Replication Profiles Research Articles

Single-cell replication profiling to measure stochastic variation in mammalian replication timing

Mammalian DNA replication is regulated via multi-replicon segments that replicate in a defined temporal order during S-phase. Further, early/late replication of RDs corresponds to active/inactive chromatin interaction compartments. Although replication origins are selected stochastically, variation in replication timing is poorly understood. Here we devise a strategy to measure variation in replication timing using DNA copy number in single mouse embryonic stem cells. We find that borders between replicated and unreplicated DNA are highly conserved between cells, demarcating active and inactive compartments of the nucleus. Fifty percent of replication events deviated from their average replication time by ± 15% of S phase. This degree of variation is similar between cells, between homologs within cells and between all domains genomewide, regardless of their replication timing. These results demonstrate that stochastic variation in replication timing is independent of elements that dictate timing or extrinsic environmental variation.

Sleeping Beauty Transposon-based System forRapid Generation of HBV-replicating Stable Cell Lines.

The stable HBV-transfected cell lines, which based on stable integration of replication-competent HBV genome into hepatic cells, are widely used in basic research and antiviral drug evaluation against HBV. However, previous reported strategies to generate HBV-replicating cell lines, which primarily rely on random integration of exogenous DNA by plasmid transfection, are inefficient and time-consuming. We newly developed an all-in-one Sleeping Beauty transposon system (denoted pTSMP-HBV vector) for robust generation of stable HBV-replicating cell lines of different genotype. The pTSMP-HBV vector contains HBV 1.3-copy genome and dual selection markers (mCherry and puromycin resistance gene), allowing rapid enrichment of stably-transfected cells via red fluorescence-activated cell sorting and puromycin antibiotic selection. In this protocol, we described the detailed procedure for constructing the HBV-replicating stable cells and systematically evaluating HBV replication and viral protein expression profiles of these cells.

Factors influencing microinjection molding replication quality

In recent years, there has been increased interest in producing and providing high-precision plastic parts that can be manufactured by microinjection molding: gears, pumps, optical grating elements, and so on. For all of these applications, the replication quality is essential. This study has two goals: (1) fabrication of high-precision parts using the conventional injection molding machine; (2) identification of robust parameters that ensure production quality. Thus, different technological solutions have been used: cavity vacuuming and the use of a mold coated with DLC or CrN deposits. AFM and SEM analyses were carried out to characterize the replication profile. The replication quality was studied in terms of the process parameters, coated and uncoated molds and crystallinity of the polymer. Specific studies were processed to quantify the replicability of injection molded parts (ABS, PC and PP). Analysis of the Taguchi experimental designs permits prioritization of the impact of each parameter on the replication quality. A discussion taking into account these new parameters and the thermal and spreading properties on the coatings is proposed. It appeared that, in general, increasing the mold temperature improves the molten polymer fill in submicron features except for the steel insert (for which the presence of a vacuum is the most important factor). Moreover, the DLC coating was the best coating to increase the quality of the replication. This result could be explained by the lower thermal diffusivity of this coating. We noted that the viscosity of the polymers is not a primordial factor of the replication quality.

Dbf4 recruitment by forkhead transcription factors defines an upstream rate-limiting step in determining origin firing timing.

Initiation of eukaryotic chromosome replication follows a spatiotemporal program. The current model suggests that replication origins compete for a limited pool of initiation factors. However, it remains to be answered how these limiting factors are preferentially recruited to early origins. Here, we report that Dbf4 is enriched at early origins through its interaction with forkhead transcription factors Fkh1 and Fkh2. This interaction is mediated by the Dbf4 C terminus and was successfully reconstituted in vitro. An interaction-defective mutant, dbf4ΔC, phenocopies fkh alleles in terms of origin firing. Remarkably, genome-wide replication profiles reveal that the direct fusion of the DNA-binding domain (DBD) of Fkh1 to Dbf4 restores the Fkh-dependent origin firing but interferes specifically with the pericentromeric origin activation. Furthermore, Dbf4 interacts directly with Sld3 and promotes the recruitment of downstream limiting factors. These data suggest that Fkh1 targets Dbf4 to a subset of noncentromeric origins to promote early replication in a manner that is reminiscent of the recruitment of Dbf4 to pericentromeric origins by Ctf19.

