Cellular localization of oriC during the cell cycle of Escherichia coli as analyzed by fluorescent in situ hybridization

The origin of replication of Escherichia coli, oriC, has been labeled by fluorescent in situ hybridization (FISH). The E. coli K12 strain was grown under steady state conditions with a doubling time of 79 min at 28 degrees C. Under these growth conditions DNA replication starts in the previous cell cycle at -33 min. At birth cells possess two origins which are visible as two separated foci in fully labeled cells. The number of foci increased with cell length. The distance of foci from the nearest cell pole has been measured in various length classes. The data suggest: i) that the two most outwardly located foci keep a constant distance to the cell pole and they therefore move apart gradually in line with cell elongation; and ii) that at the initiation of DNA replication the labeled origins occur near the center of prospective daughter cells.     

Early S phase DNA replication: A search for targets of carcinogenesis

Our studies have revealed that replicating DNA is more vulnerable to adduction than is non-replicating DNA. Contrary to our expectations that the vulnerability to neoplastic transformation induced by carcinogens in synchronized cells would parallel the rate of DNA replication, we actually found that the vulnerability was notably increased early in the S phase and more closely paralleled the rate of entry of cells into the S phase (the very beginning of S phase) rather than the overall rate of DNA synthesis. From these findings we hypothesized that there were targets for the neoplastic transformation of cells that were among the earliest replicated sequences in the genome. To test that this hypothesis was plausible we investigated the temporal order of DNA replication during the S phase and showed that the order of DNA replication was far more precisely defined than had been recognized previously. The cell synchronization techniques that made those findings possible made it feasible to demonstrate that only a relatively few sites of DNA replication are identifiable in chromosomal bands at the earliest times in the S phase. The same synchronization techniques enabled us to label DNA replicated when populations of cells were very early in S phase and to isolate and clone this DNA. The clonal elements of this library of DNA prepared in this manner have been sequenced and mapped to the human genome. Efforts are in progress to characterize the genes and sequence features associated with these regions. We have utilized methods to identify and characterize origins of DNA replication as a means of locating the earliest replicating part of these early replicating regions. We have identified several new origins of DNA replication that are activated early and late in the S phase but the features of the chromatin at the origin that determines its time of activation remain obscure. In an effort to improve our ability to identify more origins, particularly adjacent origins in genomic regions, we have combined the methods of DNA combing and FISH analysis of combed DNA to search for DNA precursor incorporation patterns characteristic of origins of DNA replication. Preliminary nascent strand abundance studies appear to have proven the existence of two origins of DNA replication predicted from the precursor incorporation studies. We have found that the combed DNA techniques can be combined with precursor incorporation studies and antibodies to sites of DNA damage to address questions of mechanisms of DNA damage and repair. For example these studies have shown recently that DNA damage is not randomly distributed in the genome and that both inhibition of replicon initiation and inhibition of strand elongation are separately distinguishable as components of the S checkpoint function.It is our hope and expectation that these results and the opportunities that they provide for future studies will enable us to identify possible targets for malignant transformation that explain our observation that cells at the start of S phase are vulnerable to the initiation of carcinogenesis.     

Effect of ultraviolet light on thymidine incorporation, DNA chain elongation and replicon initiation in wild-type and excision-deficient Chinese hamster ovary cells

Wild-type Chinese hamster ovary cells (AA8) and five excision-deficient clones derived from the AA8 line (UV-4, UV-5, UV-20, UV-24 and UV-41) were exposed to ultraviolet light and then analyzed for their ability to incorporate [3H]thymidine and to initiate as well as elongate replicon-sized DNA fragments. After exposure to ultraviolet light, all cell lines exhibited a depression in the rate of thymidine incorporation. For exposures of 4.0 J/m2 or higher the wild-type cells recovered normal rates of thymidine incorporation within a few hours, while none of the excision-deficient lines exhibited complete recovery. For fluences below 4.0 J/m2 all but the UV-5 line exhibited at least some recovery. The ability to elongate DNA chains appeared to correlate with the thymidine incorporation data, with the UV-5 line exhibiting the strongest blockage of DNA chain elongation, the AA8 line exhibiting the least blockage, and the UV-20 line exhibiting an intermediate response. All cell lines exhibited a decrease in the distance between replication origins, thus supporting models which propose that exposure to ultraviolet light results in the use of alternative sites for the initiation of replication.     

