Replicon Size Research Articles

Intracellular Positioning Systems Limit the Entropic Eviction of Secondary Replicons Toward the Nucleoid Edges in Bacterial Cells

Bacterial genomes, organized intracellularly as nucleoids, are composed of the main chromosome coexisting with different types of secondary replicons. Secondary replicons are major drivers of bacterial adaptation by gene exchange. They are highly diverse in type and size, ranging from less than 2 to more than 1000 kb, and must integrate with bacterial physiology, including to the nucleoid dynamics, to limit detrimental costs leading to their counter-selection. We show that large DNA circles, whether from a natural plasmid or excised from the chromosome tend to localize in a dynamic manner in a zone separating the nucleoid from the cytoplasm at the edge of the nucleoid. This localization is in good agreement with silico simulations of DNA circles in the nucleoid volume. Subcellular positioning systems counteract this tendency, allowing replicons to enter the nucleoid space. In enterobacteria, these systems are found in replicons above 25 kb, defining the limit with small randomly segregated plasmids. Larger replicons carry at least one of the three described family of systems, ParAB, ParRM, and StbA. Replicons above 180 kb all carry a ParAB system, suggesting this system is specifically required in the cases of large replicons. Simulations demonstrated that replicon size profoundly affects localization, compaction, and dynamics of DNA circles in the nucleoid volume. The present work suggests that presence of partition systems on the larger plasmids or chromids is not only due to selection for accurate segregation but also to counteract their unmixing with the chromosome and consequent exclusion from the nucleoid.

Fine-tuning expression of begomoviral movement and nuclear shuttle proteins confers cell-to-cell movement to mastreviral replicons in Nicotiana benthamiana leaves.

Geminiviruses are a group of small plant viruses responsible for devastating crop damage worldwide. The emergence of agricultural diseases caused by geminiviruses is attributed in part to their high rates of recombination, leading to complementary function between viral components across species and genera. We have developed a mastreviral reporter system based on bean yellow dwarf virus (BeYDV) that replicates to high levels in the plant nucleus, expressing very high levels of GFP. To investigate the potential for complementation of movement function by other geminivirus genera, the movement protein (MP) and nuclear shuttle protein (NSP) from the bipartite begomovirus Bean dwarf mosaic virus (BDMV) were produced and characterized in Nicotiana benthamiana leaves. While overexpression of MP and NSP strongly inhibited GFP expression from the mastreviral reporter and caused adverse plant symptoms, optimizing the expression levels of MP and NSP allowed functional cell-to-cell movement. Hybrid virus vectors were created that express BDMV MP and NSP from mastreviral replicons, allowing efficient cell-to-cell movement comparable to native BDMV replicons. We find that the expression levels of MP and NSP must be fine-tuned to provide sufficient MP/NSP for movement without eliciting the plant hypersensitive response or adversely impacting gene expression from viral replicons. The ability to confer cell-to-cell movement to mastrevirus replicons depended strongly on replicon size: 2.1-2.7 kb replicons were efficiently moved, while 3 kb replicons were inhibited, and 3.9 kb replicons were very strongly inhibited. Optimized expression of MP/NSP from the normally phloem-limited Abutilon mosaic virus (AbMV) allows efficient movement in non-phloem cells.

4D Visualization of replication foci in mammalian cells corresponding to individual replicons.

Since the pioneering proposal of the replicon model of DNA replication 50 years ago, the predicted replicons have not been identified and quantified at the cellular level. Here, we combine conventional and super-resolution microscopy of replication sites in live and fixed cells with computational image analysis. We complement these data with genome size measurements, comprehensive analysis of S-phase dynamics and quantification of replication fork speed and replicon size in human and mouse cells. These multidimensional analyses demonstrate that replication foci (RFi) in three-dimensional (3D) preserved somatic mammalian cells can be optically resolved down to single replicons throughout S-phase. This challenges the conventional interpretation of nuclear RFi as replication factories, that is, the complex entities that process multiple clustered replicons. Accordingly, 3D genome organization and duplication can be now followed within the chromatin context at the level of individual replicons.

