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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↗
PubMed2006

An ATR- and BRCA1-mediated Fanconi anemia pathway is required for activating the G2/M checkpoint and DNA damage repair upon rereplication.

The timely assembly of prereplicative complexes at replication origins is tightly controlled to ensure that genomic DNA is replicated once per cell cycle. The loss of geminin, a DNA replication inhibitor, causes rereplication that activates a G2/M checkpoint in human cancer cells. Fanconi anemia (FA) is an autosomal recessive and X-linked disorder associated with cancer susceptibility. Here we show that rereplication activates the FA pathway both for the activation of a G2/M checkpoint and for repair processes, like recruitment of RAD51. Both ATR and BRCA1 are required to activate the FA pathway. The G2/M checkpoint-mediated arrest of the cell cycle is critical for the prevention of both apoptosis and the accumulation of cells with rereplicated DNA, because the loss of ATR, BRCA1, or FANCA promotes apoptosis and suppresses the accumulation. The accumulation of cells with rereplicated DNA is restored by the artificial induction of a G2-phase arrest even when ATR, BRCA1, or FANCA is absent. Therefore, the ATR- and BRCA1-mediated FA pathway is required for the activation of a G2/M checkpoint and for DNA damage repair in response to the endogenous signal of rereplication. In its absence, the cells rapidly lose viability when faced with rereplication.

Apoptosis↗
PubMed2000

Specification of chromatin domains and regulation of replication and transcription during development.

It is becoming increasingly clear that transcription control is carried out at several interconnecting levels. Besides nucleosomal organization and interaction between transcription factors and gene promoters and other regulatory elements, long-range organization of chromatin in loops or domains seems to play a role in transcriptional regulation. A similar organization is likely to be crucial in the control of the timing and selection of origins of DNA replication. This review considers the implications of domain organization of eukaryotic genome in developmental control of transcription and replication.

Animals↗
PubMed1979

The effect of cell mass on the cell cycle timing and duration of S-phase in fission yeast.

Two isotopic methods for measuring DNA replication in the fission yeast Schizosaccharomyces pombe are described. The first is a method for measuring the total quantity of [3H]uracil incorporated into DNA after pulse labelling. The second is a means of detecting DNA replication in single cells by autoradiography. Both of these techniques have been used to investigate the timing and duration of S-phase in a series of mutant strains whose cell mass at division varies over a 3-fold range. The results support the hypothesis that in S. pombe there are 2 different controls over the timing of S-phase: an attainment of a critical cell mass and a dependency upon the completion of the previous mitosis coupled with a short minimum time in G1. Strains whose cell mass at birth is above this critical level initiate DNA replication almost immediately after septation, that is, very soon after the previous mitosis. Strains whose cell mass at birth is below the critical level do not initiate replication until the critical cell mass is attained. The duration of S-phase has been estimated from the proportion of cells whose nuclei are labelled after a pulse of given duration. S-phase is short in S. pombe, lasting only about 0.1 of a cell cycle in wild type. Cell mass at S-phase does not have any consistent effect on this length. We have also investigated the degree of synchrony of S-phase initiation in daughter cells, and have found that, in a cell cycle 240 min long, their S-phases are initiated within 1--2 min of each other. This result indicates that between sisters variability in the duration of the G1 phase is small compared with variability in the total cell cycle time, and argues against the hypothesis that the rate of cell cycle traverse is determined by a random transition in G1.

Ascomycota↗
PubMed1999

H2O2-induced apoptotic death in serum-deprived cultures of oligodendroglia origin is linked to cell differentiation.

When deprived of serum, oligodendroglialike (OLN 93) cells grown on poly-L-lysine-coated culture dishes cease to proliferate after 3 days and morphologically extend many fibers resembling morphologically differentiated, immature oligodendrocytes. At this time no cell death is apparent unless serum deprivation is extended for a period longer than 1 week. After 3 days in serum-deprived medium, treatment of cells with 1 mM H2O2 for 30 min facilitates apoptotic cell death, even when serum is added during the recovery period. Both serum-deprived, differentiated cells, and proliferating cells, respond to H2O2 by an initial growth arrest followed by growth resumption after 48 hr. However proliferating cells show resistance to the apoptotic effect of H2O2. This is correlated with growth arrest in the S phase at different stages of DNA replication, as well as with different timing of induced p21Waf1 expression. Thus, cells grown in serum, express elevated p21Waf1 protein levels after 4 hr, whereas serum-deprived, differentiated cells, only after 24 hr. The mRNA levels of p21Waf1 follow a similar timed pattern. Hence p21Waf1 may protect OLN 93 cells against the genotoxic effect of H2O2. The data suggest an intimate relationship between G1-arrest, morphological differentiation, and H2O2-mediated apoptosis.

