DNA replication in synchronized cultured mammalian cells. 3. Relative times of synthesis of mouse satellite and main band DNA.
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Animals↗
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The control of mammalian DNA replication: a brief history of space and timing.
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Animals↗
Regulation of deoxyribonucleic acid replication and cell division in Escherichia coli B-r.
Synchronous cultures of Escherichia coli strain B/r were used to investigate the relationship between deoxyribonucleic acid (DNA) replication and cell division. We have determined that terminal steps in division can proceed in the absence of DNA synthesis. Inhibition of DNA replication with nalidixic acid prior to the start of a new round of replication does not stop cell division, which indicates that the start of the round is not essential in triggering cell division. Inhibition of DNA replication at any time prior to the termination of a round of replication completely blocks cell division, which suggests that there may be a link between the end of the replication cycle and the commitment of the cell to divide. Studies that use a temperature-sensitive mutant which is unable to synthesize DNA at the nonpermissive temperature are in complete agreement with those that use nalidixic acid to inhibit DNA synthesis. This adds support to the idea that the treatments employed limit their action to DNA synthesis. Investigation of minicell production indicates that the production of minicells is blocked when DNA synthesis is inhibited with nalidixic acid. Although nuclear segregation is not required for cell division, DNA synthesis is still required to trigger division. The evidence presented suggests strongly that (i) DNA synthesis is essential for cell division, (ii) the end of a round of replication triggers cell division, and (iii) there is considerable time lapse (one-half generation) between the completion of a round of DNA replication and physical separation of the cells.
Carbon Isotopes↗
Participation of the human beta-globin locus control region in initiation of DNA replication.
The human beta-globin locus control region (LCR) controls the transcription, chromatin structure, and replication timing of the entire locus. DNA replication was found to initiate in a transcription-independent manner within a region located 50 kilobases downstream of the LCR in human, mouse, and chicken cells containing the entire human beta-globin locus. However, DNA replication did not initiate within a deletion mutant locus lacking the sequences that encompass the LCR. This mutant locus replicated in the 3' to 5' direction. Thus, interactions between distantly separated sequences can be required for replication initiation, and factors mediating this interaction appear to be conserved in evolution.
Animals↗
Control of DNA replication by protein synthesis at defined times during the S period in Physarum polycephalum.
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Centrifugation, Density Gradient↗
Dependence of timing of mitotic events on the rate of protein synthesis and DNA replication in sea urchin early cleavages.
To understand what processes affect the cell-cycle timing of mitotic events in early cleavage cycles of sea urchin embryos, a study was made on the effects of (a) reducing protein synthesis with emetine and (b) DNA replication with aphidicolin, on the timing of nuclear envelope breakdown, anaphase onset and cytokinesis. When protein synthesis was slightly inhibited by administration of emetine, the delay in the mitotic events increased, with an increase in the delay in accumulation of proteins up to the levels to which cells must synthesize the proteins to execute the cleavage. This indicated that protein synthesis affects the timing of mitotic events. The delay in cleavage cycles caused by a slight inhibition of DNA replication with aphidicolin was in proportion to the concentration of aphidicolin administered, suggesting that DNA replication also affects the timing of mitotic events. Furthermore, it was confirmed that accumulation of the proteins to the levels required for execution of the first cleavage precedes completion of DNA replication as a requirement for execution of the first cleavage. These results imply the existence of process(es) affected by protein synthesis that are included in a feedback control system which prevents the initiation of mitosis until after the completion of DNA replication; it is the characteristic of a cell-cycle control system that has been predicted theoretically.
Anaphase↗
Synthesis of virus-specific ribonucleic acid in KB cells infected with type 2 adenovirus.
By using the technique of deoxyribonucleic acid (DNA)-ribonucleic acid (RNA) hybridization, virus-specific RNA (cRNA) was detected 6 hr after infection in preparations of total RNA from cells infected with type 2 adenovirus in the presence of 2 mum 5-fluorodeoxyuridine. In the absence of 5-fluorodeoxyuridine, there was a continuous increase in the incorporation of (3)H-uridine into viral cRNA until 20 hr after infection, at which time approximately 40% of the (3)H-uridine entering RNA was found in virus-specific RNA. When RNA was prepared from polyribosome fractions obtained from cytoplasmic extracts of infected cells, virus-directed transcription was detected at 3 hr after infection (i.e., 3 to 4 hr before the initiation of viral DNA synthesis). Viral cRNA species synthesized at different times after infection were compared by the technique of DNA-RNA hybridization-inhibition ("presaturation" hybridization-competition). Three hybridization-inhibition techniques were compared. The techniques differed in the manner in which the DNA-RNA complex was isolated after the first hybridization reaction. Depending on the procedure employed, various degrees of inhibition were measured. The variation could be essentially eliminated if prior to hybridization the inhibitory RNA species were alkali-degraded to a uniform size of about 4S. Undegraded RNA could be used if the DNA-RNA complex was isolated by using a procedure involving rigorous washing (preferably including ribonuclease treatment) before the second hybridization with labeled RNA. When a rigorous hybridization-inhibition procedure was used, three classes of virus-specific RNA species could be distinguished: (i) early RNA class I whose synthesis began prior to viral DNA replication and stopped at some time after the initiation of viral DNA replication-it comprised about 70% of the early RNA species and was apparently degraded by 18 hr after infection; (ii) early RNA class II whose synthesis began prior to viral DNA replication and apparently continued at an enhanced rate late in infection; and (iii) late RNA whose synthesis began after the initiation of viral DNA synthesis.
