Isolated HeLa Cell Nuclei Research Articles

HeLa cell nucleus, a source of thymidine for Trichomonas vaginalis growing in vitro

Trichomonas vaginalis is a parasitic protist incapable of de novo purine and pyrimidine biosynthesis. The lack of these de novo syntheses of nucleotides is supplemented with purine and pyrimidine salvage pathways. Likewise, T. vaginalis is incapable of converting its ribonucleotides to deoxyribonucleotides. Therefore, the parasite must rely on the salvage of exogenous deoxyribonucleosides for DNA synthesis. It has been demonstrated that the parasite can incorporate external adenine and guanine in vitro, but no in vivo nucleotide source has been identified so far. Accordingly, we set out to determine if the parasite could incorporate 3H-thymidine from the nuclei of a cervical-derived cell line into its own DNA. By light and electron microscopy we found that the parasite was able to interact directly, both with mechanically isolated HeLa cell nuclei and with the nuclei released after the disruption of HeLa cell monolayers by the parasite. This study shows that T. vaginalis was capable of incorporating 3H-thymidine from labeled HeLa cells into its own DNA suggesting that the nuclei of this cervical cell line could be an in vivo source of nucleotides for T. vaginalis.

BRCT Domain-containing Protein TopBP1 Functions in DNA Replication and Damage Response

Topoisomerase IIbeta-binding protein (TopBP1), a human protein with eight BRCT domains, is similar to Saccharomyces cerevisiae Dpb11 and Schizosaccharomyces pombe Cut5 checkpoint proteins and closely related to Drosophila Mus101. We show that human TopBP1 is required for DNA replication and that it interacts with DNA polymerase epsilon. In S phase TopBP1 colocalizes with Brca1 to foci that do not represent sites of ongoing DNA replication. Inhibition of DNA synthesis leads to relocalization of TopBP1 together with Brca1 to replication forks, suggesting a role in rescue of stalled forks. DNA damage induces formation of distinct TopBP1 foci that colocalize with Brca1 in S phase, but not in G(1) phase. We also show that TopBP1 interacts with the checkpoint protein hRad9. Thus, these results implicate TopBP1 in replication and checkpoint functions.

DNA topoisomerase IIalpha interacts with CAD nuclease and is involved in chromatin condensation during apoptotic execution.

Apoptotic execution is characterized by dramatic changes in nuclear structure accompanied by cleavage of nuclear proteins by caspases (reviewed in [1]). Cell-free extracts have proved useful for the identification and functional characterization of activities involved in apoptotic execution [2-4] and for the identification of proteins cleaved by caspases [5]. More recent studies have suggested that nuclear disassembly is driven largely by factors activated downstream of caspases [6]. One such factor, the caspase-activated DNase, CAD/CPAN/DFF40 [4,7,8] (CAD) can induce apoptotic chromatin condensation in isolated HeLa cell nuclei in the absence of other cytosolic factors [6,8]. As chromatin condensation occurs even when CAD activity is inhibited, however, CAD cannot be the sole morphogenetic factor triggered by caspases [6]. Here we show that DNA topoisomerase IIalpha (Topo IIalpha), which is essential for both condensation and segregation of daughter chromosomes in mitosis [9], also functions during apoptotic execution. Simultaneous inhibition of Topo IIalpha and caspases completely abolishes apoptotic chromatin condensation. In addition, we show that CAD binds to Topo IIalpha, and that their association enhances the decatenation activity of Topo IIalpha in vitro.

A neutralizing antibody against human DNA polymerase epsilon inhibits cellular but not SV40 DNA replication.

The contribution of human DNA polymerase epsilon to nuclear DNA replication was studied. Antibody K18 that specifically inhibits DNA polymerase activity of human DNA polymerase epsilon in vitro significantly inhibits DNA synthesis both when microinjected into nuclei of exponentially growing human fibroblasts and in isolated HeLa cell nuclei. The capability of this neutralizing antibody to inhibit DNA synthesis in cells is comparable to that of monoclonal antibody SJK-132-20 against DNA polymerase alpha. Contrary to the antibody against DNA polymerase alpha, antibody K18 against DNA polymerase epsilon did not inhibit SV40 DNA replication in vitro. These results indicate that DNA polymerase epsilon plays a role in replicative DNA synthesis in proliferating human cells like DNA polymerase alpha, and that this role for DNA polymerase epsilon cannot be modeled by SV40 DNA replication.

