[DNA polymerases of animal cells: III. DNA replication and DNA polymerase alpha (author's transl)].
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Animals↗
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DNA repair replication, DNA breaks and sister-chromatid exchange in human cells treated with adriamycin in vitro.
The effects of adriamycin (AM) on DNA repair replication, the frequency of sister-chromatid exchange (SCE), the rate of cell proliferation and the frequency of DNA strand breaks were studied in human cells in vitro. No repair replication was observed in lymphocytes exposed to AM in concentrations up to 10(-3) moles/l. DNA repair replication induced by UV and alkylating agents was not affected by a concentration of AM that completely inhibited cell proliferation (10(-6) moles/l). Fibroblasts exposed to AM at 10(-4) moles/l in the presence of hydroxyurea showed an increase of strand breaks and cross-links in DNA. When AM was added to UV-irradiated fibroblasts, there was an increase of DNA strand breaks in addition to the breaks caused by UV alone. Similar effects were observed in lymphocytes. A dose-dependent increase of SCE was observed in lymphocytes exposed to low concentrations of AM (less than 10(-7) moles/l). At higher concentrations the increase of SCE levelled off, and cell proliferation became severely inhibited. There was no evidence of removal of SCE-inducing damage in cells exposed to AM during G0 or G1. The level of SCE induced in the third cell cycle after treatment with AM was not different from that induced during the first two cell cycles. These results suggest that the various genotoxic and cytotoxic effects of AM are caused by different types of cellular damage. Moreover, AM-induced DNA damage persists for several cell cycles in human cells in vitro and seems to be resistant to repair activity.
Cell Cycle↗
On the fidelity of DNA replication: herpes DNA polymerase and its associated exonuclease.
Procaryotic DNA polymerases contain an associated 3'----5' exonuclease activity which provides a proofreading function and contributes substantially to replication fidelity. DNA polymerases of the eucaryotic herpes-type viruses contain similar associated exonuclease activities. We have investigated the fidelity of polymerases purified from wild type herpes simplex virus, as well as from mutator and antimutator strains. On synthetic templates, the herpes enzymes show greater relative exonuclease activities, and greater ability to excise a terminal mismatched base, than procaryotic DNA polymerases which proofread. On a phi X174 natural DNA template, the herpes enzymes are more accurate than purified eucaryotic DNA polymerases; the error rate is similar to E. coli polymerase I. However, conditions which abnegate proofreading by E. coli polymerase I have little effect on the herpes enzymes. We conclude that either these viral polymerases are accurate in the absence of proofreading, or the conditions examined have little effect on proofreading by the herpes DNA polymerases.
DNA Replication↗
A ts mutant isolated from CHL cells: inhibition of DNA replication at nonpermissive temperature.
A mutant Chinese hamster cell clone has been isolated from an S phase culture. This clone, ts154, could not replicate DNA at nonpermissive temperature (39 degrees C), although it synthesized DNA normally at permissive temperature (33 degrees C). After transfer to 39 degrees, ts154 cells replicated DNA semiconservatively through precisely two complete rounds and divided twice before they ceased DNA synthesis and further growth. The mutation in ts154 affects DNA replication specifically, as its rates of cellular transcription, its ability to support growth of vesicular stomatitis virus, and its ribo- and deoxyribonucleoside triphosphate pools were normal at 39 degrees. Cultures of ts154 resumed DNA replication within 6h when shifted from 39 degrees to 33 degrees, even after 78 h at the nonpermissive temperature. Upon return to 33 degrees, mutant cells achieved a normal steady-state rate of DNA synthesis by 10 h, suggesting that they had come to rest within G1 phase at 39 degrees. Resumption of DNA synthesis and growth after a shift-down to 33 degrees were sensitive to cycloheximide in ts154, and thus likely required synthesis of new protein.
Animals↗
The p150 and p60 subunits of chromatin assembly factor I: a molecular link between newly synthesized histones and DNA replication.
