Unperturbed Cells Research Articles

Molecular dissection of the domain architecture and catalytic activities of human PrimPol

PrimPol is a primase–polymerase involved in nuclear and mitochondrial DNA replication in eukaryotic cells. Although PrimPol is predicted to possess an archaeo-eukaryotic primase and a UL52-like zinc finger domain, the role of these domains has not been established. Here, we report that the proposed zinc finger domain of human PrimPol binds zinc ions and is essential for maintaining primase activity. Although apparently dispensable for its polymerase activity, the zinc finger also regulates the processivity and fidelity of PrimPol's extension activities. When the zinc finger is disrupted, PrimPol becomes more promutagenic, has an altered translesion synthesis spectrum and is capable of faithfully bypassing cyclobutane pyrimidine dimer photolesions. PrimPol's polymerase domain binds to both single- and double-stranded DNA, whilst the zinc finger domain binds only to single-stranded DNA. We additionally report that although PrimPol's primase activity is required to restore wild-type replication fork rates in irradiated PrimPol−/− cells, polymerase activity is sufficient to maintain regular replisome progression in unperturbed cells. Together, these findings provide the first analysis of the molecular architecture of PrimPol, describing the activities associated with, and interplay between, its functional domains and defining the requirement for its primase and polymerase activities during nuclear DNA replication.

Plasticity in the Macromolecular-Scale Causal Networks of Cell Migration

Heterogeneous and dynamic single cell migration behaviours arise from a complex multi-scale signalling network comprising both molecular components and macromolecular modules, among which cell-matrix adhesions and F-actin directly mediate migration. To date, the global wiring architecture characterizing this network remains poorly defined. It is also unclear whether such a wiring pattern may be stable and generalizable to different conditions, or plastic and context dependent. Here, synchronous imaging-based quantification of migration system organization, represented by 87 morphological and dynamic macromolecular module features, and migration system behaviour, i.e., migration speed, facilitated Granger causality analysis. We thereby leveraged natural cellular heterogeneity to begin mapping the directionally specific causal wiring between organizational and behavioural features of the cell migration system. This represents an important advance on commonly used correlative analyses that do not resolve causal directionality. We identified organizational features such as adhesion stability and adhesion F-actin content that, as anticipated, causally influenced cell migration speed. Strikingly, we also found that cell speed can exert causal influence over organizational features, including cell shape and adhesion complex location, thus revealing causality in directions contradictory to previous expectations. Importantly, by comparing unperturbed and signalling-modulated cells, we provide proof-of-principle that causal interaction patterns are in fact plastic and context dependent, rather than stable and generalizable.

The Role of MIZ-1 in MYC-Dependent Tumorigenesis

A hallmark of MYC-transformed cells is their aberrant response to antimitogenic signals. Key examples include the inability of MYC-transformed cells to arrest proliferation in response to antimitogenic signals such as TGF-β or DNA damage and their inability to differentiate into adipocytes in response to hormonal stimuli. Given the plethora of antimitogenic signals to which a tumor cell is exposed, it is likely that the ability to confer resistance to these signals is central to the transforming properties of MYC in vivo. At the same time, the inability of MYC-transformed cells to halt cell-cycle progression on stress may establish a dependence on mutations that impair or disable apoptosis. We propose that the interaction of MYC with the zinc finger protein MIZ-1 mediates resistance to antimitogenic signals. In contrast to other interactions of MYC, there is currently little evidence that MIZ-1 associates with MYC in normal, unperturbed cells. The functional interaction of both proteins becomes apparent at oncogenic expression levels of MYC and association with MIZ-1 mediates both oncogenic functions of MYC as well as tumor-suppressive responses to oncogenic levels of MYC.

Cyclin D1 Maintains Mantle Cell Lymphoma Through CDK4-Independent Regulation Of DNA Replicative Checkpoints

