Characterization and comparison of poly(adenosine dephosphoribose) synthesis and DNA synthesis in nucleotide-permeable cells.

Using eukaryotic cells that have been rendered permeable to exogenously supplied nucleotides, we have characterized the activity of the poly(adenosine diphosphoribose) (poly(ADPR)) synthesis system and compared it to the DNA synthesis complex. The synthesis of poly(ADPR) is dependent on the presence of NAD and Mg2+. It does not require ATP, NaF or a monovalent cation. It is inhibited by N-ethylmaleimide. The reaction product conforms to the nuclease susceptibilities expected for poly(ADP ribose) in that it is degraded by venom phosphodiesterase but not by DNAase of RNAase. A comparison of the effects of inhibitors of poly(ADPR) synthesis and DNA synthesis clearly distinguishes between the two enzymatic systems. Nicotinamide, 5-methyl nicotinamide, thymidine, 5-bromo deoxyuridine, adenosine diphosphoribose, caffeine and formycin all inhibit poly(ADPR) synthesis but not DNA synthesis. In contrast, araCTP, cytembena and phosphonoacetic acid all inhibit DNA synthesis but not poly(ADPR) synthesis. Addition of DNAase to the permeable cells causes a marked stimulation of poly(ADPR) synthesis. L cells in logarithmic growth were found to have high levels of activity of the DNA synthesis complex and low levels of activity of the poly(ADPR) synthesis system. In contrast, cells at plateau phase density demonstrate a decrease in the activity of the DNA synthesis complex and a marked increase in activity of the poly(ADPR) synthesis system. When examined in the presence of added DNAase, the activity of the poly(ADPR) synthesis system is the same in cells obtained from log or plateau phase cultures. This indicates that the physiologic activity of the enzyme varies while the total amount of enzyme remains constant. When the permeable cells are allowed to synthesize both poly(ADPR) and DNA simultaneously, the synthesis of one polymer has no effect on the rate of synthesis of the other.

Relation of poly(adenosine diphosphoribose) synthesis to DNA synthesis and cell growth.

A permeable cell technique has been used to measure the synthesis of DNA and poly(adenosine diphosphoribose) (poly(ADPR)) in mouse L cells subjected to different perturbations of cell growth. Cells leaving log phase growth and entering plateau phase, showed a decrease in DNA synthesis and an associated increase in intrinsic poly(ADPR) synthesis. In contrast to the variations in intrinsic poly(ADPR) synthesis, the total poly(ADPR) synthesis activity, measured in the presence of added DNAase, remained relatively constant during the fluctuations in cell growth status. Cells subjected to acute glucose deficiency also demonstrated a decrease in DNA synthesis and an associated increase in intrinsic poly(ADPR) synthesis. Similarly, cells infected with vaccinia virus demonstrated an abrupt cessation of DNA synthesis associated with an increase in poly(ADPR) synthesis. Treatment of cells with cytosine arabinoside, inhibited cellular DNA synthesis. This was also associated with an increase in the intrinsic activity of poly(ADPR) synthesis. However, in this case, the increase in poly(ADPR) synthesis was associated with an increase in activity of the DNA synthesis complex, despite the overall inhibition of cell DNA synthesis. These studies demonstrate, that in mouse L cells, suppression of DNA synthesis by multiple different physiologic mechanisms is always associated with an increase in intrinsic activity of poly(ADPR) synthesis.