Reduction of DNA primase activity in aging but still proliferating cells

The basis of the well-known decline in cell proliferation with increasing passage number of human diploid fibroblast-like cell cultures is not known. It has been found that DNA synthesis was deficient in the remaining but still proliferating cells, but when appropriate corrections reflecting the remaining dividing cells were made, the amount of DNA polymerase alpha bound to nuclear matrices was normal [Collins and Chu: Journal of Cellular Physiology 124:165-173, 1985]. In the present study, the declining percentages of S-phase and dividing cells were determined to provide better estimates of functional culture age than passage number. The amounts of DNA polymerase alpha and DNA primase activity were determined in cell lysates, permeabilized cells, and bound to nucleoids, which are residual nuclear structures similar to nuclear matrices except that no DNase-digestion step is employed. As expected, IMR 90 DNA synthesis declined with age, even after corrections for the declining numbers of proliferating cells. DNA polymerase alpha and DNA primase activity in cell lysates, permeabilized cells, and bound to nucleoids declined with increasing age. However, after appropriate corrections for the declining fraction of proliferating cells, the only activity that declined was that of DNA primase bound to nucleoids. Thus, a decrease in the binding of DNA primase to the nuclear site of DNA synthesis may account for the decreased DNA synthesis in aging but still proliferating cells.     

Microtubule-associated protein-2 stimulates DNA synthesis catalyzed by the nuclear matrix

Microtubule-associated protein-2 (MAP-2) isolated from porcine brains stimulated DNA synthesis catalyzed by the nuclear matrix isolated from Physarum polycephalum in the presence of activated DNA as exogenous templates. The degree of the stimulation depended on the amount of the nuclear matrix, but not on that of the template. MAP-2 also stimulated DNA polymerase alpha activity solubilized from nuclei, but not DNA polymerase beta activity. These results suggest that MAP-2 stimulates DNA synthesis by interacting with the putative DNA replication machinery including DNA polymerase alpha bound to the matrix. Similar stimulation occurred in the nuclear matrix isolated from HeLa and rat ascites hepatoma cells, which strongly suggests that MAP-2 is involved in the control of DNA replication in eukaryotic cells.     

DNA synthesis by the isolated nuclear matrix from synchronized plasmodia of Physarum polycephalum

Nuclear matrices were isolated from plasmodia of a true slime mold, Physarum polycephalum, and the DNA synthetic activity in vitro was examined. These matrices isolated in S-phase catalyzed DNA synthesis requiring Mg2+, deoxyribonucleoside 5'-triphosphates and ATP, without exogenous templates. The activity changed during S-phase with the rate of in vivo DNA replication. Product analysis by gel electrophoresis revealed that the matrices produced Okazaki fragments. These results suggest that DNA synthesis partially reflects in vivo DNA replication. DNA synthesis was sensitive to aphidicolin, heparin and N-ethylmaleimide, indicating involvement of the alpha-like DNA polymerase of Physarum. Exogenous addition of activated DNA stimulated DNA synthesis 4-10-fold and suggested that only some of the existing enzymes are involved in endogenous DNA synthesis. Matrices isolated in G2-phase were also associated with a similar DNA synthetic activity, but they did not produce Okazaki fragments in vitro. It is, therefore, concluded that nuclear matrices are associated with alpha-like DNA polymerase throughout the cell cycle, and that some of the enzymes participate in in vivo DNA replication in S-phase; thus, DNA replication is possibly controlled by this process. The relationship between DNA synthetic activities by the isolated nuclei and matrices was also discussed.     

The role of DNA polymerases alpha, beta and gamma in nuclear DNA synthesis.

The effects of the inhibitors 2'3' dideoxythymidine triphosphate (ddTTP) and 1-beta-D-arabinofuranosyl cytosine triphosphate (araCTP) on DNA synthesis in isolated S-phase HeLa S3 nuclei have been examined. These effects are compared with the effects of the same inhibitors in partially purified preparations of DNA polymerases alpha and beta. The effect of ddTTP on partially purified DNA polymerase gamma was also tested. DNA polymerases beta and gamma were very sensitive to ddTTP whereas DNA polymerase alpha and DNA synthesis in isolated nuclei were quite resistant. The synthesis and subsequent ligation of primary DNA pieces ('Okazaki fragments') were not affected by the presence of this inhibitor. DNA synthesis in isolated nuclei and DNA polymerase alpha activity were very sensitive to araCTP whereas DNA polymerase beta was almost totally resistant to the inhibitor. The results indicate a major role for DNA polymerase alpha in DNA replication.     

