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...

A DNA polymerase from Ustilugo rnu-vdis has been purified to apparent homogeneity. The native enzyme possesses a subunit structure consisting of 50000 and 55 000-dalton monomers. The apparent sedimentation coefficient of the polymerase activity in the absence of salt is 8.4 S (M, = 180000-200000),thatinitspresence(0.6MNaClor0.22MKCI)being6.3 S (M, = 80000-100000). Low concentrations of EDTA also converted the 8.4-S to a 6.3-S form, whereas magnesium ions catalysed the reverse association. The enzyme has an absolute requirement for both a DNA or RNA template and a DNA primer. For homopolymer templates the primer requirement was satisfied by a short Complementary oligodeoxynucleotide, but oligoribonucleotides were extremely inefficient primers. With the template-primer poly(dA) * (dT),,, the enzyme added an average of 50 dTMP nucleotides on to each primer molecule, whereas with poly(rA). (dT)12, this figure was 300.The enzyme also possesses an associated deoxyribonuclease activity. No other DNA polymerase activity was detected in cell-free extracts of U. muydis.DNA polymerase enzymes have now been isolated and characterised from a variety of prokaryotic and eukaryotic organisms [l -291. All such enzymes catalyse the polymerisation of deoxyribonucleoside triphosphates into DNA, directed by a template and initiated at the 3'-terminus of a complementary primer. Because, however, many organisms contain multiple DNA polymerase activities, it has been necessary to employ mutant strains, which contain a defective polymerase, in order to determine its function within the cell [30-331. As yet, such an approach has not been possible with mammalian systems. Purified enzymes and other proteins have also been invaluable in constructing complexes in vitro which mimic replication in i'ivo, inbrder-to probe certain aspects of the mechanisms of DNA replication [34 -371. __ -4hhrcwtrtiorzs. 5-Bromodeoxyuridine 5'-triphosphate, dBTP; 5-broinodeoxyuridine 5'-monophosphate, dBMP. Abbreviations for synthetic polynucleotides follow CBN rules, see Eur. J . Biochem. 15, 203-208 (1970).Enzymes. DNA polymerase or deoxynucleosidetriphosphate : DNA deoxynucleotidyltransferase (EC 2.7.7.7); RNA polymerase or nucleosidetriphosphate : RNA nucleotidyltransferase (EC 2.7. 7.6); terminal transferase or deoxynucleosidetriphosphate : oligodeoxynucleotide deoxynucleotidyltransferase (EC 2.7.7.-) ; deoxyribonuclease (EC 3.1.4.5); ribonuclease H (EC 3.1.4.34); polynucleotide 5'-hydroxyLkinase (EC 2.7.1.78); bacterial alkaline phosphatase (Escherichiu coli) or orthophosphoric-monoester phosphohydrolase (EC 3.1.3.1); exonuclease 111 (E. coli) or nucleate (deoxyribonucleate) 5'-nucleotidohydrolase (EC 3.1.4.9).The basidiomycete fungus Ustilugo muydis is a simple eukaryote suitable for both biochemical and genetic studies of DNA replication [38]. The poll-1 mutant strain is temperature sensitive for nuclear DNA synthesis in vivo, and its DNA polymerase activity detected in crude extracts and after partial purification was found to be thermolabile compared t...

Iron overload has been associated with damage of the liver and other organs of patients with primary or secondary increased iron load. In order to study the effect of iron overload on the pathophysiology of kidney lysosomes, experimentally induced iron overload models were employed. Iron overload was achieved through intraperitoneal injections of Fe-dextran (Imferon) in male rats, at different final iron concentrations (825 and 1650 mg/kg, single and double dose groups respectively). Controls were injected with dextran following a similar protocol. The animals were killed at different time points after the last injection. Subcellular fractionation studies of kidney homogenates were carried out by differential centrifugation and density gradient centrifugation. The kidney iron load was increased with both doses. Iron appeared to accumulate mainly in the lysosomes, bringing about distinct changes in the behaviour of the organelles as judged by subcellular fractionation studies. Lysosomes became more fragile and showed increased density. The extent of the above changes seemed to correlate with the extent and duration of iron accumulation and could be reversed when the iron load was reduced.

The in vivo effect of sodium valproate (SV) on the activity of uridine diphosphate glucuronosyltransferase (UDP-GT) and hepatotoxicity in the mouse liver was studied. Mice were injected intraperitoneally (IP) with SV at doses varying from 50 to 800 mg/kg per day, for six consecutive days (dose-response group) or at a standard dose of 300 mg/g per day for 2-10 days (time-response group), whereas the controls were injected with normal saline. Valproic acid levels had a positive correlation to the dose (P < 0.001) and duration of drug administration (P = 0.006). A gradual increase in UDP-GT activity was observed in doses of up to approximately 400 mg/kg per day, whereas in higher doses the enzyme activity gradually decreased. The time course of UDP-GT activity at the standard dose of 300 mg/kg per day increased progressively, with a maximum up to the sixth day and then had a gradual reduction. Hepatic necrosis (which was unrelated to the dose or the duration of drug administration) was found in 13% of the SV-treated animals and in none of the controls. We conclude that at an optimal dose (300-400 mg/kg per day) and at a time course of 6 days, SV causes liver UDP-GT induction, whereas in higher doses and longer duration of administration, UDP-GT activity is gradually reduced. SV also causes hepatotoxicity unrelated to dose and time course.

No abstract