Clusters,factories and domains

During S-phase of the cell cycle, chromosomal DNA is replicated according to a complex replication timing program, with megabase-sized domains replicating at different times. DNA fibre analysis reveals that clusters of adjacent replication origins fire near-synchronously. Analysis of replicating cells by light microscopy shows that DNA synthesis occurs in discrete foci or factories. The relationship between timing domains, origin clusters and replication foci is currently unclear. Recent work, using a hybrid Xenopus/hamster replication system, has shown that when CDK levels are manipulated during S-phase the activation of replication factories can be uncoupled from progression through the replication timing program. Here, we use data from this hybrid system to investigate potential relationships between timing domains, origin clusters and replication foci. We suggest that each timing domain typically comprises several replicon clusters, which are usually processed sequentially by replication factories. We discuss how replication might be regulated at different levels to create this complex organisation and the potential involvement of CDKs in this process.     

Physical relationship between a gene and its origin of replication in Physarum polycephalum

Taking advantage of the natural synchrony of the S-phase within the plasmodium of Physarum polycephalum, we extracted highly synchronous DNA samples at precise time points in early S-phase. We then separated, by electrophoresis under denaturating conditions, the newly synthesized DNA strands of the nascent chromosomal replicons from the parental DNA template. Using the cDNA clone of the early-replicating LAV1-2 gene as a probe, we could establish by filter hybridization that the elongation rate of the replicon which encompasses this gene is constant, at a rate of 1 kb/min during the first 30 min of S-phase. The smallest replication intermediate (RI) that we have detected by probing with the LAV1-2 cDNA was 5 kb long, suggesting that the LAV1-2 gene and its origin of replication are closely associated within the chromosome. This procedure should facilitate the mapping of replication origins within the genome of Physarum.     

Temporal order of replication of mouse ribosomal RNA genes during the cell cycle

The timing of replication of mouse ribosomal RNA (rRNA) genes was determined in cultured cells by using 5-bromodeoxyuridine labeling of DNA coupled with synchronization. Two subclasses of rRNA genes were characterized that differ in their temporal order of replication during S-phase. Approximately half of the rDNA repeat units replicated primarily during the first half of S-phase and the other 50% preferentially in the second half. This difference in replication timing was consistently observed for the approximately 400 rDNA repeat units of NIH3T3 fibroblasts, but not for plasmid DNA containing fragments of rRNA genes that had been stably transfected into the genome of these cells. The rDNA fragments inserted into these transfection vectors contained the recently mapped origin of bidirectional replication with or without amplification-promoting sequences, or none of the above. Since the plasmid DNA that was integrated into the host cell genome replicated randomly during S-phase we conclude that the integrated plasmid DNA is either replicated from a chromosomal origin in the neighborhood of its integration site or that inserts are replicated from their own origins and the timing of replication is determined by flanking sequences. Received: 7 July 1997; in revised form: 1 October 1997; Accepted: 1 October 1997     

Site-specific initiation of DNA replication in Xenopus egg extract requires nuclear structure.

Previous studies have shown that Xenopus egg extract can initiate DNA replication in purified DNA molecules once the DNA is organized into a pseudonucleus. DNA replication under these conditions is independent of DNA sequence and begins at many sites distributed randomly throughout the molecules. In contrast, DNA replication in the chromosomes of cultured animal cells initiates at specific, heritable sites. Here we show that Xenopus egg extract can initiate DNA replication at specific sites in mammalian chromosomes, but only when the DNA is presented in the form of an intact nucleus. Initiation of DNA synthesis in nuclei isolated from G1-phase Chinese hamster ovary cells was distinguished from continuation of DNA synthesis at preformed replication forks in S-phase nuclei by a delay that preceded DNA synthesis, a dependence on soluble Xenopus egg factors, sensitivity to a protein kinase inhibitor, and complete labeling of nascent DNA chains. Initiation sites for DNA replication were mapped downstream of the amplified dihydrofolate reductase gene region by hybridizing newly replicated DNA to unique probes and by hybridizing Okazaki fragments to the two individual strands of unique probes. When G1-phase nuclei were prepared by methods that preserved the integrity of the nuclear membrane, Xenopus egg extract initiated replication specifically at or near the origin of bidirectional replication utilized by hamster cells (dihydrofolate reductase ori-beta). However, when nuclei were prepared by methods that altered nuclear morphology and damaged the nuclear membrane, preference for initiation at ori-beta was significantly reduced or eliminated. Furthermore, site-specific initiation was not observed with bare DNA substrates, and Xenopus eggs or egg extracts replicated prokaryotic DNA or hamster DNA that did not contain a replication origin as efficiently as hamster DNA containing ori-beta. We conclude that initiation sites for DNA replication in mammalian cells are established prior to S phase by some component of nuclear structure and that these sites can be activated by soluble factors in Xenopus eggs.     

