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PubMed1985

[DNA content and synthesis in the nuclei of rat cerebellar cells in organotypic cultures].

Using cytophotometric and autoradiographic methods, it has been shown for the first time that in condition of an organotypic culture the replicative synthesis of DNA is induced in the Purkinje neurons of the cerebellum of newborn rats completing their terminal differentiation. This synthesis is accompanied by polyploidization of the initially diploid population of these cells (4c, much more rarely 8c, and a single 16c cell appear) rather than by cell division. In constant, the granular cells mostly retain their diploid state and only a few of them synthesize DNA to H2c values. The glial cells divide actively. Hence, evidence is presented that neurons, at least those of cerebellum, retain their potential of replicative synthesis of DNA in the organotypic culture. The important point is that DNA synthesis in their nuclei proceeds simultaneously with processes of differentiation.

Animals↗

PubMed1988

Hyperthermia blocks DNA processing at the nuclear matrix.

The capacity of control and heated HeLa cells to process newly polymerized DNA at the nuclear matrix was measured. DNA which had been pulse-labeled with [3H]thymidine was enriched by a factor of up to 6 at the cell's nuclear matrix. During continuous exposure to [3H]thymidine at 37 degrees C this enrichment for pulse-labeled DNA was reversed with a half-time of 7 min. We interpret this processing of newly replicated DNA to be a distribution of newly polymerized DNA throughout replicon-sized nuclear DNA domains. Both processing of newly polymerized DNA at the nuclear matrix and ligation of replicon clusters into the interphase cell chromosome were halted by incubation of cells at temperatures at or above 43 degrees C. When HeLa cells were pulse-labeled during a 30-min incubation at 45 degrees C and replaced at 37 degrees C, the enrichment for 3H-labeled DNA at the nuclear matrix was reversed with an initial half-time of 4 h. The results indicate that exposure of cells to hyperthermic temperatures blocks ongoing nascent DNA processing at the nuclear matrix and results in a retardation of DNA processing in preheated cells replaced at 37 degrees C.

Carbon Radioisotopes↗

PubMed1992

Plasmid models for bacteriophage T4 DNA replication: requirements for fork proteins.

Bacteriophage T4 DNA replication initiates from origins at early times of infection and from recombinational intermediates as the infection progresses. Plasmids containing cloned T4 origins replicate during T4 infection, providing a model system for studying origin-dependent replication. In addition, recombination-dependent replication can be analyzed by using cloned nonorigin fragments of T4 DNA, which direct plasmid replication that requires phage-encoded recombination proteins. We have tested in vivo requirements for both plasmid replication model systems by infecting plasmid-containing cells with mutant phage. Replication of origin and nonorigin plasmids strictly required components of the T4 DNA polymerase holoenzyme complex. Recombination-dependent plasmid replication also strictly required the T4 single-stranded DNA-binding protein (gene product 32 [gp32]), and replication of origin-containing plasmids was greatly reduced by 32 amber mutations. gp32 is therefore important in both modes of replication. An amber mutation in gene 41, which encodes the replicative helicase of T4, reduced but did not eliminate both recombination- and origin-dependent plasmid replication. Therefore, gp41 may normally be utilized for replication of both plasmids but is apparently not required for either. An amber mutation in gene 61, which encodes the T4 RNA primase, did not eliminate either recombination- or origin-dependent plasmid replication. However, plasmid replication was severely delayed by the 61 amber mutation, suggesting that the protein may normally play an important, though nonessential, role in replication. We deleted gene 61 from the T4 genome to test whether the observed replication was due to residual gp61 in the amber mutant infection. The replication phenotype of the deletion mutant was identical to that of the amber mutant. Therefore, gp61 is not required for in vivo T4 replication. Furthermore, the deletion mutant is viable, demonstrating that the gp61 primase is not an essential T4 protein.

Bacteriophage T4↗

PubMedDate not supplied

Mutator phenotype in cancer: timing and perspectives.

Normal human cells replicate their DNA with exceptional accuracy. During every division cycle, each daughter cell receives a full and accurate complement of genetic information. It has been estimated that approximately one error occurs during DNA replication for each 10(9) to 10(10) nucleotides polymerized. Stem cells, the cells that are progenitors of cancer, may replicate their genes even more accurately. In contrast, the malignant cells that constitute a tumor are markedly heterogeneous and exhibit multiple chromosomal abnormalities and alterations in the nucleotide sequence of DNA. To account for the disparity between the rarity of mutations in normal cells and the large numbers of mutations present in cancer, we initially hypothesized that during tumor progression, cancer cells must exhibit a mutator phenotype. In this perspective, we summarize the evidence supporting a mutator phenotype in human cancer, analyze recent measurements of mutations in human cancer, consider the timing for the expression of a mutator phenotype, and focus on the important consequences of large numbers of random mutations in human tumors.