Role of caspases in apoptotic execution

Apoptosis is a morphologically and biochemically distinct form of cell death that plays a major role in the deletion of cells under a variety of physiological and pathological conditions. Morphologically, this process is characterized by a variable latent phase, followed by a dramatic execution phase. Apoptotic execution is accompanied by the overall shrinkage of the cell, a dramatic upregulation of motility processes (plasma membrane blebbing), and condensation of chromatin, followed by fragmentation of the nucleus and cytoplasm into multiple membraneenclosed bodies. These changes are accompanied by cleavage of nuclear proteins by specialized cell death proteases termed caspases. We have developed a cell-free approach to the analysis of the events of apoptotic execution, using cytoplasmic extracts from chicken DU249 cells at various stages along the apoptotic pathway. So-called S/M extracts from morphologically normal “committed stage” cells induce apoptotic morphology and DNA cleavage in substrate nuclei (Lazebnik, Y.A., Cole, S., Cooke, C.A., Nelson, W.G. & Earnshaw, W.C. (1993). 1. Cell RioI. 123, 7-22). These apoptotic changes appear to involve the function of multiple caspases (cysteine aspartases specialized proteases that are critical for apoptosis) acting in parallel. Affinity labeling of active caspases with Z-EK(biotin)D-aomk reveals at least seventeen distinct active caspase species in the S/M extracts (Martins, L.M., Kottke, T.J., Kaufmann, S.H. & Earnshaw, W.C. (1998). Blood 92,3042-3049). Inhibition of these caspases using WAD-chloromethylketone abrogates all signs of apoptosis. Very early during nuclear disassembly in the extracts, one or more of the extract caspases cleaves poly (ADP-ribose)polymerase (PARP) at the sequence DEVD-G, the first cleavage site determined for any caspase substrate in apoptosis (Lazebnik, Y.A., Kaufmann, S.H., Desnoyers, S., Poirier, G.G. & Earnshaw, W.C. (1994). Nature 371, 346-347). A second caspase subsequently catalyzes lamin cleavage, a reaction that is. required for disassembly of the nucleus into individual apoptotic bodies (Takahashi, A., Alnemri, E., Lazebnik, Y.A., Fernandes-Alnemri, T., Litwack, G., Moir, R.D., Goldman, R.D., Poirier, G.G., Kaufmann, S.H. & Earnshaw, W.C. (1996). PYOC. Nut. Acad. Sci. (USA) 93, 8395-8400). These experiments led to a view in which the events of apoptotic execution are largely due to the direct action of caspases on crucial cellular substrates. More recently, we have developed an alternative method for making extracts from cells in apoptotic execution. Surprisingly, these extracts induce apoptotic events in substrate nuclei in a caspase-independent manner, suggesting that nuclear disassembly is driven largely by factors activated downstream of the caspases at the onset of apoptotic execution (Samejima, K., Tone, S., Kottke, T.J., Enari, M., Sakahira, H., Cooke, C.A., Durrieu, F., Martins, L.M., Nagata, S., Kaufmann, S.H. & Earnshaw, W.C. (1998). 1. Cell Biol. 143, 225-239). We showed that one such factor, the caspase-activated DNase, CAD/CPAN/DFF40 can induce apoptotic chromatin condensation in isolated HeLa cell nuclei in the absence of other cytosolic factors. However, CAD cannot be the sole morphogenic factor triggered by caspases, since inhibition of CAD by addition of the inhibitory chaperone ICAD/DFF to execution phase extracts has no effect on the ability of those extracts to induce apoptotic events in added nuclei. These observations reveal that caspases act in an executive fashion, serving to activate downstream factors that disassemble the nucleus rather than disassembling it themselves. They also suggest that activation of the downstream factors (rather than the caspases) is the critical event that occurs at the transition from the latent

Identification and characterization of an RNA molecule that copurifies with RNase P activity from HeLa cells.