On the association of DNA polymerase alpha activity with the nuclear matrix in HeLa cells

The association of DNA polymerase alpha activity with the nuclear matrix has been reinvestigated in HeLa cells. Isolated nuclei were extracted with 2M NaCl and then digested with Dnase I and the final structures were recovered by centrifugation through a sucrose cushion. Typically over 98% of the total DNA synthesized in the matrix fraction on either endogenous matrix-associated DNA or activated calf thymus DNA was due to DNA polymerase alpha as defined by inhibition to n-ethylmaleimide or aphidicolin. DNA polymerase beta activity was absent or recovered in only trace amounts. Matrix-bound DNA polymerase alpha activity demonstrated a remarkable degree of stability: DNA synthesis was essentially linear up to 3 hours at 37 degrees C. Overall, these results substantiate previous findings from regenerating rat liver, unlike other data obtained from tissue culture cells.     

Reduction of DNA synthesis in aging but still proliferating cells

It is well known that cell proliferation (and hence, DNA synthesis) declines in human diploid fibroblast-like cells with increasing passage number. It is not clear whether DNA synthesis declines in the remaining cells that are still actively proliferating. Estimations of cell kinetic parameters permitted extrapolations to be made that reflected the declining numbers of cells still capable of DNA replication. DNA synthesis declined with culture age in intact cells, permeabilized cells, and in the isolated nuclear matrix even when corrected for declining numbers of proliferating cells. With age, DNA polymerase alpha and beta activity in cell lysates declined, but when corrected for the remaining proliferating cells, only polymerase alpha activity declined; DNA polymerase alpha and beta activity bound to the nuclear matrix declined, but when corrected for declining proliferation, no decline was apparent for either enzyme. There was an increase in the number of S1-nuclease sensitive sites and breaks in the parental DNA of the dividing cells in older cultures. It is suggested that in aging cultures, not only does overall DNA synthesis decline owing to decreasing cell proliferation, but also that DNA synthesis declines in the remaining proliferating cells, that this decline is not due to decreasing amounts of DNA polymerase bound to the nuclear matrix, and that alterations in DNA structure occur.     

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 on discontinuous templates by human DNA polymerases: implications for non-homologous DNA recombination.

DNA polymerases catalyze the synthesis of DNA using a continuous uninterrupted template strand. However, it has been shown that a 3'-->5' exonuclease-deficient form of the Klenow fragment of Escherichia coli DNA polymerase I as well as DNA polymerase of Thermus aquaticus can synthesize DNA across two unlinked DNA templates. In this study, we used an oligonucleotide-based assay to show that discontinuous DNA synthesis was present in HeLa cell extracts. DNA synthesis inhibitor studies as well as fractionation of the extracts revealed that most of the discontinuous DNA synthesis was attributable to DNA polymerase alpha. Additionally, discontinuous DNA synthesis could be eliminated by incubation with an antibody that specifically neutralized DNA polymerase alpha activity. To test the relative efficiency of each nuclear DNA polymerase for discontinuous synthesis, equal amounts (as measured by DNA polymerase activity) of DNA polymerases alpha, beta, delta (+/- PCNA) and straightepsilon (+/- PCNA) were used in the discontinuous DNA synthesis assay. DNA polymerase alpha showed the most discontinuous DNA synthesis activity, although small but detectable levels were seen for DNA polymerases delta (+PCNA) and straightepsilon (- PCNA). Klenow fragment and DNA polymerase beta showed no discontinuous DNA synthesis, although at much higher amounts of each enzyme, discontinuous synthesis was seen for both. Discontinuous DNA synthesis by DNA polymerase alpha was seen with substrates containing 3 and 4 bp single-strand stretches of complementarity; however, little synthesis was seen with blunt substrates or with 1 bp stretches. The products formed from these experiments are structurally similar to that seen in vivo for non-homologous end joining in eukaryotic cells. These data suggest that DNA polymerase alpha may be able to rejoin double-strand breaks in vivo during replication.     