Animals↗
PubMedDate not supplied

The effect of Ni(II) on DNA replication.

The cellular regulation of DNA replication is governed in part by the availability of essential metal ions. A continuous supply of Mg(II) ions is necessary for the efficient and faithful replication of parental strands during S-phase as well as during the repair of DNA damage. A metal ion such as Ni(II) may interfere with the replication process by binding to sites on proteins at which essential ions normally bind. Binding at these sites by a toxic metal ion may produce inappropriate responses from the replication proteins and thus alter the normal balance in one or more of the microsteps comprising DNA synthesis. We have studied the effect of Ni(II) on DNA replication in a reconstituted in vitro system using a HeLa cell extract as a source of polymerase activity on a template of activated calf thymus DNA. Ni(II) has an initial stimulatory effect that is followed by an overall inhibition of the incorporation of DNA precursors. These results suggest that Ni(II), similar to Mg(II) may have more than one binding site, but that the binding of Ni(II) to replication proteins may significantly alter the timing of events in DNA synthesis.

Animals↗
PubMed1995

Effect of microinjection time during postfertilization S-phase on bovine embryonic development.

Microinjection into bovine zygotes was performed to evaluate the effects of the timing of injection during the phase of DNA replication on the subsequent in vitro development of embryos and expression of injected chicken beta-actin promoter-lac Z gene construct. The period of DNA replication of bovine zygotes, determined by 3H-thymidine incorporation, begins between 12 hr and 13 hr postinsemination (hpi) of in vitro matured oocytes, reaches a maximum from 17 hpi to 19 hpi, and is complete by 21-22 hpi. Aphidicolin, an inhibitor of DNA polymerase alpha, was used to synchronize the pronuclei and the zygote population. Treatment with aphidicolin at 9-18 hpi arrested DNA replication without affecting formation of the pronuclei or embryo development. Cycloheximide, an inhibitor of protein synthesis, was used for nucleocytoplasmic resynchronization of the aphidicolin-treated zygotes. Microinjection was performed at 15 (early), 18 (mid), and 21 (late S phase) hpi. Embryonic development was affected following each of the three microinjection times. The development of zygotes injected at 18 hpi was significantly higher (P < 0.01) after 5 days of culture than those injected at 15 hpi or 21 hpi. Expression of the marker gene was observed in the higher stage of development (> 16 cells) only in the zygotes injected at 18 hpi. At the earlier stages of development, the proportions of embryos showing expression of the foreign gene were the same for all microinjection times. In aphidicolin- and cycloheximide-treated zygotes, expression of the marker gene followed the same curve as development, i.e., expression was low when injected early or late and higher (P < 0.005) when injected in the middle of zygotic S phase. The ability of the embryos to survive microinjection and to express the marker gene as a function of hpi seems to be influenced mostly in the cytoplasm processing stage rather than the pronuclei processing stage.

Animals↗
PubMed2001

Quantitative detection of hepatitis B virus DNA in serum by a new rapid real-time fluorescence PCR assay.

