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.     

Nuclear matrix support of DNA replication

In higher eukaryotic cells, DNA is tandemly arranged into 10(4) replicons that are replicated once per cell cycle during the S phase. To achieve this, DNA is organized into loops attached to the nuclear matrix. Each loop represents one individual replicon with the origin of replication localized within the loop and the ends of the replicon attached to the nuclear matrix at the bases of the loop. During late G1 phase, the replication origins are associated with the nuclear matrix and dissociated after initiation of replication in S phase. Clusters of several replicons are operated together by replication factories, assembled at the nuclear matrix. During replication, DNA of each replicon is spooled through these factories, and after completion of DNA synthesis of any cluster of replicons, the respective replication factories are dismantled and assembled at the next cluster to be replicated. Upon completion of replication of any replicon cluster, the resulting entangled loops of the newly synthesized DNA are resolved by topoisomerases present in the nuclear matrix at the sites of attachment of the loops. Thus, the nuclear matrix plays a dual role in the process of DNA replication: on one hand, it represents structural support for the replication machinery and on the other, provides key protein factors for initiation, elongation, and termination of the replication of eukaryotic DNA.     

PD184352 releases the regular hypoxic reversible DNA replication arrest in T24 cells

The oxygen dependent regulation of DNA replication is an essential property of proliferating mammalian cells. In human T24 bladder cancer cells, several hours of hypoxia leads to reversible DNA replication arrest and re-entry of oxygen induces a burst of replication initiation. This short communication provides strong evidence that PD184352 initiates DNA replication in living hypoxic cells without elevating the oxygen level. PD184352 releases the regular hypoxic replicon arrest, however, at a low intensity compared to the effect of reoxygenation. Moreover, PD184352 shows no effect on normoxically incubated as well as reoxygenated T24 cells.     

Replicon Clusters Are Stable Units of Chromosome Structure: Evidence That Nuclear Organization Contributes to the Efficient Activation and Propagation of S Phase in Human Cells

In proliferating cells, DNA synthesis must be performed with extreme precision. We show that groups of replicons, labeled together as replicon clusters, form stable units of chromosome structure. HeLa cells were labeled with 5-bromodeoxyuridine (BrdU) at different times of S phase. At the onset of S phase, clusters of replicons were activated in each of ~750 replication sites. The majority of these replication “foci” were shown to be individual replicon clusters that remained together, as stable cohorts, throughout the following 15 cell cycles. In individual cells, the same replication foci were labeled with BrdU and 5-iododeoxyuridine at the beginning of different cell cycles. In DNA fibers, 95% of replicons in replicon clusters that were labeled at the beginning of one S phase were also labeled at the beginning of the next. This shows that a subset of origins are activated both reliably and efficiently in different cycles.

The majority of replication forks activated at the onset of S phase terminated 45–60 min later. During this interval, secondary replicon clusters became active. However, while the activation of early replicons is synchronized at the onset of S phase, different secondary clusters were activated at different times. Nevertheless, replication foci pulse labeled during any short interval of S phase were stable for many cell cycles. We propose that the coordinated replication of related groups of replicons, that form stable replicon clusters, contributes to the efficient activation and propagation of S phase in mammalian cells.

     

Relationship of eukaryotic DNA replication to committed gene expression: general theory for gene control.

The historic arguments for the participation of eukaryotic DNA replication in the control of gene expression are reconsidered along with more recent evidence. An earlier view in which gene commitment was achieved with stable chromatin structures which required DNA replication to reset expression potential (D. D. Brown, Cell 37:359-365, 1984) is further considered. The participation of nonspecific stable repressor of gene activity (histones and other chromatin proteins), as previously proposed, is reexamined. The possible function of positive trans-acting factors is now further developed by considering evidence from DNA virus models. It is proposed that these positive factors act to control the initiation of replicon-specific DNA synthesis in the S phase (early or late replication timing). Stable chromatin assembles during replication into potentially active (early S) or inactive (late S) states with prevailing trans-acting factors (early) or repressing factors (late) and may asymmetrically commit daughter templates. This suggests logical schemes for programming differentiation based on replicons and trans-acting initiators. This proposal requires that DNA replication precede major changes in gene commitment. Prior evidence against a role for DNA replication during terminal differentiation is reexamined along with other results from terminal differentiation of lower eukaryotes. This leads to a proposal that DNA replication may yet underlie terminal gene commitment, but that for it to do so there must exist two distinct modes of replication control. In one mode (mitotic replication) replicon initiation is tightly linked to the cell cycle, whereas the other mode (terminal replication) initiation is not cell cycle restricted, is replicon specific, and can lead to a terminally differentiated state. Aberrant control of mitotic and terminal modes of DNA replication may underlie the transformed state. Implications of a replicon basis for chromatin structure-function and the evolution of metazoan organisms are considered.     

