The prereplication complex recruits XEco2 to chromatin to promote cohesin acetylation in Xenopus egg extracts

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Highlights? XEco2 loading and cohesin acetylation require pre-RC assembly but not DNA synthesis ? Two N-terminal motifs in XEco2 are essential for XEco2 loading and cohesion ? The PIP box in XEco2 contributes to cohesion ? Interaction of acetylated cohesin with DNA is stabilized after DNA replication     

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.     

Daunomycin disrupts nuclear assembly and the coordinate initiation of DNA replication in Xenopus egg extracts

We have used Xenopus egg extracts to investigate the effects of the antitumor drug daunomycin on DNA replication in vitro. Xenopus sperm nuclei replicated nearly synchronously in our egg extracts, thereby allowing us to determine the effects of the drug on both replication initiation and elongation. Titration experiments demonstrated that daunomycin effectively inhibited replication in the extract, with 50% inhibition at a total drug concentration of 2.7 μM. However, a high concentration of daunomycin 150 μM) also inhibited nuclear envelope assembly, a prerequisite for the initiation of replication in this system. Therefore, to bypass the effects of daunomycin on nuclear envelope assembly, sperm nuclei were preassembled in extract prior to drug addition. Initiation of replication in preassembled nuclei was also inhibited by daunomycin, with 50% inhibition at a drug concentration of 3.6 μM. At low drug concentrations, where replication did occur, the synchrony of initiations within individual nuclei was lost. This drug-induced disruption of initiation events may provide important clues regarding the mechanism(s) by which these events are coordinated in eukaryotic cells. Daunomycin also inhibited replication elongation in preassembled, preinitiated nuclei. However, the concentration of drug required for 50% inhibition of elongation was nearly fourfold higher than that required for inhibition of initiation. Taken together, these data demonstrate that Xenopus egg extract can be used to investigate the effects of DNA-binding antitumor drugs on a number of interrelated cellular processes, many of which are less tractable in whole cell systems. J. Cell. Biochem. 64:476–491. © 1997 Wiley-Liss, Inc.     

Functional analysis of Survivin in spindle assembly in Xenopus egg extracts

Survivin is a member of the inhibitor of apoptosis (IAP) protein family that serves critical roles in mitosis and cytokinesis. Many studies have suggested Survivin's involvement in spindle regulation, but direct biochemical evidence for this has been lacking. Using the cell-free system of Xenopus egg extracts, we tested whether Survivin was necessary for the assembly of metaphase spindles. Removal or inhibition of Xenopus Survivin causes the disruption in the formation of metaphase spindles. In particular, we observe the generation of microtubule (MT) asters or poorly formed shortened spindle structures. In the latter phenotype the spindle structures display a decrease pole-to-pole length and a reduction of MTs around the chromatin indicating that Survivin may promote the stabilization of MT-chromatin interactions. In addition, function analysis of Survivin's conserved phosphorylation site Thr34 (Thr43 in Xenopus) and tubulin-binding domain was also assessed in regulating spindle assembly. Treatment of Xenopus egg extracts with a recombinant Survivin mutant that contained an alanine residue substitution at Thr43 (SURT43A mutant) or that was missing the C-terminal tubulin-binding domain (SURCL mutant) produced an increased frequency of MT asters and shorten abnormal spindle structures in Xenopus egg extracts. Interestingly, a phosphomimetic mutation made at residue Thr43 of Survivin (SURT43E mutant) generated a high frequency of MT asters implying that premature 'activation' of Survivin may interfere with an early stage of spindle assembly. Taken together, we propose that Survivin is a necessary component of the mitotic spindle and its phosphorylation at residue Thr43 is important for Survivin function in spindle assembly.     

Single-molecule analysis of DNA replication in Xenopus egg extracts

The recent advent in single-molecule imaging and manipulation methods has made a significant impact on the understanding of molecular mechanisms underlying many essential cellular processes. Single-molecule techniques such as electron microscopy and DNA fiber assays have been employed to study the duplication of genome in eukaryotes. Here, we describe a single-molecule assay that allows replication of DNA attached to the functionalized surface of a microfluidic flow cell in a soluble Xenopus leavis egg extract replication system and subsequent visualization of replication products via fluorescence microscopy. We also explain a method for detection of replication proteins, through fluorescently labeled antibodies, on partially replicated DNA immobilized at both ends to the surface.     