DNA topoisomerase IIα controls replication origin cluster licensing and firing time in Xenopus egg extracts

Sperm chromatin incubated in Xenopus egg extracts undergoes origin licensing and nuclear assembly before DNA replication. We found that depletion of DNA topoisomerase IIα (topo IIα), the sole topo II isozyme of eggs and its inhibition by ICRF-193, which clamps topo IIα around DNA have opposite effects on these processes. ICRF-193 slowed down replication origin cluster activation and fork progression in a checkpoint-independent manner, without altering replicon size. In contrast, topo IIα depletion accelerated origin cluster activation, and topo IIα add-back negated overinitiation. Therefore, topo IIα is not required for DNA replication, but topo IIα clamps slow replication, probably by forming roadblocks. ICRF-193 had no effect on DNA synthesis when added after nuclear assembly, confirming that topo IIα activity is dispensable for replication and revealing that topo IIα clamps formed on replicating DNA do not block replication, presumably because topo IIα acts behind and not in front of forks. Topo IIα depletion increased, and topo IIα addition reduced, chromatin loading of MCM2-7 replicative helicase, whereas ICRF-193 did not affect MCM2-7 loading. Therefore, topo IIα restrains MCM2-7 loading in an ICRF-193-resistant manner during origin licensing, suggesting a model for establishing the sequential firing of origin clusters.

Cdc45 Limits Replicon Usage from a Low Density of preRCs in Mammalian Cells

Little is known about mammalian preRC stoichiometry, the number of preRCs on chromosomes, and how this relates to replicon size and usage. We show here that, on average, each 100-kb of the mammalian genome contains a preRC composed of approximately one ORC hexamer, 4–5 MCM hexamers, and 2 Cdc6. Relative to these subunits, ∼0.35 total molecules of the pre-Initiation Complex factor Cdc45 are present. Thus, based on ORC availability, somatic cells contain ∼70,000 preRCs of this average total stoichiometry, although subunits may not be juxtaposed with each other. Except for ORC, the chromatin-bound complement of preRC subunits is even lower. Cdc45 is present at very low levels relative to the preRC subunits, but is highly stable, and the same limited number of stable Cdc45 molecules are present from the beginning of S-phase to its completion. Efforts to artificially increase Cdc45 levels through ectopic expression block cell growth. However, microinjection of excess purified Cdc45 into S-phase nuclei activates additional replication foci by three-fold, indicating that Cdc45 functions to activate dormant preRCs and is rate-limiting for somatic replicon usage. Paradoxically, although Cdc45 colocalizes in vivo with some MCM sites and is rate-limiting for DNA replication to occur, neither Cdc45 nor MCMs colocalize with active replication sites. Embryonic metazoan chromatin consists of small replicons that are used efficiently via an excess of preRC subunits. In contrast, somatic mammalian cells contain a low density of preRCs, each containing only a few MCMs that compete for limiting amounts of Cdc45. This provides a molecular explanation why, relative to embryonic replicon dynamics, somatic replicons are, on average, larger and origin efficiency tends to be lower. The stable, continuous, and rate-limiting nature of Cdc45 suggests that Cdc45 contributes to the staggering of replicon usage throughout S-phase, and that replicon activation requires reutilization of existing Cdc45 during S-phase.

Progress towards understanding the fate of plasmids in bacterial communities

Plasmid-mediated horizontal gene transfer influences bacterial community structure and evolution. However, an understanding of the forces which dictate the fate of plasmids in bacterial populations remains elusive. This is in part due to the enormous diversity of plasmids, in terms of size, structure, transmission, evolutionary history and accessory phenotypes, coupled with the lack of a standard theoretical framework within which to investigate them. This review discusses how ecological factors, such as spatial structure and temporal fluctuations, shape both the population dynamics and the physical features of plasmids. Novel data indicate that larger plasmids are more likely to be harboured by hosts in complex environments. Plasmid size may therefore be determined by environmentally mediated fitness trade-offs. As the correlation between replicon size and complexity of environment is similar for plasmids and chromosomes, plasmids could be used as tractable tools to investigate the influence of ecological factors on chromosomes. Parallels are drawn between plasmids and bacterial facultative symbionts, including the evolution of some members of both groups to a more obligate relationship with their host. The similarity between the influences of ecological factors on plasmids and bacterial symbionts suggests that it may be appropriate to study plasmids within a classical ecological framework.

