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

Abstract

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...

Nuclear Membranes from Mammalian Liver, V. On the Question of DNA Polymerase Activities Associated with the Nuclear Envelope

“…Minor differences in activity behaviour under the assay conditions examined could be due to allotopic effects of one enzyme rather than indicative of the presence of different DNA polymerizing enzymes. It must be kept in mind, however, that a more or less substantial part of DNA polymerase activity is usually lost from the nuclei during isolation especially when an aqueous procedure is used (for further discussion of the "cytoplasmic" DNA polymerase activities see, e. g., a l so l22,2G,2S, 36,37,[39][40][41][42][43][44]. In this connection one might argue that high salt concentrations as used in this study can remove a membrane-attached DNA polymerase activity.…”

Dexyribonucleic acid polymerases of BHK-21/C13cells. Relationship to the physiological state of the cells, and to synchronous indution of synthesis of deoxyribonuleic acid

“…Although this substantial evidence implies a direct correlation between activity of DNA polymerase I and the rate of replication of DNA in vivo, there is little evidence to support its direct association with the replication machinery of the cell nucleus. Earlier work with cell cultures synchronized with respect to the cell cycle by the use of metabolic inhibitors (Littlefield et al, 1963;Gold & Helleiner, 1964;Lindsay et al, 1970) or mitotic selection (Madreiter et al, 1971) revealed an increase in the total DNApolymerizing activity associated with the nucleus in S phase. However, this work was not definitive; in no case was the DNA-polymerizing activity identified.…”

The cell cycle and related concepts in cell proliferation

“…In the early days it seemed reasonable to expect that G , was a period necessary for the synthesis of enzymes and substrates directly involved in the subsequent DNA replication, but this has not proved the case for those studied so far. DNA polymerase activity, for example, often shows no clear relationship to the initiation of S-phase (Schindler, Grieder and Maurer, 1972); although in some cases increases in parallel with DNA synthesis have been seen they do not precede it (Friedman, 1970;Madreiter, Kaden and Mittermeyer, 1971;Slor, Bustan and Lev, 1973). The situation is actually complex, because mammalian cells contain at least three DNA polymerases, but even when this distinction is made no clear picture emerges (Spardi and Weissbach, 1974).…”

“…It remains possible, however, that it starts even during S-phase. What is known is that chromosomes condense in response to some cytoplasmic factor, since unscheduled condensation can be induced by fusing mitotic cells with S-phase or G , cells (Johnson and Rao, 1970;Matsui et al, 1972). The type of condensation observed is not normal, however, and this suggests that the normal pattern depends upon some prior conformational preparations during S-phase.…”