Pyrimidine Pool Disequilibrium Induced by a Cytidine Deaminase Deficiency Inhibits PARP-1 Activity,Leading to the Under Replication of DNA

Genome stability is jeopardized by imbalances of the dNTP pool; such imbalances affect the rate of fork progression. For example, cytidine deaminase (CDA) deficiency leads to an excess of dCTP, slowing the replication fork. We describe here a novel mechanism by which pyrimidine pool disequilibrium compromises the completion of replication and chromosome segregation: the intracellular accumulation of dCTP inhibits PARP-1 activity. CDA deficiency results in incomplete DNA replication when cells enter mitosis, leading to the formation of ultrafine anaphase bridges between sister-chromatids at “difficult-to-replicate” sites such as centromeres and fragile sites. Using molecular combing, electron microscopy and a sensitive assay involving cell imaging to quantify steady-state PAR levels, we found that DNA replication was unsuccessful due to the partial inhibition of basal PARP-1 activity, rather than slower fork speed. The stimulation of PARP-1 activity in CDA-deficient cells restores replication and, thus, chromosome segregation. Moreover, increasing intracellular dCTP levels generates under-replication-induced sister-chromatid bridges as efficiently as PARP-1 knockdown. These results have direct implications for Bloom syndrome (BS), a rare genetic disease combining susceptibility to cancer and genomic instability. BS results from mutation of the BLM gene, encoding BLM, a RecQ 3’-5’ DNA helicase, a deficiency of which leads to CDA downregulation. BS cells thus have a CDA defect, resulting in a high frequency of ultrafine anaphase bridges due entirely to dCTP-dependent PARP-1 inhibition and independent of BLM status. Our study describes previously unknown pathological consequences of the distortion of dNTP pools and reveals an unexpected role for PARP-1 in preventing DNA under-replication and chromosome segregation defects.     

Lack of tension at kinetochores activates the spindle checkpoint in budding yeast

The spindle checkpoint delays the onset of anaphase until all pairs of sister chromatids are attached to the mitotic spindle. The checkpoint could monitor the attachment of microtubules to kinetochores, the tension that results from the two sister chromatids attaching to opposite spindle poles, or both. We tested the role of tension by allowing cells to enter mitosis without a prior round of DNA replication. The unreplicated chromatids are attached to spindle microtubules but are not under tension since they lack a sister chromatid that could attach to the opposite pole. Because the spindle checkpoint is activated in these cells, we conclude that the absence of tension at the yeast kinetochore is sufficient to activate the spindle checkpoint in mitosis.     

Separase is required for chromosome segregation during meiosis I in Caenorhabditis elegans.

BACKGROUND: Chromosome segregation during mitosis and meiosis is triggered by dissolution of sister chromatid cohesion, which is mediated by the cohesin complex. Mitotic sister chromatid disjunction requires that cohesion be lost along the entire length of chromosomes, whereas homolog segregation at meiosis I only requires loss of cohesion along chromosome arms. During animal cell mitosis, cohesin is lost in two steps. A nonproteolytic mechanism removes cohesin along chromosome arms during prophase, while the proteolytic cleavage of cohesin's Scc1 subunit by separase removes centromeric cohesin at anaphase. In Saccharomyces cerevisiae and Caenorhabditis elegans, meiotic sister chromatid cohesion is mediated by Rec8, a meiosis-specific variant of cohesin's Scc1 subunit. Homolog segregation in S. cerevisiae is triggered by separase-mediated cleavage of Rec8 along chromosome arms. In principle, chiasmata could be resolved proteolytically by separase or nonproteolytically using a mechanism similar to the mitotic "prophase pathway." RESULTS: Inactivation of separase in C. elegans has little or no effect on homolog alignment on the meiosis I spindle but prevents their timely disjunction. It also interferes with chromatid separation during subsequent embryonic mitotic divisions but does not directly affect cytokinesis. Surprisingly, separase inactivation also causes osmosensitive embryos, possibly due to a defect in the extraembryonic structures, referred to as the "eggshell." CONCLUSIONS: Separase is essential for homologous chromosome disjunction during meiosis I. Proteolytic cleavage, presumably of Rec8, might be a common trigger for the first meiotic division in eukaryotic cells. Cleavage of proteins other than REC-8 might be necessary to render the eggshell impermeable to solutes.     