Replication-associated strand asymmetries in vertebrate genomes and implications for replicon size, DNA replication origin, and termination

Strand compositional asymmetry has been observed in prokaryotes and used in predicting prokaryotic DNA replication origins and termini. However, it was not found in eukaryotic genomes by the same methods. We propose that transcription-associated strand asymmetries mask the replication-associated ones. By analyzing the nucleotide composition of intergenic sequences larger than 50 kb by cumulative skew diagrams (CSD), we found replication-associated strand asymmetry in vertebrate genomes. Furthermore, we found that the most common replicon sizes in vertebrates are 50–100 kb, and show evidence that the replication origin and termination regions of vertebrate genomes range from a discrete site to a broad zone.

Bromodeoxyuridine DNA fiber technology in plants: replication origins and DNA synthesis in tobacco BY-2 cells under prolonged treatment with aphidicolin.

Newly synthesized DNA can be observed on chromosomes or extended chromatin fibers after incorporation and immunodetection of bromodeoxyuridine. This technique, frequently used in animal cells, was adapted for use in BY-2 cells. For the first time, the origins of replication in plant cells could be visualized and monitored on DNA fibers without the use of radioactive traces. The replicon size for BY-2 cells was estimated to be 12.9 microm; and the fork rate, 1.17 microm/h. These values are comparable to those reported for tomato and mustard cells. Furthermore, the data confirm our previous observation that DNA synthesis is not totally blocked by aphidicolin. Bromodeoxyuridine incorporation into DNA was obvious from 24 h onwards after treatment with aphidicolin.

Paradoxes of eukaryotic DNA replication: MCM proteins and the random completion problem.

Eukaryotic DNA replication initiates at multiple origins. In early fly and frog embryos, chromosomal replication is very rapid and initiates without sequence specificity. Despite this apparent randomness, the spacing of these numerous initiation sites must be sufficiently regular for the genome to be completely replicated on time. Studies in various eukaryotes have revealed that there is a strict temporal separation of origin "licensing" prior to S phase and origin activation during S phase. This may suggest that replicon size must be already established at the licensing stage. However, recent experiments suggest that a large excess of potential origins are assembled along chromatin during licensing. Thus, a regular replicon size may result from the selection of origins during S phase. We review single molecule analyses of origin activation and other experiments addressing this issue and their general significance for eukaryotic DNA replication.

Changes in chromatin organization during early development and carcinogenesis

Similar changes in chromatin organization take place during development and carcinogenesis. The size of chromatin loop domains fixed on the nuclear skeleton (matrix) increased from 20 to approximately 200 kbp. These changes are accompanied by an increased size of replicons and altered specificity of loop attachment to the nuclear matrix. During carcinogenesis, inverse changes in the chromatin structure are observed, neoplastic cells are dedifferentiated and return to the initial state. In this review, we consider new experimental data on organization of the DFNA loops and nuclear matrix in embryogenesis and carcinogenesis.

Mapping Sites Where Replication Initiates in Mammalian Cells Using DNA Fibers

DNA replication in mammalian chromosomes takes place as a unit of replicon clusters. Here we show a powerful method to detect replication origins and fork movement on DNA fibers from mammalian cells. Cells were loaded with nucleotide analogs, DNA fibers were prepared, and replicated DNA was detected. Using this approach, we could detect origins as close as 10 kb apart and found that the average size of replicon is smaller (∼46 kb) than previously estimated. In addition, the procedure visualizes the complex structure of replicon clusters, e.g. sequential activation of origins in a cluster and flexible initiation sites in different cell cycles. Combined with fluorescence in situ hybridization, replication origins can be mapped in genomic loci including repetitive DNA and a single-copy gene.

Investigation of the potential of maize streak virus to act as an infectious gene vector in maize plants.

