Fiber DNA studies of premeiotic mouse spermatogenesis

The technique of fiber DNA measurement was used to study the possibility that the lengthening of the DNA “S” phase previously reported for mouse premeiotic spermatogonia was due to a reduced number of initiation sites. The mean replicon size of neonatal mouse preleptotene cells was similar to sizes reported for adult mouse somatic cells. A slow rate of DNA chain growth was observed in all cells from day 1 through days 10–12 of age. It was felt that the meiotic entry in male mouse germ cells may involve a slower replication fork rate and other factors which increased the time between activation and replication of replicon families.     

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.     

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).     

Effects of psoralen on replicon size and mean rate of DNA synthesis in partially synchronized cells of Pisum sativum L.

We have examined by fibre autoradiography the spacing of replicons in pea root meristems during synchronized entry into S phase from arrest at the G1/S boundary. Pretreatment with the DNA cross-linking agent, psoralen, produces a marked shortening of replicon spacing, suggesting that premature arrest of the replication fork results in the recruitment of additional initiation points within a given replicon family. This is discussed in relation to models for the control of DNA replication.     

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 μm 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 μm 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. Received: 11 October 1999 / Accepted: 23 December 1999     

Correlations between sister chromatid exchange frequencies and replicon sizes: A model for the mechanism of SCE production

Baseline sister chromatid exchanges (SCEs) increase as a function of average replicon size in a variety of human and Chinese hamster cell lines. This observation is the basis for a model in which SCEs are generated by errors in the unravelling of daughter double helices by topo isomerases. These errors cause exchanges behind the replication fork, not at the fork as several current models assume. This model also provides an explanation for SCEs induced when residual DNA damage that has been replicated interferes with the normal processes of unravelling the daughter strands.     

The human Tim/Tipin complex coordinates an Intra-S checkpoint response to UV that slows replication fork displacement

UV-induced DNA damage stalls DNA replication forks and activates the intra-S checkpoint to inhibit replicon initiation. In response to stalled replication forks, ATR phosphorylates and activates the transducer kinase Chk1 through interactions with the mediator proteins TopBP1, Claspin, and Timeless (Tim). Murine Tim recently was shown to form a complex with Tim-interacting protein (Tipin), and a similar complex was shown to exist in human cells. Knockdown of Tipin using small interfering RNA reduced the expression of Tim and reversed the intra-S checkpoint response to UVC. Tipin interacted with replication protein A (RPA) and RPA-coated DNA, and RPA promoted the loading of Tipin onto RPA-free DNA. Immunofluorescence analysis of spread DNA fibers showed that treating HeLa cells with 2.5 J/m(2) UVC not only inhibited the initiation of new replicons but also reduced the rate of chain elongation at active replication forks. The depletion of Tim and Tipin reversed the UV-induced inhibition of replicon initiation but affected the rate of DNA synthesis at replication forks in different ways. In undamaged cells depleted of Tim, the apparent rate of replication fork progression was 52% of the control. In contrast, Tipin depletion had little or no effect on fork progression in unirradiated cells but significantly attenuated the UV-induced inhibition of DNA chain elongation. Together, these findings indicate that the Tim-Tipin complex mediates the UV-induced intra-S checkpoint, Tim is needed to maintain DNA replication fork movement in the absence of damage, Tipin interacts with RPA on DNA and, in UV-damaged cells, Tipin slows DNA chain elongation in active replicons.     

Coordinated leading- and lagging-strand synthesis at the Escherichia coli DNA replication fork. I. Multiple effectors act to modulate Okazaki fragment size.