Suppression of Oncolytic Adenovirus-Mediated Hepatotoxicity by Liver-Specific Inhibition of NF-κB

Telomerase-specific replication-competent adenoviruses (Ads), i.e., TRADs, which possess an E1 gene expression cassette driven by the human telomerase reverse transcriptase promoter, are promising agents for cancer treatment. However, even though oncolytic Ads, including TRAD, are intratumorally administered, they are disseminated from the tumor to systemic circulation, causing concern about oncolytic Ad-mediated hepatotoxicity (due mainly to leaky expression of Ad genes in liver). We reported that inhibition of nuclear factor-κB (NF-κB) leads to the suppression of replication-incompetent Ad vector-mediated hepatotoxicity via reduction of the leaky expression of Ad genes in liver. Here, to develop a TRAD with an improved safety profile, we designed a TRAD that carries a liver-specific promoter-driven dominant-negative IκBα (DNIκBα) expression cassette (TRAD-DNIκBα). Compared with a conventional TRAD, TRAD-DNIκBα showed hepatocyte-specific inhibition of NF-κB signaling and significantly reduced Ad gene expression and replication in the normal human hepatocyte cell line. TRAD-induced hepatotoxicity was largely suppressed in mice following intravenous administration of TRAD-DNIκBα. However, the replication profiles and oncolytic activities of TRAD-DNIκBα were comparable with those of the conventional TRAD in human non-hepatic tumor cells. These results indicate that oncolytic Ads containing the liver-specific DNIκBα expression cassette have improved safety profiles without inhibiting oncolytic activities.

DNA Replication Profiling Using Deep Sequencing.

Profiling of DNA replication during progression through S phase allows a quantitative snap-shot of replication origin usage and DNA replication fork progression. We present a method for using deep sequencing data to profile DNA replication in S. cerevisiae.

ERP arrangement: a strategy for assembled genomic contig rearrangement based on replication profiling in bacteria

BackgroundThe reduced cost of sequencing has made de novo sequencing and the assembly of draft microbial genomes feasible in any ordinary biology lab. However, the process of finishing and completing the genome remains labor-intensive and computationally challenging in some cases, such as in the study of complete genome sequences, genomic rearrangements, long-range syntenic relationships, and structural variations.MethodsHere, we show a contig reordering strategy based on experimental replication profiling (eRP) to recapitulate the bacterial genome structure within draft genomes. During the exponential growth phase, the majority of bacteria show a global genomic copy number gradient that is enriched near the replication origin and gradually declines toward the terminus. Therefore, if genome sequencing is performed with appropriate timing, the short-read coverage reflects this copy number gradient, providing information about the contig positions relative to the replication origin and terminus.ResultsWe therefore investigated the appropriate timing for genomic DNA sampling and developed an algorithm for the reordering of the contigs based on eRP. As a result, this strategy successfully recapitulates the genomic structure of various structural mutants with draft genome sequencing.ConclusionsOur strategy was successful for contig rearrangement with intracellular DNA replication behavior mechanisms and can be applied to almost all bacteria because the DNA replication system is highly conserved. Therefore, eRP makes it possible to understand genomic structural information and long-range syntenic relationships using a draft genome that is based on short reads.

Pseudomonas aeruginosa Proteome under Hypoxic Stress Conditions Mimicking the Cystic Fibrosis Lung.