Adenoviridae↗
Sequence of DNA replication in 277 R- and Q-bands of human chromosomes using a BrdU treatment.
Replication times for all important chromosome bands, of both types R and Q (277 structures) are analysed. The R-bands form a group of structures whose DNA replicates during the early S-phase, while the DNA situated in the Q-bands replicates during the late S-phase. There may not exist overlapping between replication times of these two types of structures. The widest R-bands are those which are the earliest to replicate; in general, the most intense Q-bands are those which are the latest to replicate. Especially among these last ones, a certain asynchronism exists between the replication times. Finally the heterochromatin of chromosomes 1, 16 and Y and of the short arms of the acrocentrics could contain two types of DNA which replicate at different times.
Bromodeoxyuridine↗
The effect of 2,2'-dichlorodiethyl sulfide on DNA synthesis of a murine stratified keratinocyte culture system.
A primary stratified keratinocyte culture resembling the epidermis in situ was used as a model for studying the effects of exposure to 2,2'-dichlorodiethyl sulfide, or sulfur mustard (SM), on DNA synthesis. A method that distinguishes between semi-conservative (s.c.) DNA synthesis and repair synthesis was used to determine if the former was inhibited following treatment with SM. In this method the density of the newly synthesized DNA was increased by incorporation of 5-bromo-2-deoxyuridine. Density gradient centrifugation was then used to isolate the heavy DNA for quantification. It was demonstrated that topically applied SM in the dose range of 1-10 nmole/cm2 inhibited s.c. DNA synthesis (replication) in a dose and time related manner. Inhibition of DNA replication by SM would result in inhibition of cell division which must be preceded by s.c. DNA synthesis. This failure to replace damaged germinative cells may lead to the destruction of the basal layer which is observed in vivo and in our epidermal culture following exposure to SM. This may also be related to development of vesication observed in exposed intact human skin.
Animals↗
Specific initiation site for simian virus 40 deoxyribonucleic acid replication.
Replicating simian virus 40 (SV40) deoxyribonucleic acid (DNA) molecules have been isolated under conditions in which the newly synthesized DNA is uniformly labeled with (3)H-thymidine. These newly synthesized strands are released from the replicative intermediate molecules by alkaline treatment, and it has been possible to isolate single-stranded SV40 DNA which varies in size from 157,000 daltons (from molecules that are 10% replicated) to 1,360,000 daltons (85% replicated). The rates of duplex formation of newly synthesized DNA have been used to relate their genetic complexity to the extent of DNA replication. As DNA replication proceeds, the time required to effect 50% renaturation of the newly synthesized DNA increases at a proportional rate. The data establish that DNA replication is not initiated at random, but rather that there is a single specific initiation site for DNA replication.
Animals↗
Cell cycle parameters of Proteus mirabilis: interdependence of the biosynthetic cell cycle and the interdivision cycle.
We investigated the time periods of DNA replication, lateral cell wall extension, and septum formation within the cell cycle of Proteus mirabilis. Cells were cultivated under three different conditions, yielding interdivision times of approximately 55, 57, and 160 min, respectively. Synchrony was achieved by sucrose density gradient centrifugation. The time periods were estimated by division inhibition studies with cephalexin, mecillinam, and nalidixic acid. In addition, DNA replication was measured by thymidine incorporation, and murein biosynthesis was measured by incorporation of N-acetylglucosamine into sodium dodecyl sulfate-insoluble murein sacculi. At interdivision times of 55 to 57 min murein biosynthesis for reproduction of a unit cell lasted longer than the interdivision time itself, whereas DNA replication finished within 40 min. Surprisingly, inhibition of DNA replication by nalidixic acid did not inhibit the subsequent cell division but rather the one after that. Because P. mirabilis fails to express several reactions of the recA-dependent SOS functions known from Escherichia coli, the drug allowed us to determine which DNA replication period actually governed which cell division. Taken together, the results indicate that at an interdivision time of 55 to 57 min, the biosynthetic cell cycle of P. mirabilis lasts approximately 120 min. To achieve the observed interdivision time, it is necessary that two subsequent biosynthetic cell cycles be tightly interlocked. The implications of these findings for the regulation of the cell cycle are discussed.