Binding of the DNA polymerase alpha-DNA primase complex to the nuclear matrix in HeLa cells

It is well-known that there are multiple forms of DNA polymerase alpha. In order to determine which form(s) is (are) tightly bound, the activities were dissociated from DNA-poor nuclear matrices, with octyl beta-D-glucoside. Sucrose gradient sedimentation analysis revealed three bands with s values of 7.5, 10.5, and 13. The 7.5S form was free of DNA primase and represented only 10% of the total DNA polymerase alpha bound to the nuclear matrix. The 13S and the 10.5S forms each contained DNA primase activity. The 10.5S form comprised 85% of the DNA polymerase alpha activity and 95% of the DNA primase activity, dissociated from the nuclear matrix. Neither temperature of nuclease digestion nor various salt treatments of nuclei had significant effects on the proportions of DNA polymerase alpha and DNA primase activities bound to, or subsequently dissociated from, nuclear matrices. In a comparison of primase activity bound to the nuclear matrix, dissociated from the nuclear matrix, and in the soluble fraction, it was found that the bound activity had a lower ATP dependence, had less KCl inhibition, and was less sensitive to heat, compared to the dissociated and soluble activities. No differences in Mg2+ or pH dependence were noted. The amounts of DNA polymerase alpha and DNA primase activities bound to the nuclear matrix varied over the cell cycle of synchronized cells. Over the S phase, there were two peaks of matrix-bound DNA primase and two peaks of subsequently dissociated DNA polymerase alpha-DNA primase complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of DNA polymerase alpha by gossypol

Our earlier studies have shown that gossypol is a specific inhibitor of DNA synthesis in cultured cells at low doses. In an attempt to determine the mechanism for the inhibition of DNA synthesis by gossypol we observed that gossypol does not interact with DNA per se but may affect some of the enzymes involved in DNA replication. These studies indicated that gossypol inhibits both in vivo and in vitro the activity of DNA polymerase alpha (EC 2.7.7.7), a major enzyme involved in DNA replication, in a time- and dose-dependent manner. Kinetic analysis revealed that gossypol acts as a noncompetitive inhibitor of DNA polymerase alpha with respect to all four deoxynucleotide triphosphates and to the activated DNA template. Inhibition of DNA polymerase alpha does not appear to be due to either metal chelation or reduction of sulfhydryl groups on the enzyme. Gossypol also inhibited HeLa DNA polymerase beta in a dose-dependent manner, but had no effect on DNA polymerase gamma. These results suggest that inhibition of DNA polymerase alpha may account in part for the inhibition of DNA synthesis and the S-phase block caused by gossypol. The data also raise the possibility that gossypol may interfere with DNA repair processes as well.     

Cell cycle specific plasmid DNA replication in the nuclear extract of Saccharomyces cerevisiae: modulation by replication protein A and proliferating cell nuclear antigen

Plasmid DNA replication in nuclear extracts of Saccharomyces cerevisiae in vitro has been shown to be S-phase specific, similar to that observed in vivo. We report here a reconstituted in vitro system with partially purified replication proteins, purified replication protein A (RPA), and recombinant proliferating cell nuclear antigen (PCNA). Nuclear extracts from S-phase, G(1)-phase, and unsynchronized yeast cells were fractionated by phosphocellulose chromatography. Protein fraction (polymerase fraction) enriched with replication proteins, including DNA polymerases (alpha, delta, etc.), was isolated, which was not capable of in vitro replication of supercoiled plasmid DNA. However, when purified yeast RPA and recombinant PCNA together were added to the polymerase fraction obtained from S-phase synchronized cells, in vitro plasmid DNA replication was restored. In vitro plasmid DNA replication with polymerase fractions from unsynchronized and G(1)-phase cells could not be reconstituted upon addition of purified RPA and PCNA. RPA and PCNA isolated from various phases of the cell cycle complemented the S-phase polymerase pool to the same extent. Reconstituted systems with the S-phase polymerase pool, complemented with either the RPA- and PCNA-containing fraction or purified RPA and recombinant PCNA together, were able to produce replication intermediates (ranging in size from 50 to 1500 bp) similar to that observed with the S-phase nuclear extract. Results presented here demonstrate that both RPA and PCNA are cell cycle-independent in their ability to stimulate in vitro plasmid DNA replication, whereas replication factors in the polymerase fractions are strictly S-phase dependent.     

