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

Adams, Roger; Lindsay, J. Gordon

doi:10.1042/bj1140057p

Cited by 3 publications

(3 citation statements)

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“…I n these investigations heat-denatured DNA was used as primer. While our paper was in preparation Adams and Lindsay [56] reported results comparable to a certain degree with ours except for the preference of denatured DNA as primer. Both the nuclear enzyme preparation and the supernatant enzyme of L 929 cells released from the early-stationary phase demonstrate a considerable increase in enzyme activity in the DNA synthetic period with a maximum at 18 h for the nuclear enzyme.…”

Section: Discussionsupporting

confidence: 79%

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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Section: Discussionsupporting

confidence: 79%

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

Madreiter

Kaden

Mittermayer

1971

European Journal of Biochemistry

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“…There is also the untested possibility that the pellet contains degradative enzymes not present in the supernatant. Nonetheless, complete nuclear localization of the DNA polymerase activity has not been a general finding (Keir et al, 1962;Bach, 1962;Keir and Gold, 1963;Littlefield et al, 1963; Adams and Linsay, 1969). The sea urchin system is a possible exception (Loeb et al, 1969).…”

Section: Resultsmentioning

confidence: 99%

“…Work on the turnover time is continuing. We are also initiating study to see if the enzyme becomes inactivated in the process of becoming membrane bound and/or moving into the nucleus (Gurdon and Speight, 1969; Pansier and Loeb, 1969;Adams and Linsay, 1969). Weiss, B. G. (1969),/.…”

Section: Resultsmentioning

confidence: 99%