Mantle cell lymphoma (MCL) is rarely curable and therapy resistance often leaves few viable treatment options for patients. Previous studies have identified the importance of cyclin D1 (CCND1) translocation and overexpression in MCL pathogenesis, which leads to increased cyclin-dependent kinase 4 (CDK4) activity and accelerated cell cycle progression. However, targeting this abnormal cell cycle control, mainly through CDK4 inhibition causes only G1-phase growth arrest without significant cell death (Marzec et al. 2006). In contrast, prolonged inhibition of CCND1 with RNA interference induces apoptosis in MCL cell lines (Weinstein et al. 2012), suggesting an essential function of CCND1 independent of CDK4 activity. The mechanism of this non-catalytic role of CCND1 in maintaining MCL cell survival is largely unknown.To clarify the cell cycle role of CCND1 in addition to its CDK4-dependent function, we compared the effects of CCND1 and CDK4 silencing on MCL cell survival. MCL cell lines co-expressing GFP and doxycycline-inducible shRNA targeting CCND1 or CDK4 were generated. Cells with similar GFP expression levels were FACS sorted to normalize for shRNA expression. Both CCND1 and CDK4 silencing resulted in G1-phase arrest, but only CCND1-silenced cells demonstrated a marked increase in apoptosis. Investigation of the potential cause of apoptosis revealed significant accumulation of DNA double-strand breaks following CCND1 ablation, as measured by nuclear gamma-H2AX focus formation. Interestingly, CCND1-silenced cells exhibited a significant increase in 53BP1+ nuclear bodies in G1-phase, reminiscent of 53BP1 foci observed by Lukas and colleagues in cells undergoing aphidicolin-induced replication stress (Lukas et al. 2011). Analysis of replication fork movement in CCND1-depleted cells showed substantially reduced fork speed and increased frequency of origin firing, both of which are indicative of replication stress. In contrast, knockdown of CDK4 did not result in slower forks or increase in the frequency of origin firing. Genomic instability associated with replication stress was also apparent in CCND1-silenced cells, including increased micronucleus formation and recurrent chromatid gaps or breaks detected by cytokinesis-block assay and karyotyping, respectively.Analysis of DNA replicative and damage checkpoints revealed that both ATR-CHEK1 and ATM-CHEK2 pathways were activated by phosphorylation following CCND1 silencing in MCL cell lines, a xenograft animal model, and primary tumor samples, but not in non-MCL tumors. Interestingly, this activation (with the exception of ATM phosphorylation) was unsustainable over time and did not cause down-regulation of the downstream targets CDC25 and CDK1/2 but, instead, we observed an increase in CDC25A/B protein levels and CDK1/2 activity, indicating defective cell cycle checkpoints. Exposing CCND1-silenced cells to replication stress-inducing or DNA-damaging agents such hydroxyurea, aphidicolin, etoposide or ionizing radiation further amplified the checkpoint defects seen in unperturbed cells. We did not observe any significant difference in this checkpoint signaling in control and CDK4 knockdown cells under these conditions. Furthermore, CCND1-deficient cells were more sensitive to pharmacological inhibition of ATR and CHEK1 but not ATM, confirming a constitutive role of CCND1 in the ATR-CHEK1 pathway.In conclusion, these studies revealed an unexpected CDK4-independent role of CCND1 in maintaining DNA replicative checkpoints to prevent replication stress and genome instability in MCL cells. As most cancer treatments rely on agents that create DNA replication stress, targeting this function of CCND1 could provide a rational approach to overcome resistance to conventional therapies in MCL. Disclosures:No relevant conflicts of interest to declare.

Effective siRNA delivery to inflamed primary vascular endothelial cells by anti-E-selectin and anti-VCAM-1 PEGylated SAINT-based lipoplexes

The endothelium represents an attractive therapeutic target due to its pivotal role in many diseases including chronic inflammation and cancer. Small interfering RNAs (siRNAs) specifically interfere with the expression of target genes and are considered an important new class of therapeutics. However, due to their size and charge, siRNAs do not spontaneously enter unperturbed endothelial cells (EC). To overcome this problem, we developed novel lipoplexes for siRNA delivery that are based on the cationic amphiphilic lipid SAINT-C18. Antibodies recognizing disease induced cell adhesion molecules were employed to create cell specificity resulting in so-called antibody-SAINTargs. To improve particle stability, antibody-SAINTargs were further optimized for EC-specific siRNA-mediated gene silencing by addition of polyethylene glycol (PEG). Although PEGylated antibody-SAINTargs maintained specificity, they lost their siRNA delivery capacity. Coupling of antibodies to the distal end of PEG (so-called antibody-SAINTPEGargs), resulted in anti-E-selectin- and anti-vascular cell adhesion molecule (VCAM)-1-SAINTPEGarg that preserved their antigen recognition and their capability to specifically deliver siRNA into inflammation-activated primary endothelial cells. The enhanced uptake of siRNA by antibody-SAINTPEGargs was followed by improved silencing of the target gene VE-cadherin, demonstrating that antibody-SAINTPEGargs were capable of functionally delivering siRNA into primary endothelial cells originating from different vascular beds. In conclusion, the newly developed, physicochemically stable, and EC-specific siRNA carrying antibody-SAINTPEGargs selectively down-regulate target genes in primary endothelial cells that are generally difficult to transfect.