Disease Progression↗

PubMed1980

Chromosome replication in mouse intraspecific hybrids.

Hybrid cells (HY SS2 and HY SS6) arising from the fusion of diploid cells of the mouse lymphosarcoma LS/BL and L cells resistant to 8-azaguanine (HGPRT-) showed slower growth and a longer generation time than the parent lines. The inter- and intrachromosomal timing and patterns of early chromosome DNA replication of parent cells was preserved in the hybrid genome and was not influenced by loss of telocentric chromosomes from LS/BL or L (HGPRT-) cells. Thus DNA chromosome replication sequences are not dependent on the presence of a complete set of chromosomes of the parent cells and do not therefore seem to be a result of interaction between chromosomes not segregated in the hybrid genome.

Animals↗

PubMed1996

Characterization of replication origins flanking the 23S rRNA gene in tobacco chloroplast DNA.

Using 5' end-labeled nascent strands of tobacco chloroplast DNA (ctDNA) as a probe, replication displacement loop (D-loop) regions were identified. The strongest hybridization was observed with restriction fragments containing the rRNA genes from the inverted repeat region. Two-dimensional gel analysis of various digests of tobacco ctDNA suggested that a replication origin is located near each end of the 7.1 kb BamHI fragment containing part of the rRNA operon. Analysis of in vitro replication products indicated that templates from either of the origin regions supported replication, while the vector alone or ctDNA clones from other regions of the genome did not support in vitro replication. Sequences from both sides of the BamHI site in the rRNA spacer region were required for optimal in vitro DNA replication activity. Primer extension was used for the first time to identify the start site of DNA synthesis for the D-loop in the rRNA spacer region. The major 5' end of the D-loop was localized to the base of a stem-loop structure which contains the rRNA spacer BamHI site. Primer extension products were insensitive to both alkali and RNase treatment, suggesting that RNA primers had already been removed from the 5' end of nascent DNA. Location of an origin in the rRNA spacer region of ctDNA from tobacco, pea and Oenothera suggests that ctDNA replication origins may be conserved in higher plants.

Base Composition↗

PubMed1992

Regulation of DNA replication by the nuclear envelope.

Recent evidence suggests that the nuclear envelope is directly involved in regulating DNA replication. It does this in at least three ways. First, replication is dependent on assembly of an intact nuclear envelope capable of nuclear transport. Second, the nuclear membrane defines the nucleus as the fundamental unit of replication and determines the timing of initiation. Third, the nuclear membrane is essential for coupling DNA replication to the cell cycle. Thus, regulated DNA replication in eukaryotic cells depends on a structurally intact and functional nuclear envelope.

Animals↗

PubMed1999

Radiation down-regulates replication origin activity throughout the S phase in mammalian cells.

An asynchronous culture of mammalian cells responds acutely to ionizing radiation by inhibiting the overall rate of DNA replication by approximately 50% for a period of several hours, presumably to allow time to repair DNA damage. At low and moderate doses, this S phase damage-sensing (SDS) pathway appears to function primarily at the level of individual origins of replication, with only a modest inhibition of chain elongation per se. We have shown previously that the majority of the inhibition observed in an asynchronous culture can be accounted for by late G1cells that were within 2-3 h of entering the S period at the time of irradiation and which then fail to do so. A much smaller effect was observed on the overall rate of replication in cells that had already entered the S phase. This raised the question whether origins of replication that are activated within S phase per se are inhibited in response to ionizing radiation. Here we have used a two-dimensional gel replicon mapping strategy to show that cells with an intact SDS pathway completely down-regulate initiation in both early- and late-firing rDNA origins in human cells. We also show that initiation in mid- or late-firing rDNA origins is not inhibited in cells from patients with ataxia telangiectasia, confirming the suggestion that these individuals lack the SDS pathway.

Animals↗

PubMed1983

Mitotic cell cycle control in Physarum. Unprecedented insights via flow-cytometry.

