Mitosis DNA Research Articles

Mechanisms of eukaryotic replisome disassembly.

DNA replication is a complex process that needs to be executed accurately before cell division in order to maintain genome integrity. DNA replication is divided into three main stages: initiation, elongation and termination. One of the key events during initiation is the assembly of the replicative helicase at origins of replication, and this mechanism has been very well described over the last decades. In the last six years however, researchers have also focused on deciphering the molecular mechanisms underlying the disassembly of the replicative helicase during termination. Similar to replisome assembly, the mechanism of replisome disassembly is strictly regulated and well conserved throughout evolution, although its complexity increases in higher eukaryotes. While budding yeast rely on just one pathway for replisome disassembly in S phase, higher eukaryotes evolved an additional mitotic pathway over and above the default S phase specific pathway. Moreover, replisome disassembly has been recently found to be a key event prior to the repair of certain DNA lesions, such as under-replicated DNA in mitosis and inter-strand cross-links (ICLs) in S phase. Although replisome disassembly in human cells has not been characterised yet, they possess all of the factors involved in these pathways in model organisms, and de-regulation of many of them are known to contribute to tumorigenesis and other pathological conditions.

Targeting Vulnerabilities in Successive Cell Cycle Stages to Induce Death of PI3K-Activated Basal-Like Breast Cancer Cells

Frequent mutation of PI3K pathway genes in human tumors has stimulated large investments in therapeutic drugs targeting this pathway but clinical successes have been rare. As a result, many cancers with high PI3K pathway activity are still treated with conventional chemotherapy. By systematically analyzing responses of basal-like breast cancer cells to PI3K pathway inhibitors, we show that one drug, Torin2, is unusually effective because it inhibits both mTOR and PI3K-like kinases (PIKKs). Experiments with Torin2 and combinations of selective kinase inhibitors demonstrate that submaximal G1/S inhibition, increased S phase replication stress, and single-stranded DNA in mitosis are all involved in cell killing. Combination therapy with inhibitors of signal transduction kinases conventionally targets simultaneously active pathways. Our studies of Torin2 show that favorable combinatorial drug effects can also result from targeting successive cell cycle vulnerabilities, eliciting a cascade of deleterious effects that culminates in mitotic errors and death.

Proteomic Profiling Reveals a Specific Role for Translesion DNA Polymerase η in the Alternative Lengthening of Telomeres

SUMMARYCancer cells rely on the activation of telomerase or the alternative lengthening of telomeres (ALT) pathways for telomere maintenance and survival. ALT involves homologous recombination (HR)-dependent exchange and/or HR-associated synthesis of telomeric DNA. Utilizing proximity-dependent biotinylation (BioID), we sought to determine the proteome of telomeres in cancer cells that employ these distinct telomere elongation mechanisms. Our analysis reveals that multiple DNA repair networks converge at ALT telomeres. These include the specialized translesion DNA synthesis (TLS) proteins FANCJ-RAD18-PCNA and, most notably, DNA polymerase eta (Polη). We observe that the depletion of Polη leads to increased ALT activity and late DNA polymerase δ (Polδ)-dependent synthesis of telomeric DNA in mitosis. We propose that Polη fulfills an important role in managing replicative stress at ALT telomeres, maintaining telomere recombination at tolerable levels and stimulating DNA synthesis by Polδ.

A balanced pyrimidine pool is required for optimal Chk1 activation to prevent ultrafine anaphase bridge formation.

Cytidine deaminase (CDA) deficiency induces an excess of cellular dCTP, which reduces basal PARP-1 activity, thereby compromising complete DNA replication, leading to ultrafine anaphase bridge (UFB) formation. CDA dysfunction has pathological implications, notably in cancer and in Bloom syndrome. It remains unknown how reduced levels of PARP-1 activity and pyrimidine pool imbalance lead to the accumulation of unreplicated DNA during mitosis. We report that a decrease in PARP-1 activity in CDA-deficient cells impairs DNA-damage-induced Chk1 activation, and, thus, the downstream checkpoints. Chemical inhibition of the ATR-Chk1 pathway leads to UFB accumulation, and we found that this pathway was compromised in CDA-deficient cells. Our data demonstrate that ATR-Chk1 acts downstream from PARP-1, preventing the accumulation of unreplicated DNA in mitosis, and, thus, UFB formation. Finally, delaying entry into mitosis is sufficient to prevent UFB formation in both CDA-deficient and CDA-proficient cells, suggesting that both physiological and pathological UFBs are derived from unreplicated DNA. Our findings demonstrate an unsuspected requirement for a balanced nucleotide pool for optimal Chk1 activation both in unchallenged cells and in response to genotoxic stress.

