Fate of newly synthesized histones shortly after interruption of DNA replication

The synthesis and association of histones with chromatin were studied using MH-134SC cells in suspension culture. Cultures containing approximately equal numbers of cells were pulse-labeled with [3H]lysine at various times after the interruption of DNA synthesis with hydroxyurea. Each culture was mixed with a fixed volume of a culture generally labeled with [14C]lysine at the time of harvesting. Acid-soluble proteins extracted from different subcellular fractions of cells labeled under various conditions were compared by electrophoresis on polyacrylamide gels containing acetic acid and urea. All types of chromatin histones were labeled nearly equally as [14C]marker histones by a 15 min pulse under normal conditions, except that a considerable portion of pulse-labeled H4 was in highly acetylated forms. Addition of hydroxyurea at the start of the pulse markedly reduced the labeling of H3 and H4, but affected the labeling of the other histones only slightly. When DNA synthesis was inhibited before the start of the pulse, labeling of all histones decreased significantly. The addition of hydroxyurea was found to cause transient accumulation of newly synthesized proteins in the cytoplasmic soluble fraction; these were characterized as H3 and H4 from their metabolic properties and their electrophoretic mobilities on sodium dodecyl sulfate-polyacrylamide gels. The results suggest that association of newly synthesized H3 and H4 histones is closely coupled with ongoing DNA replication. The implications of the results for the mechanism of formation of new nucleosomes are discussed.     

Effect of hypoxia on nucleic acid and protein synthesis in different brain regions

The incorporation of [methyl-³H]thymidine into DNA, of [5-³H]uridine into RNA, and of [1-¹⁴C]leucine into proteins of cerebral hemispheres, cerebellum, and brainstem of guinea pigs after 80 hr of hypoxic treatment was measured. Both in vivo (intraventricular administration of labeled precursors) and in vitro (tissue slices incubation) experiments were performed. The labeling of macromolecules extracted from the various subcellular fractions of the above-mentioned brain regions was also determined. After hypoxic treatment the incorporation of the labeled precursors into DNA, RNA, and proteins was impaired to a different extent in the three brain regions and in the various subcellular fractions examined; DNA and RNA labeling in cerebellar mitochondria and protein labeling in microsomes of the three brain regions examined were particularly affected.     

Organization of the newly replicated chromatin in the vicinity of the replication fork

Ehrlich ascites tumour cells were pulse-labelled with [³H]thymidine for 1 min or were treated with cycloheximide and labelled with [³H]thymidine for 45 min. The kinetics of digestion with micrococcal nuclease of both pulse-labelled and cycloheximide chromatins showed that they exhibited increased susceptibility towards the enzyme. At the same time their release from the nucleus was retarted and this was interpreted to mean that, unlike the bulk of chromatin, they were tightly bound to a fixed nuclear structure. When subjected to an equilibrium metrizamide-triethanolamine density gradient centrifugation both pulse-labelled and cycloheximide chromatins banded at higher density than control chromatin, which was an indication of their higher protein to DNA ratio. After a mild trypsinization, eliminating H1 and the nonhistone proteins, the pulse-labelled chromatin sedimented to the same density as control chromatin, and the cycloheximide chromatin sedimented to a density which was intermediate between those of control chromatin and free DNA. This result showed that the newly replicated chromatin had the same, and the cycloheximide chromatin half the amount of core histones present in control chromatin.     

DNA REPLICATION: DIRECTION AND RATE OF CHAIN GROWTH IN MAMMALIAN CELLS

Using pulse labeling techniques with [³H]thymidine or [³H]cytidine, combined with DNA fiber autoradiography, we have investigated the direction and rate of DNA chain growth in mammalian cells. In general, chain elongation proceeds bidirectionally from the common origin of pairs of adjacent replication sections. This type of replication is noted whether the DNA is labeled first with [³H]thymidine of high specific activity, followed by [³H]thymidine of low specific activity or the sequence is reversed. Approximately one-fifth of the growing points have unique origins and in these replication units, chain growth proceeds in one direction only. Fluorodeoxyuridine and hydroxyurea both inhibit DNA chain propagation. Fluorodeoxyuridine exerts its effect on chain growth within 15–23 min, while the effect of hydroxyurea is evident within 15 min under conditions where the endogenous thymidine pool has been depleted by prior treatment with fluorodeoxyuridine. Puromycin has no effect on chain growth until 60 min after addition of the compound, even though thymidine incorporation is more than 50% reduced within 15 min. After 2 h of treatment with puromycin, the rate of chain growth is reduced by 50%, whereas thymidine incorporation is reduced by 75%. Cycloheximide reduces the rates of DNA chain growth and thymidine incorporation 50% within 15 min, and, on prolonged treatment, the decrease in rate of chain growth generally parallels the reduction in thymidine incorporation.     