High resolution flow-cytometric studies of isolated macroplasmodial nuclei of the myxomycete. Physarum polycephalum provide definite evidence for the persistence of natural synchrony at mitosis throughout the entire cell cycle, i.e. completely synchronous DNA replication and traverse of G2. Even if nuclei within a given macroplasmodium belong to two distinct genome size classes (mixoploidy), they cycle and traverse mitosis in strict synchrony. This cannot be explained by current models of regulation of division based solely upon nuclear size and/or nuclear/cytoplasm ratios. Constitutional DNA content variation was apparent among all tested strains, and loss of late-replicating, presumably AT-rich DNA accounts for this variation. A constant duration of the S phase is maintained, irrespective of DNA content, via differential slowdown of replication rates during the 2nd and 3rd hours of replication. A frequently described extension of nuclear replication into G2 could not be substantiated. Interference with DNA and protein synthesis provides the first evidence for a brief "G1 phase" equivalent of 3-4 min duration in asynchronous microplasmodial cultures, and temporally assigns a protein synthesis-dependent "transition point" for completion of mitosis and initiation of DNA synthesis at 5 min prior to actual division nuclei which have passed this point at the time of addition of cycloheximide replicate 5% of their DNA before they become arrested. These findings provide strong experimental support for the transition point concept of cell cycle control, and additionally are commensurate with some form of the replicon-set hypothesis in Physarum.

Cell Nucleus↗

PubMed1982

Organization of herpes simplex virus type 1 deoxyribonucleic acid during replication probed in living cells with 4,5',8-trimethylpsoralen.

The structure of herpes simplex virus type 1 (HSV-1) DNA in the nuclei of living infected cells was studied with the DNA photoaffinity probe 4,5',8-trimethylpsoralen. The rate of photobinding to HSV-1 DNA was compared to that of a suitable internal control at different times during infection. The rates of photobinding to DNA packaged in virions, capsids, and prereplicative and postreplicative DNA were characteristically different. By 4 h after infection, after the initiation of DNA replication, the rate of photobinding to HSV-1 DNA increased 4 times relative to the rate of binding to the host DNA. The enhanced rate of photobinding to HSV-1 DNA was maintained at all later times during infection and was not affected when frequent single-strand breaks were introduced in HSV-1 DNA by gamma irradiation of infected cells. The results suggest that the bulk of the replicating herpes DNA is free of torsional tension and that the differing rates of photobinding are attributable to changes in accessibility of the HSV-1 DNA. The results are compatible with previous proposals, based on in vitro studies, that intranuclear HSV-1 DNA is primarily free of nucleosomal organization and suggest that there are few, if any, unrestrained DNA supercoils averaged over the entire HSV-1 genome.

Affinity Labels↗

PubMed1997

Correlation between DNA replication and expression of cyclins A and B1 in individual MOLT-4 cells.

Cyclins A and B1 activate cyclin-dependent kinases CDK2 and CDC2, which regulate cell progression through S and G2. Expression of these cyclins is generally measured in populations of synchronized cells, by immunoblotting. Such studies neither provide information regarding intercellular variability in cyclin expression nor yield precise data on a time relationship between initiation and termination of DNA replication in relation to cyclin expression. Furthermore, cell synchronization by DNA polymerase inhibitors or excess of thymidine induces cell growth imbalance and alters expression of cyclins, thereby introducing an experimental bias. Using a novel flow cytometric method of detection of incorporated bromodeoxyuridine (BrdUrd) in the present study, we have been able to correlate expression of immunocytochemically discerned cyclins A and B1 with incorporation of BrdUrd and the cell cycle position of individual MOLT-4 cells. On the basis of differences in amount of incorporated BrdUrd and DNA content, the following cohorts of cells in narrow windows of the cell cycle were identified: (a) cells initiating and (b) terminating DNA replication during a 1-h pulse of BrdUrd; (c) cells replicating DNA throughout the duration of BrdUrd pulse; (d) G1 cells; and (e) G2 cells that remained in G2 for at least 1 h after exiting S phase. These populations were characterized with respect to expression of cyclins A and B1. Expression of cyclin A was an early event of S phase, and 84% of cells entering S phase during 1 h of exposure to BrdUrd were already cyclin A positive. More than 95% of S-phase cells, as well as the cells exiting S during BrdUrd pulse, were also cyclin A positive. The maximal rate of accumulation of cyclin A was seen during the first hour of progression through S phase. In contrast, the maximal accumulation rate of cyclin B1 showed cells during the first hour of progression through G2. A strong correlation between expression of cyclin A and the rate of DNA replication, estimated by the degree of BrdUrd incorporation (r = 0.99), was observed.

Cell Cycle↗

PubMed2002

Meiotic deletion at the BUF1 locus of the fungus Magnaporthe grisea is controlled by interaction with the homologous chromosome.

