Solution structure of YggX: a prokaryotic protein involved in Fe(II) trafficking

We report the three-dimensional structure of YggX from Salmonella enterica, determined by solution nuclear magnetic resonance (NMR) spectroscopy from protein labeled with carbon-13 and nitrogen-15 produced by Escherichia coli cells. The protein has a beta1beta2alpha1alpha2alpha3 fold that is unique to YggX and one of its homologs, a protein from Pseudomonas aeruginosa with 45% sequence identity whose X-ray structure [Protein Data Bank (PDB) 1T07] was determined by a structural genomics center. The NMR structure, which revealed that the C-terminal region of YggX is dynamically disordered, explains why electron density from the corresponding region was missing in the X-ray structure of the Pseudomonas protein. Because it has been hypothesized that YggX has a role in iron trafficking, we investigated the influence of Fe(II) on the (1)H-(15)N NMR fingerprint region of nitrogen-15-labeled YggX. Several signals shifted or broadened upon the addition of excess Fe(II) under anoxic conditions, with His81 showing the largest effect. These results indicate that Fe(II) binds weakly to this protein at a region of the sequence conserved only in the subset of the YggX proteins from organisms similar to Salmonella. The finding that iron binds only weakly to YggX, and not to a highly conserved region of the structure, suggests that the role of this protein in iron homeostasis is more complex than previously thought.     

The solution structure of the oxidative stress-related protein YggX from Escherichia coli

YggX is a highly conserved protein found only in eubacteria and is proposed to be involved in the bacterial response to oxidative stress. Here we report the solution structure of YggX from Escherichia coli determined by nuclear magnetic resonance spectroscopy. The structure of YggX displays a fold consisting of two N-terminal antiparallel beta-sheets and three alpha-helices, which shares significant structural similarity to the crystal structure of a hypothetical protein PA5148 from Pseudomonas aeruginosa. Previous studies propose YggX as an iron binding protein that is involved in cellular iron trafficking. Our data indicate that the protein alone does not bind iron in vitro, suggesting other cofactors or different conditions may be necessary for metal binding.     

Lack of YggX results in chronic oxidative stress and uncovers subtle defects in Fe-S cluster metabolism in Salmonella enterica

As components involved in Fe-S cluster metabolism are described, the challenge becomes defining the integrated process that occurs in vivo based on the individual functions characterized in vitro. Strains lacking yggX have been used here to mimic chronic oxidative stress and uncover subtle defects in Fe-S cluster metabolism. We describe the in vivo similarities and differences between isc mutants, which have a known function in cluster assembly, and mutants disrupted in four additional loci, gshA, apbC, apbE, and rseC. The latter mutants share similarities with isc mutants: (i) a sensitivity to oxidative stress, (ii) a thiamine auxotrophy in the absence of the YggX protein, and (iii) decreased activities of Fe-S proteins, including aconitase, succinate dehydrogenase, and MiaB. However, they differ from isc mutants by displaying a phenotypic dependence on metals and a distinct defect in the SoxRS response to superoxides. Results presented herein support the proposed role of YggX in iron trafficking and protection against oxidative stress, describe additional phenotypes of isc mutants, and suggest a working model in which the ApbC, ApbE, and RseC proteins and glutathione participate in Fe-S cluster repair.     

Mutagenicity of 7H-dibenzo[c,g]carbazole and its tissue specific derivatives in genetically engineered Chinese hamster V79 cell lines stably expressing cytochrome P450