Developmental changes in myelin-induced proliferation of cultured Schwann cells

Schwann cell proliferation induced by a myelin-enriched fraction was examined in vitro. Although nearly all the Schwann cells contained material that was recognized by antisera to myelin basic protein after 24 h, only 1% of the cells were synthesizing DNA. 72 h after the addition of the mitogen a maximum of 10% of the cells incorporated [3H]thymidine. If the cultures were treated with the myelin-enriched fraction for 24 h and then washed, the number of proliferating Schwann cells decreased by 75% when compared with those cells that were incubated with the mitogen continuously. When Schwann cells were labeled with [14C]thymidine followed by a pulse of [3H]thymidine 24 h later, every Schwann cell labeled with [3H]thymidine was also labeled with [14C]thymidine. Although almost every Schwann cell can metabolize the myelin membranes within 24 h of exposure, a small population of cell initially utilizes the myelin as a mitogen, and this population continues to divide only if myelin is present in the extracellular media. The percentage of the Schwann cells that initially recognize the myelin-enriched fraction as a mitogen is dependent upon the age of the animal from which the cells were prepared.     

Structural state of newly replicated closed circular simian virus 40 DNA.

We have studied the early transition of newly replicated, segregated daughter molecules of simian virus 40 (SV40) into their mature, fully supercoiled state. The DNA of SV40 replicating in African green monkey kidney CV1 cells was chronically labeled with [14C]thymidine and pulse-labeled with [3H]thymidine. The cells were lysed and the viral DNA was isolated. Density gradient centrifugation of viral DNA in cesium chloride revealed that the pulse-labeled, newly synthesized, closed circular supercoiled DNA molecules banded at a slightly higher density (delta sigma = 0.0025) than the chronically labeled DNA, suggesting that the newly completed molecules were in a different structural state. Electrophoresis of DNA in agarose gels at appropriate chloroquine concentrations demonstrated that the mobility of the pulse-labeled closed, superhelical DNA was retarded relative to that of the chronically labeled DNA. These observations indicated that the newly completed SV40 DNA molecules existed in a structural state more relaxed than that of mature DNA by one or two linking numbers.     

Fragmentation of chromatin with 125I radioactive disintegrations.

The DNA in Chinese hamster cells was labeled first for 3 h with [3H]TdR and then for 3 h with [125I]UdR. Chromatin was extracted, frozen, and stored at -30 degrees C until 1.0 X 10(17) and 1.25 X 10(17) disintegrations/g of labeled DNA occurred for 125I and 3H respectively. Velocity sedimentation of chromatin (DNA with associated chromosomal proteins) in neutral sucrose gradients indicated that the localized energy from the 125I disintegrations, which gave about 1 double-strand break/disintegration plus an additional 1.3 single strand breaks, selectively fragmented the [125I] chromatin into pieces smaller than the [3H] chromatin. In other words, 125I disintegrations caused much more localized damage in the chromatin labeled with 125I than in the chromatin labeled with 3H, and fragments induced in DNA by 125I disintegrations were not held together by the associated chromosomal proteins. Use of this 125I technique for studying chromosomal proteins associated with different regions in the cellular DNA is discussed. For these studies, the number of disintegrations required for fragmenting DNA molecules of different sizes is illustrated.     

Chromatin maturation depends on continued DNA-replication

The structure of [3H]thymidine pulse-labeled chromatin in lymphocytes differs from that of non-replicating chromatin by several operational criteria which are related to the higher nuclease sensitivity of replicating chromatin. These structural features of replicating chromatin rapidly disappear when the [3H]thymidine pulse is followed by a chase in the presence of an excess of non-radioactive thymidine. However, when the rate of DNA replication is reduced, as in cycloheximide-treated lymphocytes, chromatin maturation is retarded. No chromatin maturation is observed when nuclei from pulse-labeled lymphocytes are incubated in vitro in the absence of DNA precursors. In contrast, when these nuclei are incubated under conditions known to be optimal for DNA replication, the structure of replicating chromatin is efficiently converted to that of 'mature', non-replicating chromatin. We conclude that the properties of nascent DNA and/or the distance from the replication fork are important factors in chromatin maturation.     