Initiating the Uninitiated: Replication of damaged DNA and Carcinogenesis

Cancer is a genetic disease and carcinogenesis is the process whereby the relevant genetic alterations are acquired. Environmental carcinogens may damage DNA to induce mutations and chromosomal aberrations as permanent heritable changes in the genome that initiate carcinogenesis. For many carcinogens initiation of carcinogenesis requires the initiation of DNA replication suggesting that genetic alterations are fixed in the genome during replication of damaged DNA. It is of great interest to understand the mechanisms whereby carcinogen-induced damage to DNA causes mutations and chromosomal aberrations, and how cells may resist such events. It is clear now that cells express a complex repertoire of responses to DNA damage including several pathways of DNA repair and cell cycle checkpoints that protect against carcinogenesis. This commentary is concerned with the protective influence of DNA damage checkpoints that delay or arrest progression through the cell division cycle and especially with the responses of S phase cells to the environmental carcinogens UV and benzo[a]pyrene diolepoxide I (BPDE). Recent studies indicate that checkpoint responses may act at the very point of replication of damaged DNA to slow DNA chain elongation, inhibit replicon initiation, and suppress initiation of carcinogenesis.     

Similar replicon properties of higher plant cells with different S periods and genome sizes

Root meristematic cells of nine unrelated diploid higher plants with genome sizes that differ 82-fold and with S periods that differ 4-fold have similar replicon sizes and single replication fork rates that average 22 μm and 8 μm/h respectively. The average replicon size of 22 μm is near the 18 μm obtained by extrapolation of measurements, taken from DNA fiber autoradiograms, to zero pulse time with [³H]thymidine. The data suggest that the duration of S is determined by the minimal number of replicon families that function sequentially during DNA replication.     

Activation of replicon origins as a possible target for cytokinins in shoot meristems of Sinapis

Whilst the cytokinins are important promoters of plant cell division in vitro and in vivo, their mode of action remains unknown. Here we report the results of a study showing that a single application of a low dose of a cytokinin to the shoot apical meristem of Sinapis alba L. activates new replicon origins in chromosomal DNA, resulting in the halving of replicon size, and synchronizes the activation of replicon origins. These effects cause a 3.5-fold shortening of the duration of chromosomal DNA replication (S phase of the cell cycle). We hypothesize that one of the proteins involved in the initiation of DNA replication is a target for cytokinins.Abbreviations BA N⁶-benzyladenine - F fork rate - R size ofmost replicons - Rs time taken for replicon to replicate its allotedDNA - TdR [³H]thymidine - Ts duration of S phase C. Houssa is grateful to I.R.S.I.A. for the award of a research fellowship. This research was supported by the Belgian Government (Concerted Research Actions and FRFC).     

Replication fork stalling by bulky DNA damage: localization at active origins and checkpoint modulation

The integrity of the genome is threatened by DNA damage that blocks the progression of replication forks. Little is known about the genomic locations of replication fork stalling, and its determinants and consequences in vivo. Here we show that bulky DNA damaging agents induce localized fork stalling at yeast replication origins, and that localized stalling is dependent on proximal origin activity and is modulated by the intra-S-phase checkpoint. Fork stalling preceded the formation of sister chromatid junctions required for bypassing DNA damage. Despite DNA adduct formation, localized fork stalling was abrogated at an origin inactivated by a point mutation and prominent stalling was not detected at naturally-inactive origins in the replicon. The intra-S-phase checkpoint contributed to the high-level of fork stalling at early origins, while checkpoint inactivation led to initiation, localized stalling and chromatid joining at a late origin. Our results indicate that replication forks initially encountering a bulky DNA adduct exhibit a dual nature of stalling: a checkpoint-independent arrest that triggers sister chromatid junction formation, as well as a checkpoint-enhanced arrest at early origins that accompanies the repression of late origin firing. We propose that the initial checkpoint-enhanced arrest reflects events that facilitate fork resolution at subsequent lesions.     

