On the mechanism of hydrogen peroxide-, superoxide-, and ultraviolet light-induced DNA cleavages of inactive bleomycin-iron (III) complex

The ³²P-labeled DNA cleavage experiments showed that the biological activity of the bleomycin(BLM)-Fe(III)OH^? complex is evidently induced by addition of H₂O₂ and KO₂, or by irradiation of UV light. Hydrogen peroxide contributes to the conversion from the inactive BLM-Fe(III)OH^? complex to the active BLM-Fe(III)O₂H^? complex, and UV light to the reduction of the BLM-Fe(III)OH^? complex to the BLM-Fe(II) complex. The proposed hypothetical mechanism for cyclic function of BLM-iron complex is similar to that of certain heme-oxygenases and heme-oxidases.     

Recombinational repair of chromosomal DNA double-strand breaks generated by a restriction endonuclease

DNA double-strand break repair can be accomplished by homologous recombination when a sister chromatid or a homologous chromosome is available. However, the study of sister chromatid double-strand break repair in prokaryotes is complicated by the difficulty in targeting a break to only one copy of two essentially identical DNA sequences. We have developed a system using the Escherichia coli chromosome and the restriction enzyme EcoKI, in which double-strand breaks can be introduced into only one sister chromatid. We have shown that the components of the RecBCD and RecFOR 'pathways' are required for the recombinational repair of these breaks. Furthermore, we have shown a requirement for SbcCD, the prokaryotic homologue of Rad50/Mre11. This is the first demonstration that, like Rad50/Mre11, SbcCD is required for recombination in a wild-type cell. Our work suggests that the SbcCD-Rad50/Mre11 family of proteins, which have two globular domains separated by a long coiled-coil linker, is specifically required for the co-ordination of double-strand break repair reactions in which two DNA ends are required to recombine at one target site.     

Magnesium monoperoxophtalate: an efficient single oxygen atom donor in DNA cleavage catalyzed by metalloporphyrin

The peracid compound magnesium monoperoxophtalate (MMPP), water-soluble at physiological pH, behaves as a promising oxygen donor in the oxidative cleavage of DNA catalyzed by meso-tetrakis-(4-N-methylpyridyl)porphyrinatomanganese(III) pentaacetate. Shown on cleavage of supercoiled phi X174 DNA, MMPP is significantly more efficient than KHSO5, another recently developed single oxygen atom donor (Fouquet et al., J. Chem. Soc., Chem. Commun., 1987, 1169).     

Double-strand breaks from single photochemical events in DNA containing 5-bromouracil.

Ultraviolet irradiation of Escherichia coli cells with a low level of 5-bromouracil incorporated produces DNA double-strand breaks by single photochemical events, one such break per 100 single-strand breaks, the latter assayed in alkali-denatured DNA. About 2.5--4 double-strand breaks are produced per "lethal hit," compared with about 6 double-strand breaks per lethal hit induced by gamma rays. These results are consistent with the hypothesis that an unrepaired DNA double-strand break is a major lethal event in both cases. The increase in sensitivity to ultraviolet (measured by colony-forming ability) seems linear in the number of bromouracils incorporated (0--20% of the thymines), and the linear relationship is much the same for incorporation in one or in both strands of the DNA double helix.     

Study on DNA strand breaks induced by sodium nitroprusside, a nitric oxide donor, in vivo and in vitro

Nitric oxide (NO) as well as its donors has been shown to generate mutation and DNA damage in in vitro assays. The objective of this study was to identify that DNA single-strand breaks (SSBs) could be elicited by NO, not only in vitro but also in vivo. The alkaline single-cell gel electrophoresis (SCGE) was performed to examine the DNA damage in g12 cells and the cells isolated from the organs of mice exposed to sodium nitroprusside (SNP). A modified method, in which neither collagenase nor trypsin was necessary, was used to prepare the single-cell suspension isolated from organs of mice. Results showed that the exposure of g12 cells to 0.13-0.5 micromol/ml SNP with S9 for 1 h induced a concentration-dependent increase in DNA SSBs in g12 cells. The significant increase in DNA migration and comet frequency has appeared in the cells isolated from the spleen, thymus, and peritoneal macrophages of mice after injecting i.p. SNP in the dosage range of 0.67-6.0 mg/kg b.wt for 1 h. However, no obvious increase in DNA strand breaks was observed in the cells isolated from the liver, kidney, lung, brain and heart obtained from the same treated mice. These results suggested that DNA SSBs could be induced by NO in some cells both in vivo and in vitro. There were organ differences in sensitivity in the mice exposed to NO. Spleen, thymus, and macrophages might be the important targets of NO.     

