Ongoing replication forks delay nuclear envelope breakdown upon mitotic entry

DNA replication is a major contributor to genomic instability and protection against DNA replication perturbation is essential for normal cell division. Certain types of replication stress agents, such as aphidicolin and hydroxyurea, have been shown to cause reversible replication fork stalling, wherein replisome complexes are stably maintained with competence to restart in the S-phase of the cell cycle. If these stalled forks persist into the M-phase without a replication restart, replisomes are disassembled in a p97-dependent pathway and under-replicated DNA is subjected to mitotic DNA repair synthesis. Here, using Xenopus egg extracts, we investigated the consequences that arise when stalled forks are released simultaneously with the induction of mitosis. Ara-cytidine-5’-triphosphate (Ara-CTP)-induced stalled forks were able to restart with the addition of excess dCTPduring early mitosis before the nuclear envelope breakdown (NEB). However, stalled forks could no longer restart efficiently after NEB. Although replisome complexes were finally disassembled in a p97-dependent manner during mitotic progression whether or not fork stalling was relieved, the timing of NEB was delayed with the ongoing forks, rather than the stalled forks, and the delay was dependent on Wee1/Myt1 kinase activities. Thus, ongoing DNA replication was found to be directly linked to the regulation of Wee1/Myt1 kinases to modulate cyclin-dependent kinase (CDK) activities, owing to which DNA replication and mitosis occur in a mutually exclusive and sequential manner.

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Phosphorylation and activation of the Xenopus Cdc25 phosphatase in the absence of Cdc2 and Cdk2 kinase activity.

Molecular Biology of the Cell ◽

10.1091/mbc.6.2.215 ◽

1995 ◽

Vol 6 (2) ◽

pp. 215-226 ◽

Cited By ~ 74

Author(s):

T Izumi ◽

J L Maller

Keyword(s):

Kinase Activity ◽

Cdc2 Kinase ◽

Nuclear Envelope Breakdown ◽

Dual Specificity ◽

M Phase ◽

Egg Extracts ◽

Xenopus Egg ◽

Xenopus Egg Extracts

The M-phase inducer, Cdc25C, is a dual-specificity phosphatase that directly phosphorylates and activates the cyclin B/Cdc2 kinase complex, leading to initiation of mitosis. Cdc25 itself is activated at the G2/M transition by phosphorylation on serine and threonine residues. Previously, it was demonstrated that Cdc2 kinase is capable of phosphorylating and activating Cdc25, suggesting the existence of a positive feedback loop. In the present study, kinases other than Cdc2 that can phosphorylate and activate Cdc25 were investigated. Cdc25 was found to be phosphorylated and activated by cyclin A/Cdk2 and cyclin E/Cdk2 in vitro. However, in interphase Xenopus egg extracts with no detectable Cdc2 and Cdk2, treatment with the phosphatase inhibitor microcystin activated a distinct kinase that could phosphorylate and activate Cdc25. Microcystin also induced other mitotic phenomena such as chromosome condensation and nuclear envelope breakdown in extracts containing less than 5% of the mitotic level of Cdc2 kinase activity. These findings implicate a kinase other than Cdc2 and Cdk2 that may initially activate Cdc25 in vivo and suggest that this kinase may also phosphorylate M-phase substrates even in the absence of Cdc2 kinase.

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Chromosome architecture can dictate site-specific initiation of DNA replication in Xenopus egg extracts.

The Journal of Cell Biology ◽

10.1083/jcb.135.5.1207 ◽

1996 ◽

Vol 135 (5) ◽

pp. 1207-1218 ◽

Cited By ~ 35

Author(s):

S J Lawlis ◽

S M Keezer ◽

J R Wu ◽

D M Gilbert

Keyword(s):

