The effect of aphidicolin on DNA synthesis in isolated HeLa cell nuclei.

The effect of the inhibitor aphidicolin on DNA synthesis in isolated nuclei from HeLa cells and on the activities of partially purified DNA polymerases has been tested. Aphidicolin inhibited DNA synthesis and DNA polymerase alpha very efficiently whereas DNA polymerases beta and gamma were insensitive to the drug. The results indicate that DNA polymerase alpha is the polymerase active during elongation as well as in the gapfilling process of discontinuous DNA synthesis.     

No stimulatory effect of added histones on DNA synthesis in isolated HeLa cell nuclei

Summary Contrary to some recent reports DNA synthesis in isolated HeLa cell nuclei wasnot stimulated by the addition of low amounts of histones neither in the presence nor in the absence of cytosol. The individual histone fractions H₁, H₂A, H₂B and H₃ also failed to stimulated DNA synthesis.     

Effect of cytosol on DNA synthesis in isolated HeLa cell nuclei

Cytosol obtained by centrifugation of cytoplasm from synchronized S-phase HeLa cells at 200 000 × g for 30 min had a stimulatory effect on the rate and extent of DNA synthesis in isolated nuclei. The cytosol preserved the ability of isolated nuclei to initiate early nascent intermediates (primary DNA pieces). The stimulatory activity was partially separated from the DNA polymerase activity present in the cytosol.     

DNA synthesis in HeLa cell nuclei isolated in a non-aqueous medium.

Nuclei were isolated from synchronized HeLa cells in the S-phase by a modification of the non-aqueous method described by Kirsch et al. (Science (1970) 168, 1592-1595). The method involved lyophilization of the cells, homogenization in non-aqueous glycerol and centrifugation in a gradient of 0-35% (w/w) 3-chloro-1,2-propanediol in glycerol. Such nucleic incorporated deoxyribonucleotides into DNA when incubated in an aqueous buffer containing Mg2+, ATP, dATP, dGTP, dCTP and dTTP. The product was sensitive to DNAase and banded with bulk DNA in isopycnic centrifugation. Sedimentation of the product in alkaline sucrose gradients after labelling of the nuclei for 2 min revealed labelled material in the 5 S peak and in the 18 S area. The material in the 5 S peak moved into the 12 S area after a 13 min chase.     

RNA synthesis in isolated HeLa cell nuclei.

RNA synthesis has been studied in isolated nuclei of HeLa cells. The incubation medium has been optimized for RNA synthesis and the requirements for the presence of specific components previously used by other investigators has been examined. Nuclei isolated by centrifugation through 2 M sucrose synthesize RNA linearly for at least 1 h only at low temperature (25 degrees C). Low molecular weight RNA is found in the supernatant fraction after incubation; this RNA accounts for about 10% of the RNA synthesized. The RNA which remains within nuclei is of high molecular weight and processing of this RNA into molecules of the size of cytoplasmic mRNA does not seem to occur in isolated nuclei. We have studied the effect of an inhibitor of protein-nucleic acid interaction - aurintricarboxylic acid - on RNA synthesis by isolated nuclei. At concentrations below 0.1 mM, this drug does not inhibit RNA synthesis effectively, whereas at concentrations above 0.1 mM it inhibits RNA synthesis by about 80%. In view of the proposed mechanism of action of aurintricarboxylic acid, we suggest that completion of nucleotide chains initiated before nuclei isolation accounts for 20% of the RNA synthesized in our system by isolated nuclei, whereas nucleotide chains initiated during the in vitro incubation account for 80% of the RNA synthesized.     

RNA synthesis in isolated HeLa cell nuclei

RNA synthesis has been studied in isolated nuclei of HeLa cells. The incubation medium has been optimized for RNA synthesis and the requirements for the presence of specific components previously used by other investigators has been examined. Nuclei isolated by centrifugation through 2 M sucrose synthesize RNA linearly for at least 1 h only at low temperature (25°C). Low molecular weight RNA is found in the supernatant fraction after incubation; this RNA accounts for about 10% of the RNA synthesized. The RNA which remains within nuclei is of high molecular weight and processing of this RNA into molecules of the size of cytoplasmic mRNA does not seem to occur in isolated nuclei. We have studied the effect of an inhibitor of protein-nucleic acid interaction — aurintricarboxylic acid — on RNA synthesis by isolated nuclei. At concentrations below 0.1 mM, this drug does not inhibit RNA synthesis effectively, whereas at concentrations above 0.1 mM it inhibits RNA synthesis by about 80%. In view of the proposed mechanism of action of aurintricarboxylic acid, we suggest that completion of nucleotide chains initiated before nuclei isolation accounts for 20% of the RNA synthesized in our system by isolated nuclei, whereas nucleotide chains initiated during the in vitro incubation account for 80% of the RNA synthesized.     