DNA synthesis by Pol η promotes fragile site stability by preventing under-replicated DNA in mitosis

Human DNA polymerase η (Pol η) is best known for its role in responding to UV irradiation-induced genome damage. We have recently observed that Pol η is also required for the stability of common fragile sites (CFSs), whose rearrangements are considered a driving force of oncogenesis. Here, we explored the molecular mechanisms underlying this newly identified role. We demonstrated that Pol η accumulated at CFSs upon partial replication stress and could efficiently replicate non-B DNA sequences within CFSs. Pol η deficiency led to persistence of checkpoint-blind under-replicated CFS regions in mitosis, detectable as FANCD2-associated chromosomal sites that were transmitted to daughter cells in 53BP1-shielded nuclear bodies. Expression of a catalytically inactive mutant of Pol η increased replication fork stalling and activated the replication checkpoint. These data are consistent with the requirement of Pol η-dependent DNA synthesis during S phase at replication forks stalled in CFS regions to suppress CFS instability by preventing checkpoint-blind under-replicated DNA in mitosis.

The ABCs of CENPs

Equal distribution of DNA in mitosis requires the assembly of a large proteinaceous ensemble onto the centromeric DNA, called the kinetochore. With few exceptions, kinetochore specification is independent of the DNA sequence and is determined epigenetically by deposition at the centromeric chromatin of special nucleosomes containing an H3-related histone, CENP-A. Onto centromeric CENP-A chromatin is assembled the so-called constitutive centromere-associated network (CCAN) of 16 proteins distributed in several functional groups as follows: CENP-C, CENP-H/CENP-I/CENP-K/, CENP-L/CENP-M/CENP-N, CENP-O/CENP-P/CENP-Q/CENP-R/CENP-U(50), CENP-T/CENP-W, and CENP-S/CENP-X. One role of the CCAN is to recruit outer kinetochore components further, such as KNL1, the Mis12 complex, and the Ndc80 complex (KMN network) to which attach the spindle microtubules with their structural and regulatory proteins. Among the CENPs in CCAN, CENP-C and CENP-T are required in parallel for operational kinetochore specification and spindle attachment. This review presents discussion of the latest structural and functional data on CENP-A and CENPs from the CCAN as well as their interaction with the KMN network.

Exclusion of NFAT5 from Mitotic Chromatin Resets Its Nucleo-Cytoplasmic Distribution in Interphase

BackgroundThe transcription factor NFAT5 is a major inducer of osmoprotective genes and is required to maintain the proliferative capacity of cells exposed to hypertonic stress. In response to hypertonicity, NFAT5 translocates to the nucleus, binds to regulatory regions of osmoprotective genes and activates their transcription. Besides stimulus-specific regulatory mechanisms, the activity of transcription factors in cycling cells is also regulated by the passage through mitosis, when most transcriptional processes are downregulated. It was not known whether mitosis could be a point of control for NFAT5.Methodology/Principal FindingsUsing confocal microscopy we observed that NFAT5 was excluded from chromatin during mitosis in both isotonic and hypertonic conditions. Analysis of NFAT5 deletions showed that exclusion was mediated by the carboxy-terminal domain (CTD). NFAT5 mutants lacking this domain showed constitutive binding to mitotic chromatin independent of tonicity, which caused them to localize in the nucleus and remain bound to chromatin in the subsequent interphase without hypertonic stimulation. We analyzed the contribution of the CTD, DNA binding, and nuclear import and export signals to the subcellular localization of this factor. Our results indicated that cytoplasmic localization of NFAT5 in isotonic conditions required both the exclusion from mitotic DNA and active nuclear export in interphase. Finally, we identified several regions within the CTD of NFAT5, some of them overlapping with transactivation domains, which were separately capable of causing its exclusion from mitotic chromatin.Conclusions/SignificanceOur results reveal a multipart mechanism regulating the subcellular localization of NFAT5. The transactivating module of NFAT5 switches its function from an stimulus-specific activator of transcription in interphase to an stimulus-independent repressor of binding to DNA in mitosis. This mechanism, together with export signals acting in interphase, resets the cytoplasmic localization of NFAT5 and prevents its nuclear accumulation and association with DNA in the absence of hypertonic stress.

Universal Temporal Profile of Replication Origin Activation in Eukaryotes

Although replication proteins are conserved among eukaryotes, the sequence requirements for replication initiation differ between species. In all species, however, replication origins fire asynchronously throughout S phase. The temporal program of origin firing is reproducible in cell populations but largely probabilistic at the single-cell level. The mechanisms and the significance of this program are unclear. Replication timing has been correlated with gene activity in metazoans but not in yeast. One potential role for a temporal regulation of origin firing is to minimize fluctuations in replication end time and avoid persistence of unreplicated DNA in mitosis. Here, we have extracted the population-averaged temporal profiles of replication initiation rates for S. cerevisiae, S. pombe, D. melanogaster, X. laevis and H. sapiens from genome-wide replication timing and DNA combing data. All the profiles have a strikingly similar shape, increasing during the first half of S phase then decreasing before its end. A previously proposed minimal model of stochastic initiation modulated by accumulation of a recyclable, limiting replication-fork factor and fork-promoted initiation of new origins, quantitatively described the observed profiles without requiring new implementations.The selective pressure for timely completion of genome replication and optimal usage of replication proteins that must be imported into the cell nucleus can explain the generic shape of the profiles. We have identified a universal behavior of eukaryotic replication initiation that transcends the mechanisms of origin specification. The population-averaged efficiency of replication origin usage changes during S phase in a strikingly similar manner in a highly diverse set of eukaryotes. The quantitative model previously proposed for origin activation in X. laevis can be generalized to explain this evolutionary conservation.