Preclinical Evaluation of the WEE1 Inhibitor MK-1775 as Single-Agent Anticancer Therapy

Inhibition of the DNA damage checkpoint kinase WEE1 potentiates genotoxic chemotherapies by abrogating cell-cycle arrest and proper DNA repair. However, WEE1 is also essential for unperturbed cell division in the absence of extrinsic insult. Here, we investigate the anticancer potential of a WEE1 inhibitor, independent of chemotherapy, and explore a possible cellular context underlying sensitivity to WEE1 inhibition. We show that MK-1775, a potent and selective ATP-competitive inhibitor of WEE1, is cytotoxic across a broad panel of tumor cell lines and induces DNA double-strand breaks. MK-1775-induced DNA damage occurs without added chemotherapy or radiation in S-phase cells and relies on active DNA replication. At tolerated doses, MK-1775 treatment leads to xenograft tumor growth inhibition or regression. To begin addressing potential response markers for MK-1775 monotherapy, we focused on PKMYT1, a kinase functionally related to WEE1. Knockdown of PKMYT1 lowers the EC(50) of MK-1775 by five-fold but has no effect on the cell-based response to other cytotoxic drugs. In addition, knockdown of PKMYT1 increases markers of DNA damage, γH2AX and pCHK1(S345), induced by MK-1775. In a post hoc analysis of 305 cell lines treated with MK-1775, we found that expression of PKMYT1 was below average in 73% of the 33 most sensitive cell lines. Our findings provide rationale for WEE1 inhibition as a potent anticancer therapy independent of a genotoxic partner and suggest that low PKMYT1 expression could serve as an enrichment biomarker for MK-1775 sensitivity.

Molecular Basis of the Essential S Phase Function of the Rad53 Checkpoint Kinase

The essential yeast kinases Mec1 and Rad53, or human ATR and Chk1, are crucial for checkpoint responses to exogenous genotoxic agents, but why they are also required for DNA replication in unperturbed cells remains poorly understood. Here we report that even in the absence of DNA-damaging agents, the rad53-4AQ mutant, lacking the N-terminal Mec1 phosphorylation site cluster, is synthetic lethal with a deletion of the RAD9 DNA damage checkpoint adaptor. This phenotype is caused by an inability of rad53-4AQ to activate the downstream kinase Dun1, which then leads to reduced basal deoxynucleoside triphosphate (dNTP) levels, spontaneous replication fork stalling, and constitutive activation of and dependence on S phase DNA damage checkpoints. Surprisingly, the kinase-deficient rad53-K227A mutant does not share these phenotypes but is rendered inviable by additional phosphosite mutations that prevent its binding to Dun1. The results demonstrate that ultralow Rad53 catalytic activity is sufficient for normal replication of undamaged chromosomes as long as it is targeted toward activation of the effector kinase Dun1. Our findings indicate that the essential S phase function of Rad53 is comprised by the combination of its role in regulating basal dNTP levels and its compensatory kinase function if dNTP levels are perturbed.

A lifeline for lipid rafts?

Study finds raft-like domains in yeast vacuole membrane.

Long-range heterochromatin association is mediated by silencing and double-strand DNA break repair proteins

The eukaryotic genome is highly organized in the nucleus, and this organization affects various nuclear processes. However, the molecular details of higher-order organization of chromatin remain obscure. In the present study, we show that the Saccharomyces cerevisiae silenced loci HML and HMR cluster in three-dimensional space throughout the cell cycle and independently of the telomeres. Long-range HML-HMR interactions require the homologous recombination (HR) repair pathway and phosphorylated H2A (γ-H2A). γ-H2A is constitutively present at silenced loci in unperturbed cells, its localization requires heterochromatin, and it is restricted to the silenced domain by the transfer DNA boundary element. SMC proteins and Scc2 localize to the silenced domain, and Scc2 binding requires the presence of γ-H2A. These findings illustrate a novel pathway for heterochromatin organization and suggest a role for HR repair proteins in genomic organization.