The biological mechanisms responsible for aging remain poorly understood. We propose that increases in DNA damage and mutations that occur with age result from a reduced ability to repair DNA damage. To test this hypothesis, we have measured the ability to repair DNA damage in vitro by the base excision repair (BER) pathway in tissues of young (4-month-old) and old (24-month-old) C57BL/6 mice. We find in all tissues tested (brain, liver, spleen and testes), the ability to repair damage is significantly reduced (50-75%; P<0.01) with age, and that the reduction in repair capacity seen with age correlates with decreased levels of DNA polymerase beta (beta-pol) enzymatic activity, protein and mRNA. To determine the biological relevance of this age-related decline in BER, we measured spontaneous and chemically induced lacI mutation frequency in young and old animals. In line with previous findings, we observed a three-fold increase in spontaneous mutation frequency in aged animals. Interestingly, lacI mutation frequency in response to dimethyl sulfate (DMS) does not significantly increase in young animals whereas identical exposure in aged animals results in a five-fold increase in mutation frequency. Because DMS induces DNA damage processed by the BER pathway, it is suggested that the increased mutagenicity of DMS with age is related to the decline in BER capacity that occurs with age. The inability of the BER pathway to repair damages that accumulate with age may provide a mechanistic explanation for the well-established phenotype of DNA damage accumulation with age.     

DEHP: genotoxicity and potential carcinogenic mechanisms-a review

Di(ethylhexyl) phthalate (DEHP) is a manufactured chemical commonly added to plastics: it is a ubiquitous environmental contaminant to which humans are exposed through multiple routes. DEHP is a rodent carcinogen with an extensive data base on genotoxicity and related effects spanning several decades. Although DEHP has been reported to be negative in most non-mammalian in vitro mutation assays, most studies were performed under conditions of concurrent cytotoxicity, precipitation, or irrelevant metabolic activation. However, a number of in vitro rodent tissue assays have reported DEHP to be positive for effects on chromosomes, spindle, and mitosis. A robust database shows that DEHP increases transformation and inhibits apoptosis in Syrian hamster embryo cells. In a transgenic mouse assay, in vivo DEHP exposure increased the mutation frequency only in the liver, which is the target organ for cancer. In vitro exposure of human cells or tissues to DEHP induced DNA damage; altered mitotic rate, apoptosis, and cell proliferation; increased proliferation, tumor mobility, and invasiveness of tumor cell lines; and activated a number of nuclear receptors. DEHP has been shown to be an agonist for CAR2, a novel constitutive androstane receptor occurring only in humans. Environmental exposures of humans to DEHP have been associated with DNA damage. After taking into account study context and relevant issues affecting interpretation, in vitro studies reported that a similar DEHP concentration range induced both mutagenic and non-mutagenic effects in human tissues and, using a much more limited rodent database, transformation of embryonic rodent tissues. The human and rodent data suggest that DEHP induces cancer through multiple molecular signals, including DNA damage. The analyses presented here may provide guidance for similar data sets used in structure-activity relationships, computational-toxicology extrapolations, and attempts to extrapolate in vitro results to predict in vivo effects for hazard characterization.     

Nucleophosmin is required for DNA integrity and p19Arf protein stability

Nucleophosmin (NPM) is a nucleolar phosphoprotein that binds the tumor suppressors p53 and p19(Arf) and is thought to be indispensable for ribogenesis, cell proliferation, and survival after DNA damage. The NPM gene is the most frequent target of genetic alterations in leukemias and lymphomas, though its role in tumorigenesis is unknown. We report here the first characterization of a mouse NPM knockout strain. Lack of NPM expression results in accumulation of DNA damage, activation of p53, widespread apoptosis, and mid-stage embryonic lethality. Fibroblasts explanted from null embryos fail to grow and rapidly acquire a senescent phenotype. Transfer of the NPM mutation into a p53-null background rescued apoptosis in vivo and fibroblast proliferation in vitro. Cells null for both p53 and NPM grow faster than control cells and are more susceptible to transformation by activated oncogenes, such as mutated Ras or overexpressed Myc. In the absence of NPM, Arf protein is excluded from nucleoli and is markedly less stable. Our data demonstrate that NPM regulates DNA integrity and, through Arf, inhibits cell proliferation and are consistent with a putative tumor-suppressive function of NPM.     

