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PubMed1995

DNA-strand breaks induced by dimethylarsinic acid, a metabolite of inorganic arsenics, are strongly enhanced by superoxide anion radicals.

We previously reported that dimethylarsinic acid (DMAA), a major metabolite of inorganic arsenics, induced DNA single-strand breaks (ssb) both in vivo and in cultured alveolar type II (L-132) cells in vitro, possibly via the production of dimethylarsenic peroxyl radicals. Here, the interaction of superoxide anion radicals (O2-) in the induction of ssb in L-132 cells was investigated using paraquat, an O2(-)-producing agent. A significant enhancement of ssb formation was observed in the DMAA-exposed cells when coexposed to paraquat. This enhancement occurred even when post-exposed to DMAA after washing, suggesting that the DMAA exposure caused some modification of DNA such as DNA-adducts, which was recognized by active oxygens to form ssb. An experiment with UV-irradiation, which was likely to induce ssb at the modified region, supported the possibility of DNA modification by DMAA exposure. An ESR study indicated that O2- produced by paraquat in DMAA-exposed cells was more consumed than in non-exposed cells, assumingly through the reaction with the dimethylarsenic-modified region of DNA. The species of active oxygens were estimated by using diethyldithiocarbamate, aminotriazole, diethylmaleate, hydrogen peroxide (H2O2), gamma-irradiation and ethanol. O2- but neither H2O2 nor hydroxyl radicals was very likely to contribute to the ssb-enhancing action of paraquat.

Cacodylic Acid↗
PubMed2006

Effects of decreasing sugar-sweetened beverage consumption on body weight in adolescents: a randomized, controlled pilot study.

OBJECTIVE: The role of sugar-sweetened beverages (SSBs) in promoting obesity is controversial. Observational data link SSB consumption with excessive weight gain; however, randomized, controlled trials are lacking and necessary to resolve the debate. We conducted a pilot study to examine the effect of decreasing SSB consumption on body weight. METHODS: We randomly assigned 103 adolescents aged 13 to 18 years who regularly consumed SSBs to intervention and control groups. The intervention, 25 weeks in duration, relied largely on home deliveries of noncaloric beverages to displace SSBs and thereby decrease consumption. Change in SSB consumption was the main process measure, and change in body mass index (BMI) was the primary end point. RESULTS: All of the randomly assigned subjects completed the study. Consumption of SSBs decreased by 82% in the intervention group and did not change in the control group. Change in BMI, adjusted for gender and age, was 0.07 +/- 0.14 kg/m2 (mean +/- SE) for the intervention group and 0.21 +/- 0.15 kg/m2 for the control group. The net difference, -0.14 +/- 0.21 kg/m2, was not significant overall. However, baseline BMI was a significant effect modifier. Among the subjects in the upper baseline-BMI tertile, BMI change differed significantly between the intervention (-0.63 +/- 0.23 kg/m2) and control (+0.12 +/- 0.26 kg/m2) groups, a net effect of -0.75 +/- 0.34 kg/m2. The interaction between weight change and baseline BMI was not attributable to baseline consumption of SSBs. CONCLUSIONS: A simple environmental intervention almost completely eliminated SSB consumption in a diverse group of adolescents. The beneficial effect on body weight of reducing SSB consumption increased with increasing baseline body weight, offering additional support for American Academy of Pediatrics guidelines to limit SSB consumption.

Adolescent↗
PubMed2004

Autoimmune response in mothers of children with congenital and postnatally diagnosed isolated heart block: a population based study.