4-Hydroxy-2-Nonenal Enhances Tissue Factor Activity in Human Monocytic Cells via p38 Mitogen-Activated Protein Kinase Activation-Dependent Phosphatidylserine Exposure

4-hydroxy-2-nonenal (HNE) is one of the major aldehydes formed during lipid peroxidation and is believed to play a role in the pathogenesis of atherosclerosis. The objective of the present study is to investigate the effect of HNE on tissue factor (TF) procoagulant activity expressed on cell surfaces. TF activity and antigen levels on intact cells were measured using factor Xa generation and TF monoclonal antibody binding assays, respectively. Exposure of phosphatidylserine on the cell surface was analyzed using thrombin generation assay or by binding of a fluorescent dye-conjugated annexin V. 2',7'-dichlorodihydrofluorescein diacetate was used to detect the generation of reactive oxygen species. Our data showed that HNE increased the procoagulant activity of unperturbed THP-1 cells that express traces of TF antigen, but had no effect on unperturbed endothelial cells that express no measurable TF antigen. HNE increased TF procoagulant activity but not TF antigen of both activated monocytic and endothelial cells. HNE treatment generated reactive oxygen species, activated p38 mitogen-activated protein kinase, and increased the exposure of phosphatidylserine at the outer leaflet in THP-1 cells. Treatment of THP-1 cells with an antioxidant, N-acetyl cysteine, suppressed the above HNE-induced responses and negated the HNE-mediated increase in TF activity. Blockade of p38 mitogen-activated protein kinase activation inhibited HNE-induced phosphatidylserine exposure and increased TF activity. HNE increases TF coagulant activity in monocytic cells through a novel mechanism involving p38 mitogen-activated protein kinase activation that leads to enhanced phosphatidylserine exposure at the cell surface.

Myosin 16 levels fluctuate during the cell cycle and are downregulated in response to DNA replication stress

Myosins comprise a highly conserved superfamily of eukaryotic actin-dependent motor proteins implicated in a large repertoire of functions in both the cytoplasm and the nucleus. Class XVI myosin, MYO16, reveals expression in most somatic as well as meiotic cells with prominent localization in the nucleus, excepting the nucleolus; however, the role(s) of Myo16 in the nucleus remain unknown. In this report, we investigated Myo16 abundance during transit through the cell cycle. Immunolocalization, immunoblot, flow cytometric and quantitative RT-PCR studies performed in Rat2 cells indicate that Myo16 mRNA and protein abundance are cell cycle regulated: in the unperturbed cell cycle, each rises to peak levels in late G1 and thereon through S-phase and each decays as cells enter M-phase. Notably, RNA interference-induced Myo16 depletion results in altered cell cycle distribution as well as in large-scale cell death. In response to DNA replication stress (impaired replication fork progression as a consequence of DNA damage, lack of sufficient deoxynucleotides, or inhibition of DNA polymerases), Myo16 protein shows substantial loss. Attenuation of replication stress (aphidicolin or hydroxyurea) is followed by a recovery of Myo16 expression and resumption of S-phase progression. Collectively, these observations suggest that Myo16 may play a regulatory role in cell cycle progression.

Replication stress induces specific enrichment of RECQ1 at common fragile sites FRA3B and FRA16D

BackgroundStalled replication forks at common fragile sites are a major cause of genomic instability. RecQ helicases, a highly conserved family of DNA-unwinding enzymes, are believed to ease ‘roadblocks’ that pose challenge to replication fork progression. Among the five known RecQ homologs in humans, functions of RECQ1, the most abundant of all, are poorly understood. We previously determined that RECQ1 helicase preferentially binds and unwinds substrates that mimic DNA replication/repair intermediates, and interacts with proteins involved in DNA replication restart mechanisms.MethodWe have utilized chromatin immunoprecipitation followed by quantitative real-time PCR to investigate chromatin interactions of RECQ1 at defined genetic loci in the presence or absence of replication stress. We have also tested the sensitivity of RECQ1-depleted cells to aphidicolin induced replication stress.ResultsRECQ1 binds to the origins of replication in unperturbed cells. We now show that conditions of replication stress induce increased accumulation of RECQ1 at the lamin B2 origin in HeLa cells. Consistent with a role in promoting fork recovery or repair, RECQ1 is specifically enriched at two major fragile sites FRA3B and FRA16D where replication forks have stalled following aphidicolin treatment. RECQ1-depletion results in attenuated checkpoint activation in response to replication stress, increased sensitivity to aphidicolin and chromosomal instability.ConclusionsGiven a recent biochemical observation that RECQ1 catalyzes strand exchange on stalled replication fork structures in vitro, our results indicate that RECQ1 facilitates repair of stalled or collapsed replication forks and preserves genome integrity. Our findings provide the first evidence of a crucial role for RECQ1 at naturally occurring fork stalling sites and implicate RECQ1 in mechanisms underlying common fragile site instability in cancer.

