Premature replication of late S period DNA regions in early S nuclei transferred to late S cytoplasm by fusion in Physarum polycephalum.

Fusion of a late S period plasmodium of Physarum polycephalum to an early S period plasmodium causes premature replication of late S replicating regions in the nuclei of the early S plasmodium. The extent of ahead-of-schedule replication of late S replicating regions in early S period nuclei increases to a plateau of 16-20% for fusions with 40-70 min of phase difference, then declines for larger phase differences. The stimulatory factors for late S replicative units are present only in late S plasmodia and appear to act only on late S regions. Once replicated, early S replicating regions are not stimulated to replicate again by fusion to a plasmodium entering the S period. Our data do not discriminate between anti-termination of replication by factors of stop sites on long replicons, and a sequential initiation of replication on new, possibly non-adjacent regions, but does provide evidence that the stimulatory factors are distinct from one another and specific for certain target replicative units.     

Analysis of the replication pattern of Chinese hamster chromosomes using 5-bromodeoxyuridine suppression of 33258 Hoechst fluorescence

Chinese hamster fibroblasts were synchronized and given 5-bromodeoxyuridine for DNA synthesis except during one hour of the S phase when thymidine was present in the medium. In the next mitosis, chromosomes stained with 33258 Hoechst were banded in appearance when photographed by fluorescence microscopy. The bright regions corresponded to the chromosome segments replicated during the thymidine exposure in the S phase. The segments replicated together during any one hour produced three distinct patterns which were characteristic of early, middle, and late S phase. Most of the fluorescent regions corresponded in size and position with G-bands of these chromosomes. There was no correlation between the staining behavior of a band in G-band procedure and its time-of-replication, i.e., both light and dark G-bands were replicated during early, middle, and late S phase. However, it appears that all of the DNA within a single band is replicated together within one third of the S phase.     

Chromosomes of a cell line of Dipodomys panamintinus (kangaroo rat) a banding and autoradiographic study

The chromosomes of an established cell line of Dipodomys panamintinus have been characterised in terms of their C, G and Q banding patterns, and the distributions of silver grains in autoradiographs of chromosomes labelled in early or late S phase. No relationship could be established between C, G or Q banding regions of chromosomes and a particular S phase time of replication of the DNA in these banded regions. The implication of this result to the concept of heterochromatin is discussed.     

Recovery of acetylcholinesterase and of its multiple molecular forms in motor end-plate-free and motor end-plate-rich regions of mouse striated muscle, after irreversible inactivation by an organophosphorus compound (methyl-phosphorothiolate derivative)

Most of mouse diaphragm muscle acetylcholinesterase (AChE) is irreversibly inhibited after a single intraperitoneal injection of a methyl-phosphorothiolate derivative (MPT), an organophosphorus compound which phosphorylates the active site. The muscle recovers its AChE (de novo synthesis) and we studied the time course of reappearance of AChE and its multiple active molecular forms. After inhibition, there is an initial (3 to 15 hr) rapid recovery of total AChE (which evolves from 20-28% to 50-60% of the control values), followed by a slow phase of AChE return. After 3 days, the recovery is still incomplete (reaching 70-80% of control values). Among the main molecular forms present in diaphragm muscle (16 S, 10 S and 4 S, accompanied by minor components), the 16 S and 10 S forms are the most sensitive to MPT treatment. During the rapid initial phase of AChE recovery, the absolute rate of recovery of the 4 S form is faster than for the other forms with a correspondingly much higher relative proportion to total AChE. These observations are consistent with the hypothesized precursor role of the 4 S form. The 16 S form, which is found concentrated in the motor end-plate (MEP)-rich regions and in low amounts in MEP-free regions, is similarly partially recovered in both regions, suggesting that there is 16 S biosynthesis not only in the MEP-rich regions but also in the MEP-free regions.     

Organization of DNA replication in Physarum polycephalum. Attachment of origins of replicons and replication forks to the nuclear matrix.

We have investigated the attachment of the DNA to the nuclear matrix during the division cycle of the plasmodial slime mold Physarum polycephalum. The DNA of plasmodia was pulse labelled at different times during the S phase and the label distribution was studied by graded DNase digestion of the matrix-DNA complexes prepared from nuclei isolated by extraction with 2 M NaCl. Pulse labelled DNA was preferentially recovered from the matrix bound residual DNA at any time of the S phase. Label incorporated at the onset of the S phase remained preferentially associated with the matrix during the G2 phase and the subsequent S phase. The occurrence of the pulse label in the matrix associated DNA regions was transiently elevated at the onset of the subsequent S phase. Label incorporated at the end of the S phase was located at DNA regions which, in the G2 phase, were preferentially released from the matrix by DNase treatment. From the results and previously reported data on the distribution of attachment sites it can be concluded that origins of replicons or DNA sites very close to them are attached to the matrix during the entire nuclear cycle. The data further indicate that initiations of DNA replication occur at the same origins in successive S phases. Replicating DNA is bound to the matrix, in addition, by the replication fork or a region close to it. This binding is loosened after completion of the replication.     

