Stimulation of deoxyribonucleic acid excision repair in human fibroblasts pretreated with sodium butyrate

The effect of pretreatment with sodium butyrate on DNA excision repair was studied in intact and permeable confluent (i.e., growth-inhibited) diploid human fibroblasts. Exposure to 20 mM sodium butyrate for 48 h increased subsequent ultraviolet (UV)-induced [methyl-3H]thymidine incorporation by intact AG1518 fibroblasts by 1.8-fold and by intact IMR-90 fibroblasts by 1.2-1.3-fold. UV-induced incorporation of deoxy[5-3H]cytidine, deoxy[6-3H]cytidine, and deoxy[6-3H]uridine, however, showed lesser degrees of either stimulation or inhibition in butyrate-pretreated cells. This result suggested that measurements of butyrate's effect on DNA repair synthesis in intact cells are confounded by simultaneous changes in nucleotide metabolism. The effect of butyrate on excision repair was also studied in permeable human fibroblasts in which excision repair is dependent on exogenous nucleotides. Butyrate pretreatment stimulated UV-induced repair synthesis by 1.3-1.7-fold in permeable AG1518 cells and by 1.5-2-fold in permeable IMR-90 cells. This stimulation of repair synthesis was not due to changes in repair patch size or composition or in the efficiency of DNA damage production but rather resulted from a butyrate-induced increase in the rate of damage-specific incision of DNA. The increased rate of incision in butyrate-pretreated cells could be due either to increased levels of enzymes mediating steps in excision repair at or before incision or to alterations in chromatin structure making damage sites in DNA more accessible to repair enzymes.     

DNA synthesis and repair in permeable cells of Micrococcus radiodurans.

Cells permeable to deoxyribonucleoside triphosphate were prepared from Micrococcus radiodurans, and DNA synthesis and rejoining of strand scissions induced by gamma-rays were investigated. DNA synthesis was stimulated by ATP at an optimal concentration of 1mM. This reaction requires four deoxyribonucleoside triphosphates and MgCl2. NAD inhibited the reaction, but no rejoining of primer DNA was observed. Even in the presence of NAD, DNA which was synthesized in the unirradiated permeable cells had a peak molecular weight of only 1.3 - 10(6). DNA synthesis was stimulated by irradiation of the permeable cells with gamma-rays, but this stimulatory effect was eliminated by the addition of NAD. Both primer and synthesized DNA in the irradiated permeable cells were rejoined in vitro in the presence of NAD and deoxyribonucleoside triphosphates, while those in the unirradiated permeable cells were not rejoined.     

Inhibition by hyperthermia of repair synthesis and chromatin reassembly of ultraviolet-induced damage to DNA

We have investigated the effects of hyperthermia treatment on sequential steps of the repair of UV-induced DNA damage in HeLa cells. DNA repair synthesis was inhibited by 40% after 15 min of hyperthermia treatment at 45 degrees C; greater inhibition of repair synthesis occurred with prolonged incubation at 45 degrees C. Enzymatic digestion of repair-labeled DNA with Exonuclease III indicated that once DNA repair was initiated, the DNA repair patch was synthesized to completion and that ligation of the DNA repair patch occurred. Thus the observed inhibition of UV-induced DNA repair synthesis by hyperthermia treatment may be the result of inhibition of enzymes involved in the initiating step(s) of DNA repair. DNA repair patches synthesized in UV-irradiated cells labeled at 37 degrees C with [3H]Thd were 2.2-fold more sensitive to micrococcal nuclease digestion than was parental DNA; if the length of the labeling period was prolonged, the nuclease sensitivity of the repair patch synthesized approached that of the parental DNA. DNA repair patches synthesized at 45 degrees C, however, remained sensitive to micrococcal nuclease digestion even after long labeling periods, indicating that heat treatment inhibits the reassembly of the DNA repair patch into nucleosomal structures.     