In vivo relations between pAMβ1-encoded type I topoisomerase and plasmid replication

A number of large extrachromosomal elements encode prokaryotic type I topoisomerases of unknown functions. Here, we analysed the topoisomerase Topβ encoded by the Gram-positive broad-host-range plasmid pAMβ1. We show that this enzyme possesses the DNA relaxation activity of type I topoisomerases. Interestingly, it is active only on plasmids that use DNA polymerase I to initiate replication, such as pAMβ1, and depends on the activity of this polymerase. This is the first example, to our knowledge, of prokaryotic type I topoisomerase that is specific for a given type of replicon. During pAMβ1 replication in Bacillus subtilis cells, Topβ promotes premature arrest of DNA polymerase I, ≈190 bp downstream of the replication initiation point. We propose that Topβ acts on the early replication intermediates of pAMβ1, which contain D-loops formed by DNA polymerase I-mediated strand displacement. The possible role of the resulting DNA Pol I arrest in plasmid replication is discussed.     

Replicon size and rate of DNA replication fork movement are correlated in grasses

In eukaryotes, most nuclear DNA replication proceeds bidirectionally from multiple origins of replication. A unit of DNA, replicated by two replication forks from a single origin, is termed a replicon. Using results from DNA fiber autoradiography we show a novel positive correlation between replicon size and the rate of replication fork movement in root meristem nuclei of 13 grass species. Although there is interspecific variation in replicon size, it is balanced by similar variation in the rate of replication fork progression.     

Chromosomal replication initiates and terminates at random sequences but at regular intervals in the ribosomal DNA of Xenopus early embryos.

We have analysed the replication of the chromosomal ribosomal DNA (rDNA) cluster in Xenopus embryos before the midblastula transition. Two-dimensional gel analysis showed that replication forks are associated with the nuclear matrix, as in differentiated cells, and gave no evidence for single-stranded replication intermediates (RIs). Bubbles, simple forks and double Ys were found in each restriction fragment analysed, showing that replication initiates and terminates without detectable sequence specificity. Quantification of the results and mathematical analysis showed that the average rDNA replicon replicates in 7.5 min and is 9-12 kbp in length. This time is close to the total S phase duration, and this replicon size is close to the maximum length of DNA which can be replicated from a single origin within this short S phase. We therefore infer that (i) most rDNA origins must be synchronously activated soon in S phase and (ii) origins must be evenly spaced, in order that no stretch of chromosomal DNA is left unreplicated at the end of S phase. Since origins are not specific sequences, it is suggested that this spatially and temporally concerted pattern of initiation matches some periodic chromatin folding, which itself need not rely on DNA sequence.     

Selective and synchronous activation of early-S-phase replicons of Ehrlich ascites cells.

Twelve-hour exposure of G1 Ehrlich ascites cells to controlled hypoxia (200 ppm of O2 at 1 bar) suppressed replicon initiation. Synchronous cycling, beginning with a normal S phase, was released by reoxygenation immediately. The addition of cycloheximide at reoxygenation largely resuppressed, after a short initial burst, succeeding replicon initiations. Alkaline sedimentation analysis of growing daughter strand DNA, DNA fiber autoradiography, and analysis of the newly formed DNA demonstrated that normal chain growth and DNA maturation (replicon termination) in the initially activated replicons continued in the presence of cycloheximide. After 2 to 3 h, a low level of cycloheximide-insensitive background replication emerged out of the then-ebbing single surge of activity of the initially released replicons.     

Cloning, sequencing, and functional analysis of a Marek''s disease virus origin of DNA replication.