Unreplicated DNA in mitosis precludes condensin binding and chromosome condensation in S. cerevisiae

Condensin is the core activity responsible for chromosome condensation in mitosis. In the yeast S. cerevisiae, condensin binding is enriched at the regions where DNA replication terminates. Therefore, we investigated whether DNA replication completion determines the condensin-binding proficiency of chromatin. In order to fulfill putative mitotic requirements for condensin activity we analyzed chromosome condensation and condensin binding to unreplicated chromosomes in mitosis. For this purpose we used pGAL:CDC6 cdc15-ts cells that are known to enter mitosis without DNA replication if CDC6 transcription is repressed prior to S-phase. Both the condensation of nucleolar chromatin and proper condensin targeting to rDNA sites failed when unreplicated chromosomes were driven in mitosis. We propose that the DNA replication results in structural and/or biochemical changes to replicated chromatin, which are required for two-phase condensin binding and proper chromosome condensation.

Simultaneous expression of both MAT loci in haploid cells suppresses mutations in yeast microtubule motor genes.

The kinesin-related Cin8p and cytoplasmic dynein are microtubule-associated motor proteins required for anaphase spindle elongation in the yeast Saccharomyces cerevisiae. Cells deleted for DYN1 (the gene encoding the dynein heavy chain) and carrying the temperature-sensitive allele cin8-3 cannot grow at temperatures above 35 degrees C. Here, we report that the temperature sensitivity of haploid cin8-3 dyn1delta cells is suppressed by the simultaneous presence of the loci MATa and MATalpha, which contain the regulatory genes that determine mating-type and ploidy-dependent phenotypes. The presence of the two MAT loci also rendered haploid cells more resistant to the antimicrotubule drug benomyl. Our results suggest that, in preparation for handling double the amount of DNA in mitosis, properties of microtubules in diploid cells are modified in a pathway controlled by the mating-type regulatory genes.

A meiotic recombination checkpoint controlled by mitotic checkpoint genes.

In budding yeast, meiotic recombination occurs at about 200 sites per cell and involves DNA double-strand break (DSB) intermediates. Here we provide evidence that a checkpoint control requiring the mitotic DNA-damage checkpoint genes RAD17, RAD24 and MEC1 ensures that meiotic recombination is complete before the first meiotic division (MI). First, RAD17, RAD24 and MEC1 are required for the meiotic arrest caused by blocking the repair of DSBs with a mutation in the recA homologue DMC1. Second, mec1 and rad24 single mutants (DMC1+) appear to undergo MI before all recombination events are complete. Curiously, the mitosis-specific checkpoint gene RAD9 is not required for meiotic arrest of dmc1 mutants. This shows that although mitotic and meiotic control mechanisms are related, they differ significantly. Rad17 and Rad24 proteins may contribute directly to formation of an arrest signal by association with single-strand DNA in mitosis and meiosis.

Chloroplast division in cultured cells of the hornwortAnthoceros punctatus

Using cultured cells of the hornwortAnthoceros punctatus, the change in the relative chloroplast DNA content in each stage of chloroplast division was investigated to clarify the relationship between the division cycle of a chloroplast and a cell nucleus. Samples of cultured cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) and then observed with an epifluorescence microscope and a chromosome image analyzing system (CHIAS). A chloropiast in cultured cells duplicated DNA with an increase in size. When a chloroplast began to divide, it was constricted in the middle, taking a dumbbell shape, and then divided into two daughter chloroplasts. In cultured cells of this species, the pattern of quantitative change of chloroplast DNA, that is, the DNA replication pattern of chloroplasts, corresponded to that of cell nuclear DNA in mitosis.

The analysis of 40 kDa nuclear protein, p40, in interphase cells and mitotic cells

We previously reported that the monoclonal antibody M108 recognized a 40 kDa protein both in the nucleus and the cytoplasm. This nuclear 40 kDa antigen was located in the nuclear envelope in interphase cells and in the perichromosomal region during mitosis. Now, we have analyzed this nuclear 40 kDa protein (p40) further, through morphological and biochemical approaches. At the beginning of mitosis, the perinuclear p40 detached from the nuclear envelope and moved to surround the condensing chromatin, while in the late stage of mitosis, the perichromosomal p40 moved back to the reassembled nuclear envelope. Most of the perichromosomal p40 on the metaphase chromosome was solubilized only by DNase I treatment, not by either high salt or detergent treatment. On the other hand, the perinuclear p40 was not solubilized by DNase1 alone, or high salt detergent alone. Sequential treatments with DNase I and high salt detergent were required to extract p40 in interphase nuclei. These results suggest that p40 was associated both with the nuclear envelope and chromatin DNA in interphase nuclei, while it bound only to chromatin DNA in mitosis.