The Escherichia coli UVM response is accompanied by an SOS-independent error-prone DNA replication activity demonstrable in vitro

UVM is an SOS-independent inducible response characterized by elevated mutagenesis at a site-specific 3, N4-ethenocytosine (epsilonC) residue borne on M13 single-stranded DNA transfected into Escherichia coli cells pretreated with DNA-damaging agents. By constructing and using E. coli strain AM124 (polA polB umuDC dinB lexA1[Ind-]), we show here that the UVM response is manifested in cells deficient for SOS induction, as well as for all four of the 'non-replicative' DNA polymerases, namely DNA polymerase I (polA), II (polB), IV (dinB) and V (umuDC). These results confirm that UVM represents a novel, previously unidentified cellular response to DNA-damaging agents. To address the question as to whether the UVM response is accompanied by an error-prone DNA replication activity, we applied a newly developed in vitro replication assay coupled to an in vitro mutation analysis system. In the assay, circular M13 single-stranded DNA bearing a site-specific lesion is converted to circular double-stranded replicative-form DNA in the presence of cell extracts and nucleotide precursors under conditions that closely mimic M13 replication in vivo. The newly synthesized (minus) DNA strand is selectively amplified by ligation-mediated polymerase chain reaction (LM-PCR), followed by a multiplex sequence analysis to determine the frequency and specificity of mutations. Replication of DNA bearing a site-specific epsilonC lesion by cell extracts from uninduced E. coli AM124 cells results in a mutation frequency of about 13%. Mutation frequency is elevated fivefold (to 58%) in cell extracts from UVM-induced AM124 cells, with C --> A mutations predominating over C --> T mutations, a specificity similar to that observed in vivo. These results, together with previously reported data, suggest that the UVM response is mediated through the induction of a transient error-prone DNA replication activity and that a modification of DNA polymerase III or the expression of a previously unidentified DNA polymerase may account for the UVM phenotype.     

Hotspot sites for acridine-induced frameshift mutations in bacteriophage T4 correspond to sites of action of the T4 type II topoisomerase

The type II topoisomerase of bacteriophage T4 is a central determinant of the frequency and specificity of acridine-induced frameshift mutations. Acridine-induced frameshift mutagenesis is specifically reduced in a mutant defective in topoisomerase activity. The ability of an acridine to promote topoisomerase-dependent cleavage at specific DNA sites in vitro is correlated to its ability to produce frameshift mutations at those sites in vivo. The specific phosphodiester bonds cleaved in vitro are precisely those at which frameshifts are most strongly promoted by acridines in vivo. The cospecificity of in vitro cleavage and in vivo mutation implicate acridine-induced, topoisomerase-mediated DNA cleavages as intermediates of acridine-induced mutagenesis in T4.     

Mutation frequency is reduced in the cerebellum of Big Blue mice overexpressing a human wild type SOD1 gene

Amyotrophic lateral sclerosis (ALS) is a progressive paralytic disorder caused by motor neuron degeneration. A similar disease phenotype is observed in mice overexpressing a mutant human hSOD1 gene (G93A, 1Gurd(1)). Mice transgenic for lacI (Big Blue) and human mutant (1Gurd(1), Mut hSOD1) or wild type (2Gur, Wt hSOD1) SOD1 genes were used to examine spontaneous mutation, oxidative DNA damage, and neurodegeneration in vivo. The frequency and pattern of spontaneous mutation were determined for forebrain (90% glia), cerebellum (90% neurons) and thymus from 5-month-old male mice. Mutation frequency is not elevated significantly and mutation pattern is unaltered in Mut hSOD1 mice compared to control mice. Mutation frequency is reduced significantly in the cerebellum of Wt hSOD1 mice (1.6x10(-5); P=0.0093; Fisher's Exact Test) compared to mice without a human transgene (2.7x10(-5)). Mutation pattern is unaltered. This first report of an endogenous factor that can reduce in vivo, the frequency of spontaneous mutation suggests potential strategies for lowering mutagenesis related to aging, neurodegeneration, and carcinogenesis.     