OBJECTIVE: To study the autoimmune response in mothers of children with isolated congenital heart block (CHB) and heart block (HB) diagnosed postnatally. METHODS: We reviewed the Finnish hospital registries for patients born between 1950 and 2000 and diagnosed with isolated HB before the age of 16 years. Clinical data and sera for the determination of autoantibodies were available from 67 mothers of children with CHB and from 37 mothers of children with postnatally diagnosed HB 9.9 years and 22.6 years (mean) after the index delivery, respectively. Maternal antibodies to 52 kDa and 60 kDa SSA and 48 kDa SSB were determined by time-resolved fluoroimmunoassay (TR-FIA) and by immunoblotting. Other marker antibodies for connective tissue diseases (CTD) were determined by immunoblot and/or by immunofluorescence. The control group comprised 136 mothers with primary Sjögren's syndrome (SS), systemic lupus erythematosus (SLE), or other CTD with healthy children. RESULTS: Sixty of our 67 mothers (90%) of children with CHB had antibodies to SSA or SSB by the methods initially used in this study. When retests and tests performed previously were taken into account, only 3 (4%) of the 67 mothers did not have any autoantibodies. Two (3%) of the 67 mothers had antibodies to dsDNA and one (1%) each to Jo-1/HRS, RNP-70 kDa, and histone proteins. Of 37 mothers of children with postnatally diagnosed HB, only 3 (8%) had any autoantibodies. Increased risk of having a child with CHB was indicated by maternal primary SS and high levels of anti-SSA and anti-SSB by all assays, whereas low risk was indicated by maternal SLE or other CTD and undetectable or low levels of the antibodies. No single anti-SSA or anti-SSB test was clearly superior to others, but in general, immunoblots were more specific than TR-FIA. CONCLUSION: Maternal autoimmune disorder is almost always associated with CHB but only rarely with postnatally diagnosed HB. Anti-SSA and anti-SSB are marker antibodies for mothers of children with CHB, and an increased risk of having an affected child is indicated by maternal primary SS and high titer antibodies to SSA and SSB.

Adult↗
PubMed1992

A postsynaptic role for single-stranded DNA-binding protein in recA protein-promoted DNA strand exchange.

The single-stranded DNA-binding protein (SSB protein) is required for efficient genetic recombination in vivo. One function for SSB protein in DNA strand exchange in vitro is to remove secondary structure from single-stranded DNA (ssDNA) and thereby aid in the formation of recA protein-saturated presynaptic complexes. In the preceding paper (Lavery, P. E., and Kowalczykowski, S. C. (1992) J. Biol. Chem. 267, 9307-9314) we demonstrated that DNA strand exchange can occur in the presence of volume-occupying agents at low magnesium ion concentration, where secondary structures are reduced. Our results suggest that SSB protein is not acting during presynapsis under these conditions, yet the DNA strand exchange reaction is stimulated by the addition of SSB protein. In this study we present biochemical evidence which suggests that SSB protein stimulates DNA strand exchange by binding to the ssDNA displaced from joint molecules, thereby stabilizing them and allowing branch migration to extend the region of heteroduplex DNA. Therefore, our results indicate dual roles for SSB protein at elevated magnesium ion concentration; it functions during presynapsis, removing secondary structure from ssDNA, as indicated previously, and it also functions postsynaptically, binding to the ssDNA displaced from joint molecules.

Adenosine Triphosphatases↗
PubMed1991

Non-P-glycoprotein-mediated multidrug resistance in a small cell lung cancer cell line: evidence for decreased susceptibility to drug-induced DNA damage and reduced levels of topoisomerase II.

