Intragenomal Distribution of DNA Repair Synthesis: Repair in Satellite and Mainband DNA in Cultured Mouse Cells

DNA repair synthesis was examined in mouse satellite and mainband DNA derived from confluent Balb/c 3T3 cells damaged with ultraviolet radiation or N-acetoxy-2-acetylaminofluorene. Two different approaches were used: (i) Contact-inhibited cells were treated with hydroxyurea to reduce replicative synthesis to low levels; and (ii) bromodeoxyuridine was used to label newly replicated DNA in cells that had escaped contact inhibition. DNA was separated into mainband and satellite fractions in Ag(+)-Cs(2)SO(4) gradients. After treatment with either ultraviolet radiation or N-acetoxy-2-acetylaminofluorene, repair synthesis occurred to the same extent in mainband and satellite DNA. Repair synthesis increased over an ultraviolet radiation dose range of 30-200 erg/mm(2), and the extent of repair in the two DNA species was similar at each dose level. An analysis of the separated strands of satellite DNA from ultraviolet-irradiated cells indicated that the extent of repair is closely correlated with the availability of pyrimidines for cyclobutyl dimer formation and provided evidence that repair synthesis occurs at the site of damage. Within the precision of our experiments the results suggest that at least one group of highly repetitive, nontranslated DNA sequences is repaired to about the same extent as the rest of the genome.     

Translation In Vitro of Total Nuclear RNA from HeLa Cell Nuclei Infected with Adenovirus 2

Total heterogeneous nuclear RNA from HeLa cells infected with adenovirus for 18-20 hr stimulates amino acid incorporation into protein in a cell-free system from Ehrlich ascites tumors. This stimulation of protein synthesis by the nuclear RNA requires intact nuclei. The role of nuclei in this system is unknown, but evidence is presented that the nuclei are involved in the conversion of high-molecular-weight RNA to low-molecular-weight species. Some of the newly synthesized polypeptides appear to resemble the virion polypeptides.     

Characteristics of DNA repair induced by DNA polymerase β in hepatoma cells after γ-ray irradiation

AIM: To investigate the effects of DNA repair induced by DNA polymerase β in hepatoma cells after γ-ray irradiation.METHODS: Cell nuclei were prepared from mouse model (SMMC LTNM), in which human hepatoma cells are transplanted on nude mice. The nuclei were then irradiated with ⁶⁰Co-γ rays at different dose levels or dose rates. A selective inhibitor test was then used to detect the effects of the radiation on DNA repair using N-ethylmaleimide (NEM) and ddTTP as selective inhibitors to DNA polymerases γ and β respectively.RESULTS: ³H-TTP incorporation into irradiated nuclei or calf thymus DNA was significantly higher than that the rate at which it is incorporated into non-irradiated nuclei when either DNA polymerase β or γ was inhibited. When both NEM and ddTTP are present, the ³H-TTP incorporation in irradiated DNA was not significantly different from the non-irradiated nuclei. Furthermore, ³H-TTP incorporation into DNA of SMMC-LTNM hepatoma nuclei was higher than that of normal hepatocyte nuclei (P < 0.01). This suggests that DNA repair induced by DNA polymerase β was more active in hepatoma cell nuclei than in normal hepatocyte nuclei.CONCLUSION: DNA polymerase β may be more responsive to DNA damage in some tumor cells than that in normal cells, which may facilitate the cells to repair DNA damages from radiation more efficiently.     

Microinjected pBR322 stimulates cellular DNA synthesis in Swiss 3T3 cells.