Dna Replication ◽

Nuclear Envelope ◽

Cho Cells ◽

Chromosome Condensation ◽

G1 Phase ◽

Unusual Site ◽

Chromosome Architecture ◽

Egg Extracts ◽

Xenopus Egg ◽

Xenopus Egg Extracts

Xenopus egg extracts initiate DNA replication specifically at the dihydrofolate reductase (DHFR) origin locus with intact nuclei from late G1-phase CHO cells as a substrate, but at nonspecific sites when purified DNA is assembled by the extract into an embryonic nuclear structure. Here we show that late G1-phase CHO nuclei can be cycled through an in vitro Xenopus egg mitosis, resulting in the assembly of an embryonic nuclear envelope around G1-phase chromatin. Surprisingly, replication within these chimeric nuclei initiated at a novel specific site in the 5' region of the DHFR structural gene that does not function as an origin in cultured CHO cells. Preferential initiation at this unusual site required topoisomerase II-mediated chromosome condensation during mitosis. Nuclear envelope breakdown and reassembly in the absence of chromosome condensation resulted in nonspecific initiation. Introduction of condensed chromosomes from metaphase-arrested CHO cells directly into Xenopus egg extracts was sufficient to elicit assembly of chimeric nuclei and preferential initiation at this same site. These results demonstrate clearly that chromosome architecture can determine the sites of initiation of replication in Xenopus egg extracts, supporting the hypothesis that patterns of initiation in vertebrate cells are established by higher order features of chromosome structure.

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23REPROGRAMMING MAMMALIAN CELLS IN XENOPUS EGG EXTRACTS: ROLE OF EMBRYONIC LAMIN B3

Reproduction Fertility and Development ◽

10.1071/rdv16n1ab23 ◽

2004 ◽

Vol 16 (2) ◽

pp. 134

Author(s):

R. Alberio ◽

K.H.S. Campbell

Keyword(s):

Dna Replication ◽

Nuclear Envelope ◽

Somatic Cells ◽

Nuclear Transplantation ◽

Heterologous System ◽

Fetal Fibroblasts ◽

Egg Extracts ◽

Xenopus Egg ◽

Xenopus Egg Extracts

The generation of animals by nuclear transplantation has demonstrated that a fully differentiated cell can be reversed into totipotency when transferred into an oocyte. Identification of oocyte specific molecules responsible for the reprogramming of somatic cells may contribute to the understanding of cell differentiation and embryo development. We have developed a heterologous system to investigate the effect of lamin B3, a major component of Xenopus laevis egg cytoplasm, on DNA replication of mammalian somatic cells. Bovine fetal fibroblasts were arrested at G1/S by incubation in aphidicolin for 18h. After permeabilization with digitonin, the cells were incubated in either (1) lamin B3 depleted, or (2) whole Xenopus egg extracts (1000 cells μL−1 extract) supplemented with an energy regenerating system for a period of 3h at 21°C. Xenopus lamin B3-depleted egg extracts were prepared by three rounds of incubation with Dynabeads coated with a mouse monoclonal lamin B3 antibody (mAbLB3). Immunodepletion was confirmed by western blotting. Purified lamin B3 was obtained by dialysis of the beads after immunodepletion, and the purified lamin B3 was used for rescue experiments. DNA replication of cells incubated in the extracts was assessed by adding 25μM Biotin-11-dUTP for 3h. After treatment cells were fixed in 70% methanol at −20°C and incubated in mAbLB3 for 30min at 37°C. This was followed by incubation in FITC-conjugated sheep anti-mouse antibody and in 5mgmL−1 Texas Red-conjugated Streptavidin for 40min at 37°C. After three hours’ incubation in egg extracts, DNA replication was detected in 60% of cells and more than 95% of cells were lamin B3 positive. In contrast, DNA replication in immunodepleted extracts was significantly lower (P≤0.01, by one-way ANOVA) than in cells incubated in whole extracts and was coincident with the few lamin B3-positive cells observed. More than 95% of cells were lamin B3-negative and did not replicate DNA. When purified lamin B3 was re-added to depleted extracts, DNA replication was detected in 60% of cells. DNA synthesis resumed in 93% of control cells 3h after release from aphidicolin into culture medium at 39°C. These experiments show that somatic nuclei, which possess a nuclear envelope with somatic variants of lamins, are able to synthesize DNA in egg extracts only when Xenopus lamin B3 is incorporated into the nuclear envelope. This heterologous system provides new information on the role of an embryonic molecule, namely Xenopus lamin B3, in the reprogramming of DNA replication of somatic cells incubated in egg environment. These results open new questions as to whether embryonic lamins also exist in mammals, and whether failure in development of cloned animals is in part due to abnormal or incomplete replacement of somatic variants of proteins with their embryonic counterparts.