There has been a great deal of interest in the possibility that geminiviruses might be used as infectious gene vectors for expression of foreign proteins in plants. However, generic mastreviruses such as Maize streak virus (MSV) have no sequences which are dispensable for systemic infection of plants, and there is a strict limitation on the size of viral DNA which can be moved systemically. We attempted to complement the movement functions deleted from a wild-type-sized, replication-proficient gene replacement vector, by co-infecting plants with it and either wild type MSV, or a replication-deficient but putatively movement-proficient viral construct. While ssDNA formation by the gene replacement vector could be complemented in trans by co-transfected wild type virus, true systemic movement of either the vector, or of co-complementing constructs, did not occur. However, recombination between the two complementing viral constructs frequently occurred to generate wild-type virus genomes. The results therefore suggest that formation of ssDNA and size of the viral replicon are not the sole determinants of whether the MSV movement proteins can mobilise viral sequences and move them systemically in plants.

Inactivation of DNA replication origins by the cell cycle regulator, trigonelline, in root meristems of Lactuca sativa.

The effects of trigonelline (TRG) on the cell cycle in root meristems of Lactuca sativa L. were examined in the knowledge that TRG is a cell cycle regulator that causes cell arrest in G2, and prevents ligation of replicons in S-phase. The hypothesis was tested that continuous exposure to TRG would perturb DNA replication which, in turn, would lengthen the cell cycle and impair root elongation. Using DNA fibre autoradiography, mean replicon size was 31 and 13 microm in the TRG (3 mM) and control treatments, respectively. Trigonelline also resulted in a lengthening of both S-phase and the cell cycle and a decrease in primary root elongation. Hence, replicon inactivation was responsible for the protracted S-phase. Trigonelline treatment also resulted in a 1.6-fold increase in fork rate (13.8 microm h(-1)) compared with the control (8.4 m h(-1)). The faster fork rate in the larger replicons is in accord with the highly significant positive relationship already established between fork rate and replicon size for various unrelated higher plants.

Heterogeneity of eukaryotic replicons, replicon clusters, and replication foci.

According to the current paradigm, replication foci are discrete sites in the interphase nucleus where assemblies of DNA replication enzymes simultaneously elongate the replication forks of 10-100 adjacent replicons (each approximately 100 kbp). Here we review new results and provide alternative interpretations for old results to show that the current paradigm is in need of further development. In particular, many replicons are larger than previously thought - so large that their complete replication takes much longer (several hours) than the measured average time to complete replication at individual foci (45-60 min). In addition to this large heterogeneity in replicon size, it is now apparent that there is also a corresponding heterogeneity in the size and intensity of individual replication foci. An important property of all replication foci is that they are stable structures that persist, with constant dimensions, during all cell cycle stages including mitosis, and therefore likely represent a fundamental unit of chromatin organization. With this in mind, we present a modified model of replication foci in which many of the foci are composed of clusters of small replicons as previously proposed, but the size and number of replicons per focus is extremely heterogeneous, and a significant proportion of foci are composed of single large replicons. We further speculate that very large replicons may extend over two or more individual foci and that this organization may be important in regulating the replication of such large replicons as the cell proceeds through S-phase.

Activation of latent origins of DNA replication in florally determined shoot meristems of long-day and short-day plants: Silene coeli-rosa and Pharbitis nil

