DNA strand breakage during physiological apoptosis of the embryonic chick lens: Free 3' OH end single strand breaks do not accumulate even in the presence of a cation-independent deoxyribonuclease

Epithelial cells from the lens equator differentiate into elongated fiber cells. In the final steps of differentiation, the chromatin appears quite condensed and chromatin breakdown into nucleosmes occurs. DNA breaks due to an endodeoxyribonuclease activity corresponding to at least two polypeptides of 30 and 40 kDa have been identified. To identify the nature and the developmental appearance of initial breaks, nick translation reaction was followed both biochemically and in situ in fiber and epithelial cells from chick embryonic lenses. There is no accumulation of single-strand breaks (SSB) with 3'OH ends in lens fiber cells during embryonic development. Such damage can be increased in these cells by treatment with DNAase I indicating the absence of an inhibitor of the nick translation reaction in fiber cells. However, there are indications of the presence of DNA breaks with blocked termini when the phosphatase activity of nuclease P1 is used. The presence of breaks is also indicated by the large amounts of (ADP-ribose)_n found in lens fibers particularly at 11 days of embryonic development (E11) as ADP-ribosyl transferase binds to and is activated by DNA strand breaks. Incubation of lens cells in vitro, which causes nucleosomal fragmentation only in fiber cells, produces SSB with 3'OH ends in both epithelia and fibers. Incubation for short periods, observed in experiments in situ, induces SSB first in the central fiber nuclei, which are late in differentiation. This may indicate that these SSB play a physiological role. Long incubations produce larger numbers of SSB in epithelia than fibers. The SSB in the fibers may have been converted into double-strand breaks (D SB), seen as nucleosomal fragments, and therefore no longer act as substrates for nick translation. The nuclease activity responsible for SSB production is independent of divalent cations and could be implicated in lens terminal differentiation. © 1994 Wiley-Liss, Inc.     

Determination of human DNA polymerase utilization for the repair of a model ionizing radiation-induced DNA strand break lesion in a defined vector substrate.

Human DNA polymerase and DNA ligase utilization for the repair of a major class of ionizing radiation-induced DNA lesion [DNA single-strand breaks containing 3'-phosphoglycolate (3'-PG)] was examined using a novel, chemically defined vector substrate containing a single, site-specific 3'-PG single-strand break lesion. In addition, the major human AP endonuclease, HAP1 (also known as APE1, APEX, Ref-1), was tested to determine if it was involved in initiating repair of 3'-PG-containing single-strand break lesions. DNA polymerase beta was found to be the primary polymerase responsible for nucleotide incorporation at the lesion site following excision of the 3'-PG blocking group. However, DNA polymerase delta/straightepsilon was also capable of nucleotide incorporation at the lesion site following 3'-PG excision. In addition, repair reactions catalyzed by DNA polymerase beta were found to be most effective in the presence of DNA ligase III, while those catalyzed by DNA polymerase delta/straightepsilon appeared to be more effective in the presence of DNA ligase I. Also, it was demonstrated that the repair initiating 3'-PG excision reaction was not dependent upon HAP1 activity, as judged by inhibition of HAP1 with neutralizing HAP1-specific polyclonal antibody.     

Induction and repair of double- and single-strand DNA breaks in bacteriophage lambda superinfecting Escherichia coli

Induction and repair of double- and single-strand DNA breaks have been measured after decays of 125I and 3H incorporated into the DNA and after external irradiation with 4 MeV electrons. For the decay experiments, cells of wild type Escherichia coli K-12 were superinfected with bacteriophage lambda DNA labelled with 5'-(125I)iodo-2'-deoxyuridine or with (methyl-3H)thymidine and frozen in liquid nitrogen. Aliquots were thawed at intervals and lysed at neutral pH, and the phage DNA was assayed for double- and single-strand breakage by neutral sucrose gradient centrifugation. The gradients used allowed measurements of both kinds of breaks in the same gradient. Decays of 125I induced 0.39 single-strand breaks per double-strand break. No repair of either break type could be detected. Each 3H disintegration caused 0.20 single-strand breaks and very few double-strand breaks. The single-strand breaks were rapidly rejoined after the cells were thawed. For irradiation with 4 MeV electrons, cells of wild type E. coli K-12 were superinfected with phage lambda and suspended in growth medium. Irradiation induced 42 single-strand breaks per double-strand break. The rates of break induction were 6.75 x 10(-14) (double-strand breaks) and 2.82 x 10(-12) (single-strand breaks) per rad and per dalton. The single-strand breaks were rapidly repaired upon incubation whereas the double-strand breaks seemed to remain unrepaired. It is concluded that double-strand breaks in superinfecting bacteriophage lambda DNA are repaired to a very small extent, if at all.     