When pBR322 is manually microinjected into the nuclei of quiescent Swiss 3T3 cells it stimulates the incorporation of [3H]thymidine into DNA. The evidence clearly shows that this increased incorporation that is detected by in situ autoradiography in microinjected cells represents cellular DNA synthesis and not DNA repair or plasmid replication. The effect is due to pBR322 and not due to impurities, mechanical perturbances due to the microinjection technique, or aspecific effects. This stimulation is striking in Swiss 3T3 cells. Some NIH 3T3 cells show a slight stimulation, but hamster cells, derived from baby hamster kidney (BHK) cells, are not stimulated when microinjected with pBR322. The preliminary evidence seems to indicate that the integrity of the pBR322 genome is important for the stimulation of cellular DNA synthesis in quiescent Swiss 3T3 cells. These results, although of a preliminary nature, are of interest because they indicate that a prokaryotic genome may alter the cell cycle of mammalian cells. From a practical point of view the stimulatory effect of microinjected pBR322 on cellular DNA synthesis has a more immediate interest, because pBR322 is the vector most commonly used for molecular cloning and 3T3 cells are very frequently used for gene transfer experiments.     

Functional roles of DNA polymerases beta and gamma.

The physiological functions of DNA polymerases (deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) beta and gamma were investigated by using neuronal nuclei and synaptosomes isolated from rat brain. UV irradiation of neuronal nuclei from 60-day-old rats resulted in a 7- to 10-fold stimulation of DNA repair synthesis attributable to DNA polymerase beta which, at this developmental stage, is virtually the only DNA polymerase present in the nuclei. No repair synthesis could be elicited by treating the nuclei with N-methyl-N-nitrosourea, but this way probably due to the inability of brain tissues to excise alkylated bases from DNA. The role of DNA polymerase gamma was studied in synaptosomes by using a system mimicking in vivo mitochondrial DNA synthesis. By showing that, under these conditions, DNA replication occurs in mitochondria, and exploiting the fact that DNA polymerase gama is the only DNA polymerase present in mitochondria, evidence was obtained for a role of DNA polymerase gamma in mitochondrial DNA replication. Based on these results and on the wealth of literature on DNA polymerase alpha, we conclude that DNA polymerase alpha is mainly responsible for DNA replication in nuclei, DNA polymerase beta is involved in nuclear DNA repair, and DNA polymerase gamma is the mitochondrial replicating enzyme. However, minor roles for DNA polymerase alpha in DNA repair or for DNA polymerase beta in DNA replication cannot be excluded.     

Mitogenic hormone-induced intracellular message: assay and partial characterization of an activator of DNA replication induced by epidermal growth factor.

This paper explores the pathway from nuclear quiescence to mitogenesis. It describes an in vitro assay for an activator of DNA replication induced by epidermal growth factor (EGF) in responsive cells. Cytoplasmic extracts from EGF-treated 3T3 cells were found to contain substances that can stimulate DNA synthesis in isolated nuclei from spleen cells of adult frogs. Extracts from untreated resting 3T3 cells lack this activity, and EGF itself is incapable of stimulating DNA synthesis in these cell-free systems. The extract-induced stimulation of incorporation of [3H]dTTP into nuclear DNA is ATP dependent and requires the presence of the four deoxyribonucleoside triphosphates, suggesting the occurrence of replication rather than repair synthesis. This cell-free assay has been used to obtain some initial insights into the mechanism of induction and biochemical characterization of the intermediate in EGF action. Half-maximal induction of the active intracellular substance is achieved at about 0.08 nM EGF, a concentration that correlates well with the concentration required for half-maximal mitogenesis. Studies on the biochemical characteristics of this active substance strongly suggest that the activity is associated with a protein. The activity is nondialyzable and sensitive to trypsin and heat. Sucrose gradient centrifugation of the extract revealed three peaks of activity with molecular weights of 46,000, 110,000, and 270,000 (sedimentation coefficients: 3.7 S, 6.6 S, and 12 S, respectively). These results indicate that receptor-EGF interaction at the cell surface leads to the intracellular generation of protein that are capable of stimulating quiescent nuclei into activity.     