Chromatin assembly factor I (CAF-I) from human cell nuclei is a three-subunit protein complex that assembles histone octamers onto replicating DNA in a cell-free system. Sequences of cDNAs encoding the two largest CAF-I subunits reveal that the p150 protein contains large clusters of charged residues, whereas p60 contains WD repeats. p150 and p60 directly interact and are both required for DNA replication-dependent assembly of nucleosomes. Deletion of the p60-binding domain from the p150 protein prevents chromatin assembly. p150 and p60 form complexes with newly synthesized histones H3 and acetylated H4 in human cell extracts, suggesting that such complexes are intermediates between histone synthesis and assembly onto replicating DNA.
Amino Acid Sequence↗
Relationship of hepatic peroxisome proliferation and replicative DNA synthesis to the hepatocarcinogenicity of the peroxisome proliferators di(2-ethylhexyl)phthalate and [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid (Wy-14,643) in rats.
The mechanism of hepatocarcinogenesis caused by peroxisome proliferators (PP) is poorly understood, making it difficult to predict the carcinogenicity of PP to rodents or other species. It has been suggested that the carcinogenic potential of individual PP in rodents is correlated with the degree of PP-induced hepatic peroxisome proliferation. To evaluate this possible correlation, di(2-ethylhexyl)phthalate (DEHP) at 1.2% and [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid (Wy-14,643) at 0.1% were fed to male F-344 rats for up to 365 days and hepatocytic peroxisome proliferation and DNA replication were measured. All rats fed Wy-14,643 for 365 days had numerous grossly visible nodules in comparison to none in the livers of DEHP-fed or control rats. Despite this difference in the induction of tumors, both DEHP and Wy-14,643 increased the peroxisomal volume density 4- to 6-fold from 8 to 365 days of treatment. Peroxisomal beta-oxidation enzyme activities were increased 8-fold by both DEHP and Wy-14,643 after 18 days. At later time points (77 to 365 days), these enzyme activities were about 25% higher in livers of Wy-14,643- than DEHP-fed rats. DEHP or Wy-14,643 increased absolute liver weights 50 to 75% above controls after 18 to 365 days of feeding. Labeling of hepatocyte nuclei with a single injection of tritiated thymidine revealed a rapid burst in replicative DNA synthesis in both DEHP and Wy-14,643-fed rats, with a return to control levels by 4 days. Additional rats were implanted with 7-day osmotic pumps containing tritiated thymidine. With this more extended method of labeling a 5- to 10-fold increase in replicative DNA synthesis was observed in rats receiving Wy-14,643 for 39 to 365 days as compared to DEHP-fed rats or controls. In conclusion, when performed under conditions similar to the tumorigenicity studies, the degree of peroxisome proliferation correlated poorly with the relative hepatocarcinogenicity of DEHP and Wy-14,643. However, a strong correlation was observed between the relative hepatocarcinogenicity of DEHP and Wy-14,643 and the ability to induce a persistent increase in replicative DNA synthesis. These data emphasize the possible importance of cell replication in the mechanism of PP-induced hepatocarcinogenesis.
Animals↗
On the origin of bacteriophage phiX174 replicative form DNA replication.
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Chromosome Mapping↗
Mitochondrial DNA replication but no nuclear DNA replication during development of Dictyostelium.
Dictyostelium discoideum cells initiate development when nutrients are depleted. DNA synthesis decreases rapidly thereafter but resumes during late aggregation, only in prespore cells. This observation has been previously interpreted as indicating progression of prespore cells through the cell cycle during development. We show that developmental DNA replication occurs only in mitochondria and not in nuclei. We also show that the prestalk morphogen known as differentiation-inducing factor 1 can inhibit mitochondrial respiration. A model is proposed for cell type divergence, based on competition to become prespores, that involves mitochondrial replication in prespore cells and reduction of mitochondrial activity in prestalk cells.
Animals↗
cdc2 family kinases phosphorylate a human cell DNA replication factor, RPA, and activate DNA replication.