Pseudomonas aeruginosa is a ubiquitous Gram-negative pathogen known to inhabit hypoxic mucus plugs of cystic fibrosis (CF) patient lungs. Despite the high prevalence and related patient mortality, the protein machinery enabling the bacterium to adapt to low oxygen environment remains to be fully elucidated. We investigated this by performing both SWATH mass spectrometry and data-dependent SPS-MS3 of TMT-labeled peptides to profile the proteomes of two P. aeruginosa CF isolates, PASS2 and PASS3, and a laboratory reference strain, PAO1, grown under hypoxic stress (O2 < 1%) in media that mimic the nutrient components of the CF lung. Quantitated across all three strains were 3967 P. aeruginosa proteins, reflecting approximately 71% of predicted ORFs in PAO1 and representing the most comprehensive proteome of clinically relevant P. aeruginosa to date. Comparative analysis revealed 735, 640, and 364 proteins were altered by 2-fold or more when comparing low oxygen to aerobic growth in PAO1, PASS2, and PASS3, respectively. Strikingly, under hypoxic stress, all strains showed concurrent increased abundance of proteins required for both aerobic (cbb3-1 and cbb3-2 terminal oxidases) and anaerobic denitrification and arginine fermentation, with the two clinical isolates showing higher relative expression of proteins in these pathways. Additionally, functional annotation revealed that clinical strains portray a unique expression profile of replication, membrane biogenesis, and virulence proteins during hypoxia which may endow these bacteria with a survival advantage. These protein profiles illuminate the diversity of P. aeruginosa mechanisms to adapt to low oxygen and shows that CF isolates initiate a robust molecular response to persist under these conditions.

Analysis of DNA Replication in Fission Yeast by Combing.

DNA replication studies based on population experiments give an average estimate of replication kinetics from many cells. This average replication profile masks the stochastic nature of origin firing in eukaryotes, which is revealed by using single-molecule techniques, such as DNA combing. The analysis of replication kinetics by DNA combing involves isolating DNA from cells that have been pulse-labeled with thymidine analogs and stretching it on a silanized coverslip. The analog-labeled patches on the stretched DNA fibers can then be detected using fluorescent antibodies against the analog. Each fiber represents a part of the genome from a single cell; therefore, it is possible to study the variation in behavior of individual origins from one cell to another. Furthermore, each DNA fiber is uniformly stretched, making it possible to measure distances accurately at kilobase resolution. It is also possible to stretch a high density of fibers on coverslips enabling quantitative data collection.

Latent Profile Analysis of the Five Facet Mindfulness Questionnaire in a Sample With a History of Recurrent Depression.

Extending previous research, we applied latent profile analysis in a sample of adults with a history of recurrent depression to identify subgroups with distinct response profiles on the Five Facet Mindfulness Questionnaire and understand how these relate to psychological functioning. The sample was randomly divided into two subsamples to first examine the optimal number of latent profiles (test sample; n = 343) and then validate the identified solution (validation sample; n = 340). In both test and validation samples, a four-profile solution was revealed where two profiles mapped broadly onto those previously identified in nonclinical samples: “high mindfulness” and “nonjudgmentally aware.” Two additional subgroups, “moderate mindfulness” and “very low mindfulness,” were observed. “High mindfulness” was associated with the most adaptive psychological functioning and “very low mindfulness” with the least adaptive. In most people with recurrent depression, mindfulness skills are expressed evenly across different domains. However, in a small minority a meaningful and replicable uneven profile indicating nonjudgmental awareness is observable. Current findings require replication and future research should examine the extent to which profiles change from periods of wellness to illness in people with recurrent depression and how profiles are influenced by exposure to mindfulness-based intervention.

Deep functional analysis of synII, a 770-kilobase synthetic yeast chromosome

Here, we report the successful design, construction, and characterization of a 770-kilobase synthetic yeast chromosome II (synII). Our study incorporates characterization at multiple levels-including phenomics, transcriptomics, proteomics, chromosome segregation, and replication analysis-to provide a thorough and comprehensive analysis of a synthetic chromosome. Our Trans-Omics analyses reveal a modest but potentially relevant pervasive up-regulation of translational machinery observed in synII, mainly caused by the deletion of 13 transfer RNAs. By both complementation assays and SCRaMbLE (synthetic chromosome rearrangement and modification by loxP-mediated evolution), we targeted and debugged the origin of a growth defect at 37°C in glycerol medium, which is related to misregulation of the high-osmolarity glycerol response. Despite the subtle differences, the synII strain shows highly consistent biological processes comparable to the native strain.

