Ultrafine Anaphase Bridges Research Articles

RIF1 suppresses the formation of single-stranded ultrafine anaphase bridges via protein phosphatase 1.

Resolution of ultrafine anaphase bridges (UFBs) must be completed before cytokinesis to ensure sister-chromatid disjunction. RIF1 is involved in UFB resolution by a mechanism that is not yet clear. Here, we show that RIF1 functions in mitosis to inhibit the formation of 53BP1 nuclear bodies and micronuclei. Meanwhile, RIF1 localizes on PICH-coated double-stranded UFBs but not on RPA-coated single-stranded UFBs. Depletion of RIF1 leads to an elevated level of RPA-coated UFBs, in a BLM-dependent manner. RIF1 interacts with all three isoforms of protein phosphatase 1 (PP1) at its CI domain in anaphase when CDK1 activity declines. CDK1 negatively regulates RIF1-PP1 interaction via the CIII domain of RIF1. Importantly, depletion of PP1 phenocopies RIF1 depletion, and phosphorylation-resistant mutant of PICH shows reduced interaction with the BTR complex and bypasses the need of RIF1 in preventing the formation of single-stranded UFBs. Overall, our data show that PP1 is the effector of RIF1 in UFB resolution.

Cytidine deaminase deficiency in mice enhances genetic instability but limits the number of chemically induced colon tumors

Cytidine deaminase (CDA) catalyzes the deamination of cytidine (C) and deoxycytidine (dC) to uridine and deoxyuridine, respectively. We recently showed that CDA deficiency leads to genomic instability, a hallmark of cancers. We therefore investigated whether constitutive CDA inactivation conferred a predisposition to cancer development. We developed a novel mouse model of Cda deficiency by generating Cda-knockout mice. Cda+/+ and Cda−/− mice did not differ in lifetime phenotypic or behavioral characteristics, or in the frequency or type of spontaneous cancers. However, the frequency of chemically induced tumors in the colon was significantly lower in Cda−/− mice. An analysis of primary kidney cells from Cda−/− mice revealed an excess of C and dC associated with significantly higher frequencies of sister chromatid exchange and ultrafine anaphase bridges and lower Parp-1 activity than in Cda+/+ cells. Our results suggest that, despite inducing genetic instability, an absence of Cda limits the number of chemically induced tumors. These results raise questions about whether a decrease in basal Parp-1 activity can protect against inflammation-driven tumorigenesis; we discuss our findings in light of published data for the Parp-1-deficient mouse model.

Pich-induced nucleosome remodeling studied by dual-trap optical tweezers

In anaphase, entangled DNA between segregating sister chromatids can give rise to DNA threads known as ultrafine anaphase bridges (UFBs). To prevent genome instabilities, UFBs are resolved by a complex protein pathway. It has been proposed that a key step in UFB resolution is the remodeling of nucleosomes by the SNF2 protein PICH. However, direct evidence of nucleosome remodeling by PICH remains elusive. Here, we present a single-molecule assay that recapitulates chromatin re-organization during UFB resolution.

Identification and Analysis of Different Types of UFBs.

Ultrafine anaphase bridges (UFBs) result from a defect in sister chromatid segregation during anaphase. They arise from particular DNA structures, mostly generated at specific loci in the human genome, such as centromeres, common fragile sites, telomeres, or ribosomal DNA. Increases in UFB frequency are a marker of genetic instability, and their detection has become a classic way of detecting such genetic instability over the last decade. Here we describe a protocol to stain different types of UFBs in adherent human cells.

Topoisomerase IIα prevents ultrafine anaphase bridges by two mechanisms.

Topoisomerase IIα (Topo IIα), a well-conserved double-stranded DNA (dsDNA)-specific decatenase, processes dsDNA catenanes resulting from DNA replication during mitosis. Topo IIα defects lead to an accumulation of ultrafine anaphase bridges (UFBs), a type of chromosome non-disjunction. Topo IIα has been reported to resolve DNA anaphase threads, possibly accounting for the increase in UFB frequency upon Topo IIα inhibition. We hypothesized that the excess UFBs might also result, at least in part, from an impairment of the prevention of UFB formation by Topo IIα. We found that Topo IIα inhibition promotes UFB formation without affecting the global disappearance of UFBs during mitosis, but leads to an aberrant UFB resolution generating DNA damage within the next G1. Moreover, we demonstrated that Topo IIα inhibition promotes the formation of two types of UFBs depending on cell cycle phase. Topo IIα inhibition during S-phase compromises complete DNA replication, leading to the formation of UFB-containing unreplicated DNA, whereas Topo IIα inhibition during mitosis impedes DNA decatenation at metaphase–anaphase transition, leading to the formation of UFB-containing DNA catenanes. Thus, Topo IIα activity is essential to prevent UFB formation in a cell-cycle-dependent manner and to promote DNA damage-free resolution of UFBs.