Data obtained from clinical samples suggest that non-P-glycoprotein mechanisms of multidrug resistance are likely to be important in small cell lung cancer. The H69AR cell line was derived from the H69 small cell lung cancer cell line by selection in doxorubicin (adriamycin) and does not overexpress P-glycoprotein as detected by monoclonal antibody C219 (S.E.L. Mirski et al., Cancer Res., 47:2594, 1987). In the present study, we have used the polymerase chain reaction to verify that H69AR cells do not overexpress P-glycoprotein. Further, transport studies with radiolabeled daunomycin, VP-16, and vinblastine demonstrate that differences in net drug accumulation or efflux are not part of the resistance phenotype of H69AR cells. To determine if H69 and H69AR cells differ in their susceptibility to drug-induced DNA damage, DNA single-strand breaks (SSB) generated by VP-16 and Adriamycin were measured using the alkaline filter elution assay. Readily detectable SSB were produced in intact H69 cells by 5 microM VP-16, but 100 microM drug was required to cause similar damage in H69AR cells. H69AR cells were also resistant to SSB induction by Adriamycin. The formation of SSB by VP-16 was similarly reduced in isolated H69AR nuclei, indicating that resistance to this drug resides, at least in part, in the nucleus. No significant differences were observed in the rate or extent of repair of VP-16-induced DNA SSB in H69 and H69AR cells. The reduced susceptibility to drug-induced SSB may result from alterations in topoisomerase II, since less immunoreactive topoisomerase II was found in H69AR cells compared to H69 cells. However, changes in topoisomerase II cannot explain the resistance of H69AR cells to such drugs as the Vinca alkaloids and gramicidin D, indicating that multiple mechanisms contribute to drug resistance in this small cell lung cancer cell line.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1991

Characterization of activation and deactivation pathways of 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK) in rat hepatocytes.

We have characterized the metabolism of 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK) in cultured rat hepatocytes and have established the relationship between various metabolic pathways and single-strand breaks (SSB) in DNA. Metabolism of [5-3H]-NNK by carbonyl reduction, alpha-carbon hydroxylation and pyridine N-oxidation was linear from 0.5 to 6 h with 0.25-2 x 10(6) hepatocytes. Using an alkaline elution assay, we observed that NNK induces SSB in DNA in a dose- and time-dependent manner. SSB induced by NNK were rejoined partially within 2 h and totally by 12 h after exposure. NNK-N-oxide produced a smaller number of SSB than NNK, suggesting that pyridine N-oxidation of NNK is a deactivation pathway. Carbonyl reduction of NNK led to 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butan-1-ol (NNAl). Reaction of NNK with methyl magnesium iodide gave 4-(N-nitrosomethylamino)-1-(methyl)-1-(3- pyridyl)butanol-1-ol (1-MeNNAl) 82% yield. NNAl, but not 1-MeNNA1, can be reoxidized to NNK. Doses of 5 mM NNAl and 1-MeNNAl both induced SSB, indicating that NNAl does not require reconversion to NNK to be activated to DNA damaging intermediates. alpha-Methylene hydroxylation resulted in the formation of 4-oxo-4-(3- pyridyl)butanal. At equimolar concentration (5 mM), the aldehyde was more damaging than NNK to hepatocyte DNA. The results of this study demonstrate that NNK is activated by rat hepatocytes and that metabolites formed by alpha-carbon hydroxylation induce SSB.

Animals↗
PubMed1989

The origin of DNA single strand breaks induced by ethylating agents in mammalian cells.

Chinese hamster ovary (CHO) cells were treated with two ethylating agents, N-ethyl-N-nitrosourea (ENU) and diethylsulfate (DES), and the kinetics of DNA single strand break (ssb) induction and rejoining were determined in parallel with DNA adduct formation and removal. In the case of DES, DNA ssb as determined by alkaline elution (AE) were repaired very slowly with more than 50% of the lesions still present on DNA 3 h after treatment. In contrast, 45% of ENU-induced ssb were repaired within 10 min. From the relative concentration of the different ethylated products and their repair rates as measured by high performance liquid chromatography (HPLC) analysis of the ethylated DNA, a theoretical function was constructed that describes the number of ssb expected at each time point after exposure to the mutagen. DES-induced ssb are explained by excision repair processes active on the ethylated purines, mainly 3-ethyladenine (3-EtAde) and 7-ethylguanine (7-EtGua). On the same basis, the rapidly repaired ENU-induced ssb remain unexplained. These results are also discussed in relation to the sensitivity of the two techniques, AE and HPLC, for detecting DNA damage.

Alkylating Agents↗
PubMed1988

The induction of DNA strand breaks in normal human skin fibroblasts exposed to solar ultraviolet radiation.