Spatial distribution of DNA loop attachment and replicational sites in the nuclear matrix

Biochemical fractionation was combined with high resolution electron microscopic autoradiography to study the localization in rat liver nuclear matrix of attached DNA fragments, in vivo replicated DNA, and in vitro synthesized DNA. In particular, we determined the distribution of these DNA components with the peripheral nuclear lamina versus more internally localized structural elements of isolated nuclear matrix. Autoradiography demonstrated that the bulk of in vivo newly replicated DNA associated with the nuclear matrix (71%) was found within internal matrix regions. A similar interior localization was observed in isolated nuclei and in situ in whole liver tissue. Likewise, isolated nuclear lamina contained only a small amount (12%) of the total matrix-bound, newly replicated DNA. The structural localization of matrix-bound DNA fragments was examined following long-term in vivo labeling of the DNA. The radioactive DNA fragments were found predominantly within interior regions of the matrix structure (77%), and isolated nuclear lamina contained less than 15% of the total nuclear matrix-associated DNA. Most of the endogenous DNA template sites for the replicative enzyme DNA polymerase alpha (approximately 70%) were also sequestered within interior regions of the matrix. In contrast, a majority of the endogenous DNA template sites for DNA polymerase beta (a presumptive repair enzyme) were closely associated with the peripheral nuclear lamina. A similar spatial distribution for both polymerase activities was measured in isolated nuclei before matrix fractionation. Furthermore, isolated nuclear lamina contained only a small proportion of total matrix-bound DNA polymerase alpha endogenous and exogenous template activities (3-12%), but a considerable amount of the corresponding beta polymerase activities (47-52%). Our results support the hypothesis that DNA loops are both anchored and replicated at nuclear matrix-bound sites that are predominantly but not exclusively associated with interior components of the matrix structure. Our results also suggest that the sites of nuclear DNA polymerase beta-driven DNA synthesis are uniquely sequestered within the characteristic peripheral heterochromatin shell and associated nuclear envelope structure, where they may potentially participate in DNA repair and/or replicative functions.     

Replication of parvoviral DNA. I. Characterization of a nuclear lysate system.

We have developed a nuclear lysate system from infected, synchronized cells capable of synthesizing unit-length parvoviral DNA in vitro. It was necessary to supplement the nuclear lysates with the polyamines, spermidine and spermine, to prevent degradation of template and product DNAs. In this system RF, RI, and single-stranded progeny DNAs were synthesized. Label incorporated in viral RF DNA in vivo appeared first in RI DNA and then in single-stranded DNA during incubation in vitro. By sedimentation the product DNAs were identical to those found in infected cells. Their viral identity was confirmed by hybridization. The addition of ribonucleotides, RNase, or alpha-amanitin did not affect parvoviral DNA synthesis in this system. The results with the specific inhibitors of mammalian DNA polymerases, aphidicolin, N-ethylmaleimide, and 2',3'-dideoxythymidine 5'-triphosphate indicated that DNA polymerase alpha was required for synthesis of parvoviral DNA in the nuclear lysates. This requirement was confirmed by experiments with antibody to bovine DNA polymerase alpha.     

Effect of 2'',3''-dideoxythymidine-5''-triphosphate on HeLa cell in vitro DNA synthesis: evidence that DNA polymerase alpha is the only polymerase required for cellular DNA replication.

We have studied the effects of the nucleotide analogue, 2',3'-dideoxythymidine-5'-triphosphate (ddTTP) on replicative DNA synthesis in HeLa cell lysates. As previously demonstrated (1), such lysates carry out extensive DNA synthesis in vitro, at rates and in a fashion similar to in vivo DNA replication. We report here that all aspects of DNA synthesis in such lysates (total dNTP incorporation, elongation of continuous nascent strands, and the initiation, elongation, and joining of Okazaki pieces) are only slightly inhibited by concentrations of ddTTP as high as 100-500 micrometer when the dTTP concentration is maintained at 10 micrometer. This finding is consistent with the report by Edenberg, Anderson, and DePamphilis (2) that all aspects of replicative in vitro simian virus 40 DNA synthesis are also resistant to ddTTP. We also find, in agreement with Edenberg, Anderson, and DePamphilis (2), that DNA synthesis catalyzed by DNA polymerases beta or gamma is easily inhibited by ddTTP, while synthesis catalyzed by DNA polymerase alpha is very resistant. These observations suggest that DNA polymerase alpha may be the only DNA polymerase required for all aspects of cellular DNA synthesis.     

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.     

Involvement of deoxyribonucleic acid polymerase beta in nuclear deoxyribonucleic acid synthesis.