The DNA helicase activity of BLM is necessary for the correction of the genomic instability of bloom syndrome cells

Bloom syndrome (BS) is a rare autosomal recessive disorder characterized by growth deficiency, immunodeficiency, genomic instability, and the early development of cancers of many types. BLM, the protein encoded by BLM, the gene mutated in BS, is localized in nuclear foci and absent from BS cells. BLM encodes a DNA helicase, and proteins from three missense alleles lack displacement activity. BLM transfected into BS cells reduces the frequency of sister chromatid exchanges and restores BLM in the nucleus. Missense alleles fail to reduce the sister chromatid exchanges in transfected BS cells or restore the normal nuclear pattern. BLM complements a phenotype of a Saccharomyces cerevisiae sgs1 top3 strain, and the missense alleles do not. This work demonstrates the importance of the enzymatic activity of BLM for its function and nuclear localization pattern.     

Separase: a universal trigger for sister chromatid disjunction but not chromosome cycle progression

Separase is a protease whose liberation from its inhibitory chaperone Securin triggers sister chromatid disjunction at anaphase onset in yeast by cleaving cohesin's kleisin subunit. We have created conditional knockout alleles of the mouse Separase and Securin genes. Deletion of both copies of Separase but not Securin causes embryonic lethality. Loss of Securin reduces Separase activity because deletion of just one copy of the Separase gene is lethal to embryos lacking Securin. In embryonic fibroblasts, Separase depletion blocks sister chromatid separation but does not prevent other aspects of mitosis, cytokinesis, or chromosome replication. Thus, fibroblasts lacking Separase become highly polyploid. Hepatocytes stimulated to proliferate in vivo by hepatectomy also become unusually large and polyploid in the absence of Separase but are able to regenerate functional livers. Separase depletion in bone marrow causes aplasia and the presumed death of hematopoietic cells other than erythrocytes. Destruction of sister chromatid cohesion by Separase may be a universal feature of mitosis in eukaryotic cells.     

A new thermosensitive smc-3 allele reveals involvement of cohesin in homologous recombination in C. elegans

The cohesin complex is required for the cohesion of sister chromatids and for correct segregation during mitosis and meiosis. Crossover recombination, together with cohesion, is essential for the disjunction of homologous chromosomes during the first meiotic division. Cohesin has been implicated in facilitating recombinational repair of DNA lesions via the sister chromatid. Here, we made use of a new temperature-sensitive mutation in the Caenorhabditis elegans SMC-3 protein to study the role of cohesin in the repair of DNA double-strand breaks (DSBs) and hence in meiotic crossing over. We report that attenuation of cohesin was associated with extensive SPO-11-dependent chromosome fragmentation, which is representative of unrepaired DSBs. We also found that attenuated cohesin likely increased the number of DSBs and eliminated the need of MRE-11 and RAD-50 for DSB formation in C. elegans, which suggests a role for the MRN complex in making cohesin-loaded chromatin susceptible to meiotic DSBs. Notably, in spite of largely intact sister chromatid cohesion, backup DSB repair via the sister chromatid was mostly impaired. We also found that weakened cohesins affected mitotic repair of DSBs by homologous recombination, whereas NHEJ repair was not affected. Our data suggest that recombinational DNA repair makes higher demands on cohesins than does chromosome segregation.     

Incomplete sister chromatid separation of long chromosome arms

Chromosome segregation ensures the equal partitioning of chromosomes at mitosis. However, long chromosome arms may pose a problem for complete sister chromatid separation. In this paper we report on the analysis of cell division in primary cells from field vole Microtus agrestis, a species with 52 chromosomes including two giant sex chromosomes. Dual chromosome painting with probes specific for the X and the Y chromosomes showed that these long chromosomes are prone to mis-segregate, producing DNA bridges between daughter nuclei and micronuclei. Analysis of mitotic cells with incomplete chromatid separation showed that reassembly of the nuclear membrane, deposition of INner CENtromere Protein (INCENP)/Aurora B to the spindle midzone and furrow formation occur while the two groups of daughter chromosomes are still connected by sex chromosome arms. Late cytokinetic processes are not efficiently inhibited by the incomplete segregation as in a significant number of cell divisions cytoplasmic abscission proceeds while Aurora B is at the midbody. Live-cell imaging during late mitotic stages also revealed abnormal cell division with persistent sister chromatid connections. We conclude that late mitotic regulatory events do not monitor incomplete sister chromatid separation of the large X and Y chromosomes of Microtus agrestis, leading to defective segregation of these chromosomes. These findings suggest a limit in chromosome arm length for efficient chromosome transmission through mitosis.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Two putative acetyltransferases, san and deco, are required for establishing sister chromatid cohesion in Drosophila