The levels of apparent DNA single-strand breaks (ssb) were measured, following a 0-20 h incubation of normal human skin fibroblasts exposed to the solar uv wavelengths produced by a fluorescent sunlamp. The ssb were determined using the alkaline elution assay, which was performed either without proteinase K (proK) or in its presence, so as to eliminate any DNA-protein crosslinks that may be present in the cells. Cells were irradiated with either 3 kJ/m2 of sunlamp uv greater than 295 nm, 150 kJ/m2 of sunlamp uv greater than 315 nm, or 150 kJ/m2 of sunlamp uv greater than 320 nm. These treatments resulted in the production of 5-10 ssb/10(10) Da. For the two shorter wavelength irradiations, the levels of ssb decreased rapidly upon incubation of the cells. However, when the elutions were performed using proK, the number of ssb increased about twofold following a 2-4 h incubation. In contrast, the levels of ssb decreased in the sunlamp uv greater than 320 nm irradiated cells for elutions performed with or without proK. These results suggest that under certain irradiation conditions, ssb are formed in cells upon incubation, which are hidden by the crosslinking of protein to DNA.

DNA Damage↗
PubMed1986

Contrasting effects of Escherichia coli single-stranded DNA binding protein on synthesis by T7 DNA polymerase and Escherichia coli DNA polymerase I (large fragment). Evidence that binding protein inhibits trans-lesion synthesis by polymerase I.

The effect of Escherichia coli single-stranded DNA binding protein (SSB) on DNA synthesis by T7 DNA polymerase and E. coli DNA polymerase I (large fragment) using native or aminofluorene-modified M13 templates was evaluated by in vitro DNA synthesis assays and polyacrylamide gel electrophoresis analysis. The two polymerase enzymes displayed differential responses to the addition of SSB. T7 DNA polymerase, a enzyme required for the replication of the T7 chromosome, was stimulated by the addition of SSB whether native or modified templates were used. On the other hand, E. coli DNA polymerase I was slightly stimulated by the addition of SSB to the native template but substantially inhibited on modified templates. This result suggests that DNA polymerase I may be able to synthesize past an aminofluorene adduct but that the presence of SSB inhibited this trans-lesion synthesis. Polyacrylamide gels of the products of DNA synthesis by polymerase I supported this inference since SSB caused a substantial increase in the accumulation of shorter DNA chains induced by blockage at the aminofluorene adduct sites.

DNA Polymerase I↗
PubMed1982

Protein n, a primosomal DNA replication protein of Escherichia coli. Purification and characterization.

Protein n, essential in forming the primosome for the in vitro conversion of phi X174 single-stranded (SS) DNA to the duplex replicative form (RF), has been purified about 5000-fold to near homogeneity from Escherichia coli. Protein n is heat- and acid-resistant and N-ethyl-maleimide-sensitive. It appears to be a dimer of 12,000 (+/- 2000)-dalton polypeptides. About 80 molecules of protein n are present/cell. Protein n binding to phi X SS DNA depends on the presence of single-strand binding protein (SSB). This requirement for SSB reflects a direct interaction of protein n and SSB. About 30 protein n monomers can be bound to an SSB-coated circle. However, in forming the primosome on an SSB-coated phi X circle, an input of only 2-3 protein n monomers is required and 1 monomer bound/circle. Retention of this low level of protein n on SSB-coated phi X SS DNA is dependent upon protein n', a DNA-dependent ATPase (dATPase) that guides primosome assembly. This single protein n monomer is retained in the assembled primosome, which is conserved on the completed parental RF and participates in the next stage of the replicative cycle, production of progeny RF.

Amino Acids↗
PubMed1983

Limited proteolysis studies on the Escherichia coli single-stranded DNA binding protein. Evidence for a functionally homologous domain in both the Escherichia coli and T4 DNA binding proteins.