Distinct cellular responses to replication stress leading to apoptosis or senescence.

Replication stress (RS) is a major driver of genomic instability and tumorigenesis. Here, we investigated whether RS induced by the nucleotide analog fludarabine and specific kinase inhibitors [e.g. targeting checkpoint kinase 1 (Chk1) or ataxia telangiectasia and Rad3‐related (ATR)] led to apoptosis or senescence in four cancer cell lines differing in TP53 mutation status and expression of lamin A/C (LA/C). RS resulted in uneven chromatin condensation in all cell types, as evidenced by the presence of metaphasic chromosomes with unrepaired DNA damage, as well as detection of less condensed chromatin in the same nucleus, frequent ultrafine anaphase bridges, and micronuclei. We observed that responses to these chromatin changes may be distinct in individual cell types, suggesting that expression of lamin A/C and lamin B1 (LB1) may play an important role in the transition of damaged cells to senescence. MCF7 mammary carcinoma cells harboring wild‐type p53 (WT‐p53) and LA/C responded to RS by transition to senescence with a significant reduction of lamin B receptor and LB1 proteins. In contrast, a lymphoid cancer cell line WSU‐NHL (WT‐p53) lacking LA/C and expressing low levels of LB1 died after several hours, while lines MEC‐1 and SU‐DHL‐4, both with mutated p53, and SU‐DHL‐4 with mutations in LA/C, died at different rates by apoptosis. Our results show that, in addition to being influenced by p53 mutation status, the response to RS (apoptosis or senescence) may also be influenced by lamin A/C and LB1 status.

The Removal of Histones from Ultrafine Anaphase Bridges Studied by Optical Tweezers

During mitosis, duplicated DNA has to be faithfully segregated into two daughter cells. In anaphase, regions of entangled double-stranded DNA between segregating sister chromatids can give rise to DNA threads known as ultrafine anaphase bridges (UFBs). Failure to resolve UFBs can result in genome instabilities. It has been proposed that a key step in UFB resolution is the removal of histones, and that this is facilitated by the SNF2-family protein PICH. The mechanism of nucleosome remodeling by PICH has, however, remained elusive. Here, we present a single-molecule assay that recapitulates chromatin re-organization during UFB resolution. In vitro chromatin constructs consisting of 4 to 12 high affinity histone binding sites (601 sequences) within an 8.5 kbp DNA molecule were prepared using a combination of restriction-enzyme based cloning and Gibson assembly. After reconstitution with human histones, these constructs were manipulated within a multi-channel microfluidic flow cell using dual-trap optical tweezers. This approach allows us to measure tension-induced nucleosome unwrapping in the presence or absence of PICH. Our results suggest that, under moderate tension (10 pN), PICH facilitates the unwrapping of nucleosomes. In further experiments, we aim to correlate nucleosome stability with the dynamics of single fluorescently-labelled PICH proteins on DNA. Taken together, our single-molecule approach provides new insights into the mechanisms by which PICH is involved in anaphase UFB resolution.

Erratum to: Detection of Ultrafine Anaphase Bridges.

Erratum to: Detection of Ultrafine Anaphase Bridges.

Unresolved recombination intermediates lead to ultra-fine anaphase bridges, chromosome breaks and aberrations.

The resolution of joint molecules that link recombining sister chromatids is essential for chromosome segregation. Here, we determine the fate of unresolved recombination intermediates that arise in GEN1-/- knock-out cells depleted for MUS81, the two nucleases required for resolution. We find that intermediates persist until mitosis where they form a distinct class of anaphase bridges, which we term homologous recombination ultra-fine bridges, or HR-UFBs. The HR-UFBs are distinct from replication stress-associated UFBs, that arise at common fragile sites, and from centromeric UFBs. HR-UFBs are processed by BLM helicase to generate single-stranded RPA-coated bridges that are broken at mitosis. In the next cell cycle, DNA breaks activate the DNA damage checkpoint and chromosome fusions arise by non-homologous end joining. Consequently, the cells undergo a cell cycle delay and massive cell death. These results lead us to present a model detailing how unresolved recombination intermediates can promote DNA damage and chromosomal instability.