DNA cleavage by a Chromium(III) complex

A new Cr(III) complex with the empirical formula [Cr(Schiff base) (H(2)O)(2)]ClO(4), where the Schiff base is 2, 3-bis?[(2-hydroxy-4-diethylamino) (phenyl) (methylene)]amino?2-butenedinitrile has been synthesized and characterized spectroscopically. Binding of this complex to DNA has been studied using UV-visible spectroscopy. The complex has been found to bind to the major groove of DNA with a binding constant, K = (1.3 +/- 0.2) x 10(3) M(-1). The induced CD spectrum of the complex in the presence of DNA is also indicative of major groove binding. Gel electrophoresis of plasmid DNA in the presence of the complex shows that the complex brings about nicking of the DNA.     

A plant 3'-phosphoesterase involved in the repair of DNA strand breaks generated by oxidative damage

Two novel, structurally and functionally distinct phosphatases have been identified through the functional complementation, by maize cDNAs, of an Escherichia coli diphosphonucleoside phosphatase mutant strain. The first, ZmDP1, is a classical Mg(2+)-dependent and Li(+)-sensitive diphosphonucleoside phosphatase that dephosphorylates both 3'-phosphoadenosine 5'-phosphate (3'-PAP) and 2'-PAP without any discrimination between the 3'- and 2'-positions. The other, ZmDP2, is a distinct phosphatase that also catalyzes diphosphonucleoside dephosphorylation, but with a 12-fold lower Li(+) sensitivity, a strong preference for 3'-PAP, and the unique ability to utilize double-stranded DNA molecules with 3'-phosphate- or 3'-phosphoglycolate-blocking groups as substrates. Importantly, ZmDP2, but not ZmDP1, conferred resistance to a DNA repairdeficient E. coli strain against oxidative DNA-damaging agents generating 3'-phosphate- or 3'-phosphoglycolate-blocked single strand breaks. ZmDP2 shares a partial amino acid sequence similarity with a recently identified human polynucleotide kinase 3'-phosphatase that is thought to be involved in DNA repair, but is devoid of 5'-kinase activity. ZmDP2 is the first DNA 3'-phosphoesterase thus far identified in plants capable of converting 3'-blocked termini into priming sites for reparative DNA polymerization.     

Reactive oxygen species generated by the mitochondrial respiratory chain affect the complex III activity via cardiolipin peroxidation in beef-heart submitochondrial particles

The aim of this study was to investigate the effect of reactive oxygen species (ROS), produced by the mitochondrial respiratory chain, on the activity of complex III and on the cardiolipin content in bovine-heart submitochondrial particles (SMP). ROS were produced by treatment of nicotinamide adenine dinucleotide (NADH) respiring SMP with rotenone. This treatment resulted in a production of superoxide anion, detected by the epinephrine method, which was blocked by superoxide dismutase (SOD). Exposure of SMP to mitochondrial-mediated ROS generation resulted in a marked loss of complex III activity and in a parallel loss of mitochondrial cardiolipin content. Both these effects were completely abolished by SOD + catalase. Exogenous added cardiolipin was able to almost completely prevent the ROS-mediated loss of complex III activity. No effect was obtained with other major phospholipid components of the mitochondrial membrane such as phosphatidylcholine and phosphatidylethanolamine, or with peroxidized cardiolipin. The results demonstrate that mitochondrial-mediated ROS generation affects the activity of complex III via peroxidation of cardiolipin, which is required for the functioning of this multisubunit enzyme complex. These results may prove useful in probing molecular mechanisms of ROS-induced peroxidative damage to mitochondria, which have been proposed to contribute to those physiopathological conditions characterized by an increase in the basal production of ROS such as aging, ischemia/reperfusion and chronic degenerative diseases.     

Methoxyamine potentiates DNA single strand breaks and double strand breaks induced by temozolomide in colon cancer cells

We have previously shown that human cancer cells deficient in DNA mismatch repair (MMR) are resistant to the chemotherapeutic methylating agent temozolomide (TMZ) and can be sensitized by the base excision repair (BER) blocking agent methoxyamine (MX) [21]. To further characterize BER-mediated repair responses to methylating agent-induced DNA damage, we have now evaluated the effect of MX on TMZ-induced DNA single strand breaks (SSB) by alkaline elution and DNA double strand breaks (DSB) by pulsed field gel electrophoresis in SW480 (O6-alkylguanine-DNA-alkyltransferase [AGT]+, MMR wild type) and HCT116 (AGT+, MMR deficient) colon cancer cells. SSB were evident in both cell lines after a 2-h exposure to equitoxic doses of temozolomide. MX significantly increased the number of TMZ-induced DNA-SSB in both cell lines. In contrast to SSB, TMZ-induced DNA-DSB were dependent on MMR status and were time-dependent. Levels of 50 kb double stranded DNA fragments in MMR proficient cells were increased after TMZ alone or in combination with O6-benzylguanine or MX, whereas, in MMR deficient HCT116 cells, only TMZ plus MX produced significant levels of DNA-DSB. Levels of AP endonuclease, XRCC1 and polymerase beta were present in both cell lines and were not significantly altered after MX and TMZ. However, cleavage of a 30-mer double strand substrate by SW480 and HCT116 crude cell extracts was inhibited by MX plus TMZ. Thus, MX potentiation of TMZ cytotoxicity may be explained by the persistence of apurinic/apyrimidinic (AP) sites not further processed due to the presence of MX. Furthermore, in MMR-deficient, TMZ-resistant HCT116 colon cancer cells, MX potentiates TMZ cytotoxicity through formation of large DS-DNA fragmentation and subsequent apoptotic signalling.     