Limited proteolysis can be used to remove either 42 or 62 amino acids at the COOH terminus of the 18,873-dalton Escherichia coli single-stranded DNA binding protein (SSB). Since poly(dT), but not d(pT)16, increases the rate of this reaction, it appears that cooperative SSB binding to single-stranded DNA (ssDNA) is associated with a conformational change that increases the exposure of the COOH terminus to proteolysis. As a result of this DNA-induced conformational change, we presume that the COOH-terminal region of SSB will become more accessible for interacting with other proteins that utilize the SSB:ssDNA complex as a substrate and that are involved in E. coli DNA replication, repair, and recombination. Removal of this COOH-terminal domain from SSB results in a stronger helix-destabilizing protein which suggests this region may be important for controlling the ability of SSB to denature double-stranded DNA. Since similar results have previously been reported for the bacteriophage T4 gene 32 protein (Williams, K.R., and Konigsberg, W. (1978) J. Biol. Chem. 253, 2463-2470; Hosoda, J., and Moise, H. (1978) J. Biol. Chem. 253, 7547-7555), the acidic, COOH-terminal domains of these two single-stranded DNA binding proteins may be functionally homologous. Preliminary evidence is cited that suggests other prokaryotic and eukaryotic DNA binding proteins may contain similar functional domains essential for controlling their ability to invade double helical DNA.

Amino Acid Sequence↗
PubMed1983

On the role of single-stranded DNA binding protein in recA protein-promoted DNA strand exchange.

RecA protein-promoted DNA strand exchange is greatly stimulated by the single-stranded DNA binding protein (SSB) of Escherichia coli. Stimulation is not a consequence of the binding of SSB to excess single-stranded DNA. It results instead from stabilization of recA protein-single-stranded DNA complexes formed in the presence of ATP and SSB. In the presence of SSB, recA protein does not measurably dissociate from these complexes for up to 90 min. However, in its absence, recA protein moves rapidly between two populations of single-stranded DNA, and complete equilibration occurs with t 1/2 of 17 s. Rapid transfer of recA protein to single-stranded DNA occurs during all stages of DNA strand exchange and does not require ATP. The transfer involves an equilibrium between free and bound recA protein rather than a direct redistribution between single-stranded DNA molecules. Thus, SSB prevents dissociation of recA protein from single-stranded DNA, rendering the binding of the recA protein to single-stranded DNA irreversible. Under these conditions, the pairing phase of the strand exchange reaction is accelerated to the point that it is no longer rate-limiting. These results can explain the relative inefficiency of DNA strand exchange in the absence of SSB.

Bacterial Proteins↗
PubMed1984

Repair of DNA damage caused by formaldehyde in human cells.

The alkaline elution technique was used to study repair of DNA damage caused by formaldehyde (HCHO) in human bronchial epithelial cells and fibroblasts, skin fibroblasts, and DNA excision repair-deficient skin fibroblasts from donors with xeroderma pigmentosum. Exposure of cells to HCHO resulted in DNA-protein cross-links (DPC) and DNA single-strand breaks (SSB) in all cell types. DPC were induced at similar levels and were also removed by all cell types, with a half removal time of 2 to 3 hr. HCHO caused more SSB in the normal cell types than in the xeroderma pigmentosum fibroblasts. However, in all cell types, including the xeroderma pigmentosum cells, HCHO-induced DNA SSB and DPC were removed at comparable rates. By excision repair of HCHO-induced DNA damage, normal cells generated SSB that were also readily repaired. HCHO was only moderately cytotoxic to normal bronchial epithelial cells and fibroblasts at concentrations that induced substantial DNA damage. HCHO enhanced the cytotoxicity of both ionizing radiation and N-methyl-N-nitrosourea in both cell types. The results indicate that most DPC caused by HCHO can be removed without the involvement of DNA excision repair. Furthermore, HCHO also directly causes DNA SSB as well as SSB generated indirectly during ultraviolet-type excision repair. These studies indicate the complexity of the HCHO-induced DNA damage and its repair and that HCHO may enhance the cytotoxicity of chemical and physical carcinogens in human cells.