CTCF driven TERRA transcription facilitates completion of telomere DNA replication

Telomere repeat DNA forms a nucleo-protein structure that can obstruct chromosomal DNA replication, especially under conditions of replication stress. Transcription of telomere repeats can initiate at subtelomeric CTCF-binding sites to generate telomere repeat-encoding RNA (TERRA), but the role of transcription, CTCF, and TERRA in telomere replication is not known. Here, we have used CRISPR/Cas9 gene editing to mutate CTCF-binding sites at the putative start site of TERRA transcripts for a class of subtelomeres. Under replication stress, telomeres lacking CTCF-driven TERRA exhibit sister-telomere loss and upon entry into mitosis, exhibit the formation of ultra-fine anaphase bridges and micronuclei. Importantly, these phenotypes could be rescued by the forced transcription of TERRA independent of CTCF binding. Our findings indicate that subtelomeric CTCF facilitates telomeric DNA replication by promoting TERRA transcription. Our findings also demonstrate that CTCF-driven TERRA transcription acts in cis to facilitate telomere repeat replication and chromosome stability.

Detection of Ultrafine Anaphase Bridges.

Ultrafine anaphase bridges (UFBs) are thin DNA threads linking the separating sister chromatids in the anaphase of mitosis. UFBs are thought to form when topological DNA entanglements between two chromatids are not resolved prior to anaphase onset. In contrast to other markers of defective chromosome segregation, UFBs cannot be detected by direct staining of the DNA, but instead can be detected using immunofluorescence-based approaches. Due to the fact that they are short-lived and fragile in nature, UFBs can be challenging to detect. In this chapter, we describe methods that have been optimized for successful detection of UFBs. We also provide guidelines for the optimization of UFBs detection depending on the antibody and the cell line to be used.

Genome-Scale Genetic Interactions and Cell Imaging Confirm Cytokinesis as Deleterious to Transient Topoisomerase II Deficiency in Saccharomyces cerevisiae.

Topoisomerase II (Top2) is an essential protein that resolves DNA catenations. When Top2 is inactivated, mitotic catastrophe results from massive entanglement of chromosomes. Top2 is also the target of many first-line anticancer drugs, the so-called Top2 poisons. Often, tumors become resistant to these drugs by acquiring hypomorphic mutations in the genes encoding Top2. Here, we have compared the cell cycle and nuclear segregation of two coisogenic Saccharomyces cerevisiae strains carrying top2 thermosensitive alleles that differ in their resistance to Top2 poisons: the broadly-used poison-sensitive top2-4 and the poison-resistant top2-5. Furthermore, we have performed genome-scale synthetic genetic array (SGA) analyses for both alleles under permissive conditions, chronic sublethal Top2 downregulation, and acute, yet transient, Top2 inactivation. We find that slowing down mitotic progression, especially at the time of execution of the mitotic exit network (MEN), protects against Top2 deficiency. In all conditions, genetic protection was stronger in top2-5; this correlated with cell biology experiments in this mutant, whereby we observed destabilization of both chromatin and ultrafine anaphase bridges by execution of MEN and cytokinesis. Interestingly, whereas transient inactivation of the critical MEN driver Cdc15 partly suppressed top2-5 lethality, this was not the case when earlier steps within anaphase were disrupted; i.e., top2-5 cdc14-1. We discuss the basis of this difference and suggest that accelerated progression through mitosis may be a therapeutic strategy to hypersensitize cancer cells carrying hypomorphic mutations in TOP2.

A role for Tau protein in maintaining ribosomal DNA stability and cytidine deaminase-deficient cell survival

Cells from Bloom’s syndrome patients display genome instability due to a defective BLM and the downregulation of cytidine deaminase. Here, we use a genome-wide RNAi-synthetic lethal screen and transcriptomic profiling to identify genes enabling BLM-deficient and/or cytidine deaminase-deficient cells to tolerate constitutive DNA damage and replication stress. We found a synthetic lethal interaction between cytidine deaminase and microtubule-associated protein Tau deficiencies. Tau is overexpressed in cytidine deaminase-deficient cells, and its depletion worsens genome instability, compromising cell survival. Tau is recruited, along with upstream-binding factor, to ribosomal DNA loci. Tau downregulation decreases upstream binding factor recruitment, ribosomal RNA synthesis, ribonucleotide levels, and affects ribosomal DNA stability, leading to the formation of a new subclass of human ribosomal ultrafine anaphase bridges. We describe here Tau functions in maintaining survival of cytidine deaminase-deficient cells, and ribosomal DNA transcription and stability. Moreover, our findings for cancer tissues presenting concomitant cytidine deaminase underexpression and Tau upregulation open up new possibilities for anti-cancer treatment.