Escherichia coli RecX inhibits RecA recombinase and coprotease activities in vitro and in vivo

In Escherichia coli the RecA protein plays a pivotal role in homologous recombination, DNA repair, and SOS repair and mutagenesis. A gene designated recX (or oraA) is present directly downstream of recA in E. coli; however, the function of RecX is unknown. In this work we demonstrated interaction of RecX and RecA in a yeast two-hybrid assay. In vitro, substoichiometric amounts of RecX strongly inhibited both RecA-mediated DNA strand exchange and RecA ATPase activity. In vivo, we showed that recX is under control of the LexA repressor and is up-regulated in response to DNA damage. A loss-of-function mutation in recX resulted in decreased resistance to UV irradiation; however, overexpression of RecX in trans resulted in a greater decrease in UV resistance. Overexpression of RecX inhibited induction of two din (damage-inducible) genes and cleavage of the UmuD and LexA repressor proteins; however, recX inactivation had no effect on any of these processes. Cells overexpressing RecX showed decreased levels of P1 transduction, whereas recX mutation had no effect on P1 transduction frequency. Our combined in vitro and in vivo data indicate that RecX can inhibit both RecA recombinase and coprotease activities.     

Protein oxidation implicated as the primary determinant of bacterial radioresistance

In the hierarchy of cellular targets damaged by ionizing radiation (IR), classical models of radiation toxicity place DNA at the top. Yet, many prokaryotes are killed by doses of IR that cause little DNA damage. Here we have probed the nature of Mn-facilitated IR resistance in Deinococcus radiodurans, which together with other extremely IR-resistant bacteria have high intracellular Mn/Fe concentration ratios compared to IR-sensitive bacteria. For in vitro and in vivo irradiation, we demonstrate a mechanistic link between Mn(II) ions and protection of proteins from oxidative modifications that introduce carbonyl groups. Conditions that inhibited Mn accumulation or Mn redox cycling rendered D. radiodurans radiation sensitive and highly susceptible to protein oxidation. X-ray fluorescence microprobe analysis showed that Mn is globally distributed in D. radiodurans, but Fe is sequestered in a region between dividing cells. For a group of phylogenetically diverse IR-resistant and IR-sensitive wild-type bacteria, our findings support the idea that the degree of resistance is determined by the level of oxidative protein damage caused during irradiation. We present the case that protein, rather than DNA, is the principal target of the biological action of IR in sensitive bacteria, and extreme resistance in Mn-accumulating bacteria is based on protein protection.     

Replication and mutagenesis of UV-damaged DNA templates in human and monkey cell extracts.

We have used in vitro DNA replication systems from human HeLa cells and monkey CV-1 cells to replicate a UV-damaged simian virus 40-based shuttle vector plasmid, pZ189. We found that replication of the plasmid was inhibited in a UV fluence-dependent manner, but even at UV fluences which caused damage to essentially all of the plasmid molecules some molecules became completely replicated. This replication was accompanied by an increase (up to 15-fold) in the frequency of mutations detected in the supF gene of the plasmid. These mutations were predominantly G:C-->A:T transitions similar to those observed in vivo. Treatment of the UV-irradiated plasmid DNA with Escherichia coli photolyase to reverse pyrimidine cyclobutane dimers (the predominant UV-induced photoproduct) before replication prevented the UV-induced inhibition of replication and reduced the frequency of mutations in supF to background levels. Therefore, the presence of pyrimidine cyclobutane dimers in the plasmid template appears to be responsible for both inhibition of replication and mutation induction. Further analysis of the replication of the UV-damaged plasmid revealed that closed circular replication products were sensitive to T4 endonuclease V (a pyrimidine cyclobutane dimer-specific endonuclease) and that this sensitivity was abolished by treatment of the replicated DNA with E. coli photolyase after replication but before T4 endonuclease treatment. These results demonstrate that these closed circular replication products contain pyrimidine cyclobutane dimers. Density labeling experiments revealed that the majority of plasmid DNA synthesized in vitro in the presence of bromodeoxyuridine triphosphate was hybrid density whether or not the plasmid was treated with UV radiation before replication; therefore, replication of UV-damaged templates appears to occur by the normal semiconservative mechanism. All of these data suggest that replication of UV-damaged templates occurs in vitro as it does in vivo and that this replication results in mutation fixation.     