Induction of Mitochondrial DNA Synthesis in Monkey Cells Infected by Simian Virus 40 and (or) Treated with Calf Serum

Infection of confluent monolayer cultures of African green monkey kidney cells with simian virus 40 results in an enhanced synthesis of nuclear and mitochondrial DNA. This is demonstrated both by an increased rate of incorporation of [(3)H]thymidine into mitochondrial DNA and by detection of increased amounts of mitochondrial DNA in infected cells. With monkey BSC-1 cells, where SV40 infection does not result in a stimulation of nuclear DNA synthesis, no stimulation of mitochondrial DNA synthesis is observed. SV40 infection of mouse 3T3 cells also stimulates nuclear and mitochondrial DNA synthesis.     

Displacement and aberrant methylation in vitro of H-1 histone in rat liver nuclei after half-saturation of chromatin with polycations.

Radiomethyl incorporation in vitro into Nepsilon-methyllysine of histones from rat liver nuclei incubated in the presence of S-adenosyl[methyl-3H]methionine is stimulated if the polycations polylysines, protamines, or histones are added to the incubation mixture. Maximal stimulation occurs at a cation/nucleotide ratio of 0.5. Past this point stimulation drops, except in the case of very lysine-rich histone H-1, for which the maximal level of incorporation remains constant upon further addition of this histone. Bio-Gel P-10 chromatography, differential precipitation, and gel electrophoresis of radiomethylated histones indicate that although the usual incorporation of radiomethyl into histone H-3 is not affected, active methylation of H-1 occurs in the presence of polycations. Column chromatographic amino acid analysis reveals that the methylation of H-1 will specifically generate Nepsilon-monomethyllysine. Except for this condition, H-1 is never methylated in vivo or in incubated cell nuclei. Because H-1 is the weakest bound histone in chromatin, the above phenomena may be explained by assuming that, within the chromatin, polycations displace the lysine-rich histone towards the nucleosome, which results in its abberant methylation, assuming that the native nucleosome is the seat of the histone lysine methyltransferase.     

Regulation of DNA replication in S phase nuclei by ATP and ADP pools

Synchronized 3T6 (mouse fibroblast) ghost monolayers (isolated nuclei) were utilized to study the effects of ATP and ADP levels on DNA replication in vitro. A system yielding discontinuous semiconservative DNA replication (without any detectable repair) in synchronized S phase nuclei has been developed. Lack of initiation of new sites has been observed in isolated S phase 3T6 nuclei without the presence of cytoplasmic material; DNA synthesis is comprised only of elongation at sites where initiation had previously taken place. DNA synthesis in S phase nuclei proceeded optimally at an ATP concentration of 4-5 mM. High ATP levels as well as high ATP/ADP ratios (produced by an ATP-regenerating system at a variety of ATP concentrations) yielded marked inhibition of [(3)H]dTTP incorporation. The cellular and nuclear pools of ATP and ADP in intact synchronized 3T6 cells were accurately determined by high-pressure liquid chromatography. A good correlation with the studies on isolated nuclei has been observed. Whereas total cellular ATP pools increase during the progression of 3T6 cells from G(1) to S phase of the cell cycle, nuclear ATP pools do not increase, and the nuclear ATP/ADP ratios decrease once the cells enter the S phase of their cycle. These experiments suggest that nuclear ATP pools and ATP/ADP ratios act as S phase controls, regulating DNA elongation at sites where its synthesis has previously been initiated by cytoplasmic factors.     

DNA repair of a single UV photoproduct in a designed nucleosome

Eukaryotic DNA repair enzymes must interact with the architectural hierarchy of chromatin. The challenge of finding damaged DNA complexed with histone proteins in nucleosomes is complicated by the need to maintain local chromatin structures involved in regulating other DNA processing events. The heterogeneity of lesions induced by DNA-damaging agents has led us to design homogeneously damaged substrates to directly compare repair of naked DNA with that of nucleosomes. Here we report that nucleotide excision repair in Xenopus nuclear extracts can effectively repair a single UV radiation photoproduct located 5 bases from the dyad center of a positioned nucleosome, although the nucleosome is repaired at about half the rate at which the naked DNA fragment is. Extract repair within the nucleosome is >50-fold more rapid than either enzymatic photoreversal or endonuclease cleavage of the lesion in vitro. Furthermore, nucleosome formation occurs (after repair) only on damaged naked DNA (165-bp fragments) during a 1-h incubation in these extracts, even in the presence of a large excess of undamaged DNA. This is an example of selective nucleosome assembly by Xenopus nuclear extracts on a short linear DNA fragment containing a DNA lesion.     