Alteration of Cytokine Profiles Inhibits Efficacy of Silver Nanoparticle-based Neutralization of arenaviruses

Background: Arenaviruses are important pathogens that can cause hemorrhagic fever or meningoencephalitis. There is not an effective treatment targeting arenaviruses, which necessitates a search for therapies against this virus family, as well as others. Silver nanoparticles (Ag-NPs) have been shown to have effective antiviral activity in cell culture models against arenaviruses, and therefore it was hypothesized that they may make an effective therapeutic against viral meningitis. However, the silver nanoparticles interfered with normal cytokine profiles produced in response to the virus infection, which led to exacerbated pathology. Methods: Mice were infected with Tacaribe virus, a mouse-adapted arenavirus that causes lethal encephalitis. The virus was left untreated or treated with 10 nm Ag-NPs, which were demonstrated previously to inhibit virus replication in cell culture. Virus replication, brain pathology, and cytokine profiles (pro-inflammatory interleukins and type I interferons), as well as over morbidity and mortality were assessed in the mice with and without treatment. Results: Although the viral loads appeared to be reduced in the Ag-NP treated mice, mortality was observed in all virus-infected animals, regardless of whether or not they received nanoparticle therapy. The Ag-NP treatments also the innate immune response against the virus infection and unregulated the production of interleukin-1 beta, which likely contributed to the observed mortality in the mice. Conclusions: This study confirms why it is important to assess promising in vitro results using in vivo models. Biological barriers can impact the efficacy of therapeutics in mammals. Although Ag-NPs may still have success as an antiviral therapeutic in other areas of the body that are less constricted, in sensitive areas such as the brain, the nanoparticles can cause very adverse reactions.

Single-molecule Analysis of DNA Replication Dynamics in Budding Yeast and Human Cells by DNA Combing.

The DNA combing method allows the analysis of DNA replication at the level of individual DNA molecules stretched along silane-coated glass coverslips. Before DNA extraction, ongoing DNA synthesis is labeled with halogenated analogues of thymidine. Replication tracks are visualized by immunofluorescence using specific antibodies. Unlike biochemical and NGS-based methods, DNA combing provides unique information on cell-to-cell variations in DNA replication profiles, including initiation and elongation. Finally, this assay can be used to monitor the effect of DNA lesions on fork progression, arrest and restart.

Model-based analysis of DNA replication profiles: predicting replication fork velocity and initiation rate by profiling free-cycling cells.

Eukaryotic cells initiate DNA synthesis by sequential firing of hundreds of origins. This ordered replication is described by replication profiles, which measure the DNA content within a cell population. Here, we show that replication dynamics can be deduced from replication profiles of free-cycling cells. While such profiles lack explicit temporal information, they are sensitive to fork velocity and initiation capacity through the passive replication pattern, namely the replication of origins by forks emanating elsewhere. We apply our model-based approach to a compendium of profiles that include most viable budding yeast mutants implicated in replication. Predicted changes in fork velocity or initiation capacity are verified by profiling synchronously replicating cells. Notably, most mutants implicated in late (or early) origin effects are explained by global modulation of fork velocity or initiation capacity. Our approach provides a rigorous framework for analyzing DNA replication profiles of free-cycling cells.

Cell Panel Profiling Reveals Conserved Therapeutic Clusters and Differentiates the Mechanism of Action of Different PI3K/mTOR, Aurora Kinase and EZH2 Inhibitors.

Cancer cell line panels are important tools to characterize the in vitro activity of new investigational drugs. Here, we present the inhibition profiles of 122 anticancer agents in proliferation assays with 44 or 66 genetically characterized cancer cell lines from diverse tumor tissues (Oncolines). The library includes 29 cytotoxics, 68 kinase inhibitors, and 11 epigenetic modulators. For 38 compounds this is the first comparative profiling in a cell line panel. By strictly maintaining optimized assay protocols, biological variation was kept to a minimum. Replicate profiles of 16 agents over three years show a high average Pearson correlation of 0.8 using IC50 values and 0.9 using GI50 values. Good correlations were observed with other panels. Curve fitting appears a large source of variation. Hierarchical clustering revealed 44 basic clusters, of which 26 contain compounds with common mechanisms of action, of which 9 were not reported before, including TTK, BET and two clusters of EZH2 inhibitors. To investigate unexpected clusterings, sets of BTK, Aurora and PI3K inhibitors were profiled in biochemical enzyme activity assays and surface plasmon resonance binding assays. The BTK inhibitor ibrutinib clusters with EGFR inhibitors, because it cross-reacts with EGFR. Aurora kinase inhibitors separate into two clusters, related to Aurora A or pan-Aurora selectivity. Similarly, 12 inhibitors in the PI3K/AKT/mTOR pathway separated into different clusters, reflecting biochemical selectivity (pan-PI3K, PI3Kβγδ-isoform selective or mTOR-selective). Of these, only allosteric mTOR inhibitors preferentially targeted PTEN-mutated cell lines. This shows that cell line profiling is an excellent tool for the unbiased classification of antiproliferative compounds. Mol Cancer Ther; 15(12); 3097-109. ©2016 AACR.