Cell Cycle↗
Replication patterns of repetitive DNA sequences on the W chromosome are altered during development of the chick embryo.
A novel method was developed to study developmental changes in the replication pattern of repetitive DNA sequences on the W chromosome (W-DNA) of the female chick embryo. The amount of total nuclear DNA and W-DNA as well as 5-bromodeoxyuridine (BrdU) incorporation was successively measured on the same cells using multiparametric microfluorometry. With this method we first examined the possibility of changes in replication patterns of W-DNA during development. Measurements were conducted on various heterogeneous cell populations obtained from whole embryo on Day 0.4 and Day 1, and from pectoral muscle, neural tube, liver, and oogonium on Day 9. Parameters of W-DNA replication, duration, and timing were found to vary according to the stage of embryonic development. Developmental features of these changes were further studied on specific cell types during their critical developmental processes. In scutate scale dermis, the W-DNA replication duration showed a characteristic lengthening from around 0.45C during Day 5 through Day 7.4 to 0.9C during Day 7.7 through Day 7.9 and shortening to 0.37C during Day 8.1 through Day 12. Transient lengthening in W-DNA replication duration was also observed in erythrocytes; 0.65C ->1.0C ->0.6C during Day 0.9 through Day 2.17. Timing also shifted earlier in accord with changes in the duration. Replication rate of whole genome DNA was monitored by measuring BrdU incorporation on respective cells and found, to a large extent, comparable to that of W-DNA. The data suggest that a link might be operative between replication patterns of genes and the developmental program.
Animals↗
Intranuclear accumulation of subgenomic noninfectious human cytomegalovirus DNA in infected cells in the presence of ganciclovir.
In preparation for an attempt to elucidate some aspects of the interaction between ganciclovir and human cytomegalovirus (HCMV) DNA replication in cells infected with HCMV, we developed a dot blot DNA-DNA hybridization technique to quantify intracellular HCMV DNA replication. We studied the effect of ganciclovir on the time course of HCMV DNA replication in human fibroblasts. Ganciclovir resulted in complete cessation of the production of infectious virus, as detected by the plaque assay. However, viral DNA synthesis, as measured by dot blot DNA-DNA hybridization with cloned HCMV DNA BamHI C fragment probe, continued in the presence of ganciclovir at 10 times the 50% effective dose (i.e., 10 micrograms/ml). The continuation of viral DNA synthesis in ganciclovir-treated cultures leads to the intranuclear accumulation of short (subgenomic) HCMV DNA fragments. These DNA fragments are neither packaged nor released into the culture medium. Furthermore, the short DNA fragments were detected only by the BamHI C probe from the center of the unique long segment of the HCMV genome. The failure of the DNA probes from the termini of HCMV genome (BamHI-Q and HindIII-M) to detect the short DNA fragments and the intranuclear localization of these fragments suggest that these short fragments may lack the signal sequences necessary for packaging and release as infectious virions. These data strongly suggest that the anti-HCMV activity of ganciclovir is due mainly to the prevention of viral DNA chain elongation which results in the intranuclear accumulation of incomplete noninfectious viral DNA fragments.
Cells, Cultured↗
Altered cell cycle progression and aberrant mitosis in adenovirus-infected rodent cells.
Actively growing mouse or rat embryo cells suffered structural chromosome damage, mitotic anomalies, and polyploidy after infection by human adenovirus type 5. Chromosome damage required expression of one or more early viral genes and showed regular periodicity in its frequency. The growth cycle time of some of the infected cells was reduced by about 5 hours due to a decrease in G1, and the interval between successive waves of chromosome damage corresponded to this reduced cycle time. After infection there was a decrease in cells with G1 DNA content and an increase in cells with G2 diploid, aneuploid, and polyploid DNA contents. We suggest these effects are due to the expression in semipermissive cells fo early viral gene(s), whose function in productive infection in vivo is to alter cell cycle controls in order to maximize the number of cells able to replicate viral DNA and the time such cells spend in DNA replication.
Adenoviridae Infections↗
Managing DNA polymerases: coordinating DNA replication, DNA repair, and DNA recombination.