Partial purification of deoxyuridine triphosphate nucleotidohydrolase and its effect on DNA synthesis in isolated HeLa cell nuclei

Deoxyuridine triphosphate nucleotidohydrolase (EC 3.6.U.23) has been partially purified from HeLa S3 cells, and found to have an apparent molecular weight of 50--55 000 by gel filtration under non-denaturing conditions. The enzyme is specific for the hydrolysis of dUTP, requires Mg2+ and is inhibited by EDTA. The apparent Km for dUTP is 0.1 microM. Isolated HeLa cell nuclei were treated with dUTPase before pulse-labelling with [3H]dTTP which also had been pretreated with dUTPase. This pretreatment changed neither the total amount nor the size of the primary DNA pieces. A role for dUTP incorporation in their genesis can therefore be excluded and these primary DNA pieces are considered to be true intermediates in discontinuous DNA replication.     

DNA replication in isolated HeLa cell nuclei

DNA replication was investigated in HeLa cell nuclei isolated from different phases of the cell cycle. DNA synthesis occurred only in S-phase nuclei and was dependent on the presence of the four deoxynucleoside triphosphates, Mg⁺⁺, ATP and S-phase cytoplasm. G₁-phase cytoplasm was unable to support such DNA synthesis. A purified preparation of calf thymus DNA polymerase, however, was able to replace S-phase cytoplasm in supporting ATP dependent DNA synthesis, which suggests that the S-phase cytoplasmic factor is a DNA polymerase. G₁-phase nuclei could under no conditions be stimulated to initiate DNA replication prematurely.     

Neocarzinostatin induction of DNA repair synthesis in HeLa cells and isolated nuclei.

The antitumor antibiotic neocarzinostatin that causes DNA strand breaks in vivo and in vitro is shown to induce DNA repair synthesis in HeLa S3 cells. In the repair assay, the parental DNA was prelabeled with 32P and a density label (bromodeoxyuridine) was introduced into the new synthesized DNA. Quantitation of the repair synthesis as measured by the incorporation of [3H]thymidine into the light parental DNA at varying doses of the drug indicate that there is a significant repair response at low levels of the drug (0.2--0.5 microgram/ml) which cause DNA strand breakage and inhibition of DNA synthesis. In isolated HeLa nuclei neocarzinostatin stimulates the incorporation of dTMP many-fold. This enhancement of dTMP incorporation, which requires the presence of a sulfhydryl agent, is a consequence of the drug-induced DNA strand breakage and is in the parental DNA. These results suggest that an intact cell membrane is not required for DNA strand breakage and its subsequent repair.     

DNA synthesis in isolated HeLa cell nuclei. Optimalization of the system and characterization of the product.

DNA replication in isolated nuclei from synchronized HeLa cells has been studied in an effort to optimalize the system and characterize the product. The synthesis was highly dependent on the four deoxyribonucleoside triphosphates, ATP, and Mg2+. Optimum pH was about 7.8. The system was further stimulated by monovalent ions with NH4Cl and Tris-HCl (each 65 mM) being the most effective. The four ribonucleoside triphosphates and glycerol gave a slight but very reproducible and additive stimulation. Low concentrations of spermine and spermidine (0.2-1.5 X 10(-4) M) were also slightly stimulatory (10-15%) whereas higher concentrations were inhibitory. The reaction product was DNase sensitive, and banded at 1.699 g/ml in neutral CsCl together with bulk HeLa nuclear DNA. When studied by neutral CsCl and alkaline Cs2SO4 gradients, the incorporation of [3H]TTP was mainly (more than 85%) due to further elongation of strands initiated in vivo as evidenced by BrdUrd labeling.     

The inhibitory mode of aphidicolin on DNA synthesis in NaCl-treated HeLa cell nuclei

Isolated HeLa cell nuclei were treated with NaCl at various concentrations and inhibition by aphidicolin of DNA synthesis in the treated nuclei was studied. The inhibition was either noncompetitive or of the mixed type with respect to each dNTP when the nuclei were treated with NaCl at concentrations lower than 0.08 M. However, aphidicolin was a competitive inhibitor with respect to dCTP and a non-competitive or mixed type inhibitor with respect to the other 3 dNTPs when they were treated with NaCl at concentrations higher than 0.1 M. These results suggest the presence of nuclear factor(s) responsible for the changes in the inhibitory mode of aphidicolin on endogenous nuclear DNA synthesis.     