Undermethylation of DNA in mononucleosomes solubilized by micrococcal nuclease digestion of HeLa cell nuclei

To examine the distribution of 5-methylcytosine in chromatin DNA, DNA of HeLa cells was labeled with [3H-methyl]methionine and [14C] thymidine and analyzed after extensive digestion of the nuclei with micrococcal nuclease. When the chromatin solubilized with the nuclease was fractionated on a sucrose density gradient, DNA in mononucleosomes was considerably depleted in 5-methylcytosine, as compared with polynucleosomes. Electrophoretic separation of DNA from the chromatin also revealed the depletion of 5-methylcytosine in the mononucleosomal size of DNA. This was confirmed by the chromatographic analysis of 5-methyldeoxycytidine after enzymatic digestion of the DNA to nucleosides. Thus the DNA in mononucleosomes solubilized by extensive micrococcal nuclease digestion is depleted in 5-methylcytosine, suggesting that 5-methylcytosine is preferentially missing from the DNA in the nucleosome core particles.     

Effect of nickel(II) on DNA-protein binding,thymidine incorporation,and sedimentation pattern of chromatin fractions from intact mammalian cells

Nuclear uptake and chromatin binding of nickel(II) was investigated in Chinese hamster ovary (CHO) cells. The cytoplasmic:nuclear ratio of nickel immediately following treatment was 5:1, but by 24 and 48 hours this ratio decreased to 4:l and 2:1, respectively, indicating that nickel is retained longer in the nucleus than cytoplasmic nickel. Chromatin was fractionated by sonication and centrifugation into fast-sedimenting, magnesium-insoluble, or magnesiumsoluble components. The magnesium-insoluble portion bound more nickel ions and retained the metal longer than either the magnesium-soluble or the fastsedimenting fractions. Treatment of cells with nickel chloride (NiCl₂) decreased the amount of DNA in the magnesium-insoluble fraction but increased the amount of DNA in the fast- sedimenting chromatin fraction. The magnesium-insoluble fraction isolated from nickel-treated cells contained approximately ten times more [35-S]-methionine–labeled protein per milligram DNA compared with untreated cells. The magnesium-soluble and the fast-sedimenting fractions isolated from the nickel-treated cells did not exhibit a similar increase in [35-S]-methionine–labeled protein per milligram of DNA. Nickel treatment suppressed [14-C]-thymidine incorporation into total DNA by 30% compared with untreated cells. However, the magnesium-insoluble chromatin fraction from nickel-treated cells had a tenfold to 20-fold increase in thymidine incorporation, while the other chromatin fractions did not exhibit an increase in thymidine incorporation. These findings indicate that nickel induced widespread alterations in chromatin conformation and preferentially interacted with an Mg-insoluble component of chromatin.     

Mitochondrial DNA,RNA, and protein synthesis in different regions of developing rat brain

In vivo and in vitro (tissue slices) incorporation of labeled precursors into DNA, RNA, and proteins was measured in mitochondria obtained from cerebral hemispheres, cerebellum, and brain stem of rats at different days of postnatal development. To compare the synthesis of macromolecules in mitochondria with that in other subcellular fractions, the incorporation of labeled precursors into DNA, RNA, and proteins extracted from nuclei and into RNA and proteins extracted from microsomes and cytoplasmic soluble fractions was also measured.The results obtained showed that the incorporation of [³H]thymidine into DNA and of [¹⁴C]leucine into proteins of nuclei and mitochondria from the various brain regions examined decreased during postnatal development, however, at 30 days of age the specific radioactivity of mitochondrial DNA was higher than that of nuclear DNA. [³H]Uridine incorporation into RNA decreased from 10 to 30 days of age in nuclei while in mitochondria it was quite similar at both ages. This result may be due to a faster turnover of mitochondrial RNA compared to that of mitochondrial DNA and proteins. The results obtained suggest an active biosynthesis of macromolecules in brain mitochondria and might indicate an intense biogenesis of these organelles in rat brain during postnatal development.Preliminary reports of these results were presented at the XI FEBS Meeting, Copenhagen, August 14–19, 1977, Poster number A2-2-155-3, and at III Meeting of Italian Biochem. Soc., Siena, October 3–5, 1977, Abstract C6.     