The Magnaporthe grisea BUF1 gene suffers high-frequency mutation in certain genetic crosses, resulting in buff-colored progeny. Analysis of 16 buf1 mutants arising from a cross with a mutation frequency of 25% revealed that, in every case, the BUF1 gene was deleted. The deletions occurred in only one of the parental chromosomes and were due to intrachromosomal recombination. Tetrad analysis revealed that deletions occurred in 44% of meioses and usually affected both chromatids of the mutable chromosome. This suggests that they happen before the premeiotic round of DNA synthesis. However, they were also almost entirely restricted to heteroallelic crosses. This, together with the discovery of numerous repetitive elements that were present only in the mutable BUF1 locus, suggests that the deletion process is sensitive to pairing interactions between homologous chromosomes, such that only unpaired loci are subject to deletion. Given that karyogamy is not supposed to occur until after premeiotic DNA replication in Pyrenomycetous fungi such as M. grisea, this latter observation would place the time of deletion during, or after, DNA synthesis. These conflicting results suggest that karyogamy might actually precede DNA replication in Pyrenomycetous fungi or that parts of the genome remain unreplicated until after karyogamy and subsequent chromosome pairing have taken place.

Chromosome Deletion↗

PubMed2000

Hierarchy of S-phase-promoting factors: yeast Dbf4-Cdc7 kinase requires prior S-phase cyclin-dependent kinase activation.

In all eukaryotes, the initiation of DNA synthesis requires the formation of prereplicative complexes (pre-RCs) on replication origins, followed by their activation by two S-T protein kinases, an S-phase cyclin-dependent kinase (S-CDK) and a homologue of yeast Dbf4-Cdc7 kinase (Dbf4p-dependent kinase [DDK]). Here, we show that yeast DDK activity is cell cycle regulated, though less tightly than that of the S-CDK Clb5-Cdk1, and peaks during S phase in correlation with Dbf4p levels. Dbf4p is short-lived throughout the cell cycle, but its instability is accentuated during G(1) by the anaphase-promoting complex. Downregulating DDK activity is physiologically important, as joint Cdc7p and Dbf4p overexpression is lethal. Because pre-RC formation is a highly ordered process, we asked whether S-CDK and DDK need also to function in a specific order for the firing of origins. We found that both kinases are activated independently, but we show that DDK can perform its function for DNA replication only after S-CDKs have been activated. Cdc45p, a protein needed for initiation, binds tightly to chromatin only after S-CDK activation (L. Zou and B. Stillman, Science 280:593-596, 1998). We show that Cdc45p is phosphorylated by DDK in vitro, suggesting that it might be one of DDK's critical substrates after S-CDK activation. Linking the origin-bound DDK to the tightly regulated S-CDK in a dependent sequence of events may ensure that DNA replication initiates only at the right time and place.

Blotting, Northern↗

PubMed1978

[Replication and reassociation kinetics of nuclear matrix DNA from regenerating rat liver].

Fraction 1, containing 65--70% of nuclear DNA (nDNA) was extracted from isolated rat liver nuclei. Then nuclear matrix fraction, containing 30--35% of nDNA was extracted with 1.2 M NaCl. About 1% of nDNA war discovered in residual matrix. Specific activity of residual matrix labelled DNA within 1 and 3 min. after the injection of 14C-orotic acid into the liver portal vein (24 hours after hepatectomy) was respectively in 70 and 50 times higher, and that of matrix DNA--in 45 and 20 times as high as in fraction 1 DNA. Thus, replication begins from nuclear matrix DNA. The latter is enriched with unique sequences by 10%, as compared with total nDNA, and its reassociation kinetics does not change at different stages of the cell cycle (0 and 24 hours after partial hepatectomy). It is suggested, that DNA does not migrate with respect to nuclear matrix under replication.

Animals↗

PubMed1975

Adenovirus type 2 DNA replication. II. Termini of DNA replication.

Complete, mature adenovirus type 2 DNA molecules were isolated from virus-infected HeLa cells, pulse-labeled at 20 h postinfection in [3H]thymidine pulses shorter than the time necessary for one round of viral DNA replication. After digestion with the restriction endonucleases Eco RI, Hpa I, and Hind III, a temporal order of synthesis of different regions of the viral genome was established from the relative specific radioactivities in the restriction enzyme fragments. A comparison with the physical order of these fragments revealed the existence of two termini of DNA replication towards both the molecular right and left ends, respectively, of the viral chromosome.

Adenoviridae↗

PubMed1971

Heat induction of prophage phi 105 in Bacillus subtilis: replication of the bacterial and bacteriophage genomes.