BACKGROUND: Sister chromatid cohesion is needed for proper alignment and segregation of chromosomes during cell division. Chromatids are linked by the multiprotein cohesin complex, which binds to DNA during G(1) and then establishes cohesion during S phase DNA replication. However, many aspects of the mechanisms that establish and maintain cohesion during mitosis remain unclear.RESULTS: We found that mutations in two evolutionarily conserved Drosophila genes, san (separation anxiety) and deco (Drosophila eco1), disrupt centromeric sister chromatid cohesion very early in division. This failure of sister chromatid cohesion does not require separase and is correlated with a failure of the cohesin component Scc1 to accumulate in centromeric regions. It thus appears that these mutations interfere with the establishment of centromeric sister chromatid cohesion. Secondary consequences of these mutations include activation of the spindle checkpoint, causing metaphase delay or arrest. Some cells eventually escape the block but incur many errors in anaphase chromosome segregation. Both san and deco are predicted to encode acetyltransferases, which transfer acetyl groups either to internal lysine residues or to the N terminus of other proteins. The San protein is itself acetylated, and it associates with the Nat1 and Ard1 subunits of the NatA acetyltransferase.CONCLUSIONS: At least two diverse acetyltransferases play vital roles in regulating sister chromatid cohesion during Drosophila mitosis.     

BLM is required for faithful chromosome segregation and its localization defines a class of ultrafine anaphase bridges

Mutations in BLM cause Bloom's syndrome, a disorder associated with cancer predisposition and chromosomal instability. We investigated whether BLM plays a role in ensuring the faithful chromosome segregation in human cells. We show that BLM-defective cells display a higher frequency of anaphase bridges and lagging chromatin than do isogenic corrected derivatives that eptopically express the BLM protein. In normal cells undergoing mitosis, BLM protein localizes to anaphase bridges, where it colocalizes with its cellular partners, topoisomerase IIIalpha and hRMI1 (BLAP75). Using BLM staining as a marker, we have identified a class of ultrafine DNA bridges in anaphase that are surprisingly prevalent in the anaphase population of normal human cells. These so-called BLM-DNA bridges, which also stain for the PICH protein, frequently link centromeric loci, and are present at an elevated frequency in cells lacking BLM. On the basis of these results, we propose that sister-chromatid disjunction is often incomplete in human cells even after the onset of anaphase. We present a model for the action of BLM in ensuring complete sister chromatid decatenation in anaphase.     

Chromosome breakage and rejoining of sister chromatids in Bloom's syndrome

The occurrence of chromosome breaks and reunion of sister chromatids in lymphocytes of two patients with Bloom's syndrome has been compared with those found in X-rayed and control cells. The distribution of breaks in BS is non-random both between and within chromosomes, the centric regions of certain chromosomes being preferentially involved. The following working hypotheses are put forward: When chromosome breaks in human lymphocytes occur in G₀— G₁, practically no sister chromatid reunion (SCR) takes place, whereas ends created by an S—G₂ break show a considerable tendency to SCR. We propose further that chromosome aberrations in BS mainly result from breaks in S—G₂, including possible U-type rejoining of sister chromatid exchanges. Fragments extra to an intact chromosome complement result from a chromatid break or an asymmetrical chromatid translocation in a previous mitosis.     

The meiosis I-to-meiosis II transition in mouse oocytes requires separase activity

Faithful segregation of homologous chromosomes during the first meiotic division is essential for further embryo development. The question at issue is whether the same mechanisms ensuring correct separation of sister chromatids in mitosis are at work during the first meiotic division. In mitosis, sister chromatids are linked by a cohesin complex holding them together until their disjunction at anaphase. Their disjunction is mediated by Separase, which cleaves the cohesin. The activation of Separase requires prior degradation of its associated inhibitor, called securin. Securin is a target of the APC/C (Anaphase Promoting Complex/Cyclosome), a cell cycle-regulated ubiquitin ligase that ubiquitinates securin at the metaphase-to-anaphase transition and thereby targets it for degradation by the 26S proteasome. After securin degradation, Separase cleaves the cohesins and triggers chromatid separation, a prerequisite for anaphase. In yeast and worms, the segregation of homologous chromosomes in meiosis I depends on the APC/C and Separase activity. Yet, it is unclear if Separase is required for the first meiotic division in vertebrates because APC/C activity is thought to be dispensable in frog oocytes. We therefore investigated if Separase activity is required for correct chromosome segregation in meiosis I in mouse oocytes.     