Abstract 5236: Novel network analysis framework identifies context-specific drug combinations in melanoma.

Abstract Gene expression data can be used to identify the activation status of genes and pathways in tumor samples and to infer the structure of regulatory networks. Most tumor datasets such as The Cancer Genome Atlas focus on unperturbed samples, and subsequently, the statistical frameworks that analyze them. We hypothesized that perturbation of cells can uncover network structures that are undetectable in basal state, and reveal the complexity of cancer cells. To demonstrate this, we generated gene expression data from a panel of melanoma cell lines before and after inhibition of MEK or AKT. Here we present a new computational framework to analyze gene expression data before and after perturbations. The algorithm aims to define “differential networks1” - rewiring of network components in different tumors. The algorithm sheds light on the complex and context-specific nature of network structure, including pathways that are activated, or under the control of MAPK or AKT, only in a subset of samples. The perturbation data allowed us to assess pathway activation levels that are undetectable in unperturbed state. For example, AKT is activated in melanoma by PTEN loss, but the effect of NRAS mutation on AKT is unknown, and AKT activation is undetectable in unperturbed cells. Our algorithm identified an AKT activation signature in perturbed state, and helped define the effects of PTEN and NRAS mutations on the pathway. Additionally, the algorithm inferred the genetic status of p53 in each cell line by identifying pathway activation, changes that are, once again, detectable only after perturbations. By linking protein data with the algorithm's results, we identified cell lines with constitutive activation of the STAT3 pathway, and demonstrated the crosstalk between MAPK and STAT3 pathways. Moreover, combining the results from the algorithm with known signatures of drug-sensitivity accurately predicted which drug combinations provided a synergistic effect in each cell line. Associations between pathway activity level and drug sensitivity are known for some drugs. By using our algorithm's ability to infer pathway status in different tumors and to detect changes in those pathways after drug treatment, it correctly predicted when drug treatment sensitized cells to the effects of chemotherapy and other drugs. To conclude, we show that gene expression of perturbed cells greatly enhances the power of network structure analysis. Our results reveal context-specific changes in network structure, and predict which cell lines are sensitized to specific drugs by pathway perturbations. These results have direct applicability to the development of drug combinations in melanoma. 1 Pe'er and Hacohen, Cell 2011 Citation Format: Oren Litvin, Sarit Schwartz, Mark Rocco, Tanya Schild, Neal Rosen, Dana Pe'er. Novel network analysis framework identifies context-specific drug combinations in melanoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5236. doi:10.1158/1538-7445.AM2013-5236 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Plant WEE1 kinase is cell cycle regulated and removed at mitosis via the 26S proteasome machinery

In yeasts and animals, premature entry into mitosis is prevented by the inhibitory phosphorylation of cyclin-dependent kinase (CDK) by WEE1 kinase, and, at mitosis, WEE1 protein is removed through the action of the 26S proteasome. Although in higher plants WEE1 function has been confirmed in the DNA replication checkpoint, Arabidopsis wee1 insertion mutants grow normally, and a role for the protein in the G2/M transition during an unperturbed plant cell cycle is yet to be confirmed. Here data are presented showing that the inhibitory effect of WEE1 on CDK activity in tobacco BY-2 cell cultures is cell cycle regulated independently of the DNA replication checkpoint: it is high during S-phase but drops as cells traverse G2 and enter mitosis. To investigate this mechanism further, a yeast two-hybrid screen was undertaken to identify proteins interacting with Arabidopsis WEE1. Three F-box proteins and a subunit of the proteasome complex were identified, and bimolecular fluorescence complementation confirmed an interaction between AtWEE1 and the F-box protein SKP1 INTERACTING PARTNER 1 (SKIP1). Furthermore, the AtWEE1–green fluorescent protein (GFP) signal in Arabidopsis primary roots treated with the proteasome inhibitor MG132 was significantly increased compared with mock-treated controls. Expression of AtWEE1–YFPC (C-terminal portion of yellow fluorescent protein) or AtWEE1 per se in tobacco BY-2 cells resulted in a premature increase in the mitotic index compared with controls, whereas co-expression of AtSKIP1–YFPN negated this effect. These data support a role for WEE1 in a normal plant cell cycle and its removal at mitosis via the 26S proteasome.

