Alkaline gel electrophoresis assay to detect DNA strand breaks and repair mechanisms in Escherichia coli (original) (raw)
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Toxicology Letters, 1999
Stannous ion (Sn) has been employed in nuclear medicine and in food industry. We described that Stannous Chloride (SnCl 2 ) inactivation effect in Escherichia coli is mediated by a Fenton-like reaction. The effect of SnCl 2 was studied through: (i) the alteration of plasmid topology in neutral and acidic pH by gel electrophoresis; and (ii) the transformation efficiency of an wild type E. coli strain. Treatment of plasmid DNA pUC 9.1 with SnCl 2 , at pH 7.4, results in DNA single-strand breaks (SSB), in a dose-dependent manner. Addition of sodium benzoate partly inhibited the DNA damage, while EDTA completely abolishes DNA-SSB. Furthermore, the ability of the plasmid to transform E. coli was reduced. At pH 1.3, SnCl 2 exerts a protective effect on plasmid against HCl depurination. Our results suggest the generation of ROS, such as OH by a Fenton-like reaction, close to the site of the lesions due to a possible complexation of stannous ion to DNA.
2013
Copyright © 2010 Ellen S. Motta et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Stannous chloride (SnCl2) and UVA induce DNA lesions through ROS. The aim of this work was to study the toxicity induced by UVA preillumination, followed by SnCl2 treatment. E. coli BER mutants were used to identify genes which could play a role in DNA lesion repair generated by these agents. The survival assays showed (i) The nfo mutant was the most sensitive to SnCl2; (ii) lethal synergistic effect was observed after UVA pre-illumination, plus SnCl2 incubation, the nfo mutant being the most sensitive; (iii) wild type and nfo mutants, transformed with pBW21 plasmid (nfo +) had their survival increased following treatments. The alkaline agarose gel electrophoresis assays pointed that (i) UVA induced DNA breaks and fpg mutant was the ...
Journal of Biological Chemistry, 1988
Agents that act via oxygen-derived free radicals form DNA strand breaks with fragmented sugar residues that block DNA repair synthesis. Using a synthetic DNA substrate with a single type of sugar fragment, 3'-phosphoglycolaldehyde esters, we show that in Escherichia coli extracts the only EDTA-resistant diesterase for these damages depends on the bacterial nfo (endonuclease IV) gene. Endonuclease IV was purified to physical homogeneity (Mr = 31,000) from an E. coli strain carrying the cloned nfo gene and in which the enzyme had been induced with paraquat. Although heat-stable and routinely assayed in the presence of EDTA, endonuclease IV was inactivated in the absence of substrate at 23-50 "C by either EDTA or 1,lOphenanthroline, suggesting the presence of an essential metal tightly bound to the protein. Purified endonuclease IV released phosphoglycolaldehyde, phosphate, and intact deoxyribose 5-phosphate from the 3'-end of DNA, all with apparent K, of 5-10 nM. The optimal KC1 or NaCl concentration for 3'-phosphoglycolaldehyde release was 50-100 mM. The purified enzyme had endonuclease activity against partially depurinated DNA but lacked significant nonspecific nuclease activities. Endonuclease IV also activated H,Oz-damaged DNA for repair synthesis by DNA polymerase I. Thus, endonuclease IV can act on a variety of oxidative damages in DNA, consistent with a role for the enzyme in combating free-radical toxicity. Partially reduced oxygen species are toxic and mutagenic to cells (Halliwell, 1987). These species, notably superoxide radical anion (O;), hydrogen peroxide, and hydroxyl radical (HO), are formed by ionizing radiation (Hutchinson, 1985), antibiotics such as paraquat and streptonigrin (Hassett et al., 1987), and by normal cellular metabolism (Fridovich, 1978). Living cells have a front-line enzymatic defense system against damage by oxygen radicals in the form of superoxide dismutase and catalase-hydroperoxidase (Fridovich, 1978). When these scavenging enzymes are missing (Farr et al., 1986) or noninducible (Storz et al., 1987), the spontaneous mutation frequency in Escherichia coli and Salmonella typhimurium is elevated during aerobic growth, while overproduction of per-* This work was supported by Grants CA37831 and ES03926 from the National Institutes of Health (to B. D.