DNA methylase from HeLa cell nuclei.

A DNA methylase has been purified 270-fold from HeLa cell nuclei by chromatography on DEAE-cellulose, phosphocellulose, and hydroxyapatite. The enzyme transfers methyl groups from S-adenosyl-L-methionine to cytosine residues in DNA. The sole product of the reaction has been identified as 5-methylcytosine. The enzyme is able to methylate homologous (HeLa) DNA, although to a lesser extent than heterologous DNA. This may be due to incomplete methylation of HeLa DNA synthesized in vivo. The HeLa enzyme can methylate single-stranded DNA, and does so to an extent three times greater than that of the corresponding double-stranded DNA. In single-stranded M. luteus DNA, at least 2.4% of the cytosine residues can be methylated in vitro by the enzyme. The enzyme also can methylate poly (dG-dC-dG-dC) and poly (dG, dC). Bilateral nearest neighbors to the 5-methylcytosine have been determined with M. luteus DNA in vitro and HeLa DNA in vivo. The 5' neighbor can be either G or C while the 3' neighbor is always G and this sequence is, thus, p(G/C)pmCpG.     

DNA synthesis in isolated HeLa cell nuclei. Evidence for in vitro initiation of synthesis of small pieces of DNA and their subsequent ligation.

Optimum conditions for a DNA synthesizing system based on isolated nuclei have been described (Krokan, H., Bjorklid, E., and Prydz, H. (1975), Biochemistry, preceding paper in this issue) [3H]TTP-labeled nascent DNA produced during very short pulses was analyzed by centrifugation in alkaline sucrose gradients. More than 80% of the radioactivity appeared in 2-4S pieces (primary DNA pieces). It would therefore seem that the synthesis of DNA is discontinuous both in the 5' leads to 3' and in the 3' leads to 5' directions. The size of the primary DNA pieces increases from 2-4 S up to 14 S with increasing pulse length. Evidence is presented that this increase is not caused by ligation between 2-4S primary pieces. Pulse-chase experiments showed that in this nuclear system primary pieces were ligated to a product generally larger than 30 S. Evidence is also given for the initiation of primary DNA pieces in vitro.     

Isolated HeLa cell nuclei synthesize meaningful DNA.

DNA replicated at the beginning of S phase was labelled by incubating nuclei isolated from cells arrested at the G/S border with radioactive deoxyribonucleoside triphosphate in a reaction mixture sustaining DNA synthesis. By hybridization against ribosomal RNA bound to nitrocellulose, the fraction of the labelled DNA which was complementary to rRNA could be quantified, and the stability of the RNA-DNA hybrids could be estimated by sequential elution of DNA at increasing temperatures. The results obtained indicate that the isolated nuclei make "meaningful" DNA, as judged by the melting characteristics of the hybrids between rRNA and the in vitro replicated DNA. Hybridization of the labelled DNA against rRNA fractionated by electrophoresis and blotted onto nitrocellulose verified the presence of sequences complementary to 18 S and 28 S rRNA.     

Poly adenylic acid synthesis in vitro in isolated HeLa cell nuclei and whole cell homogenates,

Homogenates of HeLa cells and purified HeLa cell nuclei were found to synthesize poly(A) in vitro. The poly(A) is the normal adenylate-rich species seen in growing cells and is contained in large RNA molecules, which themselves have been synthesized at least in part in vitro. Poly(A) synthesis in purified nuclei shows a dependence on ATP concentration that is about 75–200 times higher than the concentration for total RNA synthesis.     

The presence of intact mitochondrial DNA in HeLa cell nuclei.

Restriction analysis of DNA labelled with [32P]dCTP in an in vitro replication system with isolated nuclei from early S phase cells showed preferential labelling of restriction fragments derived from mitochondrial DNA (mtDNA) by a replication machinery distinct from that responsible for bulk nuclear DNA replication. Use of restriction nucleases with one recognition site in mtDNA gave rise to 16.5 kbp long fragments corresponding to full-length linearized mtDNA, indicating the presence of intact mtDNA in the isolated cell nuclei. Incorporation of dNTPs into mtDNA was not restricted to the S phase of the cell cycle. We were unable to increase the labelling of mtDNA by the addition of purified mitochondria or mtDNA to the nuclear replication system. These and other results presented is evidenced that the presence of mtDNA in the isolated nuclei was not due to uptake during preparation, thus indicating its presence in the cell nucleus in vivo.