Bleomycin-induced DNA repair synthesis in permeable human fibroblasts: mediation of long-patch and short-patch repair by distinct DNA polymerases

Treatment of permeable human fibroblasts with bleomycin elicits DNA repair synthesis that is only partially sensitive to aphidicolin, an inhibitor of mammalian DNA polymerases alpha and delta. Inhibition of long-patch repair synthesis by omission of the three unlabeled deoxyribonucleoside triphosphates (dNTPs) selectively eliminates the aphidicolin-sensitive component. The majority of this residual aphidicolin-resistant repair synthesis is contained in ligated patches as revealed by resistance to exonuclease III. Determination of repair patch length by bromodeoxyuridine-induced density shift under conditions where essentially all of the repair synthesis is sensitive or resistant to aphidicolin yielded values of approximately 20 and 4 nucleotides per patch, respectively. On the basis of these data and the relative sensitivity of bleomycin-induced repair synthesis to N2-(p-n-butylphenyl)-2'-deoxyguanosine 5'-triphosphate (BuPdGTP), 2',3'-dideoxythymidine 5'-triphosphate (ddTTP), and N-ethylmaleimide (NEM), long-patch repair is attributed to DNA polymerase delta and short-patch repair to DNA polymerase beta.     

Roles of DNA ligase III and XRCC1 in regulating the switch between short patch and long patch BER

Damaged DNA bases are repaired by base excision repair (BER), which can proceed via two pathways: short patch and long patch BER. During the latter, a stretch of several nucleotides is replaced by strand displacement DNA synthesis. We recently demonstrated that the ATP concentration may govern the decision between these BER sub-pathways. Employing a reconstituted BER complex containing among others DNA polymerase beta (Pol beta), DNA ligase III (Lig III) and XRCC1, here we show that Lig III and XRCC1 are essential mediators of this regulation. XRCC1 stimulates Pol beta strand displacement activity and releases inhibition of Pol beta by DNA-bound Lig III if ligation is prevented. XRCC1 is thus able to strongly promote strand displacement and long patch BER under conditions of ATP shortage. If sufficient ATP is available, ligation by Lig III prevents strand displacement, leading to short patch BER. Ligation-inactive mutants of Lig III do not prevent strand displacement by Pol beta under the same conditions. Consequently, the preferred use of short patch BER depends on the ligation competence of Lig III. Accordingly, lowering the levels of the XRCC1/Lig III complex in HeLa cells using siRNA decreases ligation capacity but enhances Pol beta-dependent DNA synthesis.     

The ATP dependence of the incision and resynthesis steps of excision repair.

Escherichia coli made permeable by treatment with toluene can perform a mode of DNA synthesis that is stimulated by ultraviolet radiation and closely resembles the resynthesis step of excision repair. If ultraviolet-irradiated toulene-treated cells are incubated in an assay mixture with ATP but without the four deoxyribonucleoside triphosphates (dNTPs) or NAD, accumulations of single-strand breaks in the DNA are detected by alkaline sucrose gradient analysis. A second incubation with the dNTP'S and NAD but without ATP produces nonconservative DNA synthesis in strains with normal levels of DNA polymerase I. However, in PolA strains, ATP must be present during the second incubation in order to produce measurable amounts of ultraviolet-stimulated DNA synthesis. These results suggest that in strains deficient in DNA polymerase I there may be two ATP-dependent steps in this repair pathway, one required for incision and one associated with resynthesis.     

ATP for the DNA ligation step in base excision repair is generated from poly(ADP-ribose)

In mammalian cells, the base excision repair (BER) pathway is the main route to counteract the mutagenic effects of DNA lesions. DNA nicks induce, among others, DNA polymerase activities and the synthesis of poly(ADP-ribose). It is shown here that poly(ADP-ribose) serves as an energy source for the final and rate-limiting step of BER, DNA ligation. This conclusion was drawn from experiments in which the fate of [(32)P]poly(ADP-ribose) or [(32)P]NAD added to HeLa nuclear extracts was systematically followed. ATP was synthesized from poly(ADP-ribose) in a pathway that strictly depended on nick-induced DNA synthesis. NAD was used for the synthesis of poly(ADP-ribose), which, in turn, was converted to ATP by pyrophosphorylytic cleavage utilizing the pyrophosphate generated from dNTPs during DNA synthesis. The adenylyl moiety was then preferentially used to adenylate DNA ligase III, from which it was transferred to the 5'-phosphoryl end of the nicked DNA. Finally, ligation to the 3'-OH end resulted in the release of AMP. When using NAD, but not poly(ADP-ribose), in the presence of 3-aminobenzamide, the entire process was blocked, confirming poly(ADP-ribosyl)ation to be the essential initial step. Thus, poly(ADP-ribose) polymerase-1, DNA polymerase beta, and ligase III interact with x-ray repair cross-complementing protein-1 within the BER complex, which ensures that ATP is generated and specifically used for DNA ligation.     

