Chromatin proteins are covalently modified by at least five different processes; in no case has the precise physiological function been established. One of these post-synthetic, covalent modifications is effected by the enzyme poly(ADP-ribose) polymerase, which uses the coenzyme NAD+ to ADP-ribosylate chromatin proteins. The modification consists largely of mono(ADP-ribose), but long, homopolymer chains of (ADP-ribose) are also present. Various physiological functions have been suggested for (ADP-ribose)n. Here we demonstrate that one function of (ADP-ribose)n is to participate in the cellular recovery from DNA damage. Specific inhibitors of poly(ADP-ribose) polymerase prevent rejoining of DNA strand breaks caused by dimethyl sulphate and cytotoxicity is enhanced thereby. The rejoining of strand breaks is prevented also by nutritionally depleting the cells of NAD.

The DNA repair proteins XRCC1 and DNA ligase III are physically associated in human cells and directly interact in vitro and in vivo. Here, we demonstrate that XRCC1 is additionally associated with DNA polymerase-beta in human cells and that these polypeptides also directly interact. We also present data suggesting that poly (ADP-ribose) polymerase can interact with XRCC1. Finally, we demonstrate that DNA ligase III shares with poly (ADP-ribose) polymerase the novel function of a molecular DNA nick-sensor, and that the DNA ligase can inhibit activity of the latter polypeptide in vitro. Taken together, these data suggest that the activity of the four polypeptides described above may be co-ordinated in human cells within a single multiprotein complex.

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Chromosomal DNA of Schizosaccharomyces pombe contains sequences with properties analogous to ARS elements of Saccharomyces cerevisiae. Following Sau3A fragmentation of the S. pombe genome we have recovered a number of such fragments in an M13‐based shuttle vector, suitable for subsequent sequence analysis. The complete nucleotide sequence has been obtained for eight ARS+ inserts derived from the Sau3A cloning and for the ARS present in pFL20 isolated previously by Losson and Lacroute (Cell, 32, 371‐377, 1983). The Sau3A clones are single fragments between 0.8 and 1.8 kb. No ARS+ clones smaller than this were recovered even though the average size Sau3A fragment in S. pombe is approximately 200‐300 bp. The sequence analysis revealed that all clones are AT‐rich (69‐75% A + T residues), and all contain a particularly AT‐rich 11 bp core element represented by the consensus sequence 5′ (A/T)PuTT‐TATTTA(A/T) 3′. Deletion mapping indicates that the consensus in all cases is in the vicinity of a functional ARS domain. However precise excision of the consensus by in vitro mutagenesis has little effect on ARS activity as judged by the transformation assay. We argue that the association of the consensus with the ARS domain occurs too reproducibly to be explained by chance alone. We suggest that although it may not be essential for the extrachromosomal maintenance of plasmids in S. pombe, the consensus does have a function in situ in the chromosome and thus is always present as a cryptic sequence in the isolated ARS element.

Both N-methyl-N-nitrosourea and y-radiation lower cellular NAD in mouse leukaemia cells (L1210) in a dose-dependent way. The minimum NAD level is reached 2 h after a brief exposure to N-methyl-N-nitrosourea, but within 15 min of y-irradiation. The cells remain metabolically active ; they are able to recover their control NAD levels and are impermeable to trypan blue.Several inhibitors of poly(ADP-ribose) polymerase inhibit the drop in cellular NAD caused by these two agents: 2 mM 5-methylnicotinamide, 1 mM theophylline or 1 mM theobromine inhibit the effect of N-methyl-N-nitrosourea on cellular NAD level; 200 pM thymidine, 500 pM 5-methylnicotinamide, 500 pM theophylline and 500 pM theobromine prevent the lowering of cellular NAD by y-irradiation. The extent to which the drop in cellular NAD is inhibited is dependent on both the concentration of cytotoxic agent and of polymerase inhibitor. Caffeine will inhibit the drop in NAD but only at 10 mM, while nicotonic acid is ineffective even at this dose.The activity of poly(ADP-ribose) polymerase in permeabilized cells immediately after y-radiation increases with dose up to 12 krad, giving a maximal 3.4-fold stimulation of the enzyme activity, whereas the degradation of NAD under conditions optimal for NAD glycohydrolase does not change. The activity of the polymerase shows a close temporal correlation with the NAD drop following both y-radiation and N-methyl-N-nitrosourea. The enzyme activity is maximal when the NAD content is decreasing at the highest rate and has returned to normal levels when it ceases falling.In permeabilized cells we can distinguish poly(ADP-ribose) polymerase and NAD glycohydrolase activity by their differential response to inhibitors. The polymerase is sensitive to 5-methylnicotinamide, theophylline, theobromine and thymidine ; the NAD glycohydrolase is sensitive to 5-methylnicotinamide and theophylline, but not to theobromine and thymidine.We propose that the decrease in cellular NAD level produced by y-radiation and by N-methyl-Nnitrosourea is caused by an increased flux through poly(ADP-ribose) mediated by an increased activity of poly(ADP-ribose) polymerase. This consequently lowers the cellular NAD level. This hypothesis implies an involvement of (ADP-ribose), in the cellular response to cytotoxic drugs.In 1956, Roitt was the first to show that cytotoxic agents inhibit glycolysis due to a lowering of the cellular NAD level [l]. He used the ethyleneimines, a group of alkylating agents, but other workers have observed a marked decrease in cellular NAD following ionizing radiation [2 -41.Abbreviations. ADP-ribose, adenosine(S')diphospho(S)-P-D-ribose; (ADP-ribose),, mixture of ADP-ribose, oligo(ADP-ribose) and poly(ADP-ribose); EGTA, ethylenebis(oxoethylenenitri1o)tetraacetic acid ; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulphonic acid; Mes, 4-morpholineethanesulphonic acid ; S.D., standard deviation of the mean.Enzymes. NAD glycohydrolase (EC 3.2.2.6); poly(ADP-ribose) polymerase (EC 2.7.7.-).Streptozotocin, the methylnitrosourea deri...