5'-Hydroxyl polyribonucleotide kinase from HeLa cell nuclei. Purification and properties.

An enzyme, 5'-hydroxyl polyribonucleotide kinase, which catalyzes the phosphorylation of 5'-hydroxyl ends of RNA in the presence of ATP, has been isolated from extracts of HeLa cell nuclei. The kinase requires a divalent cation (Mg2+ or Mn2+) for activity, has an alkaline pH optimum, and is sensitive to the sulfhydryl antagonist N-ethylmaleimide. 5'-hydroxyl terminated polydeoxyribonucleotides are phosphorylated much less efficiently than the 5'-hydroxyl terminated polyribonucleotides, and the kinase preparation is inactive on ribonucleoside 3'-monophosphates. Enzyme activity is inhibited by ADP and by pyrophosphate. The sedimentation coefficient of the kinase is estimated to be 5.6 S from glycerol gradient centrifugation.     

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.     

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.     

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.     

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.     

Isolation of the protein B23/nucleophosmin from HeLa cell nuclei

Endogenous forms of the protein B23 were for the first time isolated from HeLa cell nuclei and their structural states were analyzed. It was demonstrated that incubation of HeLa cell nuclei in 10 mM Tris-HCl buffer (pH 7.4) led, not only to their swelling, but also to the release of several nuclear proteins, including the protein B23. PAGE of the supernatant fraction allowed nine major stained protein bands to be detected; the bands were identified by MALDI mass spectrometry (matrix-assisted laser desorption and ionization). The proteins in the range of 35-40 kDa were identified as nucleophosmin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1. Analysis of the N- and C-terminal amino acid sequences showed the presence of the isoforms B23.1 and B23.2, GAPDH, and the isoform hnRNP B1 and made it possible to describe the C- and N- terminal processing patterns and demonstrate the presence of isoform B23.2 at a protein level.     

Isolation of the protein B23/nucleophosmin from HeLa cell nuclei

Endogenous forms of the protein B23 were for the first time isolated from HeLa cell nuclei and their structural states were analyzed. It was demonstrated that incubation of HeLa cell nuclei in 10 mM Tris-HCl buffer (pH 7.4) led, not only to their swelling, but also to the release of several nuclear proteins, including the protein B23. PAGE of the supernatant fraction allowed nine major stained protein bands to be detected; the bands were identified by MALDI mass spectrometry (matrix-assisted laser desorption and ionization). The proteins in the range of 35–40 kDa were identified as nucleophosmin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1. Analysis of the N- and C-terminal amino acid sequences showed the presence of the isoforms B23.1 and B23.2, GAPDH, and the isoform hnRNP B1 and made it possible to describe the C-and N-terminal processing patterns and demonstrate the presence of isoform B23.2 at a protein level.     

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).     

Identification of a novel casein kinase activity in HeLa cell nuclei

Three casein kinase activities have been resolved by column chromatography of HeLa cell nuclear extracts. In addition to casein kinases NI and NII, which have been described in other cell types, HeLa nuclei contain a third casein kinase activity which we have named NIII. NIII is a cyclic nucleotide-independent casein kinase which uses either Mg2+ or Mn2+ as a divalent cation, but is inhibited by increasing NaCl concentrations in the presence of Mg2+ and has optimal activity at 50 mM NaCl in the presence of Mn2+. In Mg2+, NIII uses only ATP as a phosphate donor, but in Mn2+ NIII transfers phosphate from either ATP or GTP. NIII phosphorylates the serine and threonine residues of casein, but does not phosphorylate phosvitin or calf thymus histones.     

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.     

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.     

DNA-activated protein kinase in Raji Burkitt's lymphoma cells. Phosphorylation of c-Myc oncoprotein.

Autophosphorylation of a DNA-activated protein kinase (DNA-PK) in Raji Burkitt's lymphoma cells generated a band that corresponded to a phosphoprotein of about 300 kDa on SDS/PAGE. This band corresponds to a 300-350-kDa DNA-PK found previously in HeLa cells. In addition to the 300-kDa phosphoprotein, the band of a highly phosphorylated 58-kDa protein was detected by SDS/PAGE of partially purified DNA-PK preparations after the phosphorylation reaction in the presence of double-stranded DNA. This phosphoprotein was specifically immunoprecipitated by phosphoprotein nor detectable activities of other kinases, phosphorylated recombinant c-Myc proteins in the presence of DNA. The c-Myc phosphorylation by DNA-PK was markedly stimulated by relaxed, double-stranded DNA, but neither by single-stranded DNA nor by RNA. Phosphopeptide mapping and phosphoamino acid analysis indicated that DNA-PK phosphorylates c-Myc in vitro at several serine residues.     

Creatine kinase from muscle cell nuclei]

It has been firstly demonstrated that rat heart and skeletal muscle nuclei contain creatine dinase, one of the most important enzymes of energy metabolism. The nuclei isolated in concentrated sucrose were practically free from cytoplasm and mitochondrial fragments. Electrophoresis in acetyl cellulose revealed that the nuclear extracts from rat heart and skeletal muscles contain only one isoenzyme of creatine kinase similar in mobility to the mitochondrial isoenzyme. The magnitude of Km values for creatine kinase from the nuclei of both tissues was determined. It was shown histochemically that creatine kinase is localized inside the nuclei, predominantly in the sites of chromatin location. A possible role of the enzyme in nuclear metabolism is discussed.     

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.     

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.     

Characterization of a phosphoprotein phosphatase activity in ribonucleoprotein particles from HeLa cell nuclei.

Ribonucleoprotein particles containing heterogenous nuclear RNA (hnRNP) have a phosphoprotein phosphatase activity. This activity is optimal at pH 7.5, inhibited by divalent cations and by increasing ionic strength above 200 mM NaCl, stimulated by 2-mercaptoethanol and inhibited by N-ethylmaleimide. It is clearly distinct from non specific alkaline phosphatase and resembles the phosphoprotein phosphatase present in Novikoff hepatoma nucleoli (Olson et al. B.B.R.C. (1976), 70, 717–721). This enzyme may be involved in regulating the phosphorylation level of hnRNP proteins in combination with the protein kinase previously described (Blanchard et al. Eur. J. Biochem. (1977) in press).