Genetic profiles of plasmacytoid (BDCA-4 expressing) DC subtypes-clues to DC subtype function in vivo

Among the dendritic cell (DC) subsets, plasmacytoid DC’s (pDC) are thought to be important in the generation of both antiviral and antitumor responses. While pDC may be useful in developing dendritic cell-based tumor vaccines, the low frequency of these cells in the peripheral blood has hampered attempts to understand their biology. To provide better insight into the biology of pDC, we isolated these unperturbed cells from the peripheral blood of healthy donors in order to further characterize their gene expression. Using gene array technology we compared the genetic profiles of these cells to those of CD14+ monocytes isolated from the same donors and found several immune related genes upregulated in this cell population. This is the first description, to our knowledge, of gene expression in this subset of DCs obtained from the peripheral blood of adult human donors without exposure in vitro to cytokine or growth factors. Understanding the natural genetic profiles of this dendritic cell subtype as well as others such as the BDCA-1 expressing myeloid DCs may enable us to manipulate these cells ex-vivo to generate enhanced DC-based tumor vaccines inducing more robust antitumor responses.

Merging high-quality biochemical fractionation with a refined flow cytometry approach to monitor nucleocytoplasmic protein expression throughout the unperturbed mammalian cell cycle

This protocol describes a method for nucleocytoplasmic protein tracking during normal cell cycle progression using unmanipulated, asynchronous cells. In contrast with prevalent traditional methods, our approach does not require time-consuming, perturbing cell synchronization or separation. To this end, we chose a single-cell approach and developed a flow cytometry assay that is applied to whole cells and isolated nuclei. Our protocol involves a stepwise biochemical fractionation procedure to purify nuclei from whole cells, conventional DNA and indirect immunostaining techniques for the dual labeling of cells and nuclei for DNA and protein, and a refined concept of flow cytometric data processing and calculation: through the specific combination of DNA and cell size analyses, G1, S and G2/M phases of the cell cycle are further dissected to establish a high-resolution map of cell cycle progression, to which protein expression in cells or nuclei is correlated. In a final data analysis step, cell cycle-related, cytoplasmic protein expression is calculated on the basis of results obtained for whole cells and isolated nuclei. A minimum of 8 h is required to complete the procedure. As the approach does not require cell type-restricting pretreatments, numerous cell types of different origin can be readily studied. Human amniotic fluid stem cells, primary human fibroblasts, immortalized mouse fibroblasts and transformed tumor cells are analyzed at comparable efficiencies, demonstrating low intercell assay variability.

Spontaneous telomere to telomere fusions occur in unperturbed fission yeast cells

Telomeres protect eukaryotic chromosomes from illegitimate end-to-end fusions. When this function fails, dicentric chromosomes are formed, triggering breakage-fusion-bridge cycles and genome instability. How efficient is this protection mechanism in normal cells is not fully understood. We created a positive selection assay aimed at capturing chromosome-end fusions in Schizosaccharomyces pombe. We placed telomere sequences with a head to head arrangement in an intron of a selectable marker contained on a plasmid. By linearizing the plasmid between the telomere sequences, we generated a stable mini-chromosome that fails to express the reporter gene. Whenever the ends of the mini-chromosome join, the marker gene is reconstituted and fusions are captured by direct selection. Using telomerase mutants, we recovered several fusion events that lacked telomere sequences. The end-joining reaction involved specific homologous subtelomeric sequences capable of forming hairpins, suggestive of ssDNA stabilization prior to fusing. These events occurred via microhomology-mediated end-joining (MMEJ)/single-strand annealing (SSA) repair and also required MRN/Ctp1. Strikingly, we were able to capture spontaneous telomere-to-telomere fusions in unperturbed cells. Similar to disruption of the telomere regulator Taz1/TRF2, end-joining reactions occurred via non-homologous end-joining (NHEJ) repair. Thus, telomeres undergo fusions prior to becoming critically short, possibly through transient deprotection. These dysfunction events induce chromosome instability and may underlie early tumourigenesis.