Regulation of Op18 during spindle assembly in Xenopus egg extracts

Oncoprotein 18 (Op18) is a microtubule-destabilizing protein that is negatively regulated by phosphorylation. To evaluate the role of the three Op18 phosphorylation sites in Xenopus (Ser 16, 25, and 39), we added wild-type Op18, a nonphosphorylatable triple Ser to Ala mutant (Op18-AAA), and to mimic phosphorylation, a triple Ser to Glu mutant (Op18-EEE) to egg extracts and monitored spindle assembly. Op18-AAA dramatically decreased microtubule length and density, while Op18-EEE did not significantly affect spindle microtubules. Affinity chromatography with these proteins revealed that the microtubule-destabilizing activity correlated with the ability of Op18 to bind tubulin. Since hyperphosphorylation of Op18 is observed upon addition of mitotic chromatin to extracts, we reasoned that chromatin-associated proteins might play a role in Op18 regulation. We have performed a preliminary characterization of the chromatin proteins recruited to DNA beads, and identified the Xenopus polo-like kinase Plx1 as a chromatin-associated kinase that regulates Op18 phosphorylation. Depletion of Plx1 inhibits chromatin-induced Op18 hyperphosphorylation and spindle assembly in extracts. Therefore, Plx1 may promote microtubule stabilization and spindle assembly by inhibiting Op18.     

Study of the cell cycle-dependent assembly of the DNA pre-replication centres in Xenopus egg extracts.

RPA is a cellular, three-subunit, single-stranded (ss) DNA binding protein, which assists T-antigen in the assembly of the pre-priming complex in the SV40 replication system. By immunodepletion and complementation, we have identified RPA as an essential factor for cellular DNA replication in Xenopus extracts. RPA assembles post-mitotically on the decondensing chromosomes into numerous subnuclear pre-replication centres (preRCs) which serve, upon formation of the nuclear membrane, as RCs for the initiation of DNA synthesis. By a variety of experiments including the use of isolated components, we demonstrate that an inactive cdc2-cyclin B kinase complex is essential to allow post-mitotic assembly of the preRCs. In contrast, the active cdk2-cyclin A kinase does not impede or facilitate the assembly of preRCs. Digestion analysis using the single-strand-specific P1 nuclease as well as competition experiments with ssDNA, reveal that replication-associated unwinding of the DNA, assisted by RPA, requires the formation of the nuclear membrane. The p21 cdk-interacting protein Cip1 appears to inhibit DNA replication prior to the unwinding DNA step, but after assembly of preRC and nuclear reconstruction.     

The condensin complex is required for proper spindle assembly and chromosome segregation in Xenopus egg extracts

Chromosome condensation is required for the physical resolution and segregation of sister chromatids during cell division, but the precise role of higher order chromatin structure in mitotic chromosome functions is unclear. Here, we address the role of the major condensation machinery, the condensin complex, in spindle assembly and function in Xenopus laevis egg extracts. Immunodepletion of condensin inhibited microtubule growth and organization around chromosomes, reducing the percentage of sperm nuclei capable of forming spindles, and causing dramatic defects in anaphase chromosome segregation. Although the motor CENP-E was recruited to kinetochores pulled poleward during anaphase, the disorganized chromosome mass was not resolved. Inhibition of condensin function during anaphase also inhibited chromosome segregation, indicating its continuous requirement. Spindle assembly around DNA-coated beads in the absence of kinetochores was also impaired upon condensin inhibition. These results support an important role for condensin in establishing chromosomal architecture necessary for proper spindle assembly and chromosome segregation.     

Ku-dependent nonhomologous DNA end joining in Xenopus egg extracts

An extract from activated Xenopus eggs joins both matching and nonmatching ends of exogenous linear DNA substrates with high efficiency and fidelity (P. Pfeiffer and W. Vielmetter, Nucleic Acids Res. 16:907-924, 1988). In mammalian cells, such nonhomologous end joining (NHEJ) is known to require the Ku heterodimer, a component of DNA-dependent protein kinase. Here I investigated whether Ku is also required for the in vitro reaction in the egg extract. Immunological assays indicate that Ku is very abundant in the extract. I found that all NHEJ was inhibited by autoantibodies against Ku and that NHEJ between certain combinations of DNA ends was also decreased after immunodepletion of Ku from the extract. The formation of a joint between a DNA end with a 5'-protruding single strand (PSS) and an end with a 3'-PSS, between two ends with 3'-PSS, and between two blunt ends was most Ku dependent. On the other hand, NHEJ between two DNA ends bearing 5'-PSS was Ku independent. These results show that the Xenopus cell-free system will be useful to biochemically dissect the role of Ku in eukaryotic NHEJ.     