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

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

S Phase Progression in Human Cells Is Dictated by the Genetic Continuity of DNA Foci

DNA synthesis must be performed with extreme precision to maintain genomic integrity. In mammalian cells, different genomic regions are replicated at defined times, perhaps to preserve epigenetic information and cell differentiation status. However, the molecular principles that define this S phase program are unknown. By analyzing replication foci within discrete chromosome territories during interphase, we show that foci which are active during consecutive intervals of S phase are maintained as spatially adjacent neighbors throughout the cell cycle. Using extended DNA fibers, we demonstrate that this spatial continuity of replication foci correlates with the genetic continuity of adjacent replicon clusters along chromosomes. Finally, we used bioinformatic tools to compare the structure of DNA foci with DNA domains that are seen to replicate during discrete time intervals of S phase using genome-wide strategies. Data presented show that a major mechanism of S phase progression involves the sequential synthesis of regions of the genome because of their genetic continuity along the chromosomal fiber.     

Chromosome banding and DNA replication studies on a cell line of Dipodomys merriami

The chromsomes of a cell line of Dipodomys merriami are described in terms of their C-, G- and Q-banding patterns. Studies on the buoyant density of DNA made at different times in the S phase show that the replication of HSα satellite and AT-rich main band DNA occurs preferentially late in the S phase, whereas MS satellite and GC-rich main band DNAs are replicated early in the S phase. Autoradiographic studies of chromosomes labelled early or late in the S phase are used to relate the banding patterns nf particular chromosome regions to the fraction of DNA which they may contain.     

A position effect on the time of replication origin activation in yeast.

The chromosomes of eukaryotes are characterized by the mosaic nature of their replication--large regions of DNA that replicate early in S phase are interspersed with regions that replicate late. This pattern of early and late synthesis appears to be the consequence of a temporal program that activates replication origins at different times. The basis of this temporal regulation in the yeast S. cerevisiae has been investigated by changing the chromosomal locations of two origins, one activated early in the S phase (ARS1) and one activated late (ARS501). We show that the cis-acting information controlling time of activation can be separated from the element that determines origin function. For the ARS501 origin, late activation appears to be a consequence of its proximity to the telomere.     

A yeast origin of replication is activated late in S phase

The mechanism that causes large regions of eukaryotic chromosomes to remain unreplicated until late in S phase is not understood. We have found that 67 kb of telomere-adjacent DNA at the right end of chromosome V in S. cerevisiae is replicated late in S phase. An ARS element in this region, ARS501, was shown by two-dimensional gel analysis to be an active origin of replication. Kinetic analyses indicate that the rate of replication fork movement within this late region is similar to that in early replicating regions. Therefore, the delayed replication of the region is a consequence of late origin activation. The results also support the idea that the pattern of interspersed early and late replication along the chromosomes of higher eukaryotes is a consequence of the temporal regulation of origin activation.     

Replicating by the clock

The eukaryotic genome is divided into well-defined DNA regions that are programmed to replicate at different times during S phase. Active genes are generally associated with early replication, whereas inactive genes replicate late. This expression pattern might be facilitated by the differential restructuring of chromatin at the time of replication in early or late S phase.     

Temporal order of replication of genes responsible for hypoxanthine phosphoribosyl transferase and Na+/K+ ATPase in chemically transformed human fibroblasts

The cytotoxic and mutagenic effects of a direct perturbation of DNA during various portions of the DNA synthetic period (S phase) of a chemically induced, transformed line (Hut-11A cells) derived from diploid human skin fibroblasts were examined. The cells were synchronized by a period of growth in low serum with a subsequent blockage of the cells at the G1/S boundary by hydroxyurea. This method resulted in over 90% synchrony, although approximately 20% of the cells were noncycling. Synchronized cells were treated for each of four 2-h periods during the S phase with 5-bromodeoxyuridine (BrdU) followed by irradiation with near-ultraviolet (UV). The BrdU-plus-irradiation treatment was cytotoxic and mutagenic, while treatment with BrdU alone or irradiation alone was neither cytotoxic nor mutagenic. The cytotoxicity was dependent upon the periods of S phase during which treatment was administered. The highest lethality was observed for treatment in early to middle S phase, particularly in the first 2 h of S phase, whereas scare lethality was observed in late S phase. The BrdU-plus-irradiation treatment induced ouabain- and 6-thioguanine-resistant mutants, while BrdU alone or irradiation alone was not mutagenic. Ouabain-resistant mutants were induced during early S phase by the BrdU-plus-irradiation treatment. 6-Thioguanine-resistant mutants, however, were induced during middle to late S phase. These results suggest that a certain region or regions in the DNA of Hut-11A cells, as designated by their specific temporal relationship in the S phase, may be more sensitive to the DNA perturbation by BrdU treatment plus near-UV irradiation for cell survival and that gene(s) responsible for Na+/K+ ATPase is replicated during early S phase and gene(s) for hypoxanthine phosphoribosyl transferase is replicated during middle to late S phase.     

