p, a component of the SCF^Pop ubiquitin ligase in fission yeast. These interactions are mediated by two distinct regions within the N-terminal region of Cdc6p and depend on the integrity of its Cdc2p phosphorylation sites. The data suggest that disruption of re-replication control by overexpression of Cdc6p in fission yeast is a consequence of sequestration of Cdc2p and Pop2p, two factors involved in the negative regulation of Rum1p, Cdc18p and potentially other replication proteins. Received: 29 April 1999 / Accepted: 27 June 1999     

LDFF, the large molecular weight DNA fragmentation factor, is responsible for the large molecular weight DNA degradation during apoptosis in Xenopus egg extracts

DNA degradation is a biochemical hallmark in apoptosis. It has been demonstrated in many cell types that there are two stages of DNA fragmentation during the apoptotic execution. In the early stage, chromatin DNA is cut into large molecular weight DNA fragments, although the responsible nuclease(s) has not been recognized. In the late stage, the chromatin DNA is cleaved further into short oligonucleosomal fragments by a well-characterized nuclease in apoptosis,the caspase-activated DNase (CAD/DFF40). In this study, we demonstrate that large molecular weight DNA fragmentation also occurs in Xenopus egg extracts in apoptosis. We show that the large molecular weight DNA fragmentation factor (LDFF) is not the Xenopus CAD homolog XCAD. LDFF is activated by caspase-3. The large molecular weight DNA fragmentation activity of LDFF is Mg^2 -dependent and Ca^2 -independent, can occur in both acidic and neutral pH conditions and can tolerate 45℃ treatment. These results indicate that LDFF in Xenopus egg extracts might be a new DNase (or DNases) responsible for the large DNA fragmentation.     

Human replication protein Cdc6 is selectively cleaved by caspase 3 during apoptosis

In eukaryotes, the initiation of DNA replication involves the ordered assembly on chromatin of pre-replicative complexes (pre-RCs), including the origin recognition complex (ORC), Cdc6, Cdt1 and the minichromosome maintenance proteins (MCMs). In light of its indispensable role in the formation of pre-RCs, Cdc6 binding to chromatin represents a key step in the regulation of DNA replication and cell proliferation. Here, we study the human Cdc6 (HuCdc6) protein during programmed cell death (apoptosis). We find that HuCdc6, but not HuOrc2 (a member of the ORC) or HuMcm5 (one of the MCMs), is specifically cleaved in several human cell lines induced to undergo apoptosis by a variety of stimuli. Expression of caspase-uncleavable mutant HuCdc6 attenuates apoptosis, delaying cell death. Therefore, an important function for cleavage of HuCdc6 is to prevent a wounded cell from replicating and to facilitate death.     

Wee1-regulated apoptosis mediated by the crk adaptor protein in Xenopus egg extracts

Many of the biochemical reactions of apoptotic cell death, including mitochondrial cytochrome c release and caspase activation, can be reconstituted in cell-free extracts derived from Xenopus eggs. In addition, because caspase activation does not occur until the egg extract has been incubated for several hours on the bench, upstream signaling processes occurring before full apoptosis are rendered accessible to biochemical manipulation. We reported previously that the adaptor protein Crk is required for apoptotic signaling in egg extracts (Evans, E.K., W. Lu, S.L. Strum, B.J. Mayer, and S. Kornbluth. 1997. EMBO (Eur. Mol. Biol. Organ.) J. 16:230-241). Moreover, we demonstrated that removal of Crk Src homology (SH)2 or SH3 interactors from the extracts prevented apoptosis. We now report the finding that the relevant Crk SH2-interacting protein, important for apoptotic signaling in the extract, is the well-known cell cycle regulator, Wee1. We have demonstrated a specific interaction between tyrosine-phosphorylated Wee1 and the Crk SH2 domain and have shown that recombinant Wee1 can restore apoptosis to an extract depleted of SH2 interactors. Moreover, exogenous Wee1 accelerated apoptosis in egg extracts, and this acceleration was largely dependent on the presence of endogenous Crk protein. As other Cdk inhibitors, such as roscovitine and Myt1, did not act like Wee1 to accelerate apoptosis, we propose that Wee1-Crk complexes signal in a novel apoptotic pathway, which may be unrelated to Wee1's role as a cell cycle regulator.