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 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.     

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.     

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.     

A homogeneous type II DNA topoisomerase from HeLa cell nuclei

Using kinetoplast DNA networks as a substrate in a decatenation assay, we have purified to apparent homogeneity a type II DNA topoisomerase from HeLa cell nuclei. The most pure preparations contain a single polypeptide of 172,000 daltons as determined by sodium dodecyl sulfate-gel electrophoresis. The molecular weight of the native protein, based on sedimentation and gel filtration analyses, is estimated to be 309,000. These results suggest that the enzyme is a dimer of 172,0090-dalton subunits. The enzyme is a type II topoisomerase as demonstrated by its ability to change the linking number of DNA circles in steps of two and to decatenate or unknot covalently closed DNA circles. No gyrase activity is detectable. ATP is required for the relaxation, decatenation, and unknotting of DNA, and a DNA-dependent ATPase activity is present in the most pure fractions. ATP is hydrolyzed to ADP in this properties to T4 DNA topoisomerase (Liu, L. F., Liu, C. C., and Alberts, B. M. (1979) Nature 281, 456-461).     

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.     

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.     

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.     

Polyanion-induced release of polyribosomes from HeLa cell nuclei.

Intact detergent-washed HeLa nuclei contain a population of polyribisomes that were released by exposure to polyanions such as RNA or poly(U). The released material appeared by electron microscopic examination to be particles averaging about 200 to 300 angstroms in diameter. Sedimentation velocity analysis of the released particles indicated that the particles had S20,w values of 75 and 110. The particles stimulated amino acid incorporation in an ascites S-30 or S-100 extract at 2.5 mM Mg2+. Studies with a variety of antibiotics indicated that these polyribosomes were capable of elongating but not initiating protein synthesis. Although these polyribosomes may be of cytoplasmic origin, they appear unique in that agents thought to disperse chromatin are required for their release from the nucleus.     

A DNA-activated protein kinase from HeLa cell nuclei.

A DNA-activated protein kinase (DNA-PK) was purified from nuclei of HeLa cells. Activity was associated with a single high-molecular-mass (approximately-300,000 Da) polypeptide when analyzed by gel filtration, denaturing polyacrylamide gel electrophoresis, and Western immunoblotting using a monoclonal antibody that also inhibits enzyme activity. Nuclear localization was indicated by subcellular fractionation and confirmed by immunofluorescence on whole cells. Double-stranded DNA stimulated phosphorylation of the 300-kDa polypeptide in purified preparations as well as phosphorylation of the exogenous substrates alpha-casein, simian virus 40 large T antigen, and the human heat shock protein hsp90. Autophosphorylation led to inactivation of the enzyme. The phosphorylation of casein was stimulated over 30-fold by DNA and was specific for serine and threonine residues. Bovine serum albumin and histone H1 were poor substrates for DNA-PK, and no phosphorylation of immunoglobulin G or histones other than H1 was observed. Supercoiled or heat-denatured DNA and synthetic double-stranded RNA or RNA-DNA copolymers did not stimulate casein phosphorylation by DNA-PK. Interaction of the enzyme with DNA in the absence of exogenous substrates was demonstrated by thermal inactivation and gel mobility shifts. These characteristics identify DNA-PK as distinct from other protein kinases described in the literature and suggest that activation by DNA is an important feature of the enzyme's in vivo function.     

Vaccinia virus infection of HeLa cells. I. Synthesis of vaccinia DNA in host cell nuclei.

The replication of vaccinia virus is thought to take place exclusively in the cytoplasm of host cells. However, using DNA-DNA hybridization techniques, it can be shown that a significant fraction of the synthesis of vaccinia DNA takes place in the nucleus as well as the cytoplasm. The (3H) thymiding pulse-labeled vaccinia DNA synthesized in the nucleus reaches a maximum at about 3 h after infection, corresponding to the time of maximal DNA synthesis in infected cells. At this time host DNA synthesis drops to about 25% of the rate of the uninfected cells. Even with short labeling times (2 min) the nucleus is found to contain 60% of the incorporated (3H)thymidine, much of which is in vaccinia DNA. Prior inhibition of host nuclear DNA synthesis with mitomycin C, followed by removal of the antibiotic causes a subsequent inhibition of vaccinia DNA synthesis and complete suppression of mature virus. Purified nuclei, isolated from vaccinia-infected cells, also synthesize vaccinia DNA in vitro. Over 90% of the DNA synthesized in vitro by isolated nuclei contain vaccinia-specific sequences.     

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.     

Double-stranded ribonuclease activities from HeLa cell nuclei

Degradation of poly(I)·[³H] poly(C) to acid-soluble products is observed with a nuclear lysate from HeLa cells. Most of the activity is lost after Sephadex G-100 chromatography, but can be regained by mixing two fractions, FI and FII, that elute separately. FII has been further purified by chromatography on DEAE-cellulose and phosphocellulose, and is then totally dependent on FI for any activity with poly(I)·poly(C). However, FII retains some activity with poly(C); and in the presence of FI, it can be substituted by purified $\text{E. coli}$ RNase II for the degradation of poly(I)·poly(C). Thus, at least two macromolecular components participate in the bulk degradation of poly(I) poly(C) in HeLa nuclear lysates, and one of them may be a single-stranded exonuclease.     

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.     

Butylanilinouracil: a selective inhibitor of HeLa cell DNA synthesis and HeLa cell DNA polymerase alpha.

A series of 6-anilinouracils, dGTP analogues which selectively inhibit specific bacterial DNA polymerases, were examined for their capacity to inhibit purified DNA polymerases from HeLa cells. The p-n-butyl derivative (BuAU) was found to inhibit DNA polymerase alpha with a Ki of approximately 60 microM. The inhibitory effect of BuAU was reversed specifically by dGTP and was observed only for DNA polymerase alpha; polymerases beta and lambda were not inhibited by drug at concentrations as high as 1 mM. BuAU also was inhibitory in vivo in HeLa cell culture; at 100 microM it reversibly inhibited cell division and selectively depressed DNA synthesis. The results of these studies indicate that BuAU is an inhibitor with considerable potential as a specific probe with which to dissect the structure of mammalian polymerase alpha and its putative role in cellular DNA replication.     

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.     

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.     

Polyamine-activated protein phosphatase activity in HeLa cell nuclei

Protein phosphatase activity towards endogenous nuclear substrates in sonicates of isolated nuclei was activated 2-4-fold by spermine. Exogenous casein was dephosphorylated by these preparations only in the presence of spermine. Activation by spermine was half maximal at about 0.1 mM. Spermidine also activated, with half maximal stimulation at 1mM; putrescine activated poorly. Mg++ and Ca++ appeared to activate the same phosphatase activity but were only 50% as effective as spermine. Spermine activation was inhibited by 200 mM NaCl, 50 mM NaF, or 40 mM beta-glycerol phosphate. Nuclear phosphatase activity, with or without spermine, was inhibited 50% by inhibitor 2 of protein phosphatase 1. These observations suggest that protein phosphatase 1 is a major nuclear protein phosphatase and that its activity against endogenous nuclear substrates is activated by physiological concentrations of spermine.