Use of Comet-FISH in the study of DNA damage and repair: review

The Comet-FISH technique is a useful tool to detect overall and region-specific DNA damage and repair in individual cells. It combines two well-established methods, the Comet assay (single cell gel electrophoresis) and the technique of fluorescence in situ hybridization (FISH). Whereas the Comet assay allows separating fragmented from non-fragmented DNA, FISH helps to detect specifically labelled DNA sequences of interest, including whole chromosomes. Thus the combination of both techniques has been applied in particular for detection of site-specific breaks in DNA regions which are relevant for development of different diseases. This paper reviews the relevant literature and presents three examples on how Comet-FISH was used for studying the induction of DNA damage by genotoxic compounds related to oxidative stress in colon cancer-relevant genes (TP53, APC, KRAS) of a colon adenoma cell line. The accumulated evidence on relative sensitivity of these genes in comparison to global damage allows a more definite conclusion on the possible contribution of the genotoxic factors during colorectal carcinogenesis. Telomere fragility was compared in different cell lines treated with cytostatic agents, and revealed new patterns of biological activities through the drugs and different sensitivities of the cell lines that were found to be associated with their tumour origin. A third example relates to measuring repair of specific gene regions using Comet-FISH, a method that can be developed to biomarker application. Taken together, available data suggests that Comet-FISH helps to get further insights into sensitivity of specific DNA regions and consequently in mechanisms of carcinogenesis. Although the nature of the measured Comet-FISH endpoint precludes us from stating basically that damage and repair are occurring within the specific gene, it is at least possible to evaluate whether the damage and repair are occurring within the vicinity of the gene of interest.     

Coordination between chromosome replication, segregation, and cell division in Caulobacter crescentus

Progression through the Caulobacter crescentus cell cycle is coupled to a cellular differentiation program. The swarmer cell is replicationally quiescent, and DNA replication initiates at the swarmer-to-stalked cell transition. There is a very short delay between initiation of DNA replication and movement of one of the newly replicated origins to the opposite pole of the cell, indicating the absence of cohesion between the newly replicated origin-proximal parts of the Caulobacter chromosome. The terminus region of the chromosome becomes located at the invaginating septum in predivisional cells, and the completely replicated terminus regions stay associated with each other after chromosome replication is completed, disassociating very late in the cell cycle shortly before the final cell division event. Invagination of the cytoplasmic membrane occurs earlier than separation of the replicated terminus regions and formation of separate nucleoids, which results in trapping of a chromosome on either side of the cell division septum, indicating that there is not a nucleoid exclusion phenotype.     

Algorithms to Model Single Gene,Single Chromosome,and Whole Genome Copy Number Changes Jointly in Tumor Phylogenetics

We present methods to construct phylogenetic models of tumor progression at the cellular level that include copy number changes at the scale of single genes, entire chromosomes, and the whole genome. The methods are designed for data collected by fluorescence in situ hybridization (FISH), an experimental technique especially well suited to characterizing intratumor heterogeneity using counts of probes to genetic regions frequently gained or lost in tumor development. Here, we develop new provably optimal methods for computing an edit distance between the copy number states of two cells given evolution by copy number changes of single probes, all probes on a chromosome, or all probes in the genome. We then apply this theory to develop a practical heuristic algorithm, implemented in publicly available software, for inferring tumor phylogenies on data from potentially hundreds of single cells by this evolutionary model. We demonstrate and validate the methods on simulated data and published FISH data from cervical cancers and breast cancers. Our computational experiments show that the new model and algorithm lead to more parsimonious trees than prior methods for single-tumor phylogenetics and to improved performance on various classification tasks, such as distinguishing primary tumors from metastases obtained from the same patient population.     

Rearrangements of ribosomal DNA clusters in late generation telomerase-deficient<Emphasis Type="Italic"> Arabidopsis</Emphasis>

The ends of eukaryotic chromosomes are capped with special nucleoprotein structures called telomeres. Telomere shortening due to telomerase inactivation may result in fusion of homologous or heterologous chromosomes, leading to their successive breakage during anaphase movement, followed by fusion of broken ends in the next cell cycle, i.e. the breakage-fusion-bridge (BFB) cycle. Using fluorescence in situ hybridization (FISH) with 25S rDNA and specific bacterial artificial chromosome (BAC) probes we demonstrate participation of chromosomes 2 and 4 of Arabidopsis thaliana AtTERT null plants in the formation of anaphase bridges. Both homologous and non-homologous chromosomes formed transient anaphase bridges whose breakage and unequal separation led to genome rearrangement, including non-reciprocal translocations and aneuploidy. The 45S rDNA regions located at the ends of chromosomes 2 and 4 were observed in chromosome bridges at a frequency approximately ten times higher than expected in the case of random fusion events. This outcome could result from a functional association of rDNA repeats at nucleoli. We also describe increased variation in the number of nucleoli in some interphase cells with supernumerary rDNA FISH signals. These data indicate that dysfunctional telomeres in Arabidopsis lead to massive genome instability, which is induced by multiple rounds of the BFB mechanism.     