Mitotic spindle assembly around RCC1-coated beads in Xenopus egg extracts

During cell division the genetic material on chromosomes is distributed to daughter cells by a dynamic microtubule structure called the mitotic spindle. Here we establish a reconstitution system to assess the contribution of individual chromosome proteins to mitotic spindle formation around single 10 μm diameter porous glass beads in Xenopus egg extracts. We find that Regulator of Chromosome Condensation 1 (RCC1), the Guanine Nucleotide Exchange Factor (GEF) for the small GTPase Ran, can induce bipolar spindle formation. Remarkably, RCC1 beads oscillate within spindles from pole to pole, a behavior that could be converted to a more typical, stable association by the addition of a kinesin together with RCC1. These results identify two activities sufficient to mimic chromatin-mediated spindle assembly, and establish a foundation for future experiments to reconstitute spindle assembly entirely from purified components.     

Chromosome condensation by a human condensin complex in Xenopus egg extracts

13S condensin is a five-subunit protein complex that plays a central role in mitotic chromosome condensation. The condensin complex was originally identified and purified from Xenopus egg extracts and shown to have an ATP-dependent positive supercoiling activity in vitro. We report here the characterization of a human condensin complex purified from HeLa cell nuclear extracts. The human 13S complex has exactly the same composition as its Xenopus counterpart, being composed of two structural maintenance of chromosomes (human chromosome-associated polypeptide (hCAP)-C and hCAP-E) subunits and three non-structural maintenance of chromosomes (hCAP-D2/CNAP1, hCAP-G, and hCAP-H/BRRN) subunits. Human condensin purified from asynchronous HeLa cell cultures fails to reconfigure DNA structure in vitro. When phosphorylated by purified cdc2-cyclin B, however, it gains the ability to introduce positive supercoils into DNA in the presence of ATP and topoisomerase I. Strikingly, human condensin can induce chromosome condensation when added back into a Xenopus egg extract that has been immunodepleted of endogenous condensin. Thus, the structure and function of the condensin complex are highly conserved between Xenopus and humans, underscoring its fundamental importance in mitotic chromosome dynamics in eukaryotic cells.     

Investigating mitotic spindle assembly and function in vitro using Xenopus laevis egg extracts

Extracts from Xenopus laevis eggs provide a powerful system for the study of cell division processes in vitro through biochemical reconstitution and manipulation, and microscopic analysis. We provide protocols for the preparation of metaphase-arrested extracts and in vitro assays to examine the following pathways of spindle assembly: 1) Sperm nuclei added to meiotic extracts, supporting the formation of half-spindles and bipolar spindle structures around unreplicated chromosomes; 2) sperm nuclei added to extracts that cycle through interphase and form spindles that are capable of undergoing anaphase and chromosome segregation; and 3) spindle formation around chromatin-coated beads. Finally, we describe methods to inhibit a specific protein by immunodepletion or addition of an inhibitor such as a dominant-negative construct. These techniques can be used to analyze the mitotic function of a given protein. It takes approximately 1.5 h to prepare the extract, 1-3 h for spindle-assembly experiments and an additional 1-3 h if immunodepletion is performed.     

Micromanipulation studies of chromatin fibers in Xenopus egg extracts reveal ATP-dependent chromatin assembly dynamics

We have studied assembly of chromatin using Xenopus egg extracts and single DNA molecules held at constant tension by using magnetic tweezers. In the absence of ATP, interphase extracts were able to assemble chromatin against DNA tensions of up to 3.5 piconewtons (pN). We observed force-induced disassembly and opening-closing fluctuations, indicating our experiments were in mechanochemical equilibrium. Roughly 50-nm (150-base pair) lengthening events dominated force-driven disassembly, suggesting that the assembled fibers are chiefly composed of nucleosomes. The ATP-depleted reaction was able to do mechanical work of 27 kcal/mol per 50 nm step, which provides an estimate of the free energy difference between core histone octamers on and off DNA. Addition of ATP led to highly dynamic behavior with time courses exhibiting processive runs of assembly and disassembly not observed in the ATP-depleted case. With ATP present, application of forces of 2 pN led to nearly complete fiber disassembly. Our study suggests that ATP hydrolysis plays a major role in nucleosome rearrangement and removal and that chromatin in vivo may be subject to highly dynamic assembly and disassembly processes that are modulated by DNA tension.     

