DNA Polymerases from Human Cells

Weissbach, Arthur; Schlabach, A J; Fridlender, Bertold; Bolden, Arthur H.

doi:10.1038/newbio231167a0

Cited by 240 publications

(81 citation statements)

References 12 publications

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“…RESULTS Levels of DNA polymerase activity DNA polymerase (deoxynucleosidetriphosphate:DNA nucleotidyltransferase, EC 2.7.7.7) was purified from whole cell sonicates through the DEAE-cellulose gradient step, essentially as described for HeLa cells (13) ( Table 1). Two peaks of activity were obtained on the DEAE-cellulose, as reported for HeLa cells, but they both sedimented at 5.5-6 S and both had catalytic properties similar to those reported for the "cytoplasmic" enzyme, a (13). The reason for the apparent absence of "nuclear"…”

Section: Activitymentioning

confidence: 65%

Decreased fidelity of DNA polymerase activity isolated from aging human fibroblasts.

Linn¹,

Kairis²,

Holliday³

1976

Proc. Natl. Acad. Sci. U.S.A.

113

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DNA polymerase (deoxynucleosidetriphosphate:DNA nucleotidyltransferase, EC 2.7.7.7 or DNA nucleotidyltransferase) activity, isolated from late and early passage cells of the diploid human fibroblast line, MRC-5, was compared. The level of activity dropped with increasing passage. In addition, when the fidelity of polymerization was monitored with four synthetic templates under a variety of conditions, it was observed that the enzyme from late passage cells was more error-prone. The possible relation of these observations to "senescence" of the fibroblasts is discussed.Diploid human fibroblasts have a defined lifespan in culture that is somewhat related to the potential life expectancy of the individual from whom the cells were obtained (1, 2). This system has therefore been widely adopted as a model for studying the molecular bases of aging, physiological senescence, and cell death. Orgel has proposed that the fibroblast senescence results from an "error catastrophe" in macromolecular synthesis, which will inevitably lead to faulty and unregulated cellular metabolism (3, 4), and evidence has been obtained that defective enzyme molecules accumulate at increasingly higher levels in senescent fibroblasts, as well as in tissues from aging mice (5-10). Since the appearance of defective DNA polymerase could be an important component in any general breakdown of information transfer between macromolecules, we have compared the fidelity of DNA polymerase from cells in the early and late stages of cell culture during the replication of defined synthetic templates. MATERIALS AND METHODSGrowth of Cells. MRC-5 human male fetal lung fibroblasts (5) were grown in Eagle's basal medium containing 10% fetal calf serum, 100 units/ml of penicillin, 100 ,gg/ml of streptomycin, and 50,gg/ml of aureomycin. Cells were monitored to assure the absence of mycoplasmic contamination (11). For collection, cells that had not reached confluence, as judged by microscopic examination, were rinsed with 5 mM Tris-HCl (pH 7.5), 0.15 M NaCl, scraped off of the glass with a piece of soft plastic, then harvested and washed by centrifugation in the same buffer. The pellet could be stored at -70°or used immediately with no effect upon the subsequently fractionated activity.Assays for Enzyme Activity. Reaction mixtures (0.1 ml) contained 50 mM Tris-HCl (pH 8.5), 7.5 mM MgCl2, 0.1 M KCl, 0.5 mM dithiothreitol, 0.5 mg/ml of bovine serum albumin (Sigma, Fraction V), 0.13 ,gmol of "activated" salmon sperm DNA (12), and 5 nmol each of dATP, dGTP, dCTP, and dTTP. One of the triphosphates was labeled with 3H at 50-100 cpm/pmol. After 30 min at 37°, the reaction mixtures were chilled, then mixed with 0.2 ml of 0.1 M Na4P207 and 0.7 ml of 10% trichloroacetic acid. After at least 5 min, 3 ml of 1 M HCl, 0.1 M Na4P2O7 were added. The contents were filtered through a Whatman GF/C glass filter that had been soaked in 0.1 M Na4P207, then washed with the HCl-Na4P207. The filter was then successively washed 12 times with 3 ml of the HClNa4P2O7 and then with eth...

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Section: Activitymentioning

confidence: 65%

Decreased fidelity of DNA polymerase activity isolated from aging human fibroblasts.

Linn¹,

Kairis²,

Holliday³

1976

Proc. Natl. Acad. Sci. U.S.A.

113

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“…1). This enhancement suggests that the increase in DNA polymerase activity is dependent on the activity of the deoxyribonuclease, which is known to activate DNA templates (5,17). This possibility was ruled out by separating the two activities (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…In crude extracts derived from germinating embryos, the polymerase activity is found together with a deoxyribonuclease activity. Since the polymerase activity is more pronounced with "activated" calf thymus DNA than with denatured or native wheat embryo DNA (unpublished results), it can be suggested that this increase in polymerase activity, after germination has started, is somehow due to the DNase activity which in vitro wouid activate the DNA as template by partial digestion, as in the case of E. coli DNA polymerase (5) and of HeLa cells DNA polymerases (17) which are much more active with activated templates than with native or denatured templates. We therefore followed the activity level of the two enzymes during the early stages of germination.…”

Section: Enhancement Of Deoxynucleotide Triphosphate Polymerase Andmentioning

confidence: 99%

De Novo Biosynthesis of Deoxyribonucleic Acid Polymerase during Wheat Embryo Germination

Mory¹,

Chen²,

Sarid³

1975

Plant Physiol.