Metallothionein-III prevents gamma-ray-induced 8-oxoguanine accumulation in normal and hOGG1-depleted cells

Metallothioneins (MT) play an important biological role in preventing oxidative damage to cells. We have previously demonstrated that the efficiency of the protective effect of MT-III against the DNA degradation from oxidative damage was much higher than that of MT-I/II. As an extension of the latter investigation, this study aimed to assess the ability of MT-III to suppress 8-oxoguanine (8-oxoG), which is one of the major base lesions formed after an oxidative attack to DNA and the mutant frequency of the HPRT gene in human fibroblast GM00637 cells upon exposure to gamma-rays. We found that human MT-III expression decreased the level of 8-oxoG and mutation frequency in the gamma-irradiated cells. Using an 8-oxoguanine DNA glycosylase (OGG1)-specific siRNAs, we also found that MT-III expression resulted in the suppression of the gamma-radiation-induced 8-oxoG accumulation and mutation in the OGG1-depleted cells. Moreover, the down-regulation of MT in human neuroblastoma SKNSH cells induced by MT-specific siRNA led to a significant increase in the 8-oxoG level, after exposure to gamma-irradiation. These results suggest that under the conditions of gamma-ray oxidative stress, MT-III prevents the gamma-radiation-induced 8-oxoG accumulation and mutation in normal and hOGG1-depleted cells, and this suppression might, at least in part, contribute to the anticarcinogenic and neuroprotective role of MT-III.     

Microsome-stimulated activation of ferrous bleomycin in the presence of DNA

The inhibition of Fe(II)-bleomycin activation, by a large excess of DNA, is overcome by rat liver microsomes in the presence of NADPH. This release of inhibition, as indicated by increased yields of base propenal from DNA scission, is enhanced by menadione, is inhibited by superoxide dismutase, and is therefore dependent on superoxide anion. Microsomal activation of Fe(II)-bleomycin doubles the stoichiometry of base propenal yield compared to that obtained upon self-activation of the drug; 0.5 mol of base propenal is formed and 0.5 mol of NADPH is oxidized per mol of Fe(II)-bleomycin. In the presence of a large excess of DNA, Cu(II)-bleomycin is not reduced and Fe(III)-bleomycin is neither reduced nor activated by microsomes in cases where activation of Fe(II)-bleomycin is maximal. We suggest that in vivo, electron transport enzymes at or near the nucleus can stimulate the activation of Fe(II)-bleomycin under conditions where self-activation does not readily occur.     

N(2)-(1-Carboxyethyl)deoxyguanosine, a nonenzymatic glycation adduct of DNA, induces single-strand breaks and increases mutation frequencies.

N(2)-(1-Carboxyethyl)deoxyguanosine (CEdG) is a major nonenzymatic glycation product of DNA. The effect of CEdG modification, which was specifically prepared by incubation with dihydroxyacetone, on plasmid DNA topology was evaluated by gel electrophoresis. A time-dependent decrease of supercoiled plasmid-DNA was observed in parallel to the increase of CEdG adducts; the half-life time of the supercoiled plasmid-DNA was estimated to be approximately 16-18 h. CEdG-modified plasmid DNA showed a 25-fold reduced transformation efficiency. When modified DNA was used to transform Escherichia coli cells, a 6-fold increase in mutation frequency was determined by measuring loss of alpha-complementation. For the mutator strain BMH71-18mutS, an 8-fold increase in mutation frequency was observed. Although the exact mechanism of DNA damage is unclear, the occurrence of spontaneous depurination is likely. These findings suggest that a defined DNA glycation reaction can lead to DNA damage in vivo.     