Toxicology Letters, 1993
A number of repair endonucleases, viz. endonuclease III, fo~amidopyrimidin~DNA glycosylase (FPG protein). endonuclease IV, exonuciease III and UV endonuclease, is used to simultaneously quantify various types of DNA modifications, which were induced by agents that generate reactive oxygen species. Under cell-free conditions, two types of DNA damage profiles are obtained. The profiles induced by chemically generated singlet oxygen and by various photosensitizers (acridine orange, methylene blue. riboflavin, hematoporphyrin) plus light are dominated by base modifications sensitive to FPG protein, while 5,6-dihydropyrimidines (recognized by endonuclease III), sites of base loss (AP sites, recognized by endonuclease IV and exonuclease III) and strand breaks are minor lesions. In contrast, the DNA damage profile induced by hydroxyl radicals (y-rays) consists of approx. equal levels of base modifications, AP sites and strand breaks. The damage profiles induced by Fe(III)-EDTA in the presence of superoxide and by Fe~III)-nitrilotri~etate in the presence of HtOz do not differ from that by hydroxyl radicals. The damage profile induced by Cu(II)-phenanthroline deviates by high levels of AP sites that are recognized by endonuclease IV and exonuclease III-but not by those AP endonucleases which cfeave at the 3' site-and probably represent AP sites oxidized at C-l'. The damage induced by Fe(III)-bleomycin plus Hz@ deviates by an increased level of double strand breaks and the absence of endonuclease-sensitive base modifications. Cellular DNA damage profiles are obtained from bacteria, cultured mammalian cells and mammalian mitochondria after exposure to acridine orange plus visible light. A comparison with the cell-free profiles reveals that the damage in all three systems is not induced indirectly by hydroxyl radicals or an activation of cellular n&eases, but by the same mechanism that is responsible for the cell-free DNA damage.
Mutation research, 1977
Mutation frequency decline (MFD) is an irreversible loss of newly-induced suppressor mutations occurring in excision-proficient Escherichia coli during a short period of incubation in minimal medium before plating on broth- or Casamino acids-enriched selective agar. It is known that MFD of UV-induced mutations may occur before DNA containing pre-mutagenic lesions is replicated, but we conclude that MFD can also occur after the damaged DNA has been replicated on the basis of the following evidence. (1) Mutation fixation in rich medium (i.e., loss of susceptibility to mutation frequency decline) with ethyl methanesulphonate mutagenesis begins immediately, whereas with UV it is delayed for 20--30 min. (2) The delay in mutation fixation after UV can be explained neither by inhibition of DNA replication nor by a delay in the appearance of error-prone repair activity in the irradiated population. (3) MFD at later times after UV irradiation is more rapid and is less strongly inhibited by c...
Mutagenic DNA repair in Escherichia coli
MGG Molecular & General Genetics, 1976
In the non-filamenting tif-1 strain WP44 s NF trp a dramatic enhancement of both UV and gamma ray mutability to Trp + was observed when irradiated bacteria were incubated on plates at 43 °. This enhanced mutability was progressively suppressed when the initial plating density exceeded 108 bacteria per plate and was not demonstrable in liquid media. Under optimal conditions more mutants were induced by gamma radiation than could reasonably be accounted for by the initial number of radiation-induced lesionsin the DNA, implying the existence of some mechanism for amplifying the radiation effect. Moreover, the tif-enhanced mutation frequency could be obtained if incubation at restrictive temperature was delayed for up to 60 min in nutrient broth after irradiation, at a time when all known reparable DNA damage had been repaired and the number of viable bacteria had more than doubled. On plates the effect of high temperature was still fully demonstrable 120 rain after irradiation. The results are hard to reconcile with the hypothesis that incubation of tif-1 bacteria at restrictive temperature causes the induction of a repair system acting on DNA damaged by gamma radiation. A more compatible interpretation would be that radiation causes a persisting physiological disturbance in the cell and that this enhances the spontaneous mutator effect occurring in tif-1 bacteria subjected to subsequent thermal shock.
Persistent damaged bases in DNA allow mutagenic break repair in Escherichia coli
PLOS Genetics, 2017
Bacteria, yeast and human cancer cells possess mechanisms of mutagenesis upregulated by stress responses. Stress-inducible mutagenesis potentially accelerates adaptation, and may provide important models for mutagenesis that drives cancers, host pathogen interactions, antibiotic resistance and possibly much of evolution generally. In Escherichia coli repair of double-strand breaks (DSBs) becomes mutagenic, using low-fidelity DNA polymerases under the control of the SOS DNA-damage response and RpoS general stress response, which upregulate and allow the action of error-prone DNA polymerases IV (DinB), II and V to make mutations during repair. Pol IV is implied to compete with and replace highfidelity DNA polymerases at the DSB-repair replisome, causing mutagenesis. We report that up-regulated Pol IV is not sufficient for mutagenic break repair (MBR); damaged bases in the DNA are also required, and that in starvation-stressed cells, these are caused by reactive-oxygen species (ROS). First, MBR is reduced by either ROS-scavenging agents or constitutive activation of oxidative-damage responses, both of which reduce cellular ROS levels. The ROS promote MBR other than by causing DSBs, saturating mismatch repair, oxidizing proteins, or inducing the SOS response or the general stress response. We find that ROS drive MBR through oxidized guanines (8-oxo-dG) in DNA, in that overproduction of a glycosylase that removes 8-oxo-dG from DNA prevents MBR. Further, other damaged DNA bases can substitute for 8-oxo-dG because ROS-scavenged cells resume MBR if either DNA pyrimidine dimers or alkylated bases are induced. We hypothesize that damaged bases in DNA pause the replisome and allow the critical switch from high fidelity to error-prone DNA polymerases in the DSB-repair replisome, thus allowing MBR. The data imply that in addition to the indirect stress-response controlled switch to MBR, a direct cisacting switch to MBR occurs independently of DNA breakage, caused by ROS oxidation of DNA potentially regulated by ROS regulators.
