Deoxyribonucleic acid polymerase in L cells

Gold, Marvin; Helleiner, C. W.

doi:10.1016/0926-6550(64)90091-x

Cited by 32 publications

(17 citation statements)

References 14 publications

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“…As a necessary background to the presentation of our work we have developed the following nomenclature. The 6-8S DNA polymerase, found mainly in the cytoplasmic portion of cell extracts, is DNA polymerase C (1,10,11). The content of this enzyme in cells varies widely and is dependent on the growth rate of the cells (Chang, McKay, & Bollum, J. Mol.…”

Section: Mammalian Cells Contain Several Dna Polymerases (1-8)mentioning

confidence: 99%

Terminal Deoxynucleotidyl Transferase in a Case of Childhood Acute Lymphoblastic Leukemia

McCaffrey¹,

Smoler²,

Baltimore³

1973

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

206

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Cells from a patient with childhood acute lymphoblastic leukemia contain an apparent DNA polymerase activity that was not found in any other cells except thymus cells. The enzyme has the properties of terminal transferase, an enzyme known to be found in thymocytes. The cells also contain the three major DNA polymerases found in growing cells. The results suggest that these tumor cells arose from a block in the differentiation of thymocytes. Terminal transferase may be a marker for the origin of leukemic cells. Mammalian cells contain several DNA polymerases (1-8).We have been analyzing the content of soluble DNA polymerases in various types of cells, using phosphocellulose columns to separate the activities from a crude extract, and a series of synthetic template -primer combinations to help identify the enzymes. Using this methodology, we have recently observed in cells of a child with acute lymphoblastic leukemia (ALL) a DNA polymerizing activity that was not present in many other types of cells. This paper describes the observation and the evidence that this enzyme is terminal transferase, an enzyme specific to thymocytes (9).The nomenclature of mammalian DNA polymerases is confusing, since a wide range of inconsistent sets of names is used for the enzymes. From both the literature and our own analyses, there appear to be three major DNA polymerases in most mammalian cells. As a necessary background to the presentation of our work we have developed the following nomenclature. The 6-8S DNA polymerase, found mainly in the cytoplasmic portion of cell extracts, is DNA polymerase C (1, 10, 11). The content of this enzyme in cells varies widely and is dependent on the growth rate of the cells (Chang, McKay, & Bollum, J. Mol. Biol., in press). The second enzyme, which sediments at 3.3 S, is called DNA polymerase N, and is generally recovered from the nuclear fraction of cell extracts (3,5). This enzyme is detected in appreciable amounts in all cells and does not vary with the rate of cell growth (3,5,12). A similar activity, present in the cytoplasm, is assumed to be related to the nuclear enzyme. The third enzyme, assayed by measuring poly(dT) synthesis stimulated by poly(A) . oligo(dT) in the presence of Mn++ (6, 13), is called DNA polymerase A. It is present in most cells that we have examined, and has been reported by many investigators (13-15). It has been considered to be a "reverse transcriptase in uninfected cells," but there seems to be no utility in this designation. MATERIALS AND METHODS

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Section: Mammalian Cells Contain Several Dna Polymerases (1-8)mentioning

confidence: 99%

Terminal Deoxynucleotidyl Transferase in a Case of Childhood Acute Lymphoblastic Leukemia

McCaffrey¹,

Smoler²,

Baltimore³

1973

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

206

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“…Nevertheless acid hydrolysis of the total radioactive protein obtained with both systems yielded only one major labelled component as judged by paper chromatography and electrophoresis. Although this component is basic and would appear to be an L-amino acid possessing free ac-amino and carboxylic groups from its reactivity towards L-amino acid oxidase, its chromatographic properties do not resemble those of c-N-methyl-lysine, E-NN-dimethyl-lysine, guanidino-methylated arginine or oc-aminoguanidino-dimethylated arginine previously detected as products of protein methylase activity (Paik & Kim, 1967, 1968 Two reports (Littlefield, McGovern & Margeson, 1963;Gold & Helleiner, 1964) have appeared showing changes in the activity of DNA polymerase (DNA nucleotidyltransferase, EC 2.7.7.7) in cultured mouse cells as they pass round the cell cycle. Both reported a slight fall in the activity of the enzyme present in supernatant fractions at the time the cells were in the DNA-synthetic (S) phase.…”

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confidence: 99%

Changes in activity of nuclear deoxyribonucleic acid polymerase in synchronized mouse cells