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A Novel Role for Cdk1/Cyclin B in Regulating B-Raf Activation at Mitosis

Molecular Biology of the Cell ◽

10.1091/mbc.e07-07-0679 ◽

2008 ◽

Vol 19 (7) ◽

pp. 2907-2915 ◽

Cited By ~ 7

Author(s):

Sergiy I. Borysov ◽

Thomas M. Guadagno

Keyword(s):

Mapk Cascade ◽

Cyclin B ◽

Dependent Manner ◽

Mapk Activity ◽

M Phase ◽

Egg Extracts ◽

Xenopus Egg ◽

Subsequent Activation ◽

Xenopus Egg Extracts

MAPK activity is important during mitosis for spindle assembly and maintenance of the spindle checkpoint arrest. We previously identified B-Raf as a critical activator of the MAPK cascade during mitosis in Xenopus egg extracts and showed that B-Raf activation is regulated in an M-phase–dependent manner. The mechanism that mediates B-Raf activation at mitosis has not been elucidated. Interestingly, activation of 95-kDa B-Raf at mitosis does not require phosphorylation of Thr-599 and Ser-602 residues (Thr-633 and Ser-636 in Xenopus B-Raf), previously shown to be essential for B-Raf activation by Ras. Instead, we provide evidence for Cdk1/cyclin B in mediating mitotic activation of B-Raf. In particular, Cdk1/cyclin B complexes associate with B-Raf at mitosis in Xenopus egg extracts and contribute to its phosphorylation. Mutagenesis and in vitro kinase assays demonstrated that Cdk1/cyclin B directly phosphorylates B-Raf at Serine-144, which is part of a conserved Cdk1 preferential consensus site (S144PQK). Importantly, phosphorylation of Ser-144 is absolutely required for mitotic activation of B-Raf and subsequent activation of the MAPK cascade. However, substitution of a phospho-mimicking amino acid at Ser-144 failed to produce a constitutive active B-Raf indicating that, in addition of Ser-144 phosphorylation, other regulatory events may be needed to activate B-Raf at mitosis. Taken together, our data reveal a novel cell cycle mechanism for activating the B-Raf/MEK/MAPK cascade.

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DNA mismatch repair in Xenopus egg extracts: repair efficiency and DNA repair synthesis for all single base-pair mismatches.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.87.20.7883 ◽

1990 ◽

Vol 87 (20) ◽

pp. 7883-7887 ◽

Cited By ~ 34

Author(s):

I. Varlet ◽

M. Radman ◽

P. Brooks

Keyword(s):

Dna Repair ◽

Repair Synthesis ◽

Single Base ◽

Dna Mismatch ◽

Repair Efficiency ◽

Egg Extracts ◽

Xenopus Egg ◽

Single Base Pair ◽

Dna Repair Synthesis ◽

Xenopus Egg Extracts

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Membrane-associated lamins in Xenopus egg extracts: identification of two vesicle populations.

The Journal of Cell Biology ◽

10.1083/jcb.123.3.501 ◽

1993 ◽

Vol 123 (3) ◽

pp. 501-512 ◽

Cited By ~ 54

Author(s):

D Lourim ◽

G Krohne

Keyword(s):