The Action ofEscherichia coliEndonuclease III on Multiply Damaged Sites in DNA
Journal of Molecular Biology, 1995
Energy deposition by ionizing radiation can lead to the formation of Radiobiology Program clustered DNA damage, i.e. more than one lesion situated within a helical Cross Cancer Institute 11560 University Avenue turn of DNA. Among the postulated lesions are those characterized by damaged bases and abasic sites on opposite strands. Enzymatic removal of Edmonton, AB T6G 1Z2 Canada such lesions may inadvertently lead to the formation of double-strand breaks. To test this hypothesis, we have constructed model substrates containing damaged bases (5,6-dihydrothymine) or abasic sites set one, three, five and seven bases apart on opposite strands, and examined the reactivity of Escherichia coli endonuclease III towards these substrates. Endonuclease III demonstrates two activities; as a glycosylase that removes saturated pyrimidine bases, such as dihydrothymine, and as an AP lyase that cleaves DNA strands at abasic sites. Analysis of endonuclease III-treated dihydrothymidine containing plasmid DNA by agarose gel electrophoresis indicated that the enzyme generated only single-strand breaks when the base damage was set one and three base-pairs apart, and only slowly introduced double-strand breaks in the other substrates. Endonuclease III treatment of the abasic site-containing DNA, however, readily yielded double-strand breaks. Taken together, these results indicate that the glycosylase activity of the enzyme, but not the AP lyase activity, is inhibited by the presence of a closely positioned break in the opposite strand.
Use of DNA Repair Enzymes in Electrochemical Detection of Damage to DNA Bases in Vitro and in Cells
Analytical Chemistry, 2005
Electrochemical measurements at mercury or solid amalgam electrodes offer a highly sensitive detection of DNA strand breaks. On the other hand, electrochemical detection of damage to DNA bases at any electrode is usually much less sensitive. In this paper, we propose a new voltammetric method for the detection of the DNA base damage based on enzymatic conversion of the damaged DNA bases to single-strand breaks (ssb), single-stranded (ss) DNA regions, or both. Supercoiled DNA exposed to UV light was specifically cleaved by T4 endonuclease V, an enzyme recognizing pyrimidine dimers, the major products of photochemical DNA damage. Apurinic sites (formed in dimethyl sulfate-modified DNA) were determined after treating the DNA with E. coli exonuclease III, an enzyme introducing ssb at the abasic sites and degrading one of the DNA strands. The ssb or ssDNA regions, or both, were detected by adsorptive transfer stripping alternating current voltammetry at the mercury electrode. This technique offers much better sensitivity and selectivity of DNA base damage detection than any other electrochemical method. It is not limited to DNA damage in vitro, but it can detect also DNA base damage induced in living bacterial cells.
Mutagenesis, 2006
Exonuclease III (Exo III) and endonuclease IV (Endo IV) play a critical role in the base excision repair (BER) of Escherichia coli. Both are endowed with AP endonucleolytic activity, cleaving the 5 0 phosphodiester bond adjacent to spontaneous or induced abasic sites in DNA. Although mutants defective in Exo III (xthA) are usually hypersensitive to oxidative agents such as hydrogen peroxide, near-UV-light and X-rays, mutants defective in Endo IV (nfo) are not as sensitive as the xthA strain. To further investigate the roles of these AP endonucleases in DNA repair, we evaluated the sensitivity and mutagenesis of xthA and nfo strains after UVB and compared with UVC light. Our results revealed that xthA but not nfo strain was hypersensitive to UVB. The use of Fe 12 ion chelator (dipyridyl), prior to irradiation, completely protected the xthA mutant against UVB lethal lesions, suggesting the generation of toxic oxidative lesions mediated by transition metal reactions. The nfo strain displayed increased UVB-induced mutagenesis, which was significantly suppressed by pre-treatment with dipyridyl. Although xthA strain did not display increased mutagenesis after UVC and UVB treatments, this phenotype was not related to xthA mutation, but rather to an unknown secondary mutation specifying an antimutator phenotype. After UVB irradiation, the base substitution spectra of nfo strain revealed a bias towards AT!GC transitions and GC!CG transversions, which were also suppressed by previous treatment with the iron chelator. Overall, on the basis of the differential sensitivities and mutational spectra displayed after UVC and UVB treatments, we propose a role for Endo IV and Exo III to counteract DNA damage induced by the oxidative counterpart of UVB in E.coli.