Evidence for sequential and increasing activation of replication origins along replication timing gradients in the human genome

Genome-wide replication timing studies have suggested that mammalian chromosomes consist of megabase-scale domains of coordinated origin firing separated by large originless transition regions. Here, we report a quantitative genome-wide analysis of DNA replication kinetics in several human cell types that contradicts this view. DNA combing in HeLa cells sorted into four temporal compartments of S phase shows that replication origins are spaced at 40 kb intervals and fire as small clusters whose synchrony increases during S phase and that replication fork velocity (mean 0.7 kb/min, maximum 2.0 kb/min) remains constant and narrowly distributed through S phase. However, multi-scale analysis of a genome-wide replication timing profile shows a broad distribution of replication timing gradients with practically no regions larger than 100 kb replicating at less than 2 kb/min. Therefore, HeLa cells lack large regions of unidirectional fork progression. Temporal transition regions are replicated by sequential activation of origins at a rate that increases during S phase and replication timing gradients are set by the delay and the spacing between successive origin firings rather than by the velocity of single forks. Activation of internal origins in a specific temporal transition region is directly demonstrated by DNA combing of the IGH locus in HeLa cells. Analysis of published origin maps in HeLa cells and published replication timing and DNA combing data in several other cell types corroborate these findings, with the interesting exception of embryonic stem cells where regions of unidirectional fork progression seem more abundant. These results can be explained if origins fire independently of each other but under the control of long-range chromatin structure, or if replication forks progressing from early origins stimulate initiation in nearby unreplicated DNA. These findings shed a new light on the replication timing program of mammalian genomes and provide a general model for their replication kinetics.     

One size does not fit all: flexible models are required to understand animal movement across scales

1. Large data sets containing precise movement data from free-roaming animals are now becoming commonplace. One means of analysing individual movement data is through discrete, random walk-based models. 2. Random walk models are easily modified to incorporate common features of animal movement, and the ways that these modifications affect the scaling of net displacement are well studied. Recently, ecologists have begun to explore more complex statistical models with multiple latent states, each of which are characterized by a distribution of step lengths and have their own unimodal distribution of turning angles centred on one type of turn (e.g. reversals). 3. Here, we introduce the compound wrapped Cauchy distribution, which allows for multimodal distributions of turning angles within a single state. When used as a single state model, the parameters provide a straightforward summary of the relative contributions of different turn types. The compound wrapped Cauchy distribution can also be used to build multiple state models. 4. We hypothesize that a multiple state model with unimodal distributions of turning angles will best describe movement at finer resolutions, while a multiple state model using our multimodal distribution will better describe movement at intermediate temporal resolutions. At coarser temporal resolutions, a single state model using our multimodal distribution should be sufficient. We parameterize and compare the performance of these models at four different temporal resolutions (1, 4, 12 and 24 h) using data from eight individuals of Loxodonta cyclotis and find support for our hypotheses. 5. We assess the efficacy of the different models in extrapolating to coarser temporal resolution by comparing properties of data simulated from the different models to the properties of the observed data. At coarser resolutions, simulated data sets recreate many aspects of the observed data; however, only one of the models accurately predicts step length, and all models underestimate the frequency of reversals. 6. The single state model we introduce may be adequate to describe movement data at many resolutions and can be interpreted easily. Multiscalar analyses of movement such as the ones presented here are a useful means of identifying inconsistencies in our understanding of movement.     

Spatial and temporal organization of the Bacillus subtilis replication cycle

DNA replication occurs at discrete sites in the cell. To gain insight into the spatial and temporal organization of the Bacillus subtilis replication cycle, we simultaneously visualized replication origins and the replication machinery (replisomes) inside live cells. We found that the origin of replication is positioned near midcell prior to replication. After initiation, the replisome colocalizes with the origin, confirming that replication initiates near midcell. The replisome remains near midcell after duplicated origins separate. Artificially mispositioning the origin region leads to mislocalization of the replisome indicating that the location of the origin at the time of initiation establishes the position of the replisome. Time-lapse microscopy revealed that a single replisome focus reversibly splits into two closely spaced foci every few seconds in many cells, including cells that recently initiated replication. Thus, sister replication forks are likely not intimately associated with each other throughout the replication cycle. Fork dynamics persisted when replication elongation was halted, and is thus independent of the relative movement of DNA through the replisome. Our results provide new insights into how the replisome is positioned in the cell and refine our current understanding of the spatial and temporal events of the B. subtilis replication cycle.     