Presence of double-strand breaks with single-base 3' overhangs in cells undergoing apoptosis but not necrosis

Apoptotic cells in rat thymus were labeled in situ in paraffin-embedded and frozen tissue sections by ligation of double-stranded DNA fragments containing digoxigenin or Texas red. Two forms of double-stranded DNA fragments were prepared using the polymerase chain reaction: one was synthesized using Taq polymerase, which yields products with single- base 3' overhangs, and one using Pfu polymerase, which produces blunt- ended products. Both types of fragment could be ligated to apoptotic nuclei in thymus, indicating the presence in such nuclei of DNA double- strand breaks with single-base 3' overhangs as well as blunt ends. However, in nuclei with DNA damage resulting from a variety of nonapoptotic processes (necrosis, in vitro autolysis, peroxide damage, and heating) single-base 3' overhangs were either nondetectable or present at much lower concentrations than in apoptotic cells. Blunt DNA ends were present in such tissues, but at lower concentrations than in apoptotic cells. In contrast, in all of these forms of DNA damage, nuclei contained abundant 3'-hydroxyls accessible to labeling with terminal deoxynucleotidyl transferase. Thus, although single-base 3' overhangs and blunt ends are present in apoptotic nuclei, the specificity of the in situ ligation of 3'-overhang fragments to apoptotic nuclei indicates that apoptotic cells labeled in this way can readily be distinguished from cells with nonapoptotic DNA damage. These data are consistent with the involvement of an endonuclease similar to DNase I in apoptosis, which is predicted to leave short 3' overhangs as well as blunt ends in digestion of chromatin.     

In situ studies on the effect of acid extraction on the DNA template activity of mature avian erythrocytes.

Exogenous E. coli RNA polymerase was used to determine the in situ DNA template activity of ethanol/acetone fixed avian erythrocytes. No RNA polymerase-catalyzed incorporation of 3H-UTP was detected in mature avian erythrocytes while simultaneously fixed avian lymphocytes did exhibit incorporation of 3H-UTP. Nuclei of mature erythrocytes which were subjected to treatments known to remove histones showed dramatic increases in RNA polymerase-catalyzed incorporation of 3H-UTP. The chromatin of treated cells was presumed to be more accessible to RNA polymerase as determined by the increase in RNA polymerase-catalyzed incorporation of 3H-UTP. Incubation of acid-treated nuclei in poly-L-lysine prior to incubation with RNA polymerase failed to inhibit the incorporation of 3H-UTP. Possible mechanisms for the inactivation of avian erythrocyte nuclei are discussed.     

Detection and measurement of single-strand breaks in nuclear DNA in fixed lens sections

Ethanol-fixed lens sections were incubated with calf-thymus terminal deoxynucleotidyl transferase and ³H-dATP, and the time-course of ³H-dAMP incorporation was compared among various lens cell types by quantitative autoradiography. The data show that the proportion of cell nuclei containing DNA with single-strand breaks increases during fiber cell differentiation, and that the relative number of breaks per nucleus or unit nuclear area also increase in the same sequence.     

Interaction of poly-L-lysine with chromatin. Inhibition of in situ priming for Escherichia coli DNA polymerase

The priming capacity of chromatin of fixed nuclei and chromosomes for exogenous DNA polymerase can be evaluated radioautographically by the incorporation of labeled nucleotides. It had previously been reported that acid fixation or acid treatment of alcohol-fixed tissues led to increased priming when calf thymus DNA polymerases, specific for single-stranded DNA, were used. We employed Escherichia coli DNA polymerase and sequential treatments of the fixed tissue with acid and poly-L-lysine in order to elucidate the mechanism through which the acid effect is produced. Acid treatment enhanced chromatin priming for the E coli DNA polymerase, and saturation of the chromatin with poly-L-lysine strongly inhibited the reaction. This inhibition was reversible through subsequent treatment with acid. Wide differences in priming were observed between cell types of alcohol-fixed chicken blood smears: thrombocyte and lymphocyte nuclei exhibited strong priming ability whereas erythrocyte nuclei failed to support any detectable priming. We conclude that the acid effect is readily interpretable in terms of acid-mediated changes in the association between DNA and protein in the chromatin complex.     

Isolation and characterization of nonhistone chromosomal protein C-14 which stimulates RNA synthesis.

The nonhistone chromatin protein, C-14, was extracted from chromatin of Novikoff hepatoma ascites cells and isolated in high purity as shown by its migration as a single dense spot on two-dimensional polyacrylamide gels. Its mobility on sodium dodecyl sulfate gels is consistent with a molecular weight of approximately 70 000. The amino acid composition shows that protein C-14 has an acidic:basic amino acid ratio of 1.8. Its amino terminal amino acid is lysine. Protein C-14 stimulated the incorporation of [3H]UMP into RNA by approximately 30% when added to naked DNA and homologous RNA polymerase I. A 30% stimulation of [3H]UMP incorporation into RNA was also found when protein C-14 was added to an E. coli RNA polymerase system containing either E. coli or Novikoff hepatoma DNA.     