Incision of damaged DNA in the presence of an impaired Smc5/6 complex imperils genome stability

The Smc5/6 complex is implicated in homologous recombination-mediated DNA repair during DNA damage or replication stress. Here, we analysed genome-wide replication dynamics in a hypomorphic budding yeast mutant, smc6-P4. The overall replication dynamics in the smc6 mutant is similar to that in the wild-type cells. However, we captured a difference in the replication profile of an early S phase sample in the mutant, prompting the hypothesis that the mutant incorporates ribonucleotides and/or accumulates single-stranded DNA gaps during replication. We tested if inhibiting the ribonucleotide excision repair pathway would exacerbate the smc6 mutant in response to DNA replication stress. Contrary to our expectation, impairment of ribonucleotide excision repair, as well as virtually all other DNA repair pathways, alleviated smc6 mutant's hypersensitivity to induced replication stress. We propose that nucleotide incision in the absence of a functional Smc5/6 complex has more disastrous outcomes than the damage per se. Our study provides novel perspectives for the role of the Smc5/6 complex during DNA replication.

Cloning and expression analysis of innate immune genes from red sea bream to assess different susceptibility to megalocytivirus infection.

As suggested by the Office International des Epizooties (OIE), fishes belonging to the genus Oplegnathus are more sensitive to megalocytivirus infection than other fish species including red sea bream (Pagrus major). To assess the roles of the innate immune response to these different susceptibilities, we cloned the genes encoding inflammatory factors including IL-8 and COX-2, and the antiviral factor like Mx from red sea bream for the first time and performed phylogenetic and structural analysis. Analysed expression levels of IL-1β, IL-8 and COX-2 and the antiviral factor like Mx genes performed with invivo challenge experiment showed no difference in inflammatory gene expression or respiratory burst activity between red sea bream androck bream (Oplegnathus fasciatus). However, the Mx gene expression levels in red sea bream weremarkedly higher than those in rock bream, suggesting the importance of type I interferon (IFN)-induced proteins, particularly Mx, during megalocytivirus infection, rather than inflammation-related genes. The invitro challenge experiments using embryonic primary cultures derived from both fish species showed no difference in cytopathic effects (CPE), viral replication profiles, and inflammatory and Mx gene expression pattern between the two fish species.

The Replication of Frataxin Gene Is Assured by Activation of Dormant Origins in the Presence of a GAA-Repeat Expansion.

It is well known that DNA replication affects the stability of several trinucleotide repeats, but whether replication profiles of human loci carrying an expanded repeat differ from those of normal alleles is poorly understood in the endogenous context. We investigated this issue using cell lines from Friedreich’s ataxia patients, homozygous for a GAA-repeat expansion in intron 1 of the Frataxin gene. By interphase, FISH we found that in comparison to the normal Frataxin sequence the replication of expanded alleles is slowed or delayed. According to molecular combing, origins never fired within the normal Frataxin allele. In contrast, in mutant alleles dormant origins are recruited within the gene, causing a switch of the prevalent fork direction through the expanded repeat. Furthermore, a global modification of the replication profile, involving origin choice and a differential distribution of unidirectional forks, was observed in the surrounding 850 kb region. These data provide a wide-view of the interplay of events occurring during replication of genes carrying an expanded repeat.

