DNA repair deficiencies and cellular senescence are unrelated in xeroderma pigmentosum cell lines.

Senescence of skin fibroblast cultures from normal individuals occurred after 23.9 +/- 6.3 (S.

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.

Sensitivity of mitomycin C and nitrogen mustard crosslinks to extreme alkaline conditions.

DNA-DNA crosslinks in cells treated with mitomycin C, nitrogen mustard, or decarbamoyl mitomycin C were measured in alkaline isopycnic gradients as a function of pH. Crosslinks from cells treated with mitomycin C and nitrogen mustard, which react with DNA purines, could be detected at pH 12.5 but not at pH 14.

Inhibition of X-ray- and ultraviolet light-induced transformation in vitro by modifiers of poly(ADP-ribose) synthesis.

Neoplastic transformation in vitro of hamster embryo cells and mouse C3H 10T1/2 cells by X rays and ultraviolet light was suppressed by benzamide or 3-aminobenzamide, agents which inhibit poly(ADP-ribose) polymerization. Suppression was observed under conditions in which the inhibitors reduce poly(ADP-ribose) polymerization by about 75% and increase sister chromatid exchange frequencies, but have no influence on repair of X-ray and uv damage and reportedly have no detectable side effects on nucleotide precursor metabolism. These findings suggest that the mechanisms regulating neoplastic transformation differ from those regulating mutagenesis and sister chromatid exchanges and are mediated via alterations in poly(ADP-ribosylation), causing changes in gene control and expression.

Repair of psoralen adducts in human DNA: differences among xeroderma pigmentosum complementation groups.

Angelicin and 5-methylangelicin formed photoadducts in DNA after illumination with 360-nm radiation that were excised rapidly from normal cells; 80-90% of the initial angelicin adducts and 65% of the initial 5-methylangelicin adducts were excised within 24 h. Xeroderma pigmentosum group A cells excised about 20% of the angelicin adducts, group D cells excised 55-60%, and group E, 80%. This extent of excision resembles that reported for pyrimidine dimers in these complementation groups, except for group D.

Differential toxicity of 3-aminobenzamide to wild-type and 6-thioguanine-resistant Chinese hamster cells by interference with pathways of purine biosynthesis.

3-Aminobenzamide is commonly assumed to be a specific inhibitor of poly(ADP-ribose) polymerase. However, it also inhibits de novo synthesis of DNA purines at concentrations of 5 mM and above. A mutant cell line that lacks the ability to incorporate exogenous purines is therefore exceptionally sensitive to the toxic effects of 3-aminobenzamide.

Completion of excision repair patches in human cell preparations: identification of a probable mode of excision and resynthesis.

Excision repair of u.v. damage in human fibroblasts is more sensitive to inhibitors of DNA polymerase alpha (cytosine arabinoside, aphidicolin) than to an inhibitor of polymerase beta (dideoxythymidine), which indicates a greater role in repair for polymerase alpha than for polymerase beta.

Inhibitors of poly(adenosine diphosphate-ribose) synthesis: effect on other metabolic processes.

3-Aminobenzamide and benzamide, purported to be specific inhibitors of the synthesis of poly(adenosine diphosphate-ribose), were used to elucidate possible functions of this biopolymer. These compounds, at frequently used experimental concentrations, not only inhibited the action of poly(adenosine diphosphate-ribose) synthetase but also affected cell viability, glucose metabolism, and DNA synthesis. Thus, the usefulness of 3-aminobenzamide and benzamide may be severely restricted by the difficulty of finding a dose small enough to inhibit the synthetase without producing additional metabolic effects.

Inhibition of malignant transformation in vitro by inhibitors of poly(ADP-ribose) synthesis.

Malignant transformation in vitro of hamster embryo cells and mouse C3H 10T 1/2 cells by x-rays, ultraviolet light, and chemical carcinogens was inhibited by benzamide and by 3-aminobenzamide at concentrations that are specific for inhibition of poly(ADP-ribose) formation. These compounds slow the ligation stage of repair of x-ray and alkylation damage but not of ultraviolet light damage. At high concentrations they also inhibited de novo synthesis of DNA purines and DNA methylation by S-adenosylmethionine.

The action of ultraviolet light on the patterns of banding induced by restriction endonucleases in human chromosomes.

The irradiation of metaphase spreads of human cells with ultraviolet (UV) light blocked the chromosome banding induced by Alu I, Mbo I, Dde I, Hinf I, Hae III, and Rsa I restriction endonucleases. At 13 J/m2 there was moderate inhibition of the nuclease action, which was detected as an increase in the stain intensity of chromosomes (Alu I, Mbo I, Dde I, Rsa I) or as a change in the banding pattern (Hinf I, Hae III). AT 70-300 J/m2 the UV-induced blockage was complete; the chromosomes showed no banding, and stain intensity was similar to that of control slides incubated with buffer.

Restriction enzyme cleavage of ultraviolet-damaged simian virus 40 and pBR322 DNA.

Cleavage of specific DNA sequences by the restriction enzymes EcoRI, HindIII and TaqI was prevented when the DNA was irradiated with ultraviolet light. Most of the effects were attributed to cyclobutane pyrimidine dimers in the recognition sequences; the effectiveness of irradiation was directly proportional to the number of potential dimer sites in the DNA. Combining EcoRI with dimer-specific endonuclease digestion revealed that pyrimidine dimers blocked cleavage within one base-pair on the strand opposite to the dimer but did not block cleavage three to four base-pairs away on the same strand.