A Cell Cycle-Dependent Regulatory Circuit Composed of 53BP1-RIF1 and BRCA1-CtIP Controls DNA Repair Pathway Choice

DNA double-strand break (DSB) repair pathway choice is governed by the opposing activities of 53BP1 and BRCA1. 53BP1 stimulates nonhomologous end joining (NHEJ), whereas BRCA1 promotes end resection and homologous recombination (HR). Here we show that 53BP1 is an inhibitor of BRCA1 accumulation at DSB sites, specifically in the G1 phase of the cell cycle. ATM-dependent phosphorylation of 53BP1 physically recruits RIF1 to DSB sites, and we identify RIF1 as the critical effector of 53BP1 during DSB repair. Remarkably, RIF1 accumulation at DSB sites is strongly antagonized by BRCA1 and its interacting partner CtIP. Lastly, we show that depletion of RIF1 is able to restore end resection and RAD51 loading in BRCA1-depleted cells. This work therefore identifies a cell cycle-regulated circuit, underpinned by RIF1 and BRCA1, that governs DSB repair pathway choice to ensure that NHEJ dominates in G1 and HR is favored from S phase onward.

Timeless-Dependent DNA Replication-Coupled Recombination Promotes Kaposi's Sarcoma-Associated Herpesvirus Episome Maintenance and Terminal Repeat Stability

Kaposi's Sarcoma-associated herpesvirus (KSHV) is maintained as a stable episome in latently infected pleural effusion lymphoma (PEL) cells. Episome maintenance is conferred by the binding of the KSHV-encoded LANA protein to the viral terminal repeats (TR). Here, we show that DNA replication in the KSHV TR is coupled with DNA recombination and mediated in part through the cellular replication fork protection factors Timeless (Tim) and Tipin. We show by two-dimensional (2D) agarose gel electrophoresis that replication forks naturally stall and form recombination-like structures at the TR during an unperturbed cell cycle. Chromatin immunoprecipitation (ChIP) assays revealed that Tim and Tipin are selectively enriched at the KSHV TR during S phase and in a LANA-dependent manner. Tim depletion inhibited LANA-dependent TR DNA replication and caused the loss of KSHV episomes from latently infected PEL cells. Tim depletion resulted in the aberrant accumulation of recombination structures and arrested MCM helicase at TR. Tim depletion did not induce the KSHV lytic cycle or apoptotic cell death. We propose that KSHV episome maintenance requires Tim-assisted replication fork protection at the viral terminal repeats and that Tim-dependent recombination-like structures form at TR to promote DNA repeat stability and viral genome maintenance.

Illuminating Passive Permeability Barrier of Primary Cilia using Novel Diffusion Trap Technique

Primary cilia function as specialized compartments for signal transduction in many tissues. The stereotyped structure and signaling function of cilia are inextricably dependent on the selective segregation of molecules in cilia, demonstrated by the finding that damage to ciliary protein transport mechanisms lead to a family of human genetic diseases known as ciliopathies. Despite its importance, the fundamental principles governing the access of soluble proteins to primary cilia remain unresolved, primarily because it has not been possible to monitor the kinetics of protein movement into these organelles in unperturbed live cells. We have developed a methodology termed Chemically-Inducible Diffusion Trap (C-IDT) based on chemically-inducible protein dimerization and live-cell microscopy to visualize the diffusion process of a series of fluorescent proteins ranging in size from 32 to 79 A into primary cilia. Contrary to previous reports equating ciliary import to nuclear import, we did not find evidence for a fixed diffusion barrier that excludes proteins above a particular size_ the interior of the cilium was accessible to proteins as large as 79 A (∼650 kDa). However, the kinetics of ciliary accumulation of this panel of proteins was exponentially limited by their Stokes radii. Quantitative modeling from our kinetic data suggests that the diffusion barrier operates as a molecular sieve. In addition to elucidating key physical properties, our study presents a set of powerful, generally applicable tools for the quantitative monitoring of ciliary protein diffusion under both physiological and pathological conditions. (Currently under review at Nature Cell Biology)