Dependence of lethality induced by a direct DNA perturbation of synchronized human diploid fibroblasts on different periods of the DNA synthetic period (S Phase)

The cytotoxic effect of a direct perturbation of DNA during various portions of the DNA synthetic period (S phase) of cultured human diploid fibroblasts was examined. The cells were synchronized by a period of growth in low serum with a subsequent blockage of the cells at the G1/S boundary by hydroxyurea. This method resulted in over 90% synchrony, although approximately 20% of the cells were noncycling. Synchronized cells were treated for each of four 2-hour periods during the S phase with 5-bromodeoxyuridine (0.1–10 μM), followed by irradiation with near-UV (5–10 min). The 5-bromodeoxyuridine-plus-irradiation treatment was cytotoxic, while treatment with 5-bromodeoxyuridine alone or irradiation alone was not cytotoxic. The cytotoxicity was dependent upon the periods of S phase during which treatment was administered. The highest lethality was observed for treatment in early to middle S phase, particularly in the first 2 hours of S phase, whereas scarce lethality was observed in late S phase. The extent of substitution of 5-bromodeoxyuridine for thymidine in newly synthesized DNA was similar in every period of the S phase. Furthermore, no specific period during S phase was significantly more sensitive to treatment with respect to DNA damage, as determined by an induction of unscheduled DNA synthesis. These results suggest that a certain region or regions in the DNA of human diploid fibroblasts, as designated by their specific temporal relationship in the S phase, may be more sensitive to the DNA perturbation by 5-bromodeoxyuridine treatment plus near-UV irradiation for cell survival.     

Histone demethylation and timely DNA replication

It is well-established that silent regions of the genome replicate late during S phase. In this issue of Molecular Cell, Black et al. (2010) uncover a conserved role for the JMJD2 family of histone demethylases in promoting replication within silent chromatin regions that contain histone H3 lysine 9 methylation and HP1.     

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

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

DNA contents of replication without DNA density labeling

A new method for determining the timing of DNA replication in specific regions of the mammalian genome without the use of DNA density labeling and DNA density centrifugation is described. The method is based on determination of average relative DNA copy numbers in specific genomic regions as cells progress through S phase, and "time of replication" for a specific region is described in terms of the cell's DNA content when the region is replicated. DNA is isolated from synchronized populations of G1 and S phase cells, it is slot-blotted at the same DNA concentration(s) for each population, and it is hybridized with 32P-labeled DNA probes that are specific to the regions of interest. Quantitation of the slot blot autoradiograms and flow cytometric analysis allows determination of (a) average relative DNA copy numbers for the regions of interest in synchronized cell populations, and (b) the average total DNA content in each population of synchronized cells. This information and the flow cytometry histograms are then used to calculate the cellular DNA content at which each region of interest is replicated. The results have a precision of less than or equal to +/- 10% of S phase for Chinese hamster (line CHO) rhodopsin, metallothionein II, the 5'-end of dihydrofolate reductase, the telomeric repeated sequence, pHuR-093 (also located near the centromeres in CHO chromosomes), and the c-Ki-ras family.     

Replication of heterochromatin and structure of polytene chromosomes

Heterochromatin is characteristically the last portion of the genome to be replicated. In polytene cells, heterochromatic sequences are underreplicated because S phase ends before replication of heterochromatin is completed. Truncated heterochromatic DNAs have been identified in polytene cells of Drosophila and may be the discontinuous molecules that form between fully replicated euchromatic and underreplicated heterochromatic regions of the chromosome. In this report, we characterize the temporal pattern of heterochromatic DNA truncation during development of polytene cells. Underreplication occurred during the first polytene S phase, yet DNA truncation, which was found within heterochromatic sequences of all four Drosophila chromosomes, did not occur until the second polytene S phase. DNA truncation was correlated with underreplication, since increasing the replication of satellite sequences with the cycE(1672) mutation caused decreased production of truncated DNAs. Finally, truncation of heterochromatic DNAs was neither quantitatively nor qualitatively affected by modifiers of position effect variegation including the Y chromosome, Su(var)205(2), parental origin, or temperature. We propose that heterochromatic satellite sequences present a barrier to DNA replication and that replication forks that transiently stall at such barriers in late S phase of diploid cells are left unresolved in the shortened S phase of polytene cells. DNA truncation then occurs in the second polytene S phase, when new replication forks extend to the position of forks left unresolved in the first polytene S phase.