A sensitive and accurate HBV DNA quantification assay is essential for monitoring hepatitis B virus (HBV) replication. This study evaluated a real-time PCR method performed in the LightCycler analyser for quantitative HBV DNA assay. HBV DNA results with this method were compared with those obtained using a branched-chain DNA (bDNA) solution hybridization assay. Real-time PCR was performed using two adjacent fluorescently labelled probes and primers corresponding to the HBV core gene. The same standard employed in the bDNA assay was used for calibration. Serum samples came from 193 HBV surface antigen (HBsAg)-positive patients (34 HBV e antigen (HBeAg)-positive and 93 with antibody to HBeAg (anti-HBe)), and 66 asymptomatic HBV carriers. In addition, we analysed serum samples from 8 anti-HBe-positive patients who had been receiving lamivudine treatment for more than three years. A linear standard curve was seen in the range from 10(3) to 10(8) copies/mL. In the reproducibility analysis, intra-assay coefficient of variation (CVs) at two known HBV DNA concentrations were 4% and 2% and interassay CVs were 6% and 4%. The median of serum HBV DNA by real-time PCR was 9.2 x 10(8) copies/mL in HBeAg-positive patients with persistently elevated alanine aminotransferase (ALT) levels, 1.3 x 10(7) copies/mL in anti-HBe-positive cases with persistently elevated ALT levels, 3.7 x 10(4) copies/mL in anti-HBe-positive patients with fluctuating ALT levels and 10(4) copies/mL in asymptomatic HBV carriers. The differences in HBV DNA levels among the various groups studied were statistically significant (P < 0.05). The cut-off between chronic hepatitis patients and asymptomatic carriers was found to be at a serum HBV DNA concentration of 5 x 10(4) copies/mL. Of the 109 serum samples with a viral load < 7.5 x 10(5) (negative by bDNA assay) 44 (40%) were positive by real-time PCR: 24 (56%) chronic hepatitis and 20 (33%) asymptomatic carriers. There was a positive association between HBV DNA levels determined by real-time PCR and ALT levels (P < 0.05), which was not observed with the bDNA assay for HBV DNA quantification. At 12 months of lamivudine treatment, 6 patients (75%) showed HBV DNA levels < 5 x 10(4) copies/mL (range < 10(3)-2 x 10(3)), significantly lower than at baseline. At 36 months, 2 of 8 (25%) showed HBV DNA levels persistently lower than 5 x 10(4) copies/mL (1.7 x 10(3), 6 x 10(3)). The LightCycler quantitative real-time PCR is a practical, sensitive, reproducible single-tube assay with a wide dynamic range of detection. The assay is automatic except for DNA extraction and the running time is only 70 min. The LightCycler real-time PCR is useful for identifying different states of HBV infection and for evaluating the efficacy of viral therapy.

DNA, Viral↗
PubMed1981

Replication of viral DNA sequences integrated within the chromatin of SV40-transformed Chinese hamster lung cells.

To determine when during S phase integrated viral DNA sequences in several tsA SV40-transformed Chinese hamster cell clones replicate, we pulse-labeled cultures with BrdUrd and subsequently collected mitotic cells during sequential time intervals. Restriction endonuclease mapping indicated that each of the three SV40-transformed Chinese hamster lung cell clones contained a single viral DNA sequence at a different, but in each case unique, chromosomal site. DNA was extracted from each population of mitotic cells and then was resolved into dense, BrdUrd-containing and light, unsubstituted DNA fractions by cesium chloride gradient centrifugation. In each pair of samples obtained, we measured viral DNA sequences by solution hybridization using single-stranded SV40 32P-labeled DNA probes. Our results support the conclusions that specific genes within a mammalian DNA are programmed to replicate at particular times during S phase, and that the SV40 A gene product, large T antigen, programs integrated viral DNA sequences to replicate very early in S phase. The fractions of viral DNA replicated early in S phase appeared to correlate with each clone's content of functional large T antigen at permissive and nonpermissive culture temperatures.

Animals↗
PubMed1983

ARS replication during the yeast S phase.

A 1.45 kb circular plasmid derived from yeast chromosome IV contains the autonomous replication element called ARS1. Isotope density transfer experiments show that each plasmid molecule replicates once each S phase, with initiation depending on two genetically defined steps required for nuclear DNA replication. A density transfer experiment with synchronized cells demonstrates that the ARS1 plasmid population replicates early in the S phase. The sequences adjacent to ARS1 on chromosome IV also initiate replication early, suggesting that the ARS1 plasmid contains information which determines its time of replication. The times of replication for two other yeast chromosome sequences, ARS2 and a sequence referred to as 1OZ, indicate that the temporal order of replication is ARS1 leads to ARS2 leads to 1OZ. These experiments show directly that specific chromosome regions replicate at specific times during the yeast S phase. If ARS elements are origins of chromosome replication, then the experiment reveals times of activation for two origins.

DNA Replication↗
PubMed1998

Analysis in Escherichia coli of the effects of in vivo CpG methylation catalyzed by the cloned murine maintenance methyltransferase.