Replicon spacing was measured during the S-phase of the cell cycle in shoot meristems of Silene coeli-rosa L., a long-day (LD) plant, and Pharbitis nil Chois, a short-day (SD) plant to examine the hypothesis that activation of latent origins of DNA replication is a feature of floral determination. Silene coeli-rosa was germinated and grown in SD for 28 d and then exposed to either a florally inductive combination of 7 LD + 2 SD, the last day of which coincides with determination of the sepal and stamen whorls, or was germinated and grown in 37 non-inductive SD. Pharbitis nil was germinated and grown in continuous light (CL) for 5 d and then given either 48 h of inductive darkness followed by 1 d of CL, the last day of which coincides with determination of the sepal, petal and stamen whorls, or given one of two independent non-inductive treatments: 48 h dark interrupted by red light (R) + 1 d of CL, or 8 d of CL. Following these treatments, each batch of plants was exposed to tritiated [methyl-3H]thymidine for 30, 60, 90 or 120 min. Apical domes were dissected, nuclei lysed and prepared as fibre autoradiographs from which replicon size was recorded. In S. coeli-rosa, replicon size was in the range 10–15 μm in SD (non-inductive) and 0–5 μm in LD (inductive) while in P. nil it was 10–15 μm in the 48 h dark interrupted by R, 5–10 μm in CL (both non-inductive) but was reduced to 0–5 μm in the 48 h dark treatment (inductive). Therefore, the recruitment of additional initiation points for DNA replication occurred in both a LD and a SD plant immediately before the appearance of floral organs. The data are consistent in showing that a shortening of S-phase, which is a characteristic feature of florally determined shoot meristems for both species, is brought about by the activation of latent origins of DNA replication.

Regulation of mammalian replication origin usage in Xenopus egg extract

ABSTRACT Xenopus embryos initiate replication at random closely spaced sites until a certain concentration of nuclei is achieved within the embryo, after which fewer, more specific chromosomal sites are utilized as origins. We have examined the relationship between nucleo-cytosolic ratio and origin specification when Chinese hamster ovary (CHO) cell nuclei are introduced into Xenopus egg extracts. At concentrations of intact late-G1-phase nuclei that approximate early Xenopus embryos, the entire genome was duplicated nearly 4 times faster than in culture, accompanied by a de-localization of initiation sites at the dihydrofolate reductase (DHFR) locus. As the concentration of nuclei was increased, the number of initiation sites per nucleus decreased and initiation at the DHFR locus became localized to the physiologically utilized DHFR origin. Origin specification was optimal at nuclear concentrations that approximate the Xenopus mid-blastula transition (MBT). Higher concentrations resulted in an overall inhibition of DNA synthesis. By contrast, with intact early G1-phase nuclei, replication initiated at apparently random sites at all concentrations, despite an identical relationship between nucleo-cytosolic ratio and replicon size. Furthermore, permeabilization of late-G1-phase nuclei, using newly defined conditions that preserve the overall rate of replication, eliminated site-specificity, even at nuclear concentrations optimal for DHFR origin recognition. These data show that both nucleo-cytosolic ratio and nuclear structure play important but independent roles in the regulation of replication origin usage. Nucleo-cytosolic ratio clearly influences the number of replication origins selected. However, titration of cytosolic factors is not sufficient to focus initiation to specific sites. An independent mechanism, effecting changes within G1-phase nuclei, dictates which of many potential initiation sites will function as an origin.

Regulation of mammalian replication origin usage in Xenopus egg extract.

Xenopus embryos initiate replication at random closely spaced sites until a certain concentration of nuclei is achieved within the embryo, after which fewer, more specific chromosomal sites are utilized as origins. We have examined the relationship between nucleo-cytosolic ratio and origin specification when Chinese hamster ovary (CHO) cell nuclei are introduced into Xenopus egg extracts. At concentrations of intact late-G1-phase nuclei that approximate early Xenopus embryos, the entire genome was duplicated nearly 4 times faster than in culture, accompanied by a de-localization of initiation sites at the dihydrofolate reductase (DHFR) locus. As the concentration of nuclei was increased, the number of initiation sites per nucleus decreased and initiation at the DHFR locus became localized to the physiologically utilized DHFR origin. Origin specification was optimal at nuclear concentrations that approximate the Xenopus mid-blastula transition (MBT). Higher concentrations resulted in an overall inhibition of DNA synthesis. By contrast, with intact early G1-phase nuclei, replication initiated at apparently random sites at all concentrations, despite an identical relationship between nucleo-cytosolic ratio and replicon size. Furthermore, permeabilization of late-G1-phase nuclei, using newly defined conditions that preserve the overall rate of replication, eliminated site-specificity, even at nuclear concentrations optimal for DHFR origin recognition. These data show that both nucleo-cytosolic ratio and nuclear structure play important but independent roles in the regulation of replication origin usage. Nucleo-cytosolic ratio clearly influences the number of replication origins selected. However, titration of cytosolic factors is not sufficient to focus initiation to specific sites. An independent mechanism, effecting changes within G1-phase nuclei, dictates which of many potential initiation sites will function as an origin.