An RNA molecule, 340 nucleotides in length and designated H1 RNA, copurifies with RNase P activity from extracts of HeLa cells or isolated HeLa cell nuclei. When the genomic DNA of various organisms is probed with H1 cDNA in Southern hybridization assays, only mammalian DNA gives a positive signal. The gene coding for H1 RNA in human cells is present in one to three copies per cell. The nucleotide sequence of H1 RNA, which shows little homology to the known sequences of its analogs from prokaryotes and yeast, can be drawn as a two-dimensional, hydrogen-bonded structure that resembles similar structures proposed for the RNA subunit of RNase P from these other sources. Part of the hypothetical structure is virtually identical to structures that can be drawn for analogous RNAs from Saccharomyces cerevisiae, Schizosaccharomyces pombe, and S. octosporus.

U6 small nuclear RNA is transcribed by RNA polymerase III.

A DNA fragment homologous to U6 small nuclear RNA was isolated from a human genomic library and sequenced. The immediate 5'-flanking region of the U6 DNA clone had significant homology with a potential mouse U6 gene, including a "TATA box" at a position 26-29 nucleotides upstream from the transcription start site. Although this sequence element is characteristic of RNA polymerase II promoters, the U6 gene also contained a polymerase III "box A" intragenic control region and a typical run of five thymines at the 3' terminus (noncoding strand). The human U6 DNA clone was accurately transcribed in a HeLa cell S100 extract lacking polymerase II activity. U6 RNA transcription in the S100 extract was resistant to alpha-amanitin at 1 microgram/ml but was completely inhibited at 200 micrograms/ml. A comparison of fingerprints of the in vitro transcript and of U6 RNA synthesized in vivo revealed sequence congruence. U6 RNA synthesis in isolated HeLa cell nuclei also displayed low sensitivity to alpha-amanitin, in contrast to U1 and U2 RNA transcription, which was inhibited greater than 90% at 1 microgram/ml. In addition, U6 RNA synthesized in isolated nuclei was efficiently immunoprecipitated by an antibody against the La antigen, a protein known to bind most other RNA polymerase III transcripts. These results establish that, in contrast to the polymerase II-directed transcription of mammalian genes for U1-U5 small nuclear RNAs, human U6 RNA is transcribed by RNA polymerase III.

Modification of nuclear matrix proteins by ADP-ribosylation. Association of nuclear ADP-ribosyltransferase with the nuclear matrix.

Nuclear matrices were isolated by treatment of isolated HeLa cell nuclei with high DNase I, pancreatic RNase and salt concentrations. ADP-ribosylated nuclear matrix proteins were identified by electrophoresis, blotting and autoradiography. In one experimental approach nuclear matrix proteins were labeled by exposure of permeabilized cells to the labeled precursor [32P]NAD. Alternatively, the cellular proteins were prelabeled with [35S]methionine and the ADP-ribosylated nuclear matrix proteins separated by aminophenyl boronate column chromatography. By both methods bands of modified proteins, though with differing intensities, were detected at 41, 43, 46, 51, 60, 64, 69, 73, 116, 140, 220 and 300 kDa. Approximately 2% of the total nuclear ADP-ribosyltransferase activity, but only 0.07% of the nuclear DNA, was tightly associated with the isolated nuclear matrix. The matrix-associated enzyme catalyzes the incorporation of [32P]ADP-ribose into acid-insoluble products of molecular mass 116 kDa and above, in a 3-aminobenzamide-inhibited, time-dependent reaction. The possible function of ADP-ribosylation of nuclear matrix proteins and of the attachment of ADP-ribosyltransferase to the nuclear matrix in the regulation of matrix-associated biochemical processes is discussed.

Transcription boundaries of U1 small nuclear RNA.