A novel TPR-BEN domain interaction mediates PICH-BEND3 association.

PICH is a DNA translocase required for the maintenance of chromosome stability in human cells. Recent data indicate that PICH co-operates with topoisomerase IIα to suppress pathological chromosome missegregation through promoting the resolution of ultra-fine anaphase bridges (UFBs). Here, we identify the BEN domain-containing protein 3 (BEND3) as an interaction partner of PICH in human cells in mitosis. We have purified full length PICH and BEND3 and shown that they exhibit a functional biochemical interaction in vitro. We demonstrate that the PICH–BEND3 interaction occurs via a novel interface between a TPR domain in PICH and a BEN domain in BEND3, and have determined the crystal structure of this TPR–BEN complex at 2.2 Å resolution. Based on the structure, we identified amino acids important for the TPR–BEN domain interaction, and for the functional interaction of the full-length proteins. Our data reveal a proposed new function for BEND3 in association with PICH, and the first example of a specific protein–protein interaction mediated by a BEN domain.

Unreplicated DNA remaining from unperturbed S phases passes through mitosis for resolution in daughter cells

To prevent rereplication of genomic segments, the eukaryotic cell cycle is divided into two nonoverlapping phases. During late mitosis and G1 replication origins are "licensed" by loading MCM2-7 double hexamers and during S phase licensed replication origins activate to initiate bidirectional replication forks. Replication forks can stall irreversibly, and if two converging forks stall with no intervening licensed origin-a "double fork stall" (DFS)-replication cannot be completed by conventional means. We previously showed how the distribution of replication origins in yeasts promotes complete genome replication even in the presence of irreversible fork stalling. This analysis predicts that DFSs are rare in yeasts but highly likely in large mammalian genomes. Here we show that complementary strand synthesis in early mitosis, ultrafine anaphase bridges, and G1-specific p53-binding protein 1 (53BP1) nuclear bodies provide a mechanism for resolving unreplicated DNA at DFSs in human cells. When origin number was experimentally altered, the number of these structures closely agreed with theoretical predictions of DFSs. The 53BP1 is preferentially bound to larger replicons, where the probability of DFSs is higher. Loss of 53BP1 caused hypersensitivity to licensing inhibition when replication origins were removed. These results provide a striking convergence of experimental and theoretical evidence that unreplicated DNA can pass through mitosis for resolution in the following cell cycle.

Rif1 Regulates the Fate of DNA Entanglements during Mitosis.

SummaryClearance of entangled DNA from the anaphase mid-region must accurately proceed in order for chromosomes to segregate with high fidelity. Loss of Taz1 (fission yeast ortholog of human TRF1/TRF2) leads to stalled telomeric replication forks that trigger telomeric entanglements; the resolution of these entanglements fails at ≤20°C. Here, we investigate these entanglements and their promotion by the conserved replication/repair protein Rif1. Rif1 plays no role in taz1Δ fork stalling. Rather, Rif1 localizes to the anaphase mid-region and regulates the resolution of persisting DNA structures. This anaphase role for Rif1 is genetically separate from the role of Rif1 in S/G2, though both roles require binding to PP1 phosphatase, implying spatially and temporally distinct Rif1-regulated phosphatase substrates. Rif1 thus acts as a double-edged sword. Although it inhibits the resolution of taz1Δ telomere entanglements, it promotes the resolution of non-telomeric ultrafine anaphase bridges at ≤20°C. We suggest a unifying model for Rif1’s seemingly diverse roles in chromosome segregation in eukaryotes.

Early replication stress leads to abnormal mitosis and genome rearrangement

Yeast cell cycle genetics suggests that cells under replication stress generate damage signals that are sufficient to activate checkpoints and restrain mitosis. Yet in mammalian cancer cells, replication stress is not necessarily sufficient to block division. This can lead to abnormal mitotic structures including chromosome bridges, lagging chromosomes, ultrafine anaphase bridges, and micronuclei. This abnormal mitosis is a source for increased mutations including dramatic chromosome rearrangements including chromothripsis, resulting in characteristic genome instability. We have examined the response to different forms of replication stress in the fission yeast, S. pombe, employing visual methods and single‐cell live‐cell analysis.First, we observe distinct patterns of single‐strand binding protein RPA and homologous recombination protein Rad52 depending on the timing and type of stress we induce. This creates a fingerprint of different S phase‐specific stress responses which we can correlate to distinct outcomes.Second, we identify a novel stress‐associated phenotype in a temperature sensitive mutation of the Mcm4 helicase subunit. Despite under‐replicated DNA, the cells fail to arrest during temperature shift, and following release. Biochemical analysis suggests that they evade the checkpoint. Our dynamic pedigrees reveal surprising examples of division under stress including all the mitotic structures described above, including ultrafine anaphase bridges and apparent micronuclei. These abnormal mitoses are associated with genome instability, mutations, and chromosome rearrangement in the surviving cells.Our study shows that a subset of cells within a whole population can continue to divide, despite significant negative effects on genome integrity, and establishes a genetic model to study mechanisms that allow division to occur despite stress.Support or Funding InformationNIH R01 GM081418 &GM111040