Differences in the regulation by poly(ADP-ribose) of repair of DNA damage from alkylating agents and ultraviolet light according to cell type.

Inhibition of poly(ADP-ribose) synthesis by 3-aminobenzamide in various human and hamster cells influenced the responses to DNA damage from methyl methanesulfonate, but not from ultraviolet light. After exposure to methyl methanesulfonate, 3-aminobenzamide increased the strand break frequency in all cell types studied, but only stimulated repair replication in lymphoid and HeLa cells, suggesting these are independent effects. 3-Aminobenzamide also inhibited the pathway for de novo synthesis of DNA purines, suggesting that some of its effects may be due to disturbance of precursor pathways and irrelevant to the role of poly(ADP-ribose) in repair.

Effect of 3-aminobenzamide on the rate of ligation during repair of alkylated DNA in human fibroblasts.

3-Aminobenzamide, an inhibitor of polyadenosine diphosphoribose polymerase, produced rapid reversible changes in single-strand break frequencies in DNA from primary human fibroblasts damaged by alkylating agents, but it did not cause such changes in the DNA of cells damaged by ultraviolet light. The increase in single-strand peak frequencies was not due to an accumulation of blocked repair sites, such as occurs with DNA polymerase inhibitors, but to a delay in the rejoining of induced breaks. 3-Aminobenzamide increases the net break frequency that results from a dynamic balance between excision and ligation.

Structure of repaired sites in human DNA synthesized in the presence of inhibitors of DNA polymerases alpha and beta in human fibroblasts.

Excision repair of ultraviolet damage in human fibroblasts was partially inhibited by drugs that block DNA polymerases alpha or beta (cytosine arabinoside, aphidicolin and dideoxythymidine) causing a reduction in unscheduled synthesis and an accumulation of single-strand breaks. The strand breaks accumulated in the presence of aphidicolin could be resealed within 30 min after removal of the drug, but those accumulated by cytosine arabinoside took many hours. Digestion of repaired DNA with exonuclease III or S1 nuclease revealed that even the highest concentration of polymerase inhibitors, singly or in combination, that produced maximal accumulation of single-strand breaks only blocked 37-86% of repair sites.

Replicon size and excision repair as factors in the inhibition and recovery of DNA synthesis from ultraviolet damage.

Initiation of DNA replication and chain growth, analyzed by alkaline sucrose gradient sedimentation, was interrupted to different extents in different cell types by irradiation with ultraviolet light. Within the first hour of irradiation DNA replication was reduced in a manner that depended on the average number of lesions per replicating unit (replicon). At low numbers of lesions per replicon, inhibition of replicon initiation was the predominant response; at higher numbers of lesions per replicon, blockage of chain growth was also observed.

DNA excision repair in permeable human fibroblasts.

U.v. irradiation of confluent human fibroblasts activated DNA repair, aspects of which were characterized in the cells after they were permeabilized.

Poly(ADP-ribose): spectator or participant in excision repair of DNA damage.

Inhibition of poly(ADP-ribose) synthesis by 3-aminobenzamide in various human and hamster cell types influenced the responses to DNA damage from methyl methanesulfonate (MMS), but not from UV light. Excision of the major alkylation products and pyrimidine dimers was unaffected by 3-aminobenzamide. After exposure of cells to methyl methanesulfonate, 3-aminobenzamide increased the strand break frequency in all cell types studied, but stimulated repair replication only in lymphoid and HeLa cells, suggesting these are independent effects.

Critical biochemical and regulatory events in malignant transformation in vitro.

Oncogenic transformation of hamster embryo cells and mouse C3H 10T1/2 can be modified by a variety of agents and conditions which alter events at early stages of initiation and at later stages of promotion. Inhibition of poly(ADP-ribose) synthesis by low concentrations of benzamide and 3-aminobenzamide inhibits the induction of transformation by ultraviolet light, X-rays, and chemical carcinogens as well as inhibiting the enhancement of transformation by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). The suppression of transformation by the benzamides is observed under conditions where the inhibitors reduce poly(ADP-polymerization) by about 75%, have no influence on damage induced by X-rays or alkylating chemicals and enhance sister chromatid exchanges.

Rapid complementation method for classifying excision repair-defective xeroderma pigmentosum cell strains.

A rapid method has been developed that permits demonstration of complementation between different cell strains from ultraviolet-sensitive xeroderma pigmentosum patients. Combining polyethylene glycol-mediated cell fusion with low doses of ultraviolet light to eliminate unfused sensitive cells, the method permits assignment of cell strains to complementation groups by visual inspection, avoiding use of laborious methods involving autoradiography. This method can be augmented by measuring DNA repair synthesis, which shows large quantitative differences between fusions that result in complementation and those that do not.

Normal reconstruction of DNA supercoiling and chromatin structure in cockayne syndrome cells during repair of damage from ultraviolet light.

The chromatin of human cells undergoes structural rearrangements during excision repair of ultraviolet damage in DNA that were detected by transient relaxation of DNA supercoiling and increased staphylococcal nuclease digestibility of repaired sites. Inhibition of polymerization and/or ligation of repaired regions with inhibitors of DNA polymerase alpha (cytosine arabinoside and aphidicolin) resulted in the accumulation of single-strand breaks, delayed reconstruction of DNA supercoiling, and maintenance of the staphylococcal nuclease digestibility. These observations suggest that reconstruction of the native chromatin state requires completion of repaired regions with covalent ligation into the DNA strands.