Ku86 deficiency leads to reduced intrachromosomal homologous recombination in vivo in mice

Ku70 and Ku86 together with DNA-PKcs form the DNA-dependent protein kinase (DNA-PK) complex that is involved in DNA double-strand break repair by nonhomologous end joining. We investigated the effect of Ku86 mutation on intrachromosomal homologous recombination (HR) resulting in deletions in vivo in mice. We quantified such deletion events using a phenotypic pigmentation assay. Deletion of one copy of a 70 kb DNA duplication in the pink-eyed unstable (pun) allele results in reversion to the wildtype pink-eyed dilution (p) gene, allowing black pigment accumulation in cells of the retinal pigment epithelium (RPE). We found that the frequency of homologous recombination was significantly reduced in Ku86 deficient mice. Furthermore, the proliferation of cells in which recombination events occurred was reduced and developmentally delayed in the Ku86 deficient mice. These data indicate a role for Ku86 directly or indirectly in homologous recombination in vivo.     

Joining of linear plasmid DNA is reduced and error-prone in Bloom''s syndrome cells.

A linearized, replicating, shuttle vector plasmid, pZ189, was used to measure in vivo DNA joining ability of cells from patients with the cancer-prone, immunodeficient, chromosome breakage disorder, Bloom's syndrome (BS). The BS cell lines we studied were reported to contain reduced in vitro activity of DNA ligase I. We assessed in vivo joining ability by transfecting linear plasmids with overlapping or blunt ends (produced by EcoRI or StuI) into BS and normal fibroblast or lymphoblast host cells and measuring the amount of re-joined, replicated plasmids by their ability to transform bacteria. With plasmids having either overlapping or blunt ends we found a 1.3- to 3-fold lower (P less than 0.05) joining efficiency in BS cells than in the normal cells. The mutation frequency of the recovered plasmids was measured by screening for function of the suppressor tRNA contained in pZ189, for plasmid size, for presence of restriction sites, or by DNA sequencing. The spontaneous mutation frequency with the circular plasmid was 0.05-0.08% with both BS cell lines, values 2- to 21-fold higher (P less than 0.03) than with the normal cell lines. The mutation frequency with the linear plasmid passaged through both BS cell lines was 21-52%, values 1.4- to 5.4-fold higher (P less than 0.001) than with the normal lines. Detailed analysis of 210 recovered plasmids revealed an increase (P less than or equal to 0.001) in deletions, insertions or complex mutations at the joining sites, and in point mutations with the EcoRI cut plasmid with the BS cells in comparison to the normal cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nickel subsulfide is genotoxic in vitro but shows no mutagenic potential in respiratory tract tissues of BigBlue™ rats and Muta™Mouse mice in vivo after inhalation

Carcinogenic nickel compounds are known to induce promutagenic DNA lesions such as DNA strand breaks and DNA adducts in cultured mammalian cells. In standard mutation assays, in contrast, they were found to be either inactive or weakly active. In our in vitro mutation studies in a lacI transgenic embryonic fibroblast cell line, nickel subsulfide (Ni₃S₂) increased mutation frequency up to 4.5-fold. We subsequently applied the comet assay and transgenic rodent mutation assays to investigate the DNA damaging effect and mutagenic potential of nickel subsulfide in target cells of carcinogenesis. A 2-h in vitro treatment of freshly isolated mouse nasal mucosa and lung cells with nickel subsulfide clearly induced DNA fragmentation in a concentration dependent manner. The strong effect was not seen in the same cell types following inhalative treatment of mice and rats, leading only in the mouse nasal mucosa to high DNA damage. When the same inhalative treatment was applied to lacZ and lacI transgenic mice and rats, the spontaneous mutation frequency of these target genes in the respiratory tissues was not increased. These results support a recently proposed non-genotoxic model of nickel carcinogenesis, which acts through gene silencing via DNA methylation and chromatin condensation. This model may also explain our in vitro mutation data in the lacI transgenic cell line, in which nickel subsulfide increased mutation frequency, but in about one-third of the mutants, molecular analysis did not reveal any DNA sequence change in the coding region of the lacI gene despite of the phenotypic loss of its function.