Abstract 4635: Comparative cancer cell line profiling differentiates the mechanism of action of different PI3K/mTOR, Aurora kinase and EZH2 inhibitors

Abstract [purpose] Profiling of drug candidates in cell line panels is an important tool to compare the selectivity and targeting of new anti-cancer agents. In addition, comparative profiling may be used to repurpose established therapeutics by identifying new mechanisms of action or cross-selectivities. [experimental procedures] A collection of more than 120 anti-cancer agents, targeting all important oncogenic signaling pathways, including classic cytotoxic agents as well as many targeted kinase inhibitors and epigenetic modulators, was profiled on a panel of 44 or 66 parallel cell line proliferation assays (Oncolines™) [1,2]. The Oncolines™ profiles of the compounds were compared by Pearson correlations of their inhibitor responses. Inhibitor sets were clustered using hierarchical trees. Response profiles were correlated to the genetic background of the cell lines by Analysis of Variance (Anova). [results] Reproducibility of the NTRC Oncolines™ cell panel was validated by monitoring cell growth rate and the variation in IC50s of replicate profiles over a period of three years. The Pearson correlation between replicates ranged between 0.60 and 0.99 for 16 different inhibitors, depending on dose-response curve shape. Correlation analyses of the &amp;gt; 120 profiled anti-cancer agents revealed separate clusters of, a.o., taxanes, platins, topo-isomerase inhibitors, and EGFR, ABL, MEK and BRAF inhibitors. This demonstrates that the Oncolines™ profiles are an unbiased representation of the compound's mechanisms. The profile of the BTK inhibitor ibrutinib correlated with EGFR inhibitors. In biochemical experiments we showed that this due to its cross-reactivity with EGFR. The six Aurora kinase inhibitors profiled fall into two separate clusters, which are related to their biochemical selectivity. Thus, Aurora A-selective inhibitors are relatively more active in cell lines with mutations in cell cycle checkpoint-related genes such as TP53 and RB1; whereas pan-Aurora inhibitors are more active in cell lines with mutations in growth factor signaling pathways, such as NRAS. Profiling of eleven PI3 kinase and mTOR inhibitors revealed four distinct clusters. PI3Kalpha and PI3Kdelta isoform selective inhibitors each target genetically distinct subgroups of cell lines. Rapamycin-analogs, such as everolimus, specifically target PTEN-mutant cell lines. Finally, profiling of EZH2 inhibitors indicates that there are essentially two classes and that these have a cellular profile that is distinct from HDAC, DOT1L or BET inhibitors. [conclusions] Comparative cancer cell line profiling is a powerful tool to rapidly explore the pharmacogenomics of drug action in cancer cells and to identify new or previously unnoted activities of compounds. [references]: [1] Uitdehaag et al. (2014) PLOS ONE 9(3) e92146; [2] Uitdehaag et al. (2015) PLOS ONE 10(6) e0132230. Citation Format: Joost C.M. Uitdehaag, Jeroen A.D.M. de Roos, Martine B.W. Prinsen, Judith R.F. de Vetter, Jelle Dylus, Antoon M. van Doornmalen, Suzanne J.C. van Gerwen, Jos de Man, Rogier C. Buijsman, Guido J.R. Zaman. Comparative cancer cell line profiling differentiates the mechanism of action of different PI3K/mTOR, Aurora kinase and EZH2 inhibitors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4635.

Replication fork progression is paused in two large chromosomal zones flanking the DNA replication origin in Escherichia coli.

Although the speed of nascent DNA synthesis at individual replication forks is relatively uniform in bacterial cells, the dynamics of replication fork progression on the chromosome are hampered by a variety of natural impediments. Genome replication dynamics can be directly measured from an exponentially growing cell population by sequencing newly synthesized DNA strands that were specifically pulse-labeled with the thymidine analogue 5-bromo-2'-deoxyuridine (BrdU). However, a short pulse labeling with BrdU is impracticable for bacteria because of poor incorporation of BrdU into the cells, and thus, the genomewide dynamics of bacterial DNA replication remain undetermined. Using a new thymidine-requiring Escherichia coli strain, eCOMB, and high-throughput sequencing, we succeeded in determining the genomewide replication profile in bacterial cells. We also found that fork progression is paused in two ~200-kb chromosomal zones that flank the replication origin in the growing cells. This origin-proximal obstruction to fork progression was overcome by an increased thymidine concentration in the culture medium and enhanced by inhibition of transcription. These indicate that DNA replication near the origin is sensitive to the impediments to fork progression, namely a scarcity of the DNA precursor deoxythymidine triphosphate and probable conflicts between replication and transcription machineries.