Due in part to the complexity of mammalian systems, some of the proposed biological influences of mammalian DNA methylation have not been fully established. Escherichia coli cells, which normally contain negligible CpG methylation, exhibited progressive slowing of replication and lengthened generation times when expressing the murine DNA maintenance methyltransferase. Genomic analysis indicated significant amounts of CpG methylation in expressing cells which was absent from control cells. Expressing cells exposed to the cytosine demethylating agent, 5-azacytidine, rapidly reverted to propagation levels of controls. Substitution of cysteine with alanine in the carboxyl-terminal region proline-cysteine dipeptide of the methyltransferase completely inactivated methylating activity and cells expressing the inactive enzyme replicated as well as controls. These findings strongly implicate a role of epigenetic de novo CpG methylation in modulating cellular propagation, demonstrate that the maintenance methyltransferase can de novo methylate in vivo, and show that the methyltransferase requires an active site cysteine for activity.

Animals↗
PubMed1981

Poly(ADP-ribose) polymerase activity in mouse cells which exhibit temperature-sensitive DNA synthesis.

The poly(ADP-ribose) polymerase activity of wild-type mouse L cells and of Balb/C-3T3 mouse fibroblasts remained relatively unchanged (at approx. 400 nmol substrate utilized/mg DNA per h) in actively-growing cells incubated at 34 degrees C or at 38.5 degrees C for at least 72 h. A similar result was obtained with the following temperature-sensitive cells grown at the permissive temperature (34 degrees C): ts A1S9 mouse L cells, ts C1 mouse L cells and Balb/C-3T3 ts mouse fibroblasts. The poly(ADP-ribose) polymerase activity of the temperature-sensitive cells was little affected during incubation for 20-24 h at the non-permissive temperature of 38.5 degrees C under which conditions temperature-inactivation of DNA replication was complete. Thereafter, this enzyme activity was found to increase some 2-fold, at a time when normal semi-conservative DNA synthesis was totally suppressed and replaced by repair replication (Sheinin, R. and Guttman, S. (1977) Biochim. Biophys. Acta 479, 105-118; Sheinin, R., Dardick, I. and Doane, F.W. (1980) Exp. Cell. Res., in the press).

Animals↗
PubMed1999

A model for a umuDC-dependent prokaryotic DNA damage checkpoint.

The products of the Escherichia coli umuDC operon are required for translesion synthesis, the mechanistic basis of most mutagenesis caused by UV radiation and many chemicals. The UmuD protein shares homology with LexA, the repressor of SOS-regulated loci, and similarly undergoes a facilitated autodigestion on interaction with the RecA/single-stranded DNA nucleoprotein filaments formed after a cell experiences DNA damage. This cleavage, in which Ser-60 of UmuD acts as the nucleophile, produces UmuD', the form active in translesion synthesis. Expression of the noncleavable UmuD(S60A) protein and UmuC was found to increase survival after UV irradiation, despite the inability of the UmuD(S60A) protein to participate in translesion synthesis; this survival increase is uvr(+) dependent. Additional observations that expression of the UmuD(S60A) protein and UmuC delayed the resumption of DNA replication and cell growth after UV irradiation lead us to propose that the uncleaved UmuD protein and UmuC delay the resumption of DNA replication, thereby allowing nucleotide excision repair additional time to repair the damage accurately before replication is attempted. After a UV dose of 20 J/m(2), uncleaved UmuD is the predominant form for approximately 20 min, after which UmuD' becomes the predominant form, suggesting that the umuDC gene products play two distinct and temporally separated roles in DNA damage tolerance, the first in cell-cycle control and the second in translesion synthesis over unrepaired or irreparable lesions. The relationship of these observations to the eukaryotic DNA damage checkpoint is discussed.

Bacterial Proteins↗
PubMed2002

Ku affects the ataxia and Rad 3-related/CHK1-dependent S phase checkpoint response after camptothecin treatment.

Camptothecin (CPT) that targets DNA topoisomerase I is one of the most promising broad-spectrum anticancer drugs in development today. The cytotoxicity of CPT is S phase (S)-specific because the collision of advancing replication forks with CPT-topoisomerase I-DNA complexes results in DNA damage. After DNA damage, proliferating cells could actively slow down the DNA replication through an S checkpoint to provide time for repair. We report now that there is an activated S checkpoint response in CPT-treated mammalian cells. This response is regulated by Ataxia and Rad3-related (ATR)/CHK1 pathway. Compared with their wild-type counterparts, CPT-treated Ku80-/- cells showed stronger inhibition of DNA replication. This stronger inhibition had no relationship with DNA-dependent protein kinase (DNA-PK) activity but correlated with the higher activities of ATR and the higher activities of CHK1 in such cells. Not only caffeine, the nonspecific inhibitor of ATR, or UCN-01, the nonspecific inhibitor of CHK1, but also the specific CHK1 antisense oligonucleotide abolished the stronger inhibition of DNA replication in CPT-treated Ku80-/- cells. These results in aggregate indicated that the stronger S checkpoint in CPT-treated Ku80-/- cells is regulated through the highly activated ATR/CHK1 pathway.