Animals↗
PubMed1983

The oxygen enhancement ratio for single- and double-strand breaks induced by tritium incorporated in DNA of cultured human T1 cells. Impact of the transmutation effect.

The effect of oxygen, expressed as the oxygen enhancement ratio (OER), on the number of single-strand breaks (SSB) and double-strand breaks (DSB) induced in DNA by the radioactive decay of tritium was measured in human T1 cells whose DNA had been labeled with tritium at carbon atom number 6 of thymidine. Decays were accumulated in vivo under aerobic conditions at 0-1 degrees C and at -196 degrees C and in a nitrogen atmosphere at 0-1 degrees C. The number of SSB and DSB produced was analyzed by sucrose gradient centrifugation. For each tritium decay there were 0.25 DSB in cells exposed to air at 0-1 degrees C and 0.07 in cells kept under nitrogen, indicating an OER of 3.6, a value expected for such low-LET radiation. However, for each tritium decay there were 1.25 SSB in cells exposed to air at 0-1 degrees C and 0.76 in cells kept under nitrogen indicating an OER of only 1.7. The corresponding values for 60Co gamma radiation, expressed as SSB per 100 eV absorbed energy, were 4.5 and 1.0, giving an OER of 4.5. The low OER value found for SSB induced by tritium decay can be explained if 31% of the total SSB produced in air result from transmutation by a mechanism which does not produce DSB and is unaffected by oxygen.

Cell Line↗
PubMed2026

Sugar-sweetened beverage consumption and incident depression: an exploratory multi-omics analysis of candidate biological mediators.

BACKGROUND: Depression is a leading cause of mental and physical disability globally, with its onset and progression influenced by a complex interplay of dietary, psychological, and biological factors. Recent research suggests a link between sugar-sweetened beverage (SSB) consumption and depression risk, although the potential biological pathways underlying this association remain poorly understood. METHODS: This study utilized data from 192,045 participants in the UK Biobank to examine the prospective association between SSB consumption and incident depression using Cox proportional hazards models. SSBs were defined as the sum of five beverage categories assessed via the Oxford WebQ 24-hour dietary recall. Directional consistency of the association was further examined across three external supporting datasets encompassing diverse populations: NHANES, YRBSS, and the Lianyungang Municipal School Health and Risk Factor Surveillance Study Dataset. We further investigated whether proteins, metabolites, inflammatory markers, and brain imaging phenotypes may serve as candidate mediators statistically consistent with mediation of the SSB-depression association. RESULTS: High SSB consumption was associated with an 18% higher risk of incident depression compared with non-consumers (HR = 1.18; 95% CI: 1.11-1.25), with consistent directional associations observed across external supporting datasets. A plasma proteomic signature comprising 229 proteins was constructed using elastic net regularization and was associated with an increased risk of incident depression. Exploratory mediation analyses identified 72 proteins, 36 metabolites, and 5 inflammatory markers as candidate mediators, with IL1RN showing the strongest protein-level candidate mediating effect (9.6%), and Unsaturation and neutrophil count showing the strongest metabolite- and inflammatory marker-level effects, respectively. CONCLUSIONS: This study provides preliminary evidence that proteins, metabolites, and inflammatory markers may serve as candidate mediators statistically consistent with mediation of the association between SSB consumption and incident depression. These findings are exploratory and hypothesis-generating, and future experimental studies are needed to validate these candidate pathways and assess their potential as targets for dietary interventions in depression prevention.

Humans↗
PubMed2002

Escherichia coli single-stranded DNA-binding protein, a molecular tool for improved sequence quality in pyrosequencing.