Incorporation of nucleotides into DNA by mammalian DNA polymerase in the presence of a single deoxynucleoside triphosphate.

Rat ascites hepatoma cell DNA polymerases (EC 2.7.7.7), especially low molecular weight polymerase, could incorporate a significant amount of single nucleotide into acid-soluble products in the absence of the other three deoxynucleoside triphosphates when activated DNA was used as a template. This relaxed requirement for deoxynucleotides was not observed when poly[d(A-T).d(T-A)] was used as a template. Nearest-neighbour base analyses of the products formed in the presence of a single deoxynuclesode triphosphate revealed that the reaction is not of a terminal transferase-type but a very limited repair synthesis in which one or a few triphosphates are incorporated at numerous 3'-hydroxyl ends.     

Lethal heat induces single strand breaks in the DNA of bacterial spores

Lethal heating induces DNA single strand breakage in bacterial endospores as detected by the alkaline sucrose gradient centrifugation technique. Heating of spores of $\text{Bacillus}\text{subtilis}$ 168 at 90°C for 10, 30, and 60 min induced 6, 15, and 15 single strand breaks, respectively and inactivated 6%, 98.2%, and 99.974% of the spores. This is the first report to our knowledge identifying specifically single strand DNA breakage with lethal heat injury of bacterial spores.     

RNA polymerase III is required for the repair of DNA double-strand breaks by homologous recombination

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Interspecies differences in DNA single strand breaks caused by benzo(a)pyrene and marine environment

The presence of DNA single strand breaks in untreated specimens of selected species, mosquito fish Gambusia affinis, painted comber Serranus scriba, blue mussel Mytilus galloprovincialis, spiny crab Maja crispata and sea cucumber Holothuria tubulosa as well as in 10 microg/g benzo(a)pyrene (BaP) treated mosquito fish, blue mussel and spiny crab was measured, using alkaline filter elution. Interspecies differences in alkaline elution profiles were observed and attributed to different lengths of DNA from different sources and to differences in the number of strand breaks present during normal cellular events in different phyla. Spiny crab hemocytes are more sensitive to action of BaP then blue mussel hemocytes and mosquito fish hepatocytes that could be explained by differences in the rates of distinct metabolic reactions and DNA repair among the investigated species. In field study, DNA single strand breaks were measured in hepatocytes of painted comber and in hemocytes of blue mussel and spiny crab from natural population specimens collected at eight sampling sites along Istrian coast, Croatia. Spatial variations in DNA integrity for each species were detected and revealed for the first time that spiny crab is responsive to different environmental conditions. Interspecies variations in the DNA integrity due to environmental conditions, confirmed species specific susceptibility to genotoxicity of certain environment that in long-term may modify the structure of marine communities. The multi-species approach in designing biomonitoring studies was suggested.     

On the chemical yield of base lesions, strand breaks, and clustered damage generated in plasmid DNA by the direct effect of X rays

The purpose of this study was to determine the yield of DNA base damages, deoxyribose damage, and clustered lesions due to the direct effects of ionizing radiation and to compare these with the yield of DNA trapped radicals measured previously in the same pUC18 plasmid. The plasmids were prepared as films hydrated in the range 2.5 < Gamma < 22.5 mol water/mol nucleotide. Single-strand breaks (SSBs) and double-strand breaks (DSBs) were detected by agarose gel electrophoresis. Specific types of base lesions were converted into SSBs and DSBs using the base-excision repair enzymes endonuclease III (Nth) and formamidopyrimidine-DNA glycosylase (Fpg). The yield of base damage detected by this method displayed a strikingly different dependence on the level of hydration (Gamma) compared with that for the yield of DNA trapped radicals; the former decreased by 3.2 times as Gamma was varied from 2.5 to 22.5 and the later increased by 2.4 times over the same range. To explain this divergence, we propose that SSB yields produced in plasmid DNA by the direct effect cannot be analyzed properly with a Poisson process that assumes an average of one strand break per plasmid and neglects the possibility of a single track producing multiple SSBs within a plasmid. The yields of DSBs, on the other hand, are consistent with changes in free radical trapping as a function of hydration. Consequently, the composition of these clusters could be quantified. Deoxyribose damage on each of the two opposing strands occurs with a yield of 3.5 +/- 0.5 nmol/J for fully hydrated pUC18, comparable to the yield of 4.1 +/- 0.9 nmol/J for DSBs derived from opposed damages in which at least one of the sites is a damaged base.