The Mammalian DNA Replication Elongation Checkpoint: Implication of Chk1 and Relationship with Origin Firing as Determined by Single DNA Molecule and Single Cell Analyses

The regulation of DNA replication initiation is well documented, for both unperturbed and damaged cells. The regulation of elongation, or fork velocity, however, has only recently been revealed with the advent of new techniques allowing us to view DNA replication at the single cell and single DNA molecule levels. Normally in S phase, the progression of replication forks and their stability are regulated by the ATR-Claspin-Chk1 pathway. We recently showed that replication fork velocity varies across the human genome in normal and cancer cells, but that the velocity of a given fork is positively correlated with the distance between origins on the same DNA fiber. Accordingly, in DNA replication-deficient Bloom’s syndrome cells, reduced fork velocity is associated with an increased density of replication origins. Replication elongation is also regulated in response to DNA damage. In human colon carcinoma cells treated with the topoisomerase I inhibitor camptothecin, DNA replication is inhibited both at the level of initiation and at the level of elongation through a Chk1-dependent checkpoint mechanism. Together, these new findings demonstrate that replication fork velocity (fork progression) is coordinated with inter-origin distance and that it can be actively slowed down by Chk1-dependent mechanisms in response to DNA damage. Thus, we propose that the intra-S phase checkpoint consist of at least three elements: (1) stabilization of damaged replication forks; (2) suppression of firing of late origins; and (3) arrests of normal ongoing forks to prevent further DNA lesions by replication of a damaged DNA template.     

Chemotaxis: a feedback-based computational model robustly predicts multiple aspects of real cell behaviour

The mechanism of eukaryotic chemotaxis remains unclear despite intensive study. The most frequently described mechanism acts through attractants causing actin polymerization, in turn leading to pseudopod formation and cell movement. We recently proposed an alternative mechanism, supported by several lines of data, in which pseudopods are made by a self-generated cycle. If chemoattractants are present, they modulate the cycle rather than directly causing actin polymerization. The aim of this work is to test the explanatory and predictive powers of such pseudopod-based models to predict the complex behaviour of cells in chemotaxis. We have now tested the effectiveness of this mechanism using a computational model of cell movement and chemotaxis based on pseudopod autocatalysis. The model reproduces a surprisingly wide range of existing data about cell movement and chemotaxis. It simulates cell polarization and persistence without stimuli and selection of accurate pseudopods when chemoattractant gradients are present. It predicts both bias of pseudopod position in low chemoattractant gradients and--unexpectedly--lateral pseudopod initiation in high gradients. To test the predictive ability of the model, we looked for untested and novel predictions. One prediction from the model is that the angle between successive pseudopods at the front of the cell will increase in proportion to the difference between the cell's direction and the direction of the gradient. We measured the angles between pseudopods in chemotaxing Dictyostelium cells under different conditions and found the results agreed with the model extremely well. Our model and data together suggest that in rapidly moving cells like Dictyostelium and neutrophils an intrinsic pseudopod cycle lies at the heart of cell motility. This implies that the mechanism behind chemotaxis relies on modification of intrinsic pseudopod behaviour, more than generation of new pseudopods or actin polymerization by chemoattractants.     

The segregation of the Escherichia coli origin and terminus of replication

Escherichia coli chromosome replication forks are tethered to the cell centre. Two opposing models describe how the chromosomes segregate. In the extrusion-capture model, newly replicated DNA is fed bi-directionally from the forks toward the cell poles, forming new chromosomes in each cell half. Starting with the origins, chromosomal regions segregate away from their sisters progressively as they are replicated. The termini segregate last. In the sister chromosome cohesion model, replication produces sister chromosomes that are paired along much of their length. The origins and most other chromosomal regions remain paired until late in the replication cycle, and all segregate together. We use a combination of microscopy and flow cytometry to determine the relationship of origin and terminus segregation to the cell cycle. Origin segregation frequently follows closely after initiation, in strong support of the extrusion-capture model. The spatial disposition of the origin and terminus sequences also fits this model. Terminus segregation occurs extremely late in the cell cycle as the daughter cells separate. As the septum begins to invaginate, the termini of the completed sister chromosomes are transiently held apart at the cell centre, on opposite sides of the cell. This may facilitate the resolution of topological linkages between the chromosomes.     