The effects on DNA synthesis in vitro in mouse L929-cell nuclei of differential extraction of DNA polymerases alpha and beta were studied. Removal of all measurable DNA polymerase alpha and 20% of DNA polymerase beta leads to a 40% fall in the replicative DNA synthesis. Removal of 70% of DNA polymerase beta inhibits replicative synthesis by 80%. In all cases the nuclear DNA synthesis is sensitive to N-ethylmaleimide and aCTP (arabinosylcytosine triphosphate), though less so than DNA polymerase alpha. Addition of deoxyribonuclease I to the nuclear incubation leads to synthesis of high-molecular-weight DNA in a repair reaction. This occurs equally in nuclei from non-growing or S-phase cells. The former nuclei lack DNA polymerase alpha and the reaction reflects the sensitivity of DNA polymerase beta to inhibiton by N-ethylmaleimide and aCTP.     

Synthesis of viral and host DNA in isolated chromatin from herpes simplex virus-infected HeLa cells.

DNA synthesis in chromatin isolated from herpes simplex virus type 1-infected HeLa cells (HSV chromatin) was examined in vitro. The HSV chromatin was found to carry out an initial limited synthesis of DNA in vitro, 50 to 64 pmol of dTMP incorporated in 10(6) nuclei per 10 min, which is comparable to that found in nuclei isolated from HSV-infected cells. DNA synthesis in vitro proceeded for only 30 min, and both HSV DNA and host DNA were synthesized in significant amounts. The HSV and host DNA synthesis in isolated chromatin were inhibited to the same extent by anti-HSV antiserum or by phosphonoacetic acid. The results indicate that the HSV-induced DNA polymerase is most likely involved in the synthesis of host and HSV DNA in isolated chromatin, even though this chromatin contains small amounts of the host gamma-polymerase in addition to the HSV-induced DNA polymerase. The HSV chromatin contains no detectable levels of DNA polymerases alpha and beta, even though infected cells have normal, or increased, levels of these enzymes.     

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.     

Synthesis of herpes simplex virus, vaccinia virus, and adenovirus DNA in isolated HeLa cell nuclei. I. Effect of viral-specific antisera and phosphonoacetic acid.

Purified nuclei, isolated from appropriately infected HeLa cells, are shown to synthesize large amounts of either herpes simplex virus (HSV) or vaccinia virus DNA in vitro. The rate of synthesis of DNA by nuclei from infected cells is up to 30 times higher than the synthesis of host DNA in vitro by nuclei isolated from uninfected HeLa cells. Thus HSV nuclei obtained from HSV-infected cells make DNA in vitro at a rate comparable to that seen in the intact, infected cell. Molecular hybridization studies showed that 80% of the DNA sequences synthesized in vitro by nuclei from herpesvirus-infected cells are herpesvirus specific. Vaccinia virus nuclei from vaccinia virus-infected cells, also produce comparable percentages of vaccinia virus-specific DNA sequences. Adenovirus nuclei from adenovirus 2-infected HeLa cells, which also synthesize viral DNA in vitro, have been included in this study. Synthesis of DNA by HSV or vaccinia virus nuclei is markedly inhibited by the corresponding viral-specific antisera. These antisera inhibit in a similar fashion the purified herpesvirus-induced or vaccinia virus-induced DNA polymerase isolated from infected cells. Phosphonoacetic acid, reported to be a specific inhibitor of herpesvirus formation and the herpesvirus-induced DNA polymerase, is equally effective as an inhibitor of HSV DNA synthesis in isolated nuclei in vitro. However, we also find phosphonoacetic acid to be an effective inhibitor of vaccinia virus nuclear DNA synthesis and the purified vaccinia virus-induced DNA polymerase. In addition, this compound shows significant inhibition of DNA synthesis in isolated nuclei obtained from adenovirus-infected or uninfected cells and is a potent inhibitor of HeLa cell DNA polymerase alpha.     

The effect of aphidicolin on adenovirus DNA synthesis.

Aphidicolin inhibits adenovirus DNA replication in HeLa cells and in a cell-free, infected, nuclear extract in which viral DNA is elongated. The compound inhibits alpha DNA polymerase, extensively purified from HeLa cells, but has little or no effect on the beta or gamma DNA polymerases similarly purified. Aphidicolin does not affect thymidine uptake by cells nor does synthesis as it also inhibits DNA replication in uninfected cells. The inhibition by aphidicolin is reversible if the drug is removed within 18 hrs after addition to HeLa or Chinese Hamster Ovary cells but the cells are irreversibly affected if the drug remains for 48 hours.