Methylation of DNAase-digestible DNA and of RNA in chromatin from rats treated with dimethylnitrosamine.

After injecting rats with di[14C]methylnitrosamine we have prepared liver chromatin and have examined firstly, the methylation level of the DNAase I-degradable fraction of the DNA and secondly, the level of methylation and the stability of methylated sites in chromatin RNA. Our results show that the level of 7-methylguanine in the degradable DNA is about 1.3 times that of whole DNA; therefore in the 20% or so of the DNA which is undegradable by DNAase I, the level must be very low or zero. Experiments using chromatin from rats injected with unlabelled dimethylnitrosamine plus [3H]thymidine show that the specific activity is similar in the DNAase I degradable and undegradable fractions, suggesting that there is no preferential repair in the latter region. In chromatin RNA, the level of 7-methylguanine is higher than that of whole DNA and decreases fairly rapidly within 30 h after dimethylnitrosamine treatment. Our results indicate that this decrease is due to some type of excision or repair process rather than to normal turnover.     

Development and Localization of the Thymidine Phosphorylating Systems in Brain

Abstract: The development of the thymidine phosphorylating systems was studied in various regions of brain. Brain slices from cerebellum, brain stem, and forebrain of rabbits 2, 7, 14, 30, 90, 500, and 2500 days of age were incubated for various times in artificial CSF containing 3 nM-[³H]thymidine at 37°C under 95% O₂-5% CO₂. When slices from all brain regions of 2-day-old rabbits were incubated in [³H]thymidine for 30 min, tissue-to-medium ratios of ³H were between 2 and 4 and declined with age, and the percentages of the total ³H in perchloric acid homogenates of brain slices as [³H]DNA were 26–29%, declining to low levels with age. However, at all ages and in all regions studied, 41 -88% of the ³H within the slices was phosphorylated. After homogenization and subcellular fractionation of the brain slices incubated in [³H]thymidine for 30 min, the highest percentage of [³H]thymidine phosphates plus [³H]DNA was present in the nuclear (crude and purified) and mitochondrial fractions of all brain regions. The [³H]DNA content in the nuclear and mitochondrial fractions declined with age, but the percentage of [³H]thymidine phosphates did not. Thymidine phosphates were synthesized from thymidine in all brain regions tested throughout the entire life span.     

Nuclease-sensitivity of methylated DNA as a probe for chromatin reconstitution by genotoxicants

DNA in Chinese hamster ovary cells was labeled with [¹⁴C]thymidine and [methyl- ³H]-1-methionine in culture, and their nuclei were digested with micrococcal nuclease. Not until 10 percent of bulk DNA was digested did methylated DNA appear in the acid-soluble fraction. When these cells were exposed to UV-radiation, alkylating agents and intercalating agents in culture, the resistance of methylated DNA to digestion by the nuclease was largely or completely eliminated. The change in the sensitivity of methylated DNA to the nuclease indicates a conformational change in chromatin induced by the genotoxicants.     

Distribution of the core histones H2A.H2B.H3 and H4 during cell replication.

The distribution of newly synthesized core histones H2A, H2B, H3 and H4 relative to the DNA strand synthesized in the same generation has been examined in replicating Chinese Hamster ovary cells. Cells are grown for one generation in [14C]-lysine and thymidine, and then for one generation in [3H]-lysine and 5-bromodeoxyuridine (BrUdRib) and a further generation in unlabeled lysine and thymidine. This protocol produces equal amounts of unifilarly substituted and unsubstituted DNA. Monomer nucleosomes isolated from chromatin containing these two types of DNA can be distinguished by crosslinking with formaldehyde and banding to equilibrium in CsCl density gradients. The results indicate that the core histones are equally distributed between the two types of DNA. These findings are discussed in terms of current models for chromatin replication; they do not support any long term association of newly replicated histones with either the leading or lagging side of the replication fork.     

Isolation of a chromatin fraction from calf endometrium highly enriched in estradiol binding sites.

The intranuclear distribution of [3H]-estradiol binding sites was studied in highly purified nuclei isolated from calf endometrial tissue pre-incubated with the labeled hormone. The major part (approximately 85%) of the receptor bound estradiol was found associated with the extranucleolar chromatin; only a negligible amount of [3H]-estradiol (approximately 8%) sedimented with the nucleolar fraction. [3H]-estradiol labeled chromatin was then fragmented by sonication and fractionated by sucrose density gradient sedimentation under different conditions of centrifugation. The vast majority of the [3H]-estradiol was invariably found to be associated with a fast sedimenting fraction which contained only 5 to 10% of the nuclear DNA. The concentration of estradiol receptors (per weight of DNA) in this fraction was 25- to 50-fold higher than that found in the slow sedimenting major chromatin component. Chemical analysis showed this fraction to have a high protein/DNA ratio but no phospholipids were detected.     