Two early replicated,developmentally controlled genes of Physarum display different patterns of DNA replication by two-dimensional agarose gel electrophoresis

The nature of replication origins in eukaryotic chromosomes has been examined in some detail only in yeast, Drosophila, and mammalian cells. We have used highly synchronous cultures of plasmodia of the myxomycete Physarum and two-dimensional agarose gel electrophoresis to examine replication of two developmentally controlled, early replicated genes over time in S-phase. A single, discrete origin of replication was found within 4.8 kb of the LAV1-5 gene, which encodes a homolog of profilin. In contrast, the LAV1-2 gene appears to be surrounded by several origins. Two origins were identified within a 15 kb chromosomal domain and appear to be inefficiently used. Replication forks collide at preferred sites within this domain. These terminating structures are long lived, persisting for at least 2 h of the 3 h S-phase. Analysis of restriction fragment length polymorphisms (RFLPs) within the LAV1-2 domain indicates that replication of alleles on different parental chromosomes is a highly coordinated process. Our studies of the these two early replicated, plasmodium-specific genes indicate that both a fixed, narrow origin region and a broader zone containing two closely spaced origins of DNA replication occur in Physarum.     

Preferential binding of S-phase proteins to temporally-characteristic units of replication in Physarum polycephalum.

Synchronous plasmodia of Physarum polycephalum were pulse-labeled with 3H-thymidine in early or late portions of the S-phase, and the binding capacity of the replicated DNA for isochronous S-phase plasmodial proteins assessed by nitrocellulose filter binding assay. Replication units replicating during the first one-third of the S-phase preferentially bind cytosol proteins present in plasmodia engaged in early S DNA replication, while late S replicating DNA exhibits a corresponding preferential binding of plasmodial proteins present only in late S plasmodia. Temporally-characteristic nascent replication units were isolated by Hydroxylapatite column chromatography and were found to contain binding sites for isochronous proteins.     

Physical relationship between a gene and its origin of replication in Physarum polycephalum

Taking advantage of the natural synchrony of the S-phase within the plasmodium of Physarum polycephalum, we extracted highly synchronous DNA samples at precise time points in early S-phase. We then separated, by electrophoresis under denaturating conditions, the newly synthesized DNA strands of the nascent chromosomal replicons from the parental DNA template. Using the cDNA clone of the early-replicating LAV1-2 gene as a probe, we could establish by filter hybridization that the elongation rate of the replicon which encompasses this gene is constant, at a rate of 1 kb/min during the first 30 min of S-phase. The smallest replication intermediate (RI) that we have detected by probing with the LAV1-2 cDNA was 5 kb long, suggesting that the LAV1-2 gene and its origin of replication are closely associated within the chromosome. This procedure should facilitate the mapping of replication origins within the genome of Physarum.     

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.     

Identification and functional analysis of a human homologue of the monkey replication origin ors8

We previously isolated from African green monkey (CV-1) cells a replication origin, ors8, that is active at the onset of S-phase. Here, its homologous sequence (hors8, accession number: DQ230978) was amplified from human cells, using the monkey-ors8-specific primers. Sequence alignment between the monkey and the human fragment revealed a 92% identity. Nascent DNA abundance analysis, involving quantification by real-time PCR, indicated that hors8 is an active replication origin, as the abundance of nascent DNA from a genomic region containing it was 97-fold higher relative to a non-origin region in the same locus. Furthermore, the data showed that the hors8 fragment is capable of supporting the episomal replication of its plasmid, when cloned into pBlueScript (pBS), as assayed by the DpnI resistance assay after transfection of HeLa cells. A quantitative chromatin immunoprecipitation (ChIP) assay, using antibodies against Ku, Orc2, and Cdc6, showed that these DNA replication initiator proteins were associated in vivo with the human ors8 (hors8). Finally, nascent DNA abundance experiments from human cells synchronized at different phases of the cell cycle revealed that hors8 is a late-firing origin of DNA replication, having the highest activity 8 h after release from late G(1).     