Absence of BLM leads to accumulation of chromosomal DNA breaks during both unperturbed and disrupted S phases

Bloom's syndrome (BS), a disorder associated with genomic instability and cancer predisposition, results from defects in the Bloom's helicase (BLM) protein. In BS cells, chromosomal abnormalities such as sister chromatid exchanges occur at highly elevated rates. Using Xenopus egg extracts, we have studied Xenopus BLM (Xblm) during both unperturbed and disrupted DNA replication cycles. Xblm binds to replicating chromatin and becomes highly phosphorylated in the presence of DNA replication blocks. This phosphorylation depends on Xenopus ATR (Xatr) and Xenopus Rad17 (Xrad17), but not Claspin. Xblm and Xenopus topoisomerase IIIalpha (Xtop3alpha) interact in a regulated manner and associate with replicating chromatin interdependently. Immunodepletion of Xblm from egg extracts results in accumulation of chromosomal DNA breaks during both normal and perturbed DNA replication cycles. Disruption of the interaction between Xblm and Xtop3alpha has similar effects. The occurrence of DNA damage in the absence of Xblm, even without any exogenous insult to the DNA, may help to explain the genesis of chromosomal defects in BS cells.     

The acetyltransferase activity of San stabilizes the mitotic cohesin at the centromeres in a shugoshin-independent manner

Proper sister chromatid cohesion is critical for maintaining genetic stability. San is a putative acetyltransferase that is important for sister chromatid cohesion in Drosophila melanogaster, but not in budding yeast. We showed that San is critical for sister chromatid cohesion in HeLa cells, suggesting that this mechanism may be conserved in metazoans. Furthermore, although a small fraction of San interacts with the NatA complex, San appears to mediate cohesion independently. San exhibits acetyltransferase activity in vitro, and its activity is required for sister chromatid cohesion in vivo. In the absence of San, Sgo1 localizes correctly throughout the cell cycle. However, cohesin is no longer detected at the mitotic centromeres. Furthermore, San localizes to the cytoplasm in interphase cells; thus, it may not gain access to chromosomes until mitosis. Moreover, in San-depleted cells, further depletion of Plk1 rescues the cohesion along the chromosome arms, but not at the centromeres. Collectively, San may be specifically required for the maintenance of the centromeric cohesion in mitosis.     

The Slx4-Dpb11 scaffold complex: coordinating the response to replication fork stalling in S-phase and the subsequent mitosis

Replication fork stalling at DNA lesions is a common problem during the process of DNA replication. One way to allow the bypass of these lesions is via specific recombination-based mechanisms that involve switching of the replication template to the sister chromatid. Inherent to these mechanisms is the formation of DNA joint molecules (JMs) between sister chromatids. Such JMs need to be disentangled before chromatid separation in mitosis and the activity of JM resolution enzymes, which is under stringent cell cycle control, is therefore up-regulated in mitosis. An additional layer of control is facilitated by scaffold proteins. In budding yeast, specifically during mitosis, Slx4 and Dpb11 form a cell cycle kinase-dependent complex with the Mus81-Mms4 structure-selective endonuclease, which allows efficient JM resolution by Mus81. Furthermore, Slx4 and Dpb11 interact even prior to joining Mus81 and respond to replication fork stalling in S-phase. This S-phase complex is involved in the regulation of the DNA damage checkpoint as well as in early steps of template switch recombination. Similar interactions and regulatory principles are found in human cells suggesting that Slx4 and Dpb11 may have an evolutionary conserved role organizing the cellular response to replication fork stalling.     

Ultrafine anaphase bridges, broken DNA and illegitimate recombination induced by a replication fork barrier

Most DNA double-strand breaks (DSBs) in S- and G2-phase cells are repaired accurately by Rad51-dependent sister chromatid recombination. However, a minority give rise to gross chromosome rearrangements (GCRs), which can result in disease/death. What determines whether a DSB is repaired accurately or inaccurately is currently unclear. We provide evidence that suggests that perturbing replication by a non-programmed protein-DNA replication fork barrier results in the persistence of replication intermediates (most likely regions of unreplicated DNA) into mitosis, which results in anaphase bridge formation and ultimately to DNA breakage. However, unlike previously characterised replication-associated DSBs, these breaks are repaired mainly by Rad51-independent processes such as single-strand annealing, and are therefore prone to generate GCRs. These data highlight how a replication-associated DSB can be predisposed to give rise to genome rearrangements in eukaryotes.     