Two important and timely questions with respect to DNA replication, DNA recombination, and DNA repair are: (i) what controls which DNA polymerase gains access to a particular primer-terminus, and (ii) what determines whether a DNA polymerase hands off its DNA substrate to either a different DNA polymerase or to a different protein(s) for the completion of the specific biological process? These questions have taken on added importance in light of the fact that the number of known template-dependent DNA polymerases in both eukaryotes and in prokaryotes has grown tremendously in the past two years. Most notably, the current list now includes a completely new family of enzymes that are capable of replicating imperfect DNA templates. This UmuC-DinB-Rad30-Rev1 superfamily of DNA polymerases has members in all three kingdoms of life. Members of this family have recently received a great deal of attention due to the roles they play in translesion DNA synthesis (TLS), the potentially mutagenic replication over DNA lesions that act as potent blocks to continued replication catalyzed by replicative DNA polymerases. Here, we have attempted to summarize our current understanding of the regulation of action of DNA polymerases with respect to their roles in DNA replication, TLS, DNA repair, DNA recombination, and cell cycle progression. In particular, we discuss these issues in the context of the Gram-negative bacterium, Escherichia coli, that contains a DNA polymerase (Pol V) known to participate in most, if not all, of these processes.
Animals↗
Evidence for a new mechanism of cytotoxicity of 1-beta-D arabinofuranosylcytosine.
Inhibition of DNA synthesis by a pulse of 1-beta-D-arabinofuranosylcytosine (ara-C) results in reinitiation of DNA replication in DNA segments replicated earlier in that S phase and hence double replication of some DNA segments. Experiments were with tissue culture cell lines of human origin. DNA replicated early in S phase was labeled with a pulse of [3H]deoxycytidine with the cells pulsed later in S phase with ara-C. The DNA replicated after the time of the ara-C pulse was density labeled with 5-bromodeoxyuridine. Reinitiation of DNA replication in the already replicated [3H]DNA segments was demonstrated using CsCl density gradient analysis by an increase in the 3H label present in the light-heavy peak of semiconservatively replicated DNA. Also, in DNA of control cells, all of the 3H was in the same strand of the light-heavy DNA duplex as was the 5-bromodeoxyuridine, as shown by alkaline CsCl density gradient analysis of purified light-heavy DNA. However, after a pulse of ara-C, utilization of [3H]DNA strands as template strands was demonstrated by the presence of 3H label at the density of unsubstituted DNA chains in alkaline CsCl gradients of the purified light-heavy DNA. This double replication phenomenon can explain certain chromosomal abnormalities induced by ara-C.
Cell Cycle↗
Replicating units (replicons) of DNA in cultured mammalian cells.
Exponentially growing L5178Y mouse leukemic cells were incubated in the presence of 5'-bromodeoxyuridine (BUdR) for about 4 hr, transferred to the nonBUdR-containing medium for a certain period (t hours), and then pulse-labeled with TdR-(3)H for 10 min. When DNA isolated from these cells was subjected to CsCl gradient centrifugation, the (3)H-activity was found to shift gradually from the heavy BUdR-containing peak to the light nonBUdR-containing peak with increasing time t. The average time required for the complete shift of (3)H-activity from the heavy to the light DNA fraction was 2.76 hr. Taking this as the average replicating time and the size of DNA fragments in the present preparation as 1.3 x 10(7) daltons, the rate of replication was found to be 2.1 nucleotides per strand per replicon per sec. By taking the upper limit of the average replicating time as the S period (7.3 hr), various characteristics of the replicating units, such as the lower and upper limits of average size, the average replicating time, the average number of replicating units, etc., were calculated (see Table I).
Animals↗
DNA replication in Physarum polycephalum: UV photolysis of maturing 5-bromo-deoxyuridine substituted DNA.
Combinations of 5-bromodeoxyuridine (BrdUrd) and 3H-deoxyadenosine (3H-DAdo) short pulses were given in the synchronous DNA-replication period of Physarum polycephalum. After a chase period, UV-photolysis products were analyzed on alkaline sucrose gradients. This strategy has allowed the following conclusions. a) at the time of master-initiation of DNA replication, points separated by 1.1-2.2x10(7) daltons of single strand DNA may initiate DNA synthesis. b) among these, only selected groups of replicons actually proceed in DNA replication at this time, while others appear to hold (later temporal sets of replicons). The origins of the ones that proceed in replication are separated from each other by a distance corresponding to 1.1-2.x10(7) daltons. c) regions in actual replication are separated from each other by increasing distances (up to 1.5x10(8) daltons single strand DNA) at later times in S.
Bromodeoxyuridine↗