A temperature-inducible mutant of temperate Bacillus bacteriophage phi105 was isolated and used to lysogenize a thymine-requiring strain of Bacillus subtilis 168. Synthesis of phage and bacterial deoxyribonucleic acid (DNA) was studied by sucrose gradient centrifugation and density equilibrium centrifugation of DNA extracted from induced bacteria. The distribution of DNA in the gradients was measured by differential isotope and density labeling of DNA before and after induction and by measuring the biological activity of the DNA in genetic transformation, in rescue of phage markers, and in infectivity assays. At early times after induction, but after at least one round of replication, phage DNA remains associated with high-molecular-weight DNA, whereas, later in the infection, phage DNA is associated with material of decreasing molecular weight. Genetic linkage between phage and bacterial markers can be demonstrated in replicated DNA from induced cells. Prophage induction is shown to affect replication of the bacterial chromosome. The overall rate of replication of prelabeled bacterial DNA is identical in temperature-induced lysogenics and in "mock-induced" wild-type phi105 lysogenics. The rate of replication of the bacterial marker phe-1 (and also of nia-38), located close to the prophage in direction of the terminus of the bacterial chromosome, is increased in induced cells, however, relative to other bacterial markers tested. In temperature-inducible lysogenics, where the prophage also carries a ts mutation which blocks phage DNA synthesis, replication of both phage and bacterial DNA stops after about 50% of the phage DNA has replicated once. The results of these experiments suggest that the prophage is not initially excised in induced cells, but rather it is specifically replicated in situ together with adjacent parts of the bacterial chromosome.

Bacillus subtilis↗

PubMed2002

E.coli cell-cycle regulation by bacteriophage lambda.

We re-examined the old but surprising claim of Kourilsky and Knapp that transient expression of genes located downstream of the p(L) promoter of bacteriophage lambda can induce cell-cycle synchrony in a population of Escherichia coli cells. Although we were unable to reproduce a lasting synchrony, a cessation of division, followed by one or two fairly synchronous cell divisions was observed. This line up of the cell cycle was found to be due to two genetically separable events: a temporary block of cell division and, at the same time, a block to the initiation of new rounds of DNA replication. These blocks then release after about one mass doubling so that chromosome replication and cell division occur during a short time interval in all the cells in the population. The cell division block is a result of the transient expression of the lambda kil gene. The block to initiation of DNA replication requires a region that we term bin (blocks initiation) immediately upstream of the xis gene. The region consists of ea22 and ea8.5 and two small open reading frames (ORFs) that flank them. Deletion-substitution mutagenesis suggests that all four ORFs may be required for the initiation block. The ability of the phage to modify two aspects of the host cell cycle presumably reflects a stratagem that provides the phage with an advantage for lysogeny or lytic growth.

Bacterial Proteins↗

PubMed1978

Molecular suicide studies of 125I and 3H disintegration in the DNA of Chinese hamster cells.

The shape of the survival curve for cells inactivated by tritium decay in DNA is modified by the presence of halogenated pyrimidines in the DNA in a manner analogous to their effect on X-ray induced reproductive death. The large shoulder found for tritium suicide is removed completely by coincorporation of 10(-6) M IUdR. The oxygen enhancement ratio for 125I and tritium disintegrations in unsynchronized Chinese hamster cells was determined for cells permitted to accumulate damage from these events at 4 degrees C. The oxygen enhancement ratio for 125I induced damage is 1.4. This is much smaller than the OER found for tritium decay which is similar or more than that found for X-ray exposure under the same conditions. These results suggest that the nature of the lesions produced by 125I decay in DNA are analogous to those produced by high LET radiation while those lesions produced by tritium are similar to lesions produced by roentgen rays. In synchronous V79 cells the effects of 125I induced damage in different regions of the mammalian cell DNA was examined taking advantage of the fact that DNA replication in hamster nuclei follows a time-dependent three dimensional pattern. The experiments indicate that 125I decays accumulated in the G2-period of the cell cycle have different efficiences for the induction of reproductive death depending on the region of the DNA which is labeled. The efficiency for the induction of reproductive death appears to be a maximum in DNA that replicates in V79 cells near the end of the DNA replication cycle. Electron capture events are dramatically efficient in the production of lethal chromosome aberrations. In CHO cells synchronized in the G1-stage of the cell cycle stored in the frozen state the efficiency for the induction of dicentric and ring chromosomes is 0.03. The dose response curve for the induction of these aberrations is linear in contrast to the curvilinear response found for roentgen ray exposure under the same conditions. Data on this kind suggest that there may exist "critical" regions within mammalian cell nuclei where chromatin fibers from two different chromosomes are in close proximity to each other and both are damaged non-repairably by a single electron capture event.

Animals↗