Transcription-proximal stages of U1 small nuclear RNA biosynthesis were studied by 32P labeling of nascent chains in isolated HeLa cell nuclei. Labeled RNA was hybridized to nitrocellulose-immobilized, single-stranded M13 DNA clones corresponding to regions within or flanking a human U1 RNA gene. Transcription of U1 RNA was inhibited by greater than 95% by alpha-amanitin at 1 microgram/ml, consistent with previous evidence that it is synthesized by RNA polymerase II. No hybridization to DNA immediately adjacent to the 5' end of mature U1 RNA (-6 to -105 nucleotides) was detected, indicating that, like all studied polymerase II initiation, transcription of U1 RNA starts at or very near the cap site. However, in contrast to previously described transcription units for mRNA, in which equimolar transcription occurs for hundreds or thousands of nucleotides beyond the mature 3' end of the mRNA, labeled U1 RNA hybridization dropped off sharply within a very short region (approximately 60 nucleotides) immediately downstream from the 3' end of mature U1 RNA. Also in contrast to pre-mRNA, which is assembled into ribonucleoprotein (RNP) particles while still nascent RNA chains, the U1 RNA transcribed in isolated nuclei did not form RNP complexes by the criterion of reaction with a monoclonal antibody for the small nuclear RNP Sm proteins. This suggests that, unlike pre-mRNA-RNP particle formation, U1 small nuclear RNP assembly does not occur until after the completion of transcription. These results show that, despite their common synthesis by RNA polymerase II, mRNA and U1 small nuclear RNA differ markedly both in their extents of 3' processing and their temporal patterns of RNP assembly.

Transcription boundaries of U1 small nuclear RNA.

Transcription-proximal stages of U1 small nuclear RNA biosynthesis were studied by 32P labeling of nascent chains in isolated HeLa cell nuclei. Labeled RNA was hybridized to nitrocellulose-immobilized, single-stranded M13 DNA clones corresponding to regions within or flanking a human U1 RNA gene. Transcription of U1 RNA was inhibited by greater than 95% by alpha-amanitin at 1 microgram/ml, consistent with previous evidence that it is synthesized by RNA polymerase II. No hybridization to DNA immediately adjacent to the 5' end of mature U1 RNA (-6 to -105 nucleotides) was detected, indicating that, like all studied polymerase II initiation, transcription of U1 RNA starts at or very near the cap site. However, in contrast to previously described transcription units for mRNA, in which equimolar transcription occurs for hundreds or thousands of nucleotides beyond the mature 3' end of the mRNA, labeled U1 RNA hybridization dropped off sharply within a very short region (approximately 60 nucleotides) immediately downstream from the 3' end of mature U1 RNA. Also in contrast to pre-mRNA, which is assembled into ribonucleoprotein (RNP) particles while still nascent RNA chains, the U1 RNA transcribed in isolated nuclei did not form RNP complexes by the criterion of reaction with a monoclonal antibody for the small nuclear RNP Sm proteins. This suggests that, unlike pre-mRNA-RNP particle formation, U1 small nuclear RNP assembly does not occur until after the completion of transcription. These results show that, despite their common synthesis by RNA polymerase II, mRNA and U1 small nuclear RNA differ markedly both in their extents of 3' processing and their temporal patterns of RNP assembly.

Contamination by ATP of commercial dATP samples causing erroneous results in studies of DNA replication in isolated HeLa cell nuclei.

dATP at high concentrations was capable of replacing ATP required in the synthesis of Okazaki pieces in isolated HeLa cell nuclei. In addition, the levels of synthesis of high molecular weight DNA were observed to vary depending on the lot of dATP used. Analysis by HPLC revealed that dATP samples of a particular source contained ATP in the range of 0.25-0.43 mol%. With ATP-free dATP, almost no synthesis of high molecular weight DNA was observed, while with impure dATP, a small but significant amount of high molecular weight DNA was synthesized. While this observation confirmed our previous finding that dATP can replace ATP in the synthesis of Okazaki pieces but not in the joining of the pieces, it is also a warning to users of commercial dATP in biochemical and biological studies.

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.

Transcription renders chromatin resistant to micrococcal nuclease digestion

When isolated HeLa cell nuclei were preincubated under transcription conditions with excess E. coli RNA polymerase, chromatin DNA became relatively resistant to digestion by micrococcal nuclease. Quantitation of the DNA content in nuclei after enzyme digestion revealed that approximately twice as much nuclease was required to give the same levels of release of DNA fragments from transcribed as from untranscribed nuclei. Resistance increased with the amount of polymerase and with the time of preincubation. Since the resistance to nuclease was not observed in the presence of rifampicin or by preincubation without UTP, both RNA chain initiation and elongation were considered to be essential for the manifestation of resistance. However, when DNase 1 was used as a probe, such a change in chromatin DNA was not detected.

Importance of phospholipids to nitrosourea interactions with the nuclear envelope and associated nucleic acids.