RPA is a single-stranded DNA binding protein complex purified from human cells and is essential for the initiation and elongation stages of SV40 DNA replication in vitro. In both human and yeast cells, the 34 kDa polypeptide subunit of RPA is phosphorylated in the S and G2 phases of the cell cycle and not in G1. One of the major RPA kinases present in extracts of human cells was purified and shown to be the cyclin B-cdc2 complex. This purified kinase, and a closely related cyclin A associated cdc2-like kinase, phosphorylated RPA p34 on a subset of the chymotryptic peptides that were phosphorylated in vivo at the G1-S transition. Two serines near the N-terminus of RPA p34 were identified as possible sites of phosphorylation by cdc2 kinase. These same serines were necessary for RPA phosphorylation in vivo. The purified cdc2 kinase stimulated SV40 DNA replication in vitro when added to G1 cell extracts. The kinase also stimulated unwinding at the origin of replication, one of the earliest steps in DNA replication requiring RPA, but only in the presence of an additional factor present in G1 cell extracts. Thus, one or more members of the cyclin-cdc2 kinase family may be required for the initiation and maintenance of S phase, in part due to their ability to phosphorylate and activate a cellular DNA replication factor, RPA.
Adenovirus Early Proteins↗
Dependence of mammalian DNA replication on DNA supercoiling. I. Effects of ethidium bromide on DNA synthesis in permeable Chinese hamster ovary cells.
Chinese hamster ovary cells labelled with [14C]thymidine were made permeable, incubated with various concentrations of the intercalating dye ethidium bromide, and centrifuged through neutral sucrose gradients. The gradient profiles of these cells were qualitatively similar to those obtained by centrifuging DNA from untreated, lysed permeable cells through gradients containing ethidium bromide. The sedimentation distance of DNA had a biphasic dependence on the concentration of ethidium bromide, suggesting that the dye altered the amount of DNA supercoiling in situ. The effect of ethidium bromide intercalation on incorporation of [3H]dTMP into acid-precipitable material in an in vitro DNA synthesis mixture was measured. The incorporation of [3H]dTMP was unaffected by less than 1 microgram/ml of ethidium bromide, enhanced up to two-fold by 1--10 microgram/ml, and inhibited by concentrations greater than 10 micrograms/ml. Alkaline sucrose gradient analysis revealed a higher percentage of small DNA fragments (6--20 S) in the cells treated with 2 micrograms/ml ethidium bromide than in control cells. These fragments attained parental size within the same time as the fragments in control cells. In cells treated with 2 micrograms/ml ethidium bromide, a significant fraction of newly synthesized DNA resulted from new starts, whereas in untreated cells practically none of the newly synthesized DNA resulted from new starts. These results suggest that relaxation of DNA supercoiled structures ahead of the replication fork generates spurious initiations of DNA synthesis and that in intact cells the rate of chain elongation is limited by supercoiled regions ahead of the growing point.
Animals↗
In vitro DNA replication of recombinant plasmid DNAs containing the origin of progeny replicative form DNA synthesis of phage phi X174.
The origin of phage phi X174 progeny replicative form (RF) DNA synthesis has been inserted into the plasmid vector pBR322 and cloned. In direct contrast to pBR322, the recombinant superhelical plasmids can substitute for phi X174 RFI DNA as template in phi X174-specific reactions in vitro. We have shown that the recombinant plasmids: (i) are cleaved by the phi X174 A protein; (ii) support net synthesis of unit-length single-stranded circular DNA in the presence of the phi X174 A protein and Escherichia coli rep protein, DNA-binding protein, and DNA polymerase III elongation system; (iii) support replication of duplexes catalyzed by the phi X174 A protein and extracts of E. coli.
Bacterial Proteins↗
Co-operative interaction between the initiator E1 and the transcriptional activator E2 is required for replicator specific DNA replication of bovine papillomavirus in vivo and in vitro.
The E1 polypeptide from bovine papillomavirus binds to the origin of replication (ori) and possesses the activities attributed to initiator proteins. E1 is also the only viral protein required for replication in a cell-free replication system. Replication in vivo, however, absolutely requires in addition the viral transcription factor E2. We demonstrate that the basis for this distinction between in vitro and in vivo requirements is the limited sequence specificity of the E1 protein. E1 and E2, which bind the ori individually with low sequence specificity, together bind with greatly increased sequence specificity. This combinatorial effect provides a function for the involvement of transcriptional activation domains in replication and suggests common mechanisms of action for transcription factors in both transcription and replication. It also provides a possible explanation for the differential specificity that is observed for auxiliary transcription factors in vivo.