Gene replication in the presence of aphidicolin

DNA replication in the nucleus of eukaryotic cells is restricted to the S phase of the cell cycle, and different genes are duplicated at specific times, according to a well-defined temporal order. We have investigated whether activation of initiation sites, in proximity to genes that are replicated in different portions of the S phase, could be detected when synchronized 10T1/2 cells were maintained in aphidicolin (APC), an inhibitor of DNA polymerases alpha and delta. Cells released from confluence arrest into medium containing 2 micrograms/mL APC progressed into the S phase, and nascent DNA accumulated during incubations of 24 and 32 h. Exposure to APC for 40 or 48 h resulted in growth of the radiolabeled DNA into larger molecules. Replicating DNA was isolated in CsCl gradients and probed with 32P-labeled gene probes for early-replicating genes (e.g., Ha-ras, mos, and myc) and a late-replicating gene (VH Ig). DNA replicated during the 24-h incubation in APC was enriched in Ha-ras gene sequences. The VH Ig gene did not replicate in cells incubated for as long as 56 h with APC. The myc and the mos genes were detected after 32 and 40 h in APC, respectively. The myc gene is replicated in 10T1/2 cells after Ha-ras but before mos. Therefore, the order of activation of these genes was conserved in the presence of APC. The delay in replication of myc and mos correlated well with the slowing of DNA replication by APC.     

Molecular analysis of the aberrant replication banding pattern on chromosome 15 in murine T-cell lymphomas

Cytogenetic techniques revealed an altered early replication banding pattern on the distal part of chromosome 15 in some murine T-cell lymphomas. This pattern reverted back to normal replication in somatic cell hybrids that had become non-tumorigenic after fusion of leukemic cells with normal fibroblasts. The altered banding pattern was correlated with malignancy. To investigate the molecular basis of the aberrant pattern in more detail, centrifugal elutriation of cells containing bromodeoxyuridine labeled DNA was used to prepare newly replicated DNA from selected intervals of the S-phase from tumor cells, as well as from hybrid cells with the revertant phenotype. These different DNA fractions were probed for DNA sequences distributed over the distal half of chromosome 15. Only two out of ten chromosome 15 specific genes tested showed a clear change in replication timing between the two different cell lines tested. These two genes were the lymphocyte antigen-6,Ly-6, and the neighboring thyroglobulin gene,Tgn, which replicated at the beginning of S in the tumor cells and later in S in the non-tumorigenic hybrid cells.by J.A. Huberman     

Assembly of newly replicated chromatin.

Mild staphylococcal nuclease digestions under isotonic conditions release fragments of a 200 Å diameter fiber from nuclei of Drosophila melanogaster tissue culture cells. These soluble fragments have high sedimentation coefficients (30–100S) and show tightly packed nucleosomes in the electron microscope. Under the same conditions, newly replicated chromatin is released as more slowly sedimenting fragments (14S). Within 20 min after DNA replication, the nascent chromatin gradually matures into compact supranucleosomal structures which are indistinguishable from bulk chromatin on the isokinetic sucrose gradients.We have used this fractionation technique to examine the question of the fate and assembly of the new histones. After short pulses with either ³⁵S-methionine or ³H-lysine, the radioactive histones do not co-sediment with the bulk chromatin but appear instead in the fractions where the newly replicated DNA is found. Furthermore, the various nascent histones appear in different fractions on the gradient: histones H3 and H4 in 10–15S structures, histones H2A and H2B in 15–50S structures and histone H1 in 30–100S structures. These results, together with the analysis of pulse and pulse-chase experiments of both nascent DNA and histones, strongly suggest that histones H3 and H4 are deposited first on the nascent DNA (during or slightly after the DNA is replicated), histones H2A and H2B are deposited next (2–10 min later) and histone H1 is deposited last (10–20 min after DNA replication). A high turnover 20,000 dalton protein is also associated with the newly replicated chromatin.     

Topoisomerase II plays an essential role as a swivelase in the late stage of SV40 chromosome replication in vitro.

The effects of topoisomerases I and II on the replication of SV40 DNA were examined using an in vitro replication system of purified proteins that constitutes the monopolymerase system. In the presence of the two topoisomerases, two distinct nascent DNAs were formed. One product arising from the replication of the leading template strand was approximately half the size of the template DNA, whereas the other product derived from the lagging template strand consisted of short DNAs. These products were synthesized from both SV40 naked DNA and SV40 chromosomes. For the replication of SV40 naked DNA, either topoisomerase I or II maintained replication fork movement and supported complete leading strand synthesis. When SV40 chromosomes were replicated with the same proteins, reactions containing only topoisomerase I produced shorter leading strands. However, mature size DNA products accumulated in reactions supplemented with topoisomerase II, as well as in reactions containing only topoisomerase II. In the presence of crude extracts of HeLa cells, VP-16, a specific inhibitor of topoisomerase II, blocked elongation of the nascent DNA during the replication of SV40 chromosomes. These results indicate that topoisomerase II plays a crucial role as a swivelase in the late stage of SV40 chromosome replication in vitro.     