ATP-dependent selection between single nucleotide and long patch base excision repair

DNA base excision repair (BER) constitutes a major mechanism to restore the integrity of the genome following modifications of nucleobases. Although it is well established that poly(ADP-ribosylation) facilitates BER, the mechanism of this stimulation has remained unknown. Previous observations suggested that poly(ADP-ribose), which is synthesised from NAD(+), could serve as a unique source of ATP required for the ligation step in BER. This pathway of ATP generation is thought to compensate ATP shortage and relies on the release of pyrophosphate during DNA repair synthesis. Here, we present evidence that, in situations of cellular energy depletion, the synthesis of poly(ADP-ribose) is indeed stimulated. Simultaneously, single nucleotide repair is reduced. Rather, the number of nucleotides incorporated by DNA polymerase beta (Pol beta) during DNA repair synthesis is increased. Using a reconstituted system including the recombinant BER proteins Pol beta, AP endonuclease 1 (APE 1), X-ray repair cross-complementing group-1 (XRCC1), DNA ligase III (Lig III), flap endonuclease 1 (FEN 1), and poly(ADP-ribose) polymerase-1 (PARP-1), it is demonstrated that in the absence of ATP, both long patch DNA synthesis by Pol beta and poly(ADP-ribosylation) catalysed by PARP-1 are stimulated. Consequently, the preferred use of either long patch or single nucleotide BER depends on the availability of ATP. It is proposed that long patch BER is required for ATP generation from poly(ADP-ribose) and, therefore, predominant under conditions of ATP shortage.     

DNA polymerases and repair synthesis in NER in human cells

The late steps of nucleotide excision repair, following incisions to remove the damaged section of DNA, comprise repair synthesis and ligation. In vitro and in vivo studies have shown the size of the repaired patch to be about 30 nucleotides. In vitro studies implicated the replicative polymerases in repair synthesis, but recent in vivo data have shown that several DNA polymerases and ligases are involved in these steps in human cells.     

Multiple conformational states of repair patches in chromatin during DNA excision repair

In mammalian cells, newly synthesized DNA repair patches are highly sensitive to digestion by staphylococcal nuclease (SN), but with time, they acquire approximately the same nuclease resistance as the DNA in bulk chromatin. We refer to the process which restores native SN sensitivity to repaired DNA as chromatin rearrangement. We find that during repair of ultraviolet damage in human fibroblasts, repair patch synthesis and ligation occur at approximately the same rate, with ligation delayed by about 4 min, but that chromatin rearrangement is only 75% as rapid. Thus, repair-incorporated nucleotides can exist in at least three distinct states: unligated/unrearranged, ligated/unrearranged, and ligated/rearranged. Inhibition of repair patch synthesis by aphidicolin or hydroxyurea results in inhibition of both patch ligation and chromatin rearrangement, confirming that repair patch completion and/or ligation are prerequisites for rearrangement. We also analyze the kinetics of SN digestion of repair-incorporated nucleotides at various extents of rearrangement and find the data to be consistent with the existence of two or more forms of unrearranged repair patch which have different sensitivities to digestion by SN. These data indicate that the chromatin rearrangement which restores native SN sensitivity to repaired DNA is a multistep process. The multiple forms of unrearranged chromatin with different SN sensitivities may include the unligated/unrearranged and ligated/unrearranged states. If so, the differences in SN sensitivity must arise from differences in chromatin structure, because SN does not differentiate between ligated and unligated repair patches in naked DNA.     