Pyrosequencing is a four-enzyme bioluminometric DNA sequencing technique based on a DNA sequencing by synthesis principle. Currently, the technique is limited to analysis of short DNA sequences exemplified by single-nucleotide polymorphism analysis. In order to expand the field for pyrosequencing, the read length needs to be improved and efforts have been made to purify reaction components as well as add single-stranded DNA-binding protein (SSB) to the pyrosequencing reaction. In this study, we have performed a systematic effort to analyze the effects of SSB by comparing the pyrosequencing result of 103 independent complementary DNA (cDNA) clones. More detailed information about the cause of low quality sequences on templates with different characteristics was achieved by thorough analysis of the pyrograms. Also, real-time biosensor analysis was performed on individual cDNA clones for investigation of primer annealing and SSB binding on these templates. Results from these studies indicate that templates with high performance in pyrosequencing without SSB possess efficient primer annealing and low SSB affinity. Alternative strategies to improve the performance in pyrosequencing by increasing the primer-annealing efficiency have also been evaluated.

Cloning, Molecular↗
PubMed2006

Structure and enzymatic properties of a chimeric bacteriophage RB69 DNA polymerase and single-stranded DNA binding protein with increased processivity.

In vivo, replicative DNA polymerases are made more processive by their interactions with accessory proteins at the replication fork. Single-stranded DNA binding protein (SSB) is an essential protein that binds tightly and cooperatively to single-stranded DNA during replication to remove adventitious secondary structures and protect the exposed DNA from endogenous nucleases. Using information from high resolution structures and biochemical data, we have engineered a functional chimeric enzyme of the bacteriophage RB69 DNA polymerase and SSB with substantially increased processivity. Fusion of RB69 DNA polymerase with its cognate SSB via a short six amino acid linker increases affinity for primer-template DNA by sixfold and subsequently increases processivity by sevenfold while maintaining fidelity. The crystal structure of this fusion protein was solved by a combination of multiwavelength anomalous diffraction and molecular replacement to 3.2 A resolution and shows that RB69 SSB is positioned proximal to the N-terminal domain of RB69 DNA polymerase near the template strand channel. The structural and biochemical data suggest that SSB interactions with DNA polymerase are transient and flexible, consistent with models of a dynamic replisome during elongation.

Crystallization↗
PubMed1996

A role for molecular oxygen in the formation of DNA damage during the reduction of the carcinogen chromium (VI) by glutathione.

Although well-established as carcinogens, the way in which chromium (VI) compounds exert their carcinogenic, mutagenic, and DNA-damaging potential remains obscure. It is clear that inside cells chromium(VI) is activated to its ultimate carcinogenic form by reducing agents including glutathione (GSH). The present study is intended to clarify if Fenton mechanisms are likely to be important in the formation of DNA lesions by chromium(VI) in combination with GSH. In buffer solutions which were treated to remove Fenton-active metal ions as well as in those not further purified, chromate and GSH induced similar numbers of single-strand breaks (SSB) in isolated PM2 DNA. Molecular oxygen was found to be essential for the formation of SSB, but chromium(V) species arising from chromate/GSH, unless activated by oxygen, appeared to be unreactive toward DNA. Upon addition of Mn(II) to solutions of chromium(VI) and GSH a diminution of Mn(II) ESR signals was observed, good evidence for the presence of chromium(IV) species. Using gas chromatography/mass spectrometry in selective ion-monitoring mode and high-performance liquid chromatography with electrochemical detection, we were able to show that Cr(VI)/GSH failed to induce base modifications typical of hydroxyl radical attack on DNA. Experimental conditions which readily induced SSB gave rise to the formation of chromium-DNA adducts, clearly demonstrating that the generation of these two DNA lesions is not mutually exclusive. We conclude that models which ascribe the induction of chromium-DNA adducts to chromium(V) and the generation of oxidative DNA damage including SSB to hydrogen peroxide are oversimplistic. It is not necessary to invoke a mechanism requiring the presence of added hydrogen peroxide to account for the ability of Cr(VI)/GSH to cause SSB. Our findings suggest that the combination of GSH, molecular oxygen, and chromium(VI) can damage DNA via non-Fenton pathways.

Carcinogens, Environmental↗