The coordinated action of many enzymatic activities is required at the DNA replication fork to ensure the error-free, efficient, and simultaneous synthesis of the leading and lagging strands of DNA. In order to define the essential protein-protein interactions and model the regulatory pathways that control Okazaki fragment synthesis, we have reconstituted the replication fork of Escherichia coli in vitro in a rolling circle-type DNA replication system. In this system, in the presence of the single-stranded DNA binding protein, the helicase/primase function on the lagging-strand template is provided by the primosome, and the synthesis of DNA strands is catalyzed by the DNA polymerase III holoenzyme. These reconstituted replication forks synthesize equivalent amounts of leading- and lagging-strand DNA, move at rates comparable to those measured in vivo (600-800 nucleotides/s at 30 degrees C), and can synthesize leading strands in the range of 150-500 kilobases in length. Using this system, we have studied the cycle of Okazaki fragment synthesis at the replication fork. This cycle is likely to have several well defined decision points, steps in the cycle where incorrect execution by the enzymatic machinery will result in an alteration in the product of the reaction, i.e. in the size of the Okazaki fragments. Since identification of these decision points should aid in the determination of which of the enzymes acting at the replication fork control the cycle, we have endeavored to identify those reaction parameters that, when varied, alter the size of the Okazaki fragments synthesized. Here we demonstrate that some enzymes, such as the DnaB helicase, remain associated continuously with the fork while others, such as the primase, must be recruited from solution each time synthesis of an Okazaki fragment is initiated. We also show that variation of the concentration of the ribonucleoside triphosphates and the deoxyribonucleoside triphosphates affects Okazaki fragment size, that the control mechanisms acting at the fork to control Okazaki fragment size are not fixed at the time the fork is assembled but can be varied during the lifetime of the fork, and that alteration in the rate of the leading-strand DNA polymerase cannot account for the effect of the deoxyribonucleoside triphosphates.     

DNA fiber replication during a morphogenetic switch in the shoot meristematic cells of a higher plant

Chromosomal DNA fiber replication was investigated in the shoot meristem of mustard plants during the morphogenetic transition from the leaf-forming (vegetative) to the flower-forming (evoked) condition. The replicon size, determined using the modal class, was 15 micron in the vegetative meristem and shifted to 7.5 micron in the evoked meristem. The average fork rate was 1.05 micron.h-1 in the vegetative meristem and only slightly increased to 1.55 micron.h-1 during the morphogenetic switch. Replicon activation was asynchronous but the pattern of activation of replicons was the same in both kinds of meristems. Thus the shortening of the S phase at the floral transition in mustard was essentially achieved by an increase of the number of replicon origins per unit length of DNA. These results are similar to those obtained in amphibian and Drosophila embryogenesis.     

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.     

Mean rate of DNA replication and replicon size in the shoot apex ofSilene coelirosa L. During the initial 120 minutes of the first day of floral induction

Summary 28-day-old plants ofSilene coeli-rosa were exposed, at 1,700 hours, to long day (LD) conditions comprising light of low fluence rate provided by tungsten bulbs, or maintained in darkness as short day (SD) controls. All plants were exposed at 1,700 hours to tritiated-(methyl-³H)-thymidine for 30, 45, 60, 90, or 120 minutes. Apical domes were isolated and prepared as fiber autoradiographs from which replicon size and rates of DNA replication, per single replication fork were recorded. In SD, replicon size was between 15–20 m and exposure to LD conditions altered neither replicon size nor the pattern of deployment of replicons during S-phase relative to the SD controls. However, the mean rate of replication in LD was 8.7 m h^–1 compared with 5.2 m h^–1 in SD. Thus, exposure to LD resulted in a 1.7-fold increase in the rate of DNA replication relative to the SD controls. This rapid increase in replication rate, detectable within 30 minutes of the start of the LD is discussed in relation to changes known to occur to the cell cycle inSilene during the first day of floral induction.     