Nuclear Envelope ◽

Xenopus Oocyte ◽

Mitotic Cell ◽

Early Cleavage ◽

Nuclear Envelope Breakdown ◽

Nuclear Lamin ◽

Early Embryos ◽

Egg Extracts ◽

Xenopus Egg ◽

Xenopus Egg Extracts

Nuclear lamin isoforms of vertebrates can be divided into two major classes. The B-type lamins are membrane associated throughout the cell cycle, whereas A-type lamins are recovered from mitotic cell homogenates in membrane-free fractions. A feature of oogenesis in birds and mammals is the nearly exclusive presence of B-type lamins in oocyte nuclear envelopes. In contrast, oocytes and early cleavage embryos of the amphibian Xenopus laevis are believed to contain a single lamin isoform, lamin LIII, which after nuclear envelope breakdown during meiotic maturation is reported to be completely soluble. Consequently, we have reexamined the lamin complement of Xenopus oocyte nuclear envelopes, egg extracts, and early embryos. An mAb (X223) specific for the homologous B-type lamins B2 of mouse and LII of Xenopus somatic cells (Höger, T., K. Zatloukal, I. Waizenegger, and G. Krohne. 1990. Chromosoma. 99:379-390) recognized a Xenopus oocyte nuclear envelope protein biochemically distinct from lamin LIII and very similar or identical to somatic cell lamin LII. Oocyte lamin LII was detectable in nuclear envelopes of early cleavage embryos. Immunoblotting of fractionated egg extracts revealed that approximately 20-23% of lamin LII and 5-7% of lamin LIII were membrane associated. EM immunolocalization demonstrated that membrane-bound lamins LII and LIII are associated with separate vesicle populations. These findings are relevant to the interpretation of nuclear reconstitution experiments using Xenopus egg extracts.

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Topoisomerase II poisons inhibit vertebrate DNA replication through distinct mechanisms

10.1101/2021.10.12.464107 ◽

2021 ◽

Author(s):

Sabrina X. Van Ravenstein ◽

Kavi P. Mehta ◽

Tamar Kavlashvili ◽

Jo Ann Byl ◽

Runxiang Zhao ◽

...

Keyword(s):

Dna Replication ◽

Topoisomerase Ii ◽

Dependent Manner ◽

Replication Forks ◽

Dna Breaks ◽

Replication Fork Progression ◽

Topoisomerase Ii Poisons ◽

Egg Extracts ◽

Fork Stalling ◽

Major Target

AbstractTopoisomerase II (Top2) unlinks chromosomes during vertebrate DNA replication. Top2 ‘poisons’ are widely-used chemotherapeutics that stabilize Top2 complexes on DNA, leading to cytotoxic DNA breaks. However, it is unclear how these drugs affect DNA replication, which is a major target of Top2 poisons. Using Xenopus egg extracts, we show that the Top2 poisons etoposide and doxorubicin both inhibit DNA replication through different mechanisms. Etoposide induces Top2-dependent DNA breaks and induces Top2-dependent fork stalling by trapping Top2 behind replication forks. In contrast, doxorubicin does not lead to appreciable break formation and instead intercalates into parental DNA to inhibit replication fork progression. In human cells, etoposide stalls replication forks in a Top2-dependent manner, while doxorubicin stalls forks independently of Top2. However, both drugs exhibit Top2-dependent cytotoxicity. Thus, despite shared genetic requirements for cytotoxicity etoposide and doxorubicin inhibit DNA replication through distinct mechanisms.

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Preventing re-replication of DNA in a single cell cycle: evidence for a replication licensing factor

The Journal of Cell Biology ◽

10.1083/jcb.122.5.993 ◽

1993 ◽

Vol 122 (5) ◽

pp. 993-1002 ◽

Cited By ~ 114

Author(s):

JJ Blow

Keyword(s):

Cell Cycle ◽

Dna Replication ◽

Nuclear Envelope ◽

Kinase Inhibitor ◽

Chromatin Modification ◽

Replication Factor ◽

Licensing Factor ◽

Egg Extracts ◽

Nuclear Envelope Assembly ◽

Xenopus Egg Extracts

Xenopus egg extracts treated with the protein kinase inhibitor 6-dimethylaminopurine (6-DMAP) are unable to support the initiation of DNA replication. Nuclei assembled in 6-DMAP extracts behave as though they are in G2, and will not undergo another round of DNA replication until passage through mitosis. 6-DMAP extracts are functionally devoid of a replication factor that modifies chromatin in early G1 before nuclear envelope assembly, but which is itself incapable of crossing the nuclear envelope. This chromatin modification is capable of supporting only a single round of semiconservative replication. The behavior of this replication factor is sufficient to explain why eukaryotic DNA is replicated once and only once in each cell cycle, and conforms to the previous model of a Replication Licensing Factor. Cell cycle analysis shows that this putative Licensing Factor is inactive during metaphase, but becomes rapidly activated on exit from metaphase when it can modify chromatin before nuclear envelope assembly is complete.

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