Nuclease digestion of triton isolated HeLa cell nuclei has resulted in nuclear envelopes containing less than 10% of the nuclear macromolecule. Alkylation and carbamoylation of the nuclear envelope (NE) fraction by chloroethylnitrosoureas (HNU) was disproportionately high (approximately 35% of total nuclear alkylation and approximately 55% carbamoylation) suggesting that the nucleophilic species in the envelope were preferential targets for drug binding. Digestion of the envelope fraction with phospholipase C (PLC), followed by buoyant density gradient separation in Percoll (70%):0.15 M NaCl (30%), demonstrated that some envelope:nucleic acid attachments were destabilized as a result of phospholipid cleavage. In addition, the buoyant density of HNU-modified NE macromolecules was slightly altered by PLC digestion, suggesting that cleavage of polar phospholipids did not completely destabilize the chloroethylated products from the nuclear envelope.

Discrimination in the uptake of soluble proteins by isolated nuclei

ABSTRACT The uptake of radioactive cytosol proteins has been studied in isolated HeLa cell nuclei. The process is temperature- and pH-dependent, is enhanced by divalent metal ions, and is saturable with respect to the protein concentration of the cytosol. Nuclei prepared by washing in either detergent or sucrose buffers are similar in their abilities to accumulate protein, indicating that the outer nuclear membrane does not affect the process. The uptake is neither inhibited to any great extent by cyanide or 2,4-dinitrophenol nor significantly stimulated by glucose, glucose 6-phosphate, D-lactate, succinate, L-glutamate, NADH, phosphoenolpyruvate, cyclic AMP, or ATP. In fact, ATP is inhibitory, apparently due to its ability to chelate magnesium ions. Fractionation of the cytosol preparations on DEAE-cellulose shows that, as a class, those proteins eluting at higher salt concentrations are taken up better than those eluting at lower salt concentrations. Incubation of nuclei with protein fractions obtained from the chromatography of cytosol extracts on Sephadex G-150 indicates that the uptake of proteins in the molecular weight range from 35000 to 145000 is inversely proportional to molecular weight, but the nuclear accumulation of very small proteins (Mr 15000) was less than would be expected if molecular weight were the only factor determining nuclear uptake. Polyacrylamide gel electrophoresis shows that several protein classes in the cytosol are taken up by the nucleus and that in some cases the amount of protein in the nucleus reflects its relative concentration in the cytosol. Exceptions, however, are the drastic reduction in the nuclei of a class of proteins with approximate molecular weights of 55000 to 70000, and the increase in the nuclei of a protein class in the molecular weight range from 30000 to 40000. Proteins released from the nucleus are taken up by nuclei in vitro more efficiently than are cytosol proteins. In the presence of excess nuclei only 5-10 % of the available protein is taken up. Purified histones are rapidly taken up by nuclei, bovine serum albumin is not accumulated at all, and actin is taken up at an intermediate rate. When nuclei are fractionated following incubation, ribosomal proteins are found associated with the nucleolus to a greater extent than are cytosolic proteins. Thus, the nuclear uptake of soluble proteins in this cell-free system exhibits characteristics and specificity similar to that previously reported for in vivo systems and suggests that isolated nuclei may be useful for studying various aspects of nucleus-cytoplasm interaction.

The Inhibitory Mode of Aphidicolin on DNA Synthesis in NaCl-Treated HeLa Cell Nuclei1

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.

Metabolism of dITP in HeLa cell extracts, incorporation into DNA by isolated nuclei and release of hypoxanthine from DNA by a hypoxanthine-DNA glycosylase activity.

dITP may be generated from dATP by a slow, nonenzymatic hydrolysis. While [3H]dITP was degraded rapidly to [3H]deoxyinosine by HeLa cell nuclear extracts, no net degradation of [3H]dITP was observed in the presence of physiological concentrations of ATP, apparently because the extract contained deoxynucleoside diphosphate kinase activity that regenerated [3H]dITP from [3H]dIDP. Isolated HeLa cell nuclei, as well as partially purified DNA polymerase alpha, incorporated [3H]dITP into DNA at 50-60% of the rate of [3H]dGTP incorporation. No rapid release of the incorporated radioactivity was observed. The molecular weight of nascent DNA containing dIMP residues, however, decreased slightly after prolonged incubation in the presence of EDTA, suggesting that a repair process is initiated in dIMP-containing chromatin. Furthermore, release of free [3H]hypoxanthine from [3H]dIMP-containing DNA was detected after incubation with nuclear extracts in the presence of EDTA, suggesting the presence of hypoxanthine-DNA glycosylase activity in HeLa cell nuclei.