DNA synthesis in vivo and in vitro in Escherichia coli irradiated with ultraviolet light

DNA synthesis was followed in vivo and in permeable Escherichia coli after ultraviolet light irradiation, irradiation and incubation in a growth medium containing chloramphenicol and in unirradiated cells. In vitro, replicative type DNA synthesis was partially restored after incubation of cells in medium containing chloramphenicol, but not in vivo. The DNA was pulse-labeled in permeable cells in the presence of deoxyribonucleoside triphosphates and ribonucleoside triphosphates. dCTP was replaced by 5-Hg-dCTP as a substrate for DNA synthesis. Hg-DNA was separated from cellular nucleic acids on thiol-agarose affinity columns. The 5' termini of newly synthesized DNA were analyzed after treatment with alkaline phosphatase and rephosphorylation with polynucleotide kinase and [gamma-32P]ATP. DNA synthesis in unirradiated permeable E. coli represents a replicative process dependent on ATP and inhibited by novobiocin. About 70% of the nascent DNA carried terminally labeled RNA moiety at its 5' end. In vitro DNA synthesis in irradiated cells was suppressed and hardly influenced by the presence of ATP or novobiocin. The 5'-RNA content of this cell population was less than 5%.     

Repair of a synthetic abasic site involves concerted reactions of DNA synthesis followed by excision and ligation.

A synthetic analog of an abasic site in DNA is efficiently repaired by a short-patch repair mechanism in soluble extracts of Xenopus laevis oocytes (Y. Matsumoto and D. F. Bogenhagen, Mol. Cell. Biol. 9:3750-3757, 1989). We present a detailed analysis of the repair mechanism, using extracts depleted of endogenous nucleotide pools. ATP was required for repair with a sharp optimal concentration of 5 mM. The initial rate of repair was increased by preincubation of the DNA in the extract in the presence of ATP. During this preincubation, the DNA was cleaved on the 5' side of the lesion by a class II apurinic-apyrimidinic endonuclease, but removal of the abasic sugar residue was not observed prior to addition of deoxynucleotides to the reaction. Immediately following DNA synthesis, excision and ligation proceeded in a coordinated manner to complete repair. DNA preincubated in the extract in the absence of deoxynucleotides remained associated with repair enzymes during gel filtration. These observations suggest that the enzymes involved in concerted repair of the abasic site form a complex on DNA.     

Relation of the poly(ADP-ribosylation) of proteins to the formation and repair of DNA single-strand breaks in gamma-irradiated permeable Zajdela hepatoma cells

A study was made of the effect of poly(ADP-ribosylation) of proteins on the formation and repair of single-strand DNA breaks in gamma-irradiated (50 Gy) permeable Zajdela ascites hepatoma cells permeabilized by the treatment with 0.05% triton X-100. Incubation of gamma-irradiated permeable cells in conditions promoting DNA synthesis and providing ADP-ribosylation (in the presence of 1 mM NAD) did not cause any substantial changes in the formation of single-strand DNA breaks and did not influence their repair.     

Nicotinamide stimulates repair of DNA damage in human lymphocytes

Nicotinamide stimulates the amount of DNA repair synthesis that occurs when freshly isolated, normal human lymphocytes are treated with UV irradiation, N-methyl-N′-nitro-N-nitroso guanidine, or dimethyl sulfate. Stimulation of DNA repair synthesis is concentration dependent and reaches a maximum between 2 to 5 mM nicotinamide. In contrast, DNA synthesis in cells that have not been subjected to DNA damage is not affected by nicotinamide at concentrations below 2 mM and is inhibited by concentrations between 2 to 5 mM. In the same concentration range, nicotinic acid has no effect on the rate of DNA synthesis in the presence or absence of DNA damage.     

Organic hydroperoxides at high concentrations cause energization and activation of ATP synthesis in mitochondria

The addition of high concentrations of cumene or tert-butyl hydroperoxide to previously deenergized mitochondria results in the energization of these mitochondria and activation of ATP synthesis. The energization effect was observed in the presence of 0.5-0.7 mM cumene hydroperoxide or 2.0-2.5 mM tert-butyl hydroperoxide. This energization of mitochondria and activation of oxidative phosphorylation by organic hydroperoxides required the presence of ADP in the mitochondrial matrix and does not depend upon the method of deenergization of the mitochondria.     