TPX2 is required for postmitotic nuclear assembly in cell-free Xenopus laevis egg extracts

Cell division in many metazoa is accompanied by the disassembly of the nuclear envelope and the assembly of the mitotic spindle. These dramatic structural rearrangements are reversed after mitosis, when the mitotic spindle is dismantled and the nuclear envelope reassembles. The targeting protein for XKlp2 (TPX2) plays important roles in mitotic spindle assembly. We report that TPX2 depletion from nuclear assembly extracts prepared from Xenopus laevis eggs results in the formation of nuclei that are only about one fifth the size of control nuclei. TPX2-depleted nuclei assemble nuclear envelopes, nuclear pore complexes, and a lamina, and they perform nuclear-specific functions, including DNA replication. We show that TPX2 interacts with lamina-associated polypeptide 2 (LAP2), a protein known to be required for nuclear assembly in interphase extracts and in vitro. LAP2 localization is disrupted in TPX2-depleted nuclei, suggesting that the interaction between TPX2 and LAP2 is required for postmitotic nuclear reformation.     

DNA replication initiates at multiple sites on plasmid DNA in Xenopus egg extracts.

Cell-free extracts of Xenopus eggs will replicate plasmid DNA molecules under normal cell cycle control. We have used the neutral/neutral 2-D gel technique to map the sites at which DNA replication initiates in this system. Three different plasmids were studied: one containing the Xenopus rDNA repeat, one containing single copy Xenopus genomic DNA, and another containing the yeast 2 microns replication origin. 2-D gel profiles show that many potential sites of initiation are present on each plasmid, and are randomly situated at the level of resolution of this technique (500-1000 bp). Despite the abundance of sites capable of supporting the initiation of replication, pulse-chase experiments suggest that only a single randomly situated initiation event occurs on each DNA molecule. Once initiation has taken place, conventional replication forks appear to move away from this site at a rate of about 10nt/second, similar to the rate observed in vivo.     

DNA is a co-factor for its own replication in Xenopus egg extracts

Soluble Xenopus egg extracts efficiently replicate added plasmids using a physiological mechanism, and thus represent a powerful system to understand vertebrate DNA replication. Surprisingly, DNA replication in this system is highly sensitive to plasmid concentration, being undetectable below ∼10 pM and highly efficient above ∼75 pM. DNA replication at the high plasmid concentration does not require plasmid–plasmid contacts, since replication is not inhibited when plasmids are immobilized in agarose prior to addition of egg extract. The absence of replication at low plasmid concentration is due to a defect in the assembly of pre-replication complexes (pre-RCs). pre-RC assembly requires contact-independent communication between plasmids. Our results show that in Xenopus egg extracts, aggregation of multiple replication forks is not required for efficient replication of plasmid DNA, and they suggest that DNA functions as a co-factor for its own duplication.     

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.     

Mismatch repair-dependent metabolism of O6-methylguanine-containing DNA in Xenopus laevis egg extracts

The cytotoxicity of S_N1-type alkylating agents such as N-methyl-N′-nitrosourea (MNU), N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), or the cancer chemotherapeutics temozolomide, dacarbazine and streptozotocin has been ascribed to the persistence of O⁶-methylguanine (^meG) in genomic DNA. One hypothesis posits that ^meG toxicity is caused by futile attempts of the mismatch repair (MMR) system to process ^meG/C or ^meG/T mispairs arising during replication, while an alternative proposal suggests that the latter lesions activate DNA damage signaling, cell cycle arrest and apoptosis directly. Attempts to elucidate the molecular mechanism of ^meG-induced cell killing in vivo have been hampered by the fact that the above reagents induce several types of modifications in genomic DNA, which are processed by different repair pathways. In contrast, defined substrates studied in vitro did not undergo replication. We set out to re-examine this phenomenon in replication-competent Xenopus laevis egg extracts, using either phagemid substrates containing a single ^meG residue, or methylated sperm chromatin. Our findings provide further support for the futile cycling hypothesis.