Inhibition and recovery of the rate of DNA synthesis in V79 Chinese hamster cells following ultraviolet light irradiation. Variation in the rate of movement of the replication fork

Chinese hamster fibroblasts (V79 cell line) exhibit the phenomenon of recovery of DNA synthesis from the initial inhibition observed after ultraviolet light irradiation, in the absence of significant excision of pyrimidine dimers. In an attempt to determine whether the initial inhibition and subsequent recovery can be accounted for by parallel variations in the rate of movement of the replication fork, the cells were pulse-labeled with radioactive bromodeoxyuridine at different times following irradiation and their DNA centrifuged in neutral CsCl density gradients. When DNA synthesis inhibition was at a maximum, an accumulation of DNA, of density intermediate between hybrid and nonsubstituted DNA, was noticed in the density-distribution profiles. This intermediate-density DNA has been previously shown to correspond to fork structures, and thus it seems that inhibition of DNA synthesis after irradiation is to a great extent caused by the forks pausing at the lesions. Later on, when recovery in the rate of DNA synthesis occurs, the accumulation of intermediate-density DNA is no longer observed. The density distribution of DNA along the gradient can thus provide an estimate of the rate of movement of the replication fork, and the results indicate that most of the variation in the overall rate of DNA synthesis can be accounted for by a parallel variation in the rate of fork movement.     

Regulation of DNA replication on subchromosomal units of mammalian cells

The regulation of DNA replication at a subchromosomal level in mammalian cells has been investigated. DNA fiber autoradiographs were prepared from mouse L-929 cells pulse labeled with (3H)thymidine. Initiation events and subsequent chain growth occurring over short stretches (up to three replication units in length) of chromosomal DNA were analyzed. The results show that adjacent units usually initiate replication synchronously and that this synchrony is related to the proximity of initiation sites. In addition, adjacent units are of similar size and the rates of replication fork progression within units and on adjacent units are similar. The rate of fork progression increases with increasing replication unit size. Finally, no evidence for fixed termination sites for the units has been found. These observations suggest that despite large variations in size of replication units, timing of initiation events, and rates of fork progression found in chromosomal DNA as a whole, these processes are closely regulated within subchromosomal clusters of active replication units.     

Changes in the rate of DNA replication fork movement during S phase in mammalian cells.

DNA fiber autoradiography was used to measure the rate of replication fork movement and the size of replication units as a function of time during the S phase of synchronized Chinese hamster ovary cells. The rate of fork movement increased by about threefold from early S to later S phase, with the most dramatic change occurring in the first hour of S phase. On the other hand, the size of replication units did not vary significantly during S phase.     

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

Summary. 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 µm; and the fork rate, 1.17 µm/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.Correspondence and reprints: Department of Biology, Universitaire Instelling Antwerpen, Universiteitsplein 1, 2610 Wilrijk, Belgium.     

The effect of staphylococcal alpha-toxin on DNA replication in human cells]

The influence of Staphylococcus alpha-toxin has been investigated on the duration of S-phase of lymphocyte mitotic cycle and on DNA replication in human fibroblasts in vitro. The duration of the S-phase of lymphocytes was measured by counting labeled metaphases and by making replication curves. Alpha-toxin in a dose of 3 micrograms/ml enhances the onset of S-phase, which is inhibited at a dose of 33 micrograms/ml of alpha-toxin. The action of alpha-toxin resulted in a decreased rate of replication fork and in a progressive activation of replicon groups. This effect was most prominent at 33 micrograms/ml of alpha-toxin. The data obtained allow to suggest that immunodeficiency of the second order, so characteristic of the staphylococcal sepsis, may be due, in many respects, to suppression of DNA replication.     

Replicon properties of chromosomal DNA fibers and the duration of DNA synthesis of sunflower root-tip meristem cells at different temperatures

Chromosomal DNA fiber autoradiography was used to examine the replicon properties of root-tip meristem cells of Helianthus annuus intact seedlings grown at temperatures from 10 to 38° C and those of root-tip cells grown in vitro at 23°. The average replicon size was approximately 22 m and it did not change with temperature nor when the roots were grown in culture. The average fork rate was 6 m/h at 10° and it rose gradually to 12 m/h at 38°. The responses of replication fork movement and of the duration of S to temperature were of three types: those in which change in fork rate was primarily (more than 90%) responsible for change in the duration of S, those in which the fork rate remained constant while S increased nearly twofold, and those in which the duration of S increased even though the replication forks were moving faster. The first type of response listed was observed at temperatures from 20 to 35°, the second type listed was observed at 10 to 15°, and the third, was produced at 38°.