Animals↗
Evidence that a high molecular weight replicative DNA polymerase is conserved during evolution.
Using a technique developed recently to detect DNA polymerase activity in situ after NaDodSO4 gel electrophoresis (Spanos, A., Sedgwick, S. G., Yarranton, g. T., Hübscher, U. & Banks, G. R. (1981) Nucleic Acids Res. 9, 1825-1839), we present evidence that a high Mr (greater than or equal to 125,000) polypeptide is responsible for chromosomal DNA replication in prokaryotes, lower eukaryotes and high eukaryotes. Not only extracts from Escherichia coli, Ustilago maydis, Drosophila melanogaster, rat neurones, calf thymus, human fibroblast, and HeLa cells possess such high Mr activities, but also highly purified E. coli DNA polymerase III core enzyme, U. maydis DNA polymerase, and D. melanogaster embryo and calf thymus DNA alpha polymerases. The evidence that these activities are responsible for chromosomal DNA replication is genetical (E. coli, U. maydis, and D. melanogaster); also, the high Mr activity disappears from rat neurones during differentiation from an actively dividing precursor cell to a postmitotically mature neurone. Furthermore, when limited proteolysis is allowed to occur, a defined and remarkably similar pattern of intermediate Mr activities is generated in lower eukaryotic and high eukaryotic extracts and, to some extent, in prokaryotic extracts. In higher eukaryotic extracts, a low Mr activity of approximately 35,000 is also generated. Protease inhibitors can retard formation of these catalytically active proteolytic fragments. We propose that the replicative DNA polymerase complex of both prokaryotes and eukaryotes contains a high Mr polypeptide responsible for chain elongation which might be conserved during evolution and which is extremely sensitive to proteolytic cleavage.
Animals↗
Suppressor mutations (rin) that specifically suppress the recA+ dependence of stable DNA replication in Escherichia coliK-12.
The sdrA102 mutation confers upon cells the ability to replicate DNA in the absence of protein synthesis. This mutation was combined with the recA200 mutation, which renders the recA protein thermolabile, and had little effect on normal replication. However, the sdrA102 recA200 double mutant exhibited temperature-sensitive stable DNA replication: it replicated DNA continuously in the presence of chloramphenicol at 30 degrees C, whereas at 42 degrees C DNA replication ceased after the DNA content increased only 40-45%. Suppressor mutants (rin; recA-independent) capable of stable DNA replication at 42 degrees C were isolated from the double mutant. The suppressor mutant retained all other recA- characteristics, i.e., deficient general recombination, severe UV-sensitivity, and incapability of prophage induction in lysogens. This indicates that the rin mutation specifically suppresses the recA+ dependency of stable DNA replication. It is suggested that the recA+ protein stabilizes a specific structure, similar to an intermediate in recombination, which may function in the initiation of stable DNA replication.
Bacterial Proteins↗
Replicative DNA synthesis and unscheduled DNA synthesis in permeable sarcoma cells studied by nuclease digestion.
About 20% of DNA replicated in vitro in permeable mouse ascites sarcoma cells showed higher sensitivity to staphylococcal nuclease than the sensitivity of bulk DNA, and the remaining part showed the same nuclease sensitivity as that of parental chromatin DNA. The sensitivity of DNA replicated in permeable cells was higher than that of DNA newly replicated in vivo in intact cells, and close to that of DNA newly replicated in vivo in the presence of cycloheximide. Bleomycin-induced unscheduled DNA synthesis in permeable cells was highly sensitive to the nuclease. The results suggest that DNA replicated in vitro and parental nuclear protein form immature nucleosomes, probably in the same way as in vivo chromatin replication in the presence of protein synthesis inhibitors. It also appears that bleomycin-induced, unscheduled DNA synthesis occurs largely in the internucleosomal region.
Animals↗
Induction of UV-resistant DNA replication in Escherichia coli: induced stable DNA replication as an SOS function.