The effect of micrococcal nuclease and DNAase I on bulk and 5 S RNA syntheses in isolated HeLa cell nuclei

The effect of micrococcal nuclease and DNAase I on bulk and 5 S RNA syntheses was studied using isolated HeLa cell nuclei. Chromatin DNA isolated from micrococcal nuclease-digested nuclei ran as a series of bands that are multiples of a smallest size on agarose gel. According to the increment of the nuclease used, length of DNA has been shown to converge into mononucleosomal size. To measure transcriptional activity of thus-treated nuclei, EGTA, an effective chelating agent for calcium ion that is an essential component for micrococcal nuclease to exert its activity but not for RNA polymerases, was employed. The level of RNA syntheses stays rather constant up to the digestion with 125 units/ml of the nuclease, but at higher concentrations both bulk and 5 S RNA synthesis decreased. The proportion of 5 S RNA in bulk RNA synthesized by nuclease-treated nuclei remained constant. The size of 5 S RNA sequence as measured by Sephadex G-100 gel filtration in combination with hybridization with cloned Xenopus 5 S DNA coincided with that of authentic in vivo 5 S RNA. On the other hand, chromatin DNA derived from DNAase I-digested nuclei showed rather smear-like pattern. Transcriptional activity was also inhibited as the digestion proceeded. Both bulk and 5 S RNA syntheses appeared to be inhibited to the same extent and the invariable ratio was again observed. The inhibition of transcription was, however, observed already at a very low concentration of DNAase I at which there is no remarkable change in nuclear DNA size from that of untreated control, indicating that active class III genes are also preferentially digested by DNAase I like many class II genes. The majority of 5 S RNA sequences produced by partially DNAase I-digested nuclei was again 5 S in size. These results suggested that micrococcal nuclease and DNAase I inhibit the nuclear transcriptional activity equally effectively, irrespective of size of RNA species to be transcribed, and that long intact structure might be required for the transcription of even small genes.

Aphidicolin inhibits DNA synthesis by DNA polymerase alpha and isolated nuclei by a similar mechanism.

Aphidicolin is a selective inhibitor of DNA polymerase alpha. In contrast to earlier reports, the drug was found to inhibit DNA synthesis catalyzed by DNA polymerase alpha and isolated HeLa cell nuclei by a similar mechanism. For both systems aphidicolin primarily competed with dCTP incorporation. However, the apparent Vmax for dCTP incorporation was reduced by 50-60% at relatively low concentrations of aphidicolin, thus the mechanism of inhibition is complex. Furthermore, a 2-5 fold increase in apparent Km for dTTP was observed in the presence of aphidicolin, but the apparent Km values for dATP and dGTP were essentially unaltered. This, together with additional evidence, suggested that the mechanism of action of aphidicolin involves a strong competition with dCMP incorporation, a weaker competition with dTMP incorporation and very little, if any, competition with dGMP and dAMP incorporation.

ATP requirement for the processes of DNA replication in isolated HeLa cell nuclei.

The ATP requirement for two steps of DNA replication, the synthesis and subsequent joining of Okazaki fragments, was investigated by using isolated HeLa cell nuclei. Among adenine nucleotides tested, high levels of dATP and ADP stimulated DNA synthesis. In the presence of high levels of ATP, the addition of high levels of dATP or ADP resulted in about 70% inhibition of DNa synthesis. The optimal concentration of ATP for the stimulation of DNA synthesis varied depending on the magnesium ion concentration. When the molar ratio of magnesium ion to ATP was approximately 1, maximal stimulation was attained. Product analysis by sedimentation in an alkaline sucrose gradient revealed that both Okazaki fragments and high molecular weight DNA were synthesized in the presence of high levels of ATP, whereas in the case of dATP and ADP, little high molecular weight DNA was synthesized. The ability to synthesize high molecular weight DNA was restored to nuclei by adding low levels of ATP in the presence of high levels of ADP but not of dATP.