Regulation of replicon size in Xenopus egg extracts.

Once a specific number of cells have been produced in the early Xenopus laevis embryo, replicon size during the S phase of the cell cycle increases. Here, it is reported that similar increase in replicon size occurred when the concentration of nuclei in replication-competent Xenopus egg extracts exceeded a critical threshold. In this system, the origin recognition complex (ORC) did not become stoichiometrically limiting for initiation, and similar amounts of this complex bound to chromatin regardless of replicon size. These data suggest that in early development, an unidentified factor controls how many preformed ORC-DNA complexes initiate DNA replication.

Visualization of elementary DNA replication units in human nuclei corresponding in size to DNA loop domains.

Newly synthesized DNA in mammalian nuclei is concentrated in discrete nuclear granules called replication foci. These foci may be visualized using antibodies against 5-bromodeoxyuridine. In the early S-phase cells 100-250 foci are usually detected. On average, individual foci range between 0.5 and 2 microns in diameter and can be seen as clusters of more than ten average-sized (60-100 kb) synchronously activated replicons. In this study, employing minor modifications of the previous methods, we report the visualization of small replication foci of about 0.3 micron diameter (mini-foci). Some foci are clustered into folded chains consisting of 2-40 subunits. DNA content of one mini-focus is estimated to be 50-120 kb and there are 500-1500 mini-foci per cell in the early S-phase. Experimentally induced decrease in replicon size does not affect the size of mini-foci, suggesting that these represent elementary units of DNA replication in mammalian nuclei and are probably identical to the basic structural DNA loop domains.

Non-random deletions at the dihydrofolate reductase locus of Chinese hamster ovary cells induced by alpha-particles simulating radon.

This study presents the physical characterization of mutants induced in mammalian cells by high linear energy transfer alpha-particle radiation that simulates exposure to radon daughters. Alpha-Particles from accelerated 4He at 150 keV/microns were used to induce 20 Chinese hamster ovary mutants that are deficient in dihydrofolate reductase (DHFR) activity. Parental cells were the hemizygous UA21 line. Cell survival decreased exponentially in response to radiation dose from 0.25 to 1.75 Gy. Mutants were obtained at 1.0 (17/20) and 1.25 Gy (3/20); treatments at 1.50 Gy failed to yield mutants. The induced frequency of mutation was 2.3 x 10(-6) at the 1.0 Gy dose, approximately 18-fold greater than the spontaneous mutation rate at this locus. DNA of the 20 confirmed null mutants were examined for alterations in the 25 kb DHFR gene by Southern blotting using a mixed probe that scans a continuous 34 kb of sequence. Deletions were the most prevalent induced change (18/20). Of the two point mutants, DNA sequencing showed that one carries a T:A-->G:C base substitution that changed Val135 to Gly in exon 5; carcinogen-induced reversion to a DHFR+ phenotype at a frequency of 3 x 10(-6) confirmed that the other also carried a single base change. The distribution of deletion break sites in the DHFR locus was non-random. In half of the mutants deletion break sites were clustered within a single 9.4 kb DHFR intron. Fine mapping within the gene of 14 mutants by Southern blotting localized 10 distinct break sites to small restriction fragments (< 2 kb). Results of this mapping indicated that three unequivocally independent mutants apparently arose by the same deletion and that others shared single break sites. Deletion sizes in these mutants were determined by Southern analysis using six cosmids and two plasmid probes that together span approximately 500 kb of sequence in the region of the DHFR locus. Probing blots with the cosmids defined a maximal deletion size in 15/17 mutants and confirmed that deletions extended < 150 kb in 11, a qualitatively different result from that previously obtained after a similar analysis of gamma-ray-induced DHFR- mutants. Since deletions were non-randomly distributed, typically less than replicon size and spared regions containing matrix attachment sites, the results suggest a model whereby alpha-particles induce double-strand breaks in accessible chromatin loops.