Do inhibitor studies demonstrate a role for poly(ADP-ribose) in DNA repair?

3-Aminobenzamide, an inhibitor of poly(ADP-ribose) synthesis, has been commonly used in attempts to demonstrate a regulatory role for the polymer during a late stage of repair. When a range of inhibitor concentrations was used paradoxical results were obtained. Up to 1 mM, 3-aminobenzamide appeared to reduce DNA break frequencies in cells damaged by methyl methane sulfonate; at doses of 2 mM and above, it appeared to increase break frequencies. In the high concentration range, many nonspecific side effects and cellular toxicity predominate. Evidence used to assert a role for poly(ADP-ribose) synthesis during ligation has usually been derived from experiments using high concentrations of 3-aminobenzamide, but these may be attributed to toxic side effects. 3-Aminobenzamide stimulates a large increase in repair replication which does not result from increased excision of damaged sites or an increased patch length but may be attributable to other cellular effects such as endogenous nuclease attack on DNA. The cellular effects of 3-aminobenzamide are therefore complicated by nonspecific effects over a commonly used concentration range and evidence for a specific regulatory role of poly(ADP-ribose) in DNA repair is weak.     

Activation of nonselective cation channels by physiological cholecystokinin concentrations in mouse pancreatic acinar cells.

The activation of the nonselective cation channels in mouse pancreatic acinar cells has been assessed at low agonist concentrations using patch-clamp whole cell, cell-attached patch, and isolated inside-out patch recordings. Application of acetylcholine (ACh) (25-1,000 nM) and cholecystokinin (CCK) (2-10 pM) evoked oscillatory responses in both cation and chloride currents measured in whole cell experiments. In cell-attached patch experiments we demonstrate CCK and ACh evoked opening of single 25-pS cation channels in the basolateral membrane. Therefore, at least a component of the whole cell cation current is due to activation of cation channels in the basolateral acinar cell membrane. To further investigate the reported sensitivity of the cation channel to intracellular ATP and calcium we used excised inside-out patches. Micromolar Ca2+ concentrations were required for significant channel activation. Application of ATP and ADP to the intracellular surface of the patch blocked channel opening at concentrations between 0.2 and 4 mM. The nonmetabolizable ATP analogue, 5'-adenylylimidodiphosphate (AMP-PNP, 0.2-2 mM), also effectively blocked channel opening. The subsequent removal of ATP caused a transient increase in channel activity not seen with the removal of ADP or AMP-PNP. Patches isolated into solutions containing 2 mM ATP showed channel activation at micromolar Ca2+ concentrations. Our results show that ATP has two separate effects. The continuous presence of the nucleotide is required for operation of the cation channels and this action seems to depend on ATP hydrolysis. ATP can also close the channel and this effect can be demonstrated in excised inside-out patches when ATP is added to the bath after a period of exposure to an ATP-free solution. This action does not require ATP hydrolysis. Under physiological conditions hormonal stimulation can open the nonselective cation channels and this can be explained by the rise in the intracellular free Ca2+ concentration.     

Effect of 3-aminobenzamide on the process of ultraviolet-induced DNA excision repair

The effect of 3-aminobenzamide, a potent inhibitor of poly(ADP-ribosyl)ation, on UV-induced DNA excision repair was investigated. HeLa cells were treated with DNA replication inhibitors, hydroxyurea (HU) and 1-beta-D-arabinofuranosyl cytosine (araCyt), before and after ultraviolet light (UV) irradiation, to accumulate DNA single-strand breaks. The activity of poly(ADP-ribosyl)ation measured in the permeable cell system of HeLa cells was enhanced in a UV dose-dependent manner after the combined treatment with HU and araCyt in vivo. However, DNA repair synthesis in vitro was not affected by addition of 1 mM 3-aminobenzamide or nicotinamide, while incorporation of [3H]NAD in the same system was completely inhibited. Furthermore, neither the magnitude of UV-induced DNA single-strand breaks accumulated by the combined treatment of HU and araCyt nor the rate of their rejoining after release from the HU and araCyt block were influenced even in the presence of 10 mM 3-aminobenzamide. As the cytotoxicity of UV irradiation was significantly potentiated by 5 mM 3-aminobenzamide, these results suggest that poly(ADP-ribosyl)ation is involved in a process other than DNA excision repair induced by UV irradiation.     