An evolving tail of centromere histone variant CENP-A

We have recently shown that replication forks pause near origins in normal human fibroblasts (NHF1-hTERT) but not glioblastoma T98G cells. This observation led us to question whether other differences in the replication program may exist between these cell types that may relate to their genetic integrity. To identify differences, we detected immunoflourescently the sequential incorporation of the nucleotide analogs IdU and CldU into replicating DNA at the start of every hour of a synchronized S phase. We then characterized the patterns of labeled replicating DNA tracks and quantified the percentages and lengths of the tracks found at these hourly intervals. From the directionality of labeling in single extended replicating DNA fibers, tracks were categorized as single bidirectional origins, unidirectional elongations, clusters of origins firing in tandem, or merging forks (terminations). Our analysis showed that the start of S phase is enriched in single bidirectional origins in NHF1-hTERT cells, followed by an increase in clustering during mid S phase and an increase in merging forks during late S phase. Early S phase in T98G cells also largely consisted of single bidirectional origin initiations; however, an increase in clustering was delayed until an hour later, and clusters were shorter in mid/late S phase than in NHF1-hTERT cells. The spike in merging forks also did not occur until an hour later in T98G cells. Our observations suggest models to explain the temporal replication of single and clustered origins, and suggest differences in the replication program in a normal and cancer cell line.     

Constitutive heterochromatin DNA fragments are demethylated and decondensed in senescent primary fibroblasts MRC5 and malignant A431 cell line

It is commonly accepted that satellite DNA (satDNA) is highly condensed in the interphase. We checked localization, the degree of condensation, and methylation level of centromeric (CEN) and pericentromeric (periCEN) satDNA fragments by immunofluorescent in situ hybridization (immuno-FISH). An antibody against 5-methylcytosine was used for the immunostaining, and satDNA probes were used for FISH. Cells from the normal somatic tissues (placenta cells and lymphocytes), a primary fibroblast cell line (MRC5), and a malignant cell line (A431) were analyzed. CEN satDNA was condensed and highly methylated in all studied cell types. PeriCEN human satellite 3 from chromosome 1 (HS3-1) was condensed in lymphocytes, placenta cells, and in young cells of the primary culture. In senescent fibroblasts and in the malignant cell line A431, the unfolded HS3-1 was observed. An antibody against methylated DNA stained compact patches of the periCEN satDNA and did not stain the unfolded regions. Thus, we observed the unfolding of the HS3-1 in senescent MRC5 and malignant A431. The unfolding was accompanied by partial demethylation of the satDNA that belongs to the constitutive heterochromatin.     

A Cdc7 kinase inhibitor restricts initiation of DNA replication and has antitumor activity

Cdc7 is an essential kinase that promotes DNA replication by activating origins of replication. Here, we characterized the potent Cdc7 inhibitor PHA-767491 (1) in biochemical and cell-based assays, and we tested its antitumor activity in rodents. We found that the compound blocks DNA synthesis and affects the phosphorylation of the replicative DNA helicase at Cdc7-dependent phosphorylation sites. Unlike current DNA synthesis inhibitors, PHA-767491 prevents the activation of replication origins but does not impede replication fork progression, and it does not trigger a sustained DNA damage response. Treatment with PHA-767491 results in apoptotic cell death in multiple cancer cell types and tumor growth inhibition in preclinical cancer models. To our knowledge, PHA-767491 is the first molecule that directly affects the mechanisms controlling initiation as opposed to elongation in DNA replication, and its activities suggest that Cdc7 kinase inhibition could be a new strategy for the development of anticancer therapeutics.     

Actin-like proteins MreB and Mbl from Bacillus subtilis are required for bipolar positioning of replication origins

Actin-like proteins MreB and Mbl are required for proper cell shape and for viability in B. subtilis and form dynamic helical filaments underneath the cell membrane. We have found that depletion of MreB and Mbl proteins leads to a rapid defect in chromosome segregation before a defect in cell shape becomes detectable. Under these conditions, the SMC chromosome segregation complex that is essential for proper chromosome arrangement and segregation loses its specific subcellular localization, and replication origins fail to localize in a regular bipolar manner as in wild type cells. Time-lapse microscopy showed that during depletion of MreB, origin regions can move towards the same cell pole, showing that bipolar orientation of origin separation is lost. Contrarily, depletion of three other cell shape determinants, MreC, MreD, or MreBH (the third B. subtilis actin homolog) had no effect on chromosome segregation but varying effects on cell morphology. Depletion of MreC and MreD resulted in formation of round cells, while depletion of MreBH led to formation of vibrio-shaped cells. The data show that actin proteins Mbl and MreB are required for proper chromosome segregation and that Mre proteins affect different aspects in cell shape.