Preferential incorporation of [3H]thymidine into polypyrimidine-containing regions in DNA following ultraviolet irradiation of human cells

Ultraviolet irradiation of cells causes damage to DNA, principally to pyrimidine bases. Regions of DNA which are very rich in pyrimidines are therefore likely to be more susceptible to damage by this agent. Polypyrimidines (pyrimidine tracts > 25 nucleotides in length) are known to occur in DNA from many higher organisms. We investigated whether these tracts are particularly sensitive to ultraviolet light by irradiating human diploid flbroblasts and preparing DNA which was labelled with [³H]thymidine during post-irradiation repair replication. Polypyrimidine-containing regions of the DNA were isolated and their content of [³H]thymidine (principally arising from repair synthesis) was compared to their content of [¹⁴C]thymidine (representing bulk DNA). It was found that polypyrimidine-containing regions were enriched (approximately twofold) in ³H compared to ¹⁴C, probably because of greater susceptibility of pyrimidine-rich regions in DNA to ultraviolet light.     

The effect of microwave radiation on the cell genome

Cultured V79 Chinese hamster cells were exposed to continuous radiation, frequency 7.7 GHz, power density 30 mW/cm2 for 15, 30, and 60 min. The parameters investigated were the incorporation of [3H]thymidine and the frequency of chromosome aberrations. Data obtained by 2 methods (the incorporation of [3H]thymidine into DNA and autoradiography) showed that the inhibition of [3H]thymidine incorporation took place by complete prevention of DNA from entering into the S phase. The normal rate of incorporation of [3H]thymidine was recovered within 1 generation cycle of V79 cells. Mutagenic tests performed concurrently showed that even DNA macromolecules were involved in the process. In comparison with the control samples there was a higher frequency of specific chromosome lesions in cells that had been irradiated. Results discussed in this study suggest that microwave radiation causes changes in the synthesis as well as in the structure of DNA molecules.     

The involvement of RNA in the initiation of DNA synthesis in mammalian cells. Artifacts arising during caesium-salt density-gradient centrifugation which simulate a covalent attachment of RNA to newly synthesized DNA.

Artifacts were encountered during caesium salt density gradient centrifugation which simulated results expected if newly synthesized DNA is covalently attached to RNA. Newly synthesized DNA (in baby hamster kidney cells, BHK-21/C13), pulse-labelled with [3H]thymidine for 10 min at temperatures below 25 degrees C, banded at a greater buoyant density than mature [14C]DNA (heat-denatured nucleic acids) when centrifuged to equilibrium in caesium chloride gradients. Some of the newly synthesized RNA, labelled with [3H]uridine, banded at a buoyant density slightly greater than DNA in caesium sulphate gradients. These results were not obtained when nucleic acids were pulse-labelled at 37 degrees C, nor when samples were heat-denatured in the presence of formaldehyde. This suggests that nucleic acids can aggregate during centrifugation; this is discussed in relation to the molecular size of the DNA.     

Degradation of proteins in steady-state cultures of Escherichia coli.

The degradation of proteins in Escherichia coli was investigated in cells grown under steady-state conditions in a glucose-limited chemostat. During the first 24 h, approximately 25% of pulse-labeled proteins were degraded and after 72 h up to 58% of the proteins were broken down. To examine the stability of subcellular components steady-state cultures were labeled with an initial pulse of [14C]leucine, 24 h were allowed for turnover of these proteins, and the cells were then labeled with a short pulse of [3H]leucine. By this double-label protocol, the labile proteins were preferentially labeled with [H]leucine and had high 3H/14C ratios, while the more stable proteins had lower 3//14C ratios. The 3/-labeled proteins were degraded approximately five times as rapidly as the 14C-labeled proteins in exponentially growing cells. The relative stability of subcellular fractions was determined by comparing their 3H/14C ratios to the ratio of the cells at harvest. The soluble fraction contained the most labile proteins, while the ribosomal and membrane fractions were at least as stable as the average cell protein.