Temporal organization of replication in DNA fibers of mammalian cells

The extent of coordinate control over the multiple initiation events in DNA replication has been investigated in three mammalian cell lines by DNA fiber autoradiography. Quantitative estimates have been obtained of the degree of synchrony among initiations occurring on stretches of DNA. Synchrony decreases markedly with increasing distance between initiation sites in MDBK (bovine) and L929 (mouse) cells, but only slightly in muntjac cells. Possible control mechanisms for the initiation process are discussed.     

Calmodulin-specific monoclonal antibodies inhibit DNA replication in mammalian cells.

The involvement of calmodulin in the proliferation of Chinese hamster embryo fibroblast cells has been studied with a specific monoclonal antibody to calmodulin. We observed that calmodulin levels increase 2-fold in the late G1 period in these cells, and this coincides with the increase in DNA polymerase alpha activity as the cells progress synchronously from a quiescent state in the G1 to the S phase. However, there is a concurrent 10-fold enhancement of thymidine kinase activity, which is tightly coupled to the entry of cells into the S phase. Incubation of permeabilized S-phase cells with calmodulin-specific murine monoclonal antibody resulted in a dose-dependent inhibition of DNA replication. This inhibitory effect of anti-calmodulin antibodies on DNA replication is completely reversed by the addition of exogenously purified calmodulin. These observations provide evidence for the involvement of calmodulin in DNA replication and, therefore, in cell proliferation during the S phase.     

Delayed DNA methylation is an integral feature of DNA replication in mammalian cells

In the majority of sites of methylation in the DNA of mammalian cells, the symmetry of methylation is restored within a few minutes of the passage of a replication fork. However, it has been shown that daughter strand methylation in immortalised cell lines is delayed in a substantial minority of sites for up to several hours after replication. We report here the results of two new approaches to the determination of the functional significance of delayed DNA methylation in mammalian cells. Firstly, we demonstrate that normal, nontransformed cells (human peripheral lymphocytes in short-term primary culture) have comparable proportions of delayed DNA methylation to many immortalised cell lines, showing that delayed DNA methylation is not just a secondary consequence of abnormally high methionine requirements commonly observed in transformed cells and that delayed DNA methylation would be unlikely not to occur in vivo. Secondly, we have used 5-aza-2'-deoxycytidine (5azadCyd) to derive subclones of cells from the Chinese hamster ovary cell line which have stably hypomethylated DNA. In three of these subclones which had lost on average one fourth of the methylation sites from their genomes, the proportion of daughter strand methylation which was delayed after replication was reduced by less than 10%. If delayed DNA methylation were site-specific, this implies that of the order of twice the number of "immediate" methylation sites than delayed methylation sites had been lost from the genomes of these hypomethylated subclones. Thus, delayed DNA methylation is an integral part of the process whereby replicating mammalian cells maintain the pattern of methylation in their genomes. These observations are discussed in relation to the significance of delayed DNA methylation for the accurate maintenance of methylation patterns in the genome and the consequent implications for the possible role of methylated deoxycytidines in mammalian gene control.     

Distribution of DNA replication origins between matrix-attached and loop DNA in mammalian cells

Using a previously developed procedure (Gencheva et al. [1996] J Biol Chem 271:2608-2614), we isolated a DNA fraction consisting of short fragments originating from the regions of initiation of DNA synthesis from exponentially growing Chinese hamster ovary cells. This fraction arbitrarily designated as "collective origin fraction" was labeled in vitro and used to probe the abundance of origin containing sequences in preparations of matrix-attached and loop DNA isolated by two different procedures from Chinese hamster ovary cells. Alternatively, an individual DNA replication origin sequence - a 478-bp long DNA fragment located at about 17-kb downstream of the dihydrofolate reductase gene - was used to probe the same matrix-attached and loop DNA fractions. The results with both the collective and individual DNA replication origins showed that there was random distribution of the origin sequences between DNA attached to the matrix and DNA from the loops.     

Analysis of metal-induced DNA lesions and DNA-repair replication in mammalian cells

The potency of several metal compounds in causing lesions in DNA either directly or by exposure of intact cultured cells has been examined using the neutral conditions of nucleoid gradient sedimentation. HgCl2 was clearly the most potent inducer of single-strand breakage when added to isolated nucleoids or when nucleoids were prepared from cells treated with this compound. CaCrO4 , however, caused DNA-strand breaks in nucleoids isolated from cells treated with this agent but did not induce DNA strand breaks when added directly to nucleoids. Although less potent than HgCl2, NiCl2 also caused significant single strand breakage in isolated nucleoids or in nucleoids prepared from cells treated with this metal. Since strand breakage of DNA in intact cells may occur secondary to activation of DNA-dependent nucleases during repair replication, CsCl gradient density sedimentation was utilized to examine whether repair processes were induced by exposure of cells to NiCl2, HgCl2 and CaCrO4 . CaCrO4 and NiCl2 induced substantial DNA-repair activity at concentrations and exposure times where DNA lesions could not be detected whereas HgCl2 induced a 10-fold lower level of DNA-repair activity compared to CaCrO4 at optimal concentrations which again were below the concentrations of this metal that produced measurable DNA lesions. Both the induction of DNA-repair activity and DNA-strand breakage by these metals was concentration- and time-dependent. These results demonstrate some unique aspects of the interaction of HgCl2, NiCl2 and CaCrO4 with the DNA of intact cells and point to the possible important correlation of induction of DNA repair to carcinogenesis since nickel and chromate have clearly been implicated as carcinogens and induce considerable repair whereas HgCl2 is not considered a carcinogen and induces the least DNA repair despite its potency in producing DNA lesions.     

