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Deoxyribonucleic acid synthesis in isolated nuclei from baby-hamster kidney cells (BHK-21/C13). Characterization of the system.

ABSTRACT: A comparative study of some commonly employed laboratory procedures for studying DNA synthesis in isolated nuclei was carried out. Nuclei isolated from baby-hamster kidney (BHK-21/C13) cells synthesize DNA for 30-60min at 37 degrees C in a reaction requiring uni- and bi-valent cations, ATP and all four deoxyribonucleoside 5'-triphosphates. The addition of either ribonucleotides or cytosol from S-phase cells had no effect, but DNA synthesis was stimulated by some dextrans (mol.wt. 5x10(6)). The extent of synthesis was influenced by apparently minor variations in experimental conditions. For example, DNA synthesis by nuclei in Tris/HCl, pH7.5, was only 50% of that observed in Hepes/NaOH, pH7.5; the presence of detergents Triton X-100, Triton N-101, Nonidet P-40, Brij 58 and Tween 80 in the incubation medium altered the amount of synthesis to different extents. Although most detergents inhibited synthesis, a stimulation occurred with Tween 80 (150% of controls). These effects were reversed on washing the nuclei, except that of Brij 58, which inhibited DNA synthesis by 90-95% irreversibly. Anomalous sucrose-density-gradient sedimentation behaviour of the DNA, and of precursor [(3)H]-dTTP, was observed when nuclei were lysed with solutions of sodium dodecyl sulphate/Mg(2+) or with Sarkosyl/Mg(2+), but consistent results, showing that the DNA synthesized in vitro sedimented exclusively at about 4S, were obtained when nuclei were lysed with sodium dodecyl sulphate (without Mg(2+))/EDTA, digested with proteinase K and heated at 100 degrees C with 11% (v/v) formaldehyde to prevent macromolecular association. These results, coupled with density-labelling studies with bromodeoxyuridine and CsCl-density-gradient analysis, showed that DNA synthesis in these nuclei was replicative and was restricted to a covalent extension of Okazaki pieces previously initiated in vivo. No new initiations were observed, and the DNA was not ligated into larger molecules. The cessation of DNA synthesis after about 60 min was due to the complete utilization of available primer/template DNA.

SUBMITTER: Burke JF

PROVIDER: S-EPMC1186561 | biostudies-other | 1979 Mar

REPOSITORIES: biostudies-other

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Project description:Some properties of ADP-ribose transferase, and its reaction product, from BHK-21/C13 cells are described. Enzyme activity was found almost exclusively in nuclei (90%), with the remaining 10% located in the cytosolic fraction. The nuclear enzyme is chromatin-bound and requires bivalent cations, preferably Mg2+, a pH of 8.0 and a temperature of 25 degrees C for optimal activity. Chromatin preparations incorporated radioactivity from [14C]NAD+ into acid-insoluble material for about 60 min. Kinetics for substrate NAD+ utilization were not of Michaelis--Menten type; biphasic kinetics were shown from a double-reciprocal plot (1/reaction velocity against 1/[NAD+]) and from a 'Hofstee' plot (reaction velocity/[NAD+] against reaction velocity). The transferase is unstable in the absence of Mg2+ ions. It is inhibited by thymidine, nicotinamide and nicotinamide analogues, but not by ATP, which stimulates it at concentrations of 5 mM and above. The enzyme requires thiol groups for activity; it is readily inhibited by N-ethylmaleimide at 0.5 mM. The product of the reaction is stable under acid conditions at temperatures up to 25 degrees C, but it is hydrolysed by HClO4 at 70 degrees C. It is resistant to NaOH, but is cleaved from its attachment to protein with alkali into trichloroacetic acid-insoluble and -soluble components. On the basis of Cs2SO4- density-gradient analysis under denaturing conditions (gradients included urea and guanidinium hydrochloride), and analysis of the reaction product directly on hydroxyapatite, we conclude that most of the radioactive ADP-ribose residues are firmly bound to protein, presumably in covalent linkage. Hydroxyapatite-chromatographic analysis of ADP-ribose residues released from protein by alkaline digestion showed a spectrum of molecular sizes including mono-, oligo- and poly-(ADP-ribose), when chromatin was incubated initially with [14C]NAD+ for 10 min and then for a further 30 min after addition of excess non-radioactive NAD+, only about 10% of the radioactive mono-(ADP-ribose) could be 'chased' into longer-chain molecules. Hydroxyapatite analysis was also used to show that, whereas all ADP-ribose residues were released from protein with NaOH, only 50% of them were susceptible to hydroxylamine. These hydroxylamine-sensitive residues included all size classes, although mono-(ADP-ribose) predominated. Finally, there was an approximately equal distribution of ADP-ribose incorporated into HCl-soluble proteins (including the histones) and HCl-insoluble proteins (including the non-histone proteins) when chromatin was incubated with NAD+ up to 0.5 mM, but at higher NAD+ concentrations more ADP-ribose was incorporated into the HCl-soluble fraction (82% at 4.0 mM-NAD+).

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Deoxyribonucleic acid synthesis in isolated nuclei from baby-hamster kidney cells (BHK-21/C13). Characterization of the system.

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