In vivo formation of single-strand breaks in DNA by hydrogen peroxide is mediated by the Haber-Weiss reaction

Phenanthroline and bipyridine, strong chelators of iron, protect DNA from single-strand break formation by H2O2 in human fibroblasts. This fact strongly supports the concept that these DNA single-strand breaks are produced by hydroxyl radicals generated by a Fenton-like reaction between intracellular Fe2+ and H2O2: H2O2 + Fe2+----Fe3+ + OH- + OH: Corroborating this idea is the fact that thiourea, an effective OH radical scavenger, prevents the formation of DNA single-strand breaks by H2O2 in nuclei from human fibroblasts. The copper chelator diethyldithiocarbamate, a strong inhibitor of superoxide dismutase, greatly enhances the in vivo production of DNA single-strand breaks by H2O in fibroblasts. This supports the idea that Fe3+ is reduced to Fe2+ by superoxide ion: O divided by 2 + Fe3+----O2 + Fe2+; and therefore that the sum of this reaction and the Fenton reaction, namely the so-called Haber-Weiss reaction, H2O2 + O divided by 2----O2 + OH- + OH; represents the mode whereby OH radical is produced from H2O2 in the cell. EDTA completely protects DNA from single-strand break formation in nuclei. The chelator therefore removes iron from the chromatin, and although the Fe-EDTA complex formed is capable of reacting with H2O2, the OH radical generated under these conditions is not close enough to hit DNA. Therefore iron complexed to chromatin functions as catalyst for the Haber-Weiss reaction in vivo, similarly to the role played by Fe-chelates in vitro.     

Time Scale for Rejoining of Bacteriophage λ Deoxyribonucleic Acid Molecules in Superinfected pol+ and polA1 Strains of Escherichia coli After Exposure to 4 MeV Electrons

The time scale for rejoining of radiation-induced deoxyribonucleic acid (DNA) single-strand breaks was measured in the presence and absence of oxygen. The involvement of DNA polymerase I in this repair process was studied. Formation and rejoining of DNA strand breaks were measured in lambda DNA infecting lysogenic pol(+) and polA1 strains of Escherichia coli irradiated by 4 MeV electrons under identical conditions. Irradiation and transfer to alkaline detergent could be completed in less than 180 ms. The initial yields of DNA strand breaks were identical in pol(+) and polA1 host cells and four- to fivefold higher in the presence of oxygen than in nitrogen anoxia. Evidence for the existence of a very fast repair process, independent of DNA polymerase I, was not found, since no rejoining of radiation-induced DNA strand breaks was observed during incubation from 45 ms to 3 s. In pol(+) host cells most of the strand breaks produced in the presence of oxygen were rejoined within the first 30 to 40 s of incubation, whereas no rejoining could be detected within the same period of time in anoxic cells. Since no rejoining of broken lambda DNA molecules was observed in polA1 host cells, it is concluded that the synthetase activity of DNA polymerase I is involved in the rejoining of DNA breaks induced by radiation in the presence of oxygen.     

Identification of apoptotic cells by formamide-induced dna denaturation in condensed chromatin.

In this article we describe a novel effect of formamide on DNA of apoptotic nuclei and present a method for specific detection of apoptotic cells based on this effect. Our observations show that formamide induces DNA denaturation in apoptotic nuclei but has no such effect on DNA of non-apoptotic cells. Formamide-induced DNA denaturation combined with detection of denatured DNA with a monoclonal antibody (MAb) against single-stranded DNA made it possible to specifically identify the apoptotic cells. This procedure produced intense staining of the condensed chromatin in the apoptotic nuclei. In contrast, necrotic cells from cultures treated with sodium azide, saponin, or hyperthermia did not bind this antibody, demonstrating the specificity of the formamide-MAb assay for the apoptotic cells. However, TUNEL stained 90-100% of necrotic cells in all three models of necrosis. Because the MAb did not stain cells with single- or double-stranded DNA breaks in the absence of apoptosis, we conclude that staining of the apoptotic nuclei is not influenced by DNA breaks and is induced by specific changes in condensed chromatin, such as damage to the DNA-histone interactions. Importantly, the formamide-MAb technique identified apoptotic cells in frozen sections and in histological sections of formalin-fixed, paraffin-embedded tissues.     