Opposite effects of D-fructose on total versus cytosolic ATP/ADP ratio in pancreatic islet cells

D-fructose (10 mM) augments, in rat pancreatic islets, insulin release evoked by 10 mM D-glucose. Even in the absence of D-glucose, D-fructose (100 mM) displays a positive insulinotropic action. It was now examined whether the insulinotropic action of D-fructose could be attributed to an increase in the ATP content of islet cells. After 30-60 min incubation in the presence of D-glucose and/or D-fructose, the ATP and ADP content was measured by bioluminescence in either rat isolated pancreatic islets (total ATP and ADP) or the supernatant of dispersed rat pancreatic islet cells exposed for 30 s to digitonine (cytosolic ATP and ADP). D-fructose (10 and 100 mM) was found to cause a concentration-related decrease in the total ATP and ADP content and ATP/ADP ratio below the basal values found in islets deprived of exogenous nutrient. Moreover, in the presence of 10 mM D-glucose, which augmented both the total ATP content and ATP/ADP ratio above basal value, D-fructose (10 mM) also lowered these two parameters. The cytosolic ATP/ADP ratio, however, was increased in the presence of D-glucose and/or D-fructose. Under the present experimental conditions, a sigmoidal relationship was found between such a cytosolic ATP/ADP ratio and either (86)Rb net uptake by dispersed islet cells or insulin release from isolated islets. These data provide, to our knowledge, the first example of a dramatic dissociation between changes in total ATP content or ATP/ADP ratio and insulin release in pancreatic islets exposed to a nutrient secretagogue. Nevertheless, the cationic and insulinotropic actions of d-glucose and/or d-fructose were tightly related to the cytosolic ATP/ADP ratio.     

Effect of ATP on the Friend Murine leukemia virus-associated endonuclease activity and the endonuclease activity of the avian myeloblastosis virus RNA-directed DNA polymerase

The Mn2+-dependent endonuclease activity associated with the avian myeloblastosis virus RNA-directed DNA polymerase has been shown to be activated by ATP in the presence of Mg2+. In the presence of Mn2+ the endonucleolytic activity was stimulated about 3-fold by the addition of ATP. The earlier identified Mr = 40,000 Friend murine leukemia virus (F-MuLV)-associated endonuclease which functions in the presence of both Mg2+ and Mn2+ has also been shown to be similarly stimulated by ATP. For both endonuclease activities stimulation was only observed at ATP concentrations above 0.5 mM, and it did not increase upon elevating the ATP concentration above 2.5 mM. ADP and dATP also stimulated both activities, although not to the same extent as ATP. GTP had no apparent effect and AMP seemed to inhibit both activities. The effect ATP analogs had on the F-MuLV associated endonuclease activity could suggest that the endonuclease reaction in the presence of ATP might involve the cleavage of beta-gamma phosphate bonds in ATP. Neither adenyl-5'-yl imidodiphosphate nor (beta, gamma-methylene)adenosine 5'-triphosphate stimulated the activity, whereas significant stimulation was observed in the presence of (alpha, beta-methylene)adenosine 5'-triphosphate. Although no ATPase activity could be detected in the purified F-MuLV endonuclease preparation, the data do not exclude the possibility that ATP may be cleaved in amounts which are equivalent to the number of nicks introduced into DNA by the virus-associated endonuclease. In the presence of ATP and Mg2+ the F-MuLV-associated endonuclease nicked both supercoiled and linear DNA duplexes extensively, although the former was nicked more readily than the latter. Single-stranded DNA functioned poorly as a substrate. The nicks introduced by the enzyme contained a 5'-phosphoryl terminus and a 3'-hydroxyl group.