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.     

In situ localization of mitochondrial DNA replication in intact mammalian cells

Nearly all of the known activities required for mitochondrial DNA (mtDNA) replication and expression are nuclear-encoded gene products, necessitating communication between these two physically distinct intracellular compartments. A significant amount of both general and specific biochemical information about mtDNA replication in mammalian cells has been known for almost two decades. Early studies achieved selective incorporation of the thymidine analog 5-Bromo-2-deoxy-Uridine (BrdU) into mtDNA of thymidine kinase-deficient (TK[-]) cells. We have revisited this approach from a cellular perspective to determine whether there exist spatiotemporal constraints on mtDNA replication. Laser-scanning confocal microscopy was used to selectively detect mtDNA synthesis in situ in cultured mammalian cells using an immunocytochemical double-labeling approach to visualize the incorporation of BrdU into mtDNA of dye-labeled mitochondria. In situ detection of BrdU-incorporated mtDNA was feasible after a minimum of 1- 2 h treatment with BrdU, consistent with previous biochemical studies that determined the time required for completion of a round of mtDNA replication. Interestingly, the pattern of BrdU incorporation into the mtDNA of cultured mammalian cells consistently radiated outward from a perinuclear position, suggesting that mtDNA replication first occurs in the vicinity of nuclear-provided materials. Newly replicated mtDNA then appears to rapidly distribute throughout the dynamic cellular mitochondrial network.     

The rate of fork movement during DNA replication in mammalian cells

DNA fiber autoradiography was used to measure the rate of replication fork progression along replication units in human diploid cells. The rate in different replication units differs very significantly and lies within the range 0.1 to 1.2 m/min. However, no significant changes were found in the rate of fork movement along single replication units operating during long intervals of S phase. Moreover, the fork progression rate is constant in many replication units of human cells.     

Nuclear organization of DNA replication initiation proteins in mammalian cells.

Origin recognition complex (ORC), CDC6, and MCM proteins assemble sequentially to form prereplication chromatin. However, their organization remains largely unclear in mammalian cells. Here we show that ORC1 proteins are associated with non-chromatin nuclear structures and assemble in nuclear foci in mammalian cells using an in vivo chemical cross-linking method. CDC6 proteins were also found to assemble in nuclear foci on non-chromatin nuclear structures, although their physical association with ORC1 has been undetectable. In contrast to the situation in yeast cells, CDC6 was found to remain associated with non-chromatin nuclear structures even after cells entered into S phase. Instead, ORC1 proteins were found to be degraded by a proteasome-dependent pathway during S phase. We also found that some ORC2 proteins are associated with non-chromatin nuclear structures like ORC1, although the remainder binds to nuclease-sensitive chromatin. Further analyses indicate that ORC2 physically interacts with ORC1 on non-chromatin nuclear structures. On the other hand, our results suggest that although a small proportion of MCM complexes are loaded onto chromatin regions near ORC foci, most of them are more widely distributed. Possible relations between such organization of prereplication chromatin and complicated origin specification in higher eukaryotic cells are discussed.     

A distinct first replication cycle of DNA introduced in mammalian cells

Many mutation events in microsatellite DNA sequences were traced to the first embryonic divisions. It was not known what makes the first replication cycles of embryonic DNA different from subsequent replication cycles. Here we demonstrate that an unusual replication mode is involved in the first cycle of replication of DNA introduced in mammalian cells. This alternative replication starts at random positions, and occurs before the chromatin is fully assembled. It is detected in various cell lines and primary cells. The presence of single-stranded regions increases the efficiency of this alternative replication mode. The alternative replication cannot progress through the A/T-rich FRA16B fragile site, while the regular replication mode is not affected by it. A/T-rich microsatellites are associated with the majority of chromosomal breakpoints in cancer. We suggest that the alternative replication mode may be initiated at the regions with immature chromatin structure in embryonic and cancer cells resulting in increased genomic instability. This work demonstrates, for the first time, differences in the replication progression during the first and subsequent replication cycles in mammalian cells.     

Direction of SPP1 DNA replication in transfected B. subtilis cells

Summary The origin and the direction of replication of the SPP1 chromosome, which has a unique, nonpermuted sequence of markers, was established by determination of the frequency distribution of various markers along the SPP1 map. For this purpose replicating DNA was isolated from transfected competent B. subtilis cells. Marker frequencies were measured by means of helper mediated transfection. In the range defined by the genetic map, replication is unidirectional, originating from a point in the left part of the map. Shearing the DNA into halves prior to transfection permits only one round of replication of that half molecule which carries the origin.     