Repair of Radiation-Induced Damage in Escherichia coli II. Effect of rec and uvr Mutations on Radiosensitivity, and Repair of X-Ray-Induced Single-Strand Breaks in Deoxyribonucleic Acid

Strains of Escherichia coli K-12 mutant in the genes controlling excision repair (uvr) and genetic recombination (rec) have been studied with reference to their radiosensitivity and their ability to repair X-ray-induced single-strand breaks in deoxyribonucleic acid (DNA). Mutations in the rec genes appreciably increase the radiosensitivity of E. coli K-12, whereas uvr mutations produce little if any increase in radiosensitivity. For a given dose of X-rays, the yield of single-strand breaks has been shown by alkaline sucrose gradient studies to be largely independent of the presence of rec or uvr mutations. The rec(+) cells (including those carrying the uvrB5 mutation) could efficiently rejoin X-ray-induced single-strand breaks in DNA, whereas recA56 mutants could not repair these breaks to any great extent. The recB21 and recC22 mutants showed some indication of repair capacity. From these studies, it is concluded that a correlation exists between the inability to repair single-strand breaks and the radiosensitivity of the rec mutants of E. coli K-12. This suggests that unrepaired single-strand breaks may be lethal lesions in E. coli.     

Deoxyribonucleic Acid Damage by Monofunctional Mitomycins and Its Repair in Escherichia coli

Exposure of Escherichia coli to the antibiotic mitomycin C (MTC) at a concentration of 0.5 mug/ml caused cross-linkage between complementary strands of deoxyribonucleic acid (DNA). Derivatives of mitomycin, 7-methoxymitosene (7-MMT) and decarbamoyl mitomycin C (DCMTC), at a level as high as 20 mug/ml formed no cross-links between DNA strands. Ultraviolet light-sensitive mutants of E. coli K-12 bearing uvrA, uvrB, uvrC, or recA mutations were more sensitive to the lethal action of 7-MMT and of DCMTC than was the wild-type strain. Treatment of wild-type cells with these antibiotics resulted in the production of single-strand breaks in DNA, which were repaired upon incubation in a growth medium. Such breaks in DNA were not produced in the uvrA and the uvrB mutants. In the uvrC mutant, single-strand breaks were produced by 7-MMT or by DCMTC, but these breaks were not repaired upon incubation. These results are discussed in connection with the mechanism for removal of pyrimidine dimers in ultraviolet-irradiated bacteria.     

Sensitization of Escherichia coli C to gamma-radiation by 5-bromouracil incorporation.

Escherichia coli C cells, unifilarly substituted with 5-bromouracil (BrUra) were 2-25 times as sensitive as unsubstituted cells to killing by gamma-irradiation under aerobic conditions. The yield of DNA double-strand breaks in BrUra-substituted cells was increased by a factor only 1-55, suggesting that other lesions also contribute to cell-killing. Alkaline sucrose density gradient analysis of the 3H-thymine labelled DNA strand showed there was less repair of gamma-ray-induced single-strand breaks when BrUra was in the complementary strand. Since there are more of these unrepaired breaks than can be accounted for by BrUra-induced DNA double-strand breakage, some fraction of the lethal events in BrUra-substituted E. coli cells may be unrepaired DNA single-strand breaks.     

Hydrogen peroxide-induced DNA damage in bovine lens epithelial cells

The present investigation was undertaken to determine the types and extent of DNA damage resulting from incubation of primary cultures of bovine lens epithelial cells with hydrogen peroxide. Significant numbers of DNA single-strand breaks were detected by alkaline elution after exposure to as little as 25 microM H2O2 for 5 min at 37 degrees C. The extent of single-strand breakage was concentration dependent and linear from 25 to 200 microM H2O2. The observed single-strand breaks appear primarily due to the action of the hydroxyl radical via a Fenton reaction as both an iron chelator, 1,10-phenanthroline and OH. scavengers, including DMSO, KI and glycerol, significantly inhibited the DNA-damaging effect of H2O2. Diethyldithiocarbamate, an inhibitor of superoxide dismutase, further potentiated the DNA-damaging effects of H2O2, presumably by increasing the steady-state concentration of Fe2+. DNA-protein cross-linking was not observed. In addition, significant levels of 5,6-saturated thymine residues or pyrimidine dimers were not detected after modification of the alkaline elution methodology to allow the use of either E. coli endonuclease III or bacteriophage T4 endonuclease V, respectively. No double-strand breaks were detected after incubation of epithelial cell cultures with H2O2 concentrations of up to 400 microM for 10 min and subsequent neutral filter elution. Since, in vivo, the lens epithelium contains populations of both quiescent and dividing cells, the degree of susceptibility to oxidative damage was also studied in actively growing and plateau-phase cultures. Reduced levels of single-strand breakage were observed when plateau-phase cultures were compared to actively growing cells. In contrast, essentially no differences in repair rates were noted at equitoxic doses of H2O2. The above results suggest that lens epithelial cells may be particularly sensitive to oxidative damage and thus are a good model system in which to study the effects of oxidative stress.