Adams

Lindsay

1969

Biochemical Journal

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“…Contrary to earlier published investigations on DNA polymerase of L cells [4,51] in which synchronization of cells was performed by chemical inhibition of DNA synthesis, we used a synchronization method which does not result in an "unbalanced" growth of the cell going through the mitotic cycle [52-551. The different synchronization procedure and the slightly modified preparation method of the two enzyme fractions could possibly in part explain the different results.…”

Section: Discussionmentioning

confidence: 99%

“…The different synchronization procedure and the slightly modified preparation method of the two enzyme fractions could possibly in part explain the different results. Littlefield [4] and Gold [51] reported a drop in the activity of the enzyme present in the supernatant fraction after release of the DNA block, while simultaneously an increase of enzyme activity was occurring in the "particulate" fraction. I n these investigations heat-denatured DNA was used as primer.…”

Section: Discussionmentioning

confidence: 99%

DNA Polymerase, Triphosphates and Deoxyribonuclease in a Systemof Synchronized L Cells

Madreiter

Kaden

Mittermayer

1971

European Journal of Biochemistry

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I n a system of synchronously dividing mouse fibroblasts (L cells), the activity of several enzymes was determined in relation to initiation of DNA synthesis. Synchrony of DNA synthesis and cell division was obtained by selective mechanical detachment of cells in mitosis, thereby avoiding the use of blocking agents. The activity of DNA polymerase under assay conditions permitting terminal or replicative activity to occur shows characteristic fluctuations throughout the life cycle of the cell: whereas the terminal enzyme activity exhibits a peak at about 12 h, the replicative form of the enzyme has its maximum 16 h after mitosis. DNA synthesis starts at about 9 h and reaches its peak at 16 h after mitosis. The two enzyme fractions examined (particulate and supernatant) prefer native DNA as primer. The supernatant enzyme exhibits exclusively terminal activity with heat-denatured DNA as primer. The degradation of TTP by TTPase is subject to characteristic fluctuations in the mitotic cycle, whereby a decrease of TTPase activity of the particulate enzyme fraction in the S-phase (phase of DNA synthesis) may contribute to a preservation of the TTP pool, thus acting as an additional regulatory mechanism. DNase activity remains nearly constant throughout the mitotic cycle.The regulation of DNA synthesis is essential for an understanding of the phenomenon of cell proliferation. DNA synthesis depends in part on the presence of precursors, the availability of DNA primer, and the activity of the polymerizing enzyme. The present paper deals with the activity of DNA polymerase and degrading enzymes affecting the precursor pool (TTPase) and the primer DNA itself (DNase). This investigation presents 8 gross and certainly incomplete view of some factors operating in the complex process of DNA synthesis i n vitro.The use of a mechanically synchronized cell system prevents disturbance of metabolic processes and therefore yields perhaps a more correct picture of metabolic events in relation to the life cycle than can be obtained in studies using blocking agents, EXPERImNTAL PROCEDURES ME!l!HODS Cell ProcessingMonolayers of L cells used in these studies were grown a t 37" in a modified Eagle medium suppleUnusual Abbreviations. GI-phase, presynthetic phase; S-phase, phase of DNA synthesis; G2-phase, postsynthetic phase; TTPase, thymidine triphosphate nucleotidohydrolase.Enzymes. DNA polymerase or DNA nucleotidyltransferase (EC 2.7.7.7); RNase or ribonuclease (EC 2.7.7.16); DNase or deoxyribonuclease I (EC 3.1.4.5). To ascertain the degree of cell synchrony an aliquot of cells a t different stages of the cell cycle after pulse labelling with [3H]thymidine (1 pC/ml medium, 10 min pulse) was processed for autoradiography and for measuring the DNA amount by means of Feulgen microphotometry [3].Cells in the logarithmic phase of growth were harvested by being scraped and washed three times in 5 ml ice-cold buffer A (0.02 M Tris, pH 7.5; 0.1 M KC1; 0.1 mM EDTA); the cells were resuspended in 1 ml cold buffer B (0.02 M Tria, pH 7.5; 0.01 M KC...

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Deoxyribonucleic acid polymerase in L cells

Cited by 32 publications

References 14 publications

Terminal Deoxynucleotidyl Transferase in a Case of Childhood Acute Lymphoblastic Leukemia

Terminal Deoxynucleotidyl Transferase in a Case of Childhood Acute Lymphoblastic Leukemia

Changes in activity of nuclear deoxyribonucleic acid polymerase in synchronized mouse cells

DNA Polymerase, Triphosphates and Deoxyribonuclease in a Systemof Synchronized L Cells

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