PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “SOS Response, Genetics”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 397 records · Page 22Linked to original sources
PubMed1996

In vivo stability of the Umu mutagenesis proteins: a major role for RecA.

The Escherichia coli Umu proteins play critical roles in damage-inducible SOS mutagenesis. To avoid any gratuitous mutagenesis, the activity of the Umu proteins is normally kept to a minimum by tight transcriptional and posttranslational regulation. We have, however, previously observed that compared with an isogenic recA+ strain, the steady-state levels of the Umu proteins are elevated in a recA730 background (R. Woodgate and D. G. Ennis, Mol. Gen. Genet. 229:10-16, 1991). We have investigated this phenomenon further and find that another coprotease-constitutive (recA*) mutant, a recA432 strain, exhibits a similar phenotype. Analysis revealed that the increased steady-state levels of the Umu proteins in the recA* strains do indeed reflect an in vivo stabilization of the proteins. We have investigated the basis for the phenomenon and find that the mutant RecA* protein stabilizes the Umu proteins by not only converting the labile UmuD protein to the much more stable (and mutagenically active) UmuD' protein but by directly stabilizing UmuD' itself. In contrast, UmuC does not appear to be directly stabilized by RecA* but is instead dramatically stabilized in the presence of UmuD'. On the basis of