The Slx4-Dpb11 scaffold complex: coordinating the response to replication fork stalling in S-phase and the subsequent mitosis

Replication fork stalling at DNA lesions is a common problem during the process of DNA replication. One way to allow the bypass of these lesions is via specific recombination-based mechanisms that involve switching of the replication template to the sister chromatid. Inherent to these mechanisms is the formation of DNA joint molecules (JMs) between sister chromatids. Such JMs need to be disentangled before chromatid separation in mitosis and the activity of JM resolution enzymes, which is under stringent cell cycle control, is therefore up-regulated in mitosis. An additional layer of control is facilitated by scaffold proteins. In budding yeast, specifically during mitosis, Slx4 and Dpb11 form a cell cycle kinase-dependent complex with the Mus81-Mms4 structure-selective endonuclease, which allows efficient JM resolution by Mus81. Furthermore, Slx4 and Dpb11 interact even prior to joining Mus81 and respond to replication fork stalling in S-phase. This S-phase complex is involved in the regulation of the DNA damage checkpoint as well as in early steps of template switch recombination. Similar interactions and regulatory principles are found in human cells suggesting that Slx4 and Dpb11 may have an evolutionary conserved role organizing the cellular response to replication fork stalling.     

Mechanisms of whole chromosome gains in tumors--many answers to a simple question

Whole chromosome gain is the most common type of gross genomic abnormality observed in human tumors. It is particularly frequent in lympho-haematopoietic and embryonic neoplasms, where trisomies and tetrasomies are typically present together with few or no other cytogenetic imbalances, resulting in hyperdiploid chromosome numbers. Despite the high prevalence of whole chromosome gains in neoplastic cells, their mechanism of origin remains disputed. Here, 4 potential models for the generation of whole chromosome gains are reviewed: (1) loss of chromosomes from the tetraploid level, (2) sequential sister chromatid non-disjunction, (3) multipolar mitosis coupled to sister chromatid non-disjunction, and (4) multipolar mitosis coupled to incomplete cytokinesis. Each of these mechanisms may in theory result in the generation of hyperdiploid neoplastic clones, but none of them were single-handedly able to reproduce the scenario of chromosome copy number alterations in tumors when cell populations resulting from these models were simulated in silico and compared to published cytogenetic data. To develop models for the generation of whole chromosome gains further, it is critical to improve our knowledge of the principles of clonal selection in tumors and of the baseline rate of chromosome segregation errors in human cells. To illustrate this, a model combining multipolar mitosis coupled to incomplete cytokinesis with a low rate of baseline sister chromatid non-disjunction was shown readily to reproduce copy number distributions in hyperdiploid karyotypes from human tumors.     

Replication-Dependent Sister Chromatid Recombination in Rad1 Mutants of Saccharomyces Cerevisiae

Homolog recombination and unequal sister chromatid recombination were monitored in rad1-1/rad1-1 diploid yeast cells deficient for excision repair, and in control cells, RAD1/rad1-1, after exposure to UV irradiation. In a rad1-1/rad1-1 diploid, UV irradiation stimulated much more sister chromatid recombination relative to homolog recombination when cells were irradiated in the G(1) or the G(2) phases of the cell cycle than was observed in RAD1/rad1-1 cells. Since sister chromatids are not present during G(1), this result suggested that unexcised lesions can stimulate sister chromatid recombination events during or subsequent to DNA replication. The results of mating rescue experiments suggest that unexcised UV dimers do not stimulate sister chromatid recombination during the G(2) phase, but only when they are present during DNA replication. We propose that there are two types of sister chromatid recombination in yeast. In the first type, unexcised UV dimers and other bulky lesions induce sister chromatid recombination during DNA replication as a mechanism to bypass lesions obstructing the passage of DNA polymerase, and this type is analogous to the type of sister chromatid exchange commonly observed cytologically in mammalian cells. In the second type, strand scissions created by X-irradiation or the excision of damaged bases create recombinogenic sites that result in sister chromatid recombination directly in G(2). Further support for the existence of two types of sister chromatid recombination is the fact that events induced in rad1-1/rad1-1 were due almost entirely to gene conversion, whereas those in RAD1/rad1-1 cells were due to a mixture of gene conversion and reciprocal recombination.     

Cohesin SMC1 beta is required for meiotic chromosome dynamics, sister chromatid cohesion and DNA recombination

Sister chromatid cohesion ensures the faithful segregation of chromosomes in mitosis and in both meiotic divisions. Meiosis-specific components of the cohesin complex, including the recently described SMC1 isoform SMC1 beta, were suggested to be required for meiotic sister chromatid cohesion and DNA recombination. Here we show that SMC1 beta-deficient mice of both sexes are sterile. Male meiosis is blocked in pachytene; female meiosis is highly error-prone but continues until metaphase II. Prophase axial elements (AEs) are markedly shortened, chromatin extends further from the AEs, chromosome synapsis is incomplete, and sister chromatid cohesion in chromosome arms and at centromeres is lost prematurely. In addition, crossover-associated recombination foci are absent or reduced, and meiosis-specific perinuclear telomere arrangements are impaired. Thus, SMC1 beta has a key role in meiotic cohesion, the assembly of AEs, synapsis, recombination, and chromosome movements.