Deoxyribonucleic acid synthesis in cultured adult rat pancreatic B cells

Previous studies in rodent islets have suggested the existence of a small number of proliferating islet cells. Since islet tissue is composed of endocrine as well as nonendocrine cells, we examined whether the DNA synthesis that is detectable in intact islets in vitro corresponds to an activity of islet B cells, islet endocrine non-B cells and/or nonendocrine islet cells. DNA synthesis was quantified by [3H]thymidine incorporation in trichloracetic acid precipitable material, by nuclear thymidine labeling in autoradiographs, and by nuclear bromodeoxyuridine fluorescence. Adult islet endocrine purified B cells, as well as other endocrine islet cells, incorporated 1 to 2 fmol thymidine/1000 cells, which is 3 times lower than intact islet tissue and 30 times lower than nonendocrine islet cells. Addition of 10% fetal calf serum did not increase DNA synthesis in purified endocrine islet cells but doubled it in intact islets and enhanced it 8-fold in nonendocrine islet cells. The higher thymidine incorporation in intact islets was due to the presence of nonendocrine cells. An increase in medium glucose concentration from 100 to 200 mg/100 ml doubled the thymidine incorporation in purified islet B cells, but not in other endocrine islet cells; no concomitant increase in the number of thymidine or bromodeoxyuridine-labeled nuclei was observed. A phenomenon of glucose-stimulated DNA repair was not excluded. Using three different methods, we have found no evidence for a proliferating activity of adult rat islet B cells under the selected in vitro conditions of this study.     

Noncoincidence of histone and DNA synthesis in cleavage cycles of early development.

The times of histone and DNA synthesis do not coincide in early cleavage of the sea urchin embryo. In fact, the production of histones increases during the interval after DNA synthesis (G2). Enucleate merogones, parthenogenetically activated, synthesize histones encoded upon maternal histone messenger RNA. The pattern of protein synthesis changes following fertilization, in part, but not solely, due to the increasing synthesis of histones relative to other proteins. Regulation of histone synthesis and the loading of newly replicated DNA with histones must themselves undergo change at the time of transition from cleavage cycles to cycles more typical of somatic cells.     

Selective effects of inhibitors of protein synthesis on metabolism of nuclear anc cytoplasmic proteins: evidence for coordinate synthesis of non-histone chromosomal proteins.

We have compared the effects of inhibitors of protein synthesis on the metabolism of nuclear and cytoplasmic proteins of HeLa S-3 cells. L-1-tosylamido-2-phenylethyl chloromethyl ketone, a potent inhibitor of polypeptide chain initiation, was shown to preferentially inhibit the synthesis of cytoplasmic proteins and of histones at concentrations that permit continued amino acid incorporation into nuclear non-histone proteins. Comparisons of the molecular weight distributions of newly synthesized proteins in the presence and absence of L-1-tosylamido-2-phenylethyl chloromethyl ketone have revealed striking differences between nuclear anc cytoplasmic protein fractions. Differential effects on the synthesis of cytoplasmic proteins, acid-soluble nuclear proteins, and residual nuclear proteins have also been obtained with the antibiotic, pactamycin, another inhibitor of polypeptide chain initiation. The incorporation of radioactive amino acids into nuclear non-histone proteins shows resistance to inhibition by pactamycin, but is strongly inhibited by agents such as puromycin and cycloheximide which block chain elongation. The possibility that proliferating cells have developed specialized mechanisms for the coordinate synthesis of chromosomal proteins, possibly involving polycistronic messenger RNAs, is tested and discussed.     