Impaired Spindle Assembly Checkpoint In Vivo Promotes MDS/AML in a Novel Mouse Model of Fanconi Anemia

The Fanconi anemia (FA/BRCA) signaling network prevents bone marrow failure and cancer by protecting genomic integrity. Biallelic germline mutations within this gene network result in Fanconi anemia, an inherited bone marrow failure syndrome characterized by genomic instability and a predisposition to bone marrow failure, myelodysplasia and cancer, particularly acute myelogenous leukemia (AML). Heterozygous inborn mutations in the BRCA branch of FA network increase risk of breast and ovarian cancers as well as other tumors, and somatic mutations of FA/BRCA genes occur in malignancies in non-Fanconi patients. Thus, disruption of FA/BRCA signaling promotes malignancies in the inherited genetic syndromes and in the general population.The FA/BRCA network functions as a genome gatekeeper throughout the cell cycle. In interphase, the FA/BRCA network provides a crucial line of defense against mutagenesis by coordinating DNA damage response to a variety of genotoxic insults, from endogenous aldehydes to replication errors and mutagen exposure. Less is known about the role of the FA/BRCA pathway during mitosis. However, FA signaling has recently been implicated in multiple aspects of cell division, including the spindle assembly checkpoint (SAC) that ensures high-fidelity chromosome segregation at metaphase to anaphase transition; cytokinesis; centrosome maintenance and repair of ultrafine anaphase bridges. Although chromosomal instability due to mitotic errors is a hallmark of cancer, the in vivo contribution of abnormal mitosis to malignant transformation of FA-deficient hematopoietic cells remains unknown.To determine whether error-prone chromosome segregation upon loss of FA signaling contributes to abnormal hematopoiesis and cancer, we generated a novel murine FA model by genetically weakening the SAC in the FA-deficient background. The resulting mice were viable and born at expected Mendelian ratios, but exhibited increased baseline in vivo chromosomal instability evidenced by elevated red blood cell micronucleation, increased frequency of chromosome missegregation and DNA breakage in microscopy-based cytome assays, and augmented bone marrow karyotype instability. Importantly, unlike FA or SAC control animals, the FA-SAC mice were prone to premature death due to the development of myelodysplasia and AML at young age, recapitulating disease manifestations of human Fanconi anemia.This study provides the in vivo evidence supporting the essential role of compromised chromosome segregation in the development of myelodysplasia and acute leukemia due to impaired FA signaling. Our observations provide novel insights into complex mechanisms of genomic instability and carcinogenesis due to FA deficiency. Impaired mitosis is a well-established therapeutic target, and our independent ex vivo experiments using FA patient-derived primary cells show that exposure to antimitotic chemotherapeutics is synthetic lethal with loss of the FA network. Thus, our findings may have implications for future precision strategies against FA-deficient, chromosomally unstable hematopoietic cancers. The FA-SAC mouse model offers a preclinical platform to systematically test this hypothesis in vivo. DisclosuresNo relevant conflicts of interest to declare.

Elevated levels of TRF2 induce telomeric ultrafine anaphase bridges and rapid telomere deletions.

The shelterin protein TRF2 is essential for chromosome-end protection. Depletion of TRF2 causes chromosome end-to-end fusions, initiating genomic instability that can be cancer promoting. Paradoxically, significant increased levels of TRF2 are observed in a subset of human cancers. Experimental overexpression of TRF2 has also been shown to induce telomere shortening, through an unknown mechanism. Here we report that TRF2 overexpression results in replication stalling in duplex telomeric repeat tracts and the subsequent formation of telomeric ultrafine anaphase bridges (UFBs), ultimately leading to stochastic loss of telomeric sequences. These TRF2 overexpression-induced telomere deletions generate chromosome fusions resembling those detected in human cancers and in mammalian cells containing critically shortened telomeres. Therefore, our findings have uncovered a second pathway by which altered TRF2 protein levels can induce end-to-end fusions. The observations also provide mechanistic insight into the molecular basis of genomic instability in tumour cells containing significantly increased TRF2 levels.

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.