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An 8-fold enhancement in the activity of a DNA-dependent DNA polymerase was found in extracts from germinating wheat (Triticum vulgare var. Florence) embryos, as compared to the activity found in extracts from ungerminated embryos. The enhancement of this activity during the first hours of germination is concomitant to the increase of a DNase activity. The two activities could be separated and the increased level of the DNA polymerase upon germination was observed in an enzymatic fraction which contains very low DNase activity. Addition of the protein synthesis inhibitor, blasticidin S, to germinating wheat embryos, reduced the increase in DNA polymerase. Incorporation of radioactive amino acids into a phosphocellulose preparation, which contains the DNA polymerase starts during the first 6 hours of germination. The amount of radioactivity incorporated is doubled in the next 6 hours, and the incorporation is continued between 12 and 18 hours of germination.

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“…1 (a) the 100000g supernatant fluid from uninfected HeLa $3 cells was resolved into two peaks, one in the unbound fraction and a second major peak eluting in the 0.2 M step. Comparison with the results of other workers would suggest that the unbound activity is DNA polymerase fl and the second peak is DNA polymerase ct (Weissbach et al, 1971(Weissbach et al, , 1975. Fig.…”

mentioning

confidence: 91%

The DNA Polymerase Activity of Vaccinia Virus 'Virosomes': Solubilization and Properties

Tomley

Cooper

1984

Journal of General Virology

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SUMMARYIntracellular DNA-protein complexes ('virosomes') of vaccinia virus have been isolated. The solubilization of the 'virosome'-bound DNA polymerase activity was attempted by a variety of high-salt extraction procedures. The most efficient of these used 0.3 M-ammonium sulphate followed by brief sonication. The solubilized DNA polymerase activity from the 'virosomes', together with the DNA polymerases from 100000 g supernatant fluids from the cytoplasm of infected and uninfected cells were chromatographed on DEAE-cellulose and their properties compared. The 'virosome' DNA polymerase activity differed from the soluble vaccinia virus-induced DNA polymerase activity in its requirements for divalent cations and in respect of pH optimum, Km for the deoxyribonucleoside triphosphates and the effect of Nethylmaleimide.Vaccinia virus DNA replication takes place in nucleoprotein structures ('factories') in the cytoplasm of infected cells (Cairns, 1960). Crude preparations of similar structures ('virosomes') were found to contain low levels of DNA polymerase activity by Jungwirth & Joklik (1965) and more recently by Lambert & Magee (1977). In this paper we describe a method for the solubilization of this 'virosome' DNA polymerase activity and compare its properties with those of the soluble vaccinia virus-induced DNA polymerase.Suspension cultures of HeLa $3 cells were infected by vaccinia virus WR essentially as described by Joklik & Becker (1964). The infected or mock-infected cells were harvested 3.5 h after addition of virus and broken to give nuclei and cytoplasm by homogenization, the nuclei being removed by centrifugation at 200 g for 2 min. The vaccinia virus 'virosomes' were isolated by the method of Polisky & Kates (1976) except that the detergent was omitted. Briefly, 10 ml of cytoplasm containing the 'virosomes' were layered on top of 35 ml of a 35 to 50 % (w/v) sucrose gradient which in turn had been formed on top of an 80% (w/v) sucrose cushion. After centrifugation in an SW25.2 rotor at 100000 g for 2 h on the Beckman L2 ultracentrifuge, the 'virosomes' were clearly visible as an opaque band on top of the sucrose cushion. The 'virosomes' were removed from the gradient, diluted twofold in hypotonic buffer (0-01 M-KC1, 0-005 M-EDTA, 0-005 M-2-mercaptoethanol, 0.01 M-Tris-HCI pH 8.0 at 4 °C) and deposited by centrifugation at 1000 g for 5 min. The 'virosomes' were resuspended in a small volume of hypotonic buffer and either used immediately or stored at -70 °C.Five different methods were tested for their ability to solubilize the DNA polymerase activity of the isolated 'virosomes'. All the methods involved the use of high ionic strength and two of them combined this with detergent. The method eventually chosen was first described by Roeder & Rutter (1969) for the solubilization of i~NA polymerases from nuclei. The isolated 'virosomes' were made 0-3 M with ammonium sulphate and sonicated with a probe for three bursts of 20 s each with cooling in ice. The suspension was then immediately diluted threefold into...

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DNA Polymerases from Human Cells

Cited by 240 publications

References 12 publications

Decreased fidelity of DNA polymerase activity isolated from aging human fibroblasts.

Decreased fidelity of DNA polymerase activity isolated from aging human fibroblasts.

De Novo Biosynthesis of Deoxyribonucleic Acid Polymerase during Wheat Embryo Germination

The DNA Polymerase Activity of Vaccinia Virus 'Virosomes': Solubilization and Properties

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