Previously, we isolated a replicon from a defective Marek's disease virus (MDV), analogous to defective herpes simplex viruses (amplicons). Defective viruses contain cis-acting elements required for DNA synthesis and virus propagation such as an origin of DNA replication and a packaging-cleavage signal site. In this report, the MDV replicon was utilized to locate an origin of MDV DNA replication. A comparison of MDV replicon sequences with other herpesvirus replication origin sequences revealed a 90-bp sequence containing 72% identity to the lytic origin (oris) of herpes simplex virus type 1. This 90-bp sequence displayed no similarity to betaherpesvirus or gammaherpesvirus replication origins. The 90-bp sequence is arranged as an imperfect palindrome centered around an A+T-rich region. This sequence also contains a 9-bp motif (5'CGTTCGCAC3') highly conserved in alphaherpesvirus replication origins. To test functionality of the 90-bp putative MDV replication origin, we conducted DpnI replication assays with subclones generated from the 4-kbp MDV replicon. A 700-bp MDV replicon subfragment containing the 90-bp putative MDV replication origin sequence is capable of replicating in chicken embryo fibroblast cells cotransfected with helper virus DNA. In conclusion, we identified a functional origin of DNA replication in MDV. Similarity of MDV origin sequences to those of alphaherpesviruses supports the current contention that MDV is more closely related to alphaherpesviruses than to gammaherpesviruses.     

The effects of different thymidine concentrations on DNA replication in pea-root cells synchronized by a protracted 5-fluorodeoxyuridine treatment

Single-cell and DNA fiber autoradiography, cytophotometry and velocity sedimentation in alkaline sucrose gradients were used to analyse DNA replication and nascent replicon maturation in 5-fluorodeoxyuridine (FUdR)-synchronized cells of Pisum sativum. The replicon size was not significantly changed by the protracted FUdR treatment. When the synchronized cells were released from the inhibitor, labeled with [³H]TdR for 30 min, and chased in medium containing 1 × 10⁻⁶ M or lower concentrations of cold thymidine, DNA replication stopped after approx. 25% of the genome had replicated, and the nascent strands failed to grow above 9–12 × 10⁶ D single-stranded (ss) DNA. When the cells were chased in medium with 1 × 10⁻⁵ M cold thymidine, the DNA content of the labeled cells steadily increased with time and the size of the nascent molecules grew continuously until replicon size was achieved; then they were accumulated at replicon size until the cells arrived in late S or G2. When the FUdR-synchronized cells were chased in medium containing 1 × 10⁻⁴ M cold thymidine, the size of the nascent strands increased continuously with time, indicating that some neighbouring nascent replicons were joined as soon as they completed their replication. These observations led us to postulate that in FUdR-synchronized cells the rates of chain elongation, cell progression through the S phase and nascent replicon maturation are controlled by thymidine availability.     

A novel replicon occurring naturally in Escherichia coli is a phage-plasmid hybrid.

A novel DNA replicon in Escherichia coli was identified. It is the smallest natural isolate (1282 bp) found so far. In the presence of phage M13 it grows as a filamentous single-stranded DNA phage. Contrary to previously identified mini-phages this replicon displays sequence homology only to parts of the M13 viral and complementary strand origin. In the absence of M13 this DNA replicates autonomously. The only gene (arp) of the replicon encodes a 32-kd protein, which is essential for autonomous replication. The host rep gene required for replication of single-stranded DNA phages is dispensable. Distinct replication mechanisms are thus involved during growth as defective phage or as autonomous plasmid.     

Early replication signals in nuclei of Chinese hamster ovary cells

Summary DNA replication sites generally known as replicon domains were resolved as individual replication signals in interphase nuclei of permeabilized Chinese hamster ovary cells by immunofluorescent microscopy. Biotin-11-dUTP was utilized as a tool to label newly replicated DNA in permeable cells and to study the distribution of nascent DNA in pulselabel and in pulsechase experiments. Active sites of DNA replication were visualized in exponentially growing cells and in synchronized cultures throughout the S phase. Fluorescent images of replication sites were analyzed by standard fluorescense microscopy and in three dimensions by confocal laser scanning microscopy. The rapid increase in number of discrete foci of newly replicated DNA is an indication that DNA synthesis starts at limited number of sites in mammalian nuclei rather than at thousands of foci at the same time.     

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 ( approximately 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.