The striking similarity between the treatments that induce SOS functions and those that result in stable DNA replication (continuous DNA replication in the absence of protein synthesis) prompted us to examine the possibility of stable DNA replication being a recA+ lexA+-dependent SOS function. In addition to the treatments previously reported, ultraviolet (UV) irradiation or treatment with mitomycin C was also found to induce stable DNA replication. The thermal treatment of tif-1 strains did not result in detectable levels of stable DNA replication, but nalidixic acid readily induced the activity in these strains. The induction of stable DNA replication with malidixic acid was severely suppressed in tif-1 lexA mutant strains. The inhibitory activity of lexA3 was negated by the presence of the spr-51 mutation, an intragenic suppressor of lexA3. Induced stable DNA replication was found to be considerably more resistant to UV irradiation than normal replication both in a uvrA6 strain and a uvr+ strain. The UV-resistant replication occurred mostly in the semiconservative manner. The possible roles of stable DNA replication in repair of damaged DNA are discussed.
Bacterial Proteins↗
Human papillomavirus DNA replication compartments in a transient DNA replication system.
Many DNA viruses replicate their genomes at nuclear foci in infected cells. Using indirect immunofluorescence in combination with fluorescence in situ hybridization, we colocalized the human papillomavirus (HPV) replicating proteins E1 and E2 and the replicating origin-containing plasmid to nuclear foci in transiently transfected cells. The host replication protein A (RP-A) was also colocalized to these foci. These nuclear structures were identified as active sites of viral DNA synthesis by bromodeoxyuridine (BrdU) pulse-labeling. Unexpectedly, the great majority of RP-A and BrdU incorporation was found in these HPV replication domains. Furthermore, E1, E2, and RP-A were also colocalized to nuclear foci in the absence of an origin-containing plasmid. These observations suggest a spatial reorganization of the host DNA replication machinery upon HPV DNA replication or E1 and E2 expression. Alternatively, viral DNA replication might be targeted to host nuclear domains that are active during the late S phase, when such domains are limited in number. In a fraction of cells expressing E1 and E2, the promyelocytic leukemia protein, a component of nuclear domain 10 (ND10), was either partially or completely colocalized with E1 and E2. Since ND10 structures were recently hypothesized to be sites of bovine papillomavirus virion assembly, our observation suggests that HPV DNA amplification might be partially coupled to virion assembly.
Cell Nucleus↗
Ability of N-methyl-N'-nitro-N-nitrosoguanidine, 4-nitroquinoline 1-oxide, dimethylnitrosamine, and NaCl to induce unscheduled DNA synthesis, stimulate replicative DNA synthesis, and produce DNA single-strand breaks in pyloric mucosa of rat stomach.
Male F344 rats were given test chemicals orally, and samples of their pyloric mucosa were incubated in vitro. Induction of unscheduled DNA synthesis (UDS) and stimulation of replicative DNA synthesis in the pyloric mucosa were then examined by addition of [3H]thymidine and simultaneous determinations of DNA synthesis in the presence and absence of hydroxyurea, an inhibitor of replicative DNA synthesis. DNA damage was also examined by the alkaline elution method with DNA single-strand scission as a marker. The results showed four types of abilities of the chemicals to affect UDS and replicative DNA synthesis in the pyloric mucosa of rat stomach 1-2 h after their administration: (1) induction of UDS and stimulation of replicative DNA synthesis by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a glandular stomach carcinogen, (2) induction of only UDS by 4-nitroquinoline 1-oxide (4NQO), a glandular stomach carcinogen, (3) stimulation of only replicative DNA synthesis by NaCl, a glandular stomach tumor promoter, and (4) neither induction of UDS nor stimulation of replicative DNA synthesis by dimethylnitrosamine (DMN), a liver carcinogen. DNA single-strand scission was induced by MNNG and 4NQO, being maximal 2 h after their administration, but was not induced by NaCl or DMN. Thus it correlated well with the induction of UDS. The present results indicate four types of inductive abilities of chemicals on UDS and replicative DNA synthesis in rat stomach pyloric mucosa and show that this method can detect differences in the action mechanisms and organ specificities of glandular stomach carcinogens.
4-Nitroquinoline-1-oxide↗