Analysis of DNA replication profiles in budding yeast and mammalian cells using DNA combing

DNA combing is a powerful method developed by Bensimon and colleagues to stretch DNA molecules on silanized glass coverslips. This technique provides a unique way to monitor the activation of replication origins and the progression of replication forks at the level of single DNA molecules, after incorporation of thymidine analogs, such as 5-bromo-2'-deoxyuridine (BrdU), 5-iodo-2'-deoxyuridine (IdU) and 5-chloro-2'-deoxyuridine (CldU) in newly-synthesized DNA. Unlike microarray-based approaches, this assay gives access to the variability of replication profiles in individual cells. It can also be used to monitor the effect of DNA lesions on fork progression, arrest and restart. In this review, we propose standard DNA combing methods to analyze DNA replication in budding yeast and in human cells. We also show that 5-ethynyl-2'-deoxyuridine (EdU) can be used as a good alternative to BrdU for DNA combing analysis, as unlike halogenated nucleotides, it can be detected without prior denaturation of DNA.     

A model for the spatio-temporal organization of DNA replication in mammalian cells

The spatio-temporal organization of chromosomal DNA replication was analyzed using a model based on a "DNA unit" (or decondensation unit) hypothesis. The model is an extension of the fork movement theory of Huberman & Riggs (1968) and can account for a partially deterministic and partially stochastic order of DNA replication in chromosomes. It presumes that each chromosome is composed of DNA units that are arranged in sequence and that are replicated in parallel. A deterministic wave of chromatin decondensation propagates along the DNA unit continuously and progressively providing a field for the random activation of replication origin. Assignment of replication times to DNA compartments by a Monte Carlo method was programmed based on the model and the program was used to stimulate DNA synthesis rate curves that can be measured by the method of Dolbeare et al. (1983, 1985). The shape of the curve is shown to constrain possible parameter values of the model, which include the rate of fork movement, the fraction of chromatin that is decondensed at the start of S-phase, the initial number of origins activated, the rate at which new origins are activated, etc. The chromosomal organization that controls the molecular level of DNA replication is briefly reviewed and its relevance to the model is also discussed.     

Effects of Friedreich's ataxia GAA repeats on DNA replication in mammalian cells

Friedreich's ataxia (FRDA) is a common hereditary degenerative neuro-muscular disorder caused by expansions of the (GAA)n repeat in the first intron of the frataxin gene. The expanded repeats from parents frequently undergo further significant length changes as they are passed on to progeny. Expanded repeats also show an age-dependent instability in somatic cells, albeit on a smaller scale than during intergenerational transmissions. Here we studied the effects of (GAA)n repeats of varying lengths and orientations on the episomal DNA replication in mammalian cells. We have recently shown that the very first round of the transfected DNA replication occurs in the lack of the mature chromatin, does not depend on the episomal replication origin and initiates at multiple single-stranded regions of plasmid DNA. We now found that expanded GAA repeats severely block this first replication round post plasmid transfection, while the subsequent replication cycles are only mildly affected. The fact that GAA repeats affect various replication modes in a different way might shed light on their differential expansions characteristic for FRDA.     

Effects of inhibition of protein synthesis on DNA replication in cultured mammalian cells

We have investigated the effects of inhibiting protein synthesis on the overall rate of DNA synthesis and on the rate of replication fork movement in mammalian cells. In order to test the validity of using [³H]thymidine incorporation as a measure of the overall rate of DNA synthesis during inhibition of protein synthesis, we have directly measured the size and specific radioactivity of the cells' [³H]dTTP pool. In three different mammalian cell lines (mouse L, Chinese hamster ovary, and HeLa) nearly complete inhibition of protein synthesis has little effect on pool size (±26%) and even less effect on its specific radioactivity (±11%). Thus [³H]thymidine incorporation can be used to measure accurately changes in rate of DNA synthesis resulting from inhibition of protein synthesis.Using the assay of [³H]thymidine incorporation to measure rate of DNA synthesis, and the assay of [¹⁴C]leucine or [¹⁴C]valine incorporation to measure rate of protein synthesis, we have found that eight different methods of inhibiting protein synthesis (cycloheximide, puromycin, emetine, pactamycin, 2,4-dinitrophenol, the amino acid analogs canavanine and 5-methyl tryptophan, and a temperature-sensitive leucyl-transfer tRNA synthetase) all cause reduction in rate of DNA synthesis in mouse L, Chinese hamster ovary, or HeLa cells within two hours to a fairly constant plateau level which is approximately the same as the inhibited rate of protein synthesis.We have used DNA fiber autoradiography to measure accurately the rate of replication fork movement. The rate of movement is reduced at every replication fork within 15 minutes after inhibiting protein synthesis. For the first 30 to 60 minutes after inhibiting protein synthesis, the decline in rate of fork movement (measured by fiber autoradiography) satisfactorily accounts for the decline in rate of DNA synthesis (measured by [³H]thymidine incorporation). At longer times after inhibiting protein synthesis, inhibition of fork movement rate does not entirely account for inhibition of overall DNA synthesis. Indirect measurements by us and direct measurements suggest that the additional inhibition is the result of decline in the frequency of initiation of new replicons.