High resolution analysis of replication foci by conventional fluorescent microscopy. I. A study of complexity and DNA content of the foci

Newly replicated DNA segments (RDS) have been shown to form discrete foci in the mammalian nucleus. Comparison of the number of such foci in formaldehyde-fixed cell nucleus with estimated number of simultaneously active replication forks (RF) suggests that each replication focus contains a cluster of about 10 to 20 closely associated RF. That implied the cluster of synchronously activated replicons as the primary unit of mammalian DNA replication. It still remains unclear whether such clustering of RF does mean adjacency of the replicons in a genomic location (structural clustering, model 1), or it arises from transient clustering of the replicons from different DNA domains at the functioning replication machinery (functional clustering, model 2). In this study we used conventional fluorescence microscopy of the hypotonically treated nuclei preparations to investigate replication foci at the optical resolution limit. Human K562 cells were labeled with 5'-iododeoxyuridine for different time periods. We synchronized the cell culture with hydroxyurea to be able to measure an average increase in DNA content during labeling period using DNA cytometry. Under these conditions, RDS appear as multiple small foci (mini-foci, MF). Further studies revealed that most of such mini-foci of replication represent optical diffraction spots, which are standard in size and different in brightness. The number of the "spots" and variation of their brightness mostly depend on the extent of hypotonic treatment. Flow cytometry control of the synchronized cells peak movement allowed us to measure mean DNA content of the MF. In case of most effective hypotonic treatment, a MF contains about 40 Kbp of labeled DNA, and the general number of the MF approaches the number of replicons that are simultaneously active in a given moment of S-phase. Influence of the effect of hypotonic treatment on overall number of observed MF suggests that replication foci in early and mid S-phase cells do not represent stable structures, but rather arise from functional clustering of comparatively distant replicating regions, thus supporting model 2.     

Transitions in replication timing in a 340 kb region of human chromosomal R-Band 1p36.1

DNA replication is initiated within a few chromosomal bands as normal human fibroblasts enter the S phase. In the present study, we determined the timing of replication of sequences along a 340 kb region in one of these bands, 1p36.13, an R band on chromosome 1. Within this region, we identified a segment of DNA (approximately 140 kb) that is replicated in the first hour of the S phase and is flanked by segments replicated 1-2 h later. Using a quantitative PCR-based assay to measure sequence abundance in size-fractionated (900-1,700 nt) nascent DNA, we mapped two functional origins of replication separated by 54 kb and firing 1 h apart. One origin was found to be functional during the first hour of S and was located within a CpG island associated with a predicted gene of unknown function (Genscan NT_004610.2). The second origin was activated in the second hour of S and was mapped to a CpG island near the promoter of the aldehyde dehydrogenase 4A1 (ALDH4A1) gene. At the opposite end of the early replicating segment, a more gradual change in replication timing was observed within the span of approximately 100 kb. These data suggest that DNA replication in adjacent segments of band 1p36.13 is organized differently, perhaps in terms of replicon number and length, or rate of fork progression. In the transition areas that mark the boundaries between different temporal domains, the replication forks initiated in the early replicated region are likely to pause or delay progression before replication of the 340 kb contig is completed.     

Fine Structural in Situ Analysis of Nascent DNA Movement Following DNA Replication

Nascent DNA (newly replicated DNA) was visualized in situ with regard to the position of the previously replicated DNA and to chromatin structure. Localization of nascent DNA at the replication sites can be achieved through pulse labeling of cells with labeled DNA precursors during very short periods of time. We were able to label V79 Chinese Hamster cells for as shortly as 2 min with BrdU; Br-DNA, detected by immunoelectron microscopy, occurs at the periphery of dense chromatin, at individual dispersed chromatin fibers, and within dispersed chromatin areas. In these regions DNA polymerase α was also visualized. After a 5-min BrdU pulse, condensed chromatin also became labeled. When the pulse was followed by a chase, a larger number of gold particles occurred on condensed chromatin. Double-labeling experiments, consisting in first incubating cells with IdU for 20 min, chased for 10 min and then labeled for 5 min with CldU, reveal CldU-labeled nascent DNA on the periphery of condensed chromatin, while previously replicated IdU-labeled DNA has been internalized into condensed chromatin. Altogether, these results show that the sites of DNA replication correspond essentially to perichromatin regions and that the newly replicated DNA moves rapidly from replication sites toward the interior of condensed chromatin areas.