Nuclear-cytoplasmic interactions affecting DNA synthesis during induced cardiac muscle growth in the rat

Nuclear-cytoplasmic interactions affecting DNA synthesis during induced cardiac muscle growth were examined in 29 to 46 day old rats. DNA synthesis was examined in vitro using isolated nuclei from rat heart and adult X. laevis spleen. Cytoplasmic extract (CE) was obtained from a 105 000 g supernatant of rat heart and fetal liver homogenates. To measure DNA synthesis we utilised DNA within the isolated quiescent nucleus as the template and measured the effect of CE on the incorporation of 3H-TTP into an acid precipitable product. In a homologous system of rat heart nuclei from weanling rats and CE from cardiac muscle undergoing induced growth, no stimulation of 3H-TTP incorporation was observed. Cardiac muscle CE however, did possess stimulatory factor(s) since quiescent X. laevis nuclei could be stimulated with the rat heart CE. Furthermore, CE from hearts undergoing induced growth had greater activity than extract from control hearts. While cardiac muscle nuclei were not stimulated by heart CE, they showed substantial stimulation by CE from fetal rat liver, which contains a large population of proliferating cells. Stimulation by fetal rat liver was greater with nuclei obtained from hearts undergoing induced growth than from control hearts. Stimulatory factor(s) in CE was distinct from DNA polymerase-alpha activity, as shown by separation of the two activities on a 5 to 15% glycerol gradient.     

Chromatin and DNA synthesis associated with nuclear membrane in germinating cotton.

The synthesis of nuclear DNA and possible attachment sites of chromatin in the cells of cotton (Gossypium barbadense) radicles during germination was investigated. Biochemical analysis of nuclear membrane fragments or Sarkosyl-magnesium-membrane complexes indicates that the DNA, including newly replicated DNA, is attached to the nuclear membranes during periods of active synthesis. Electron micrographs of nuclear membrane fragments indicate a physical association between chromatin fibers and the membranes. The attachment site appears to be proteinaceous, since the chromatin is released by protein degradative enzymes as evidenced by biochemical techniques and electron microscopic observations. Short-term labeling results in incorporation into a membrane-associated product indistinguishable from the bulk of nuclear DNA. DNA polymerase activity is also associated with nuclear membrane preparations in which [3H]thymidine triphosphate is incorporated into an acid-insoluble. DNase-sensitive product.     

RBC-mediated microinjection of chromatin components into cultured mammalian cells

Radiolabeled DNA fragments or nuclear proteins were encapsulated within human erythrocytes, and the erythrocytes were then fused with cultured mammalian cells using Sendai virus. Autoradiography revealed that 125I-labeled DNA fragments remained dispersed in the cytoplasm and disappeared with a half-life of 24 hours. In contrast, the nuclear proteins, HMG1, HMG2, HMG17 and histone H1, rapidly localized within HeLa nuclei and exhibited half lives greater than 80 hours. Several biochemical criteria indicate that the association of the injected nuclear proteins with chromatin faithfully mimics the behavior of their endogenous counterparts.     

Increased RNA Synthesis in Nuclear Monolayers of WI-38 Cells Stimulated to Proliferate

Nuclear monolayers of WI-38 cells prepared by the method of Tsai and Green were used to determine RNA synthesis in isolated nuclei in situ. In nuclear monolayers, incorporation of [(3)H]UTP into RNA is dependent on the presence of the other three nucleotide triphosphate and is abolished by actinomycin D. The extent of RNA synthesis under these conditions was measured in density-inhibited WI-38 human diploid fibroblasts at various intervals after cell proliferation was stimulated by a change of medium.RNA synthesis increases 15 min after the nutritional change and reaches a peak at 18 hr, which is also the peak of DNA synthesis. Thereafter RNA synthesis declines. Essentially similar results are obtained whether the endogenous RNA polymerase or a bacterial polymerase is used. Replacement of the stimulating medium by conditioned medium stops the increase in RNA synthesis that occurs in cultures subject to continuous stimulation. Finally, RNA synthesis in nuclear monolayers, using the endogenous RNA polymerase, occurs by chain elongation only, while re-initiation occurs with the bacterial RNA polymerase.