Animals↗
PubMed1995

The effects of 5-azacytidine and 5-azadeoxycytidine on chromosome structure and function: implications for methylation-associated cellular processes.

5-Azacytidine (5-aza-C) analogs demonstrate a remarkable ability to induce heritable changes in gene and phenotypic expression. These cellular processes are associated with the demethylation of specific DNA sequences. On the other hand, 5-aza-C analogs have dramatic effects on chromosomes, leading to decondensation of chromatin structure, chromosomal instability and an advance in replication timing. Condensation inhibition of genetically inactive chromatin occurs when the DNA is still hemimethylated or fully methylated. In cell cultures prolonged for several replication cycles, chromosomal rearrangements and instability affect the 5-aza-C-sensitive regions. Moreover, the normally late-replicating inactive chromatin undergoes a transient temporal shift to an earlier DNA replication, characteristic of activatable chromatin. zThe induced alterations of chromosome structure and behavior may trigger the 5-aza-C-dependent process of cellular reprogramming. Apart from their differentiating and gene-modifying effects, 5-aza-C analogs can tumorigenically transform cells and modulate their metastatic potential. High doses of 5-aza-C analogs have cytotoxic and antineoplastic activities.

Animals↗
PubMed1978

Simian virus 40 DNA replication in isolated replicating viral chromosomes.

Three subnuclear systems capable of continuing many aspects of simian virus 40 (SV40) DNA replication were characterized in an effort to define the minimum requirements for "normal" DNA replication in vitro. Nuclear extracts, prepared by incubating nuclei isolated from SV40-infected CV-1 cells in a hypotonic buffer to release both SV40 replicating and mature chromosomes, were either centrifuged to separate the total SV40 nucleoprotein complexes from the soluble nucleosol or fractionated on sucrose gradients to provide purified SV40 replicating chromosomes. With nuclear extracts, CV-1 cell cytosol stimulated total DNA synthesis, elongation of nascent DNA chains, maturation and joining of "Okazaki pieces," and the conversion of replicating viral DNA into covalently closed, superhelical DNA. Nucleoprotein complexes responded similarly, but frequently the response was reduced by 10 to 30%. In contrast, isolated replicating chromosomes in the presence of cytosol appeared only to complete and join Okazaki pieces already present on the template; without cytosol, Okazaki pieces incorporated alpha-(32)P-labeled deoxynucleoside triphosphates but failed to join. Consequently, replicating chromosomes failed to extensively continue nascent DNA chain growth, and the conversion of viral replicating DNA into mature DNA was seven to eight times less than that observed in nuclear extracts. Addition of neither cytosol nor nucleosol corrected this problem. In the presence of cytosol, nonspecific endonuclease activity was not a problem in any of the three in vitro systems. Extensive purification of replicating chromosomes was limited by three as yet irreversible phenomena. First, replicating chromosomes isolated in a low-ionic-strength medium had a limited capability to continue DNA synthesis. Second, diluting either nuclear extracts or replicating chromosomes before incubation in vitro stimulated total DNA synthesis but was accompanied by the simultaneous appearance of small-molecular-weight nascent DNA not associated with intact viral DNA templates and a decrease in the synthesis of covalently closed viral DNA. Although this second phenomenon appeared similar to the first, template concentration alone could not account for the failure of purified replicating chromosomes to yield covalently closed DNA. Finally, preparation of nucleoprotein complexes in increasing concentrations of NaCl progressively decreased their ability to continue DNA replication. Exposure to 0.3 M NaCl removed one or more factors required for DNA synthesis which could be replaced by addition of cytosol. However, higher NaCl concentrations yielded nucleoprotein complexes that had relatively no endogenous DNA synthesis activity and that no longer responded to cytosol. These data demonstrate that continuation of endogenous DNA replication in vitro requires both the soluble cytosol fraction and a complex nucleoprotein template whose ability to continue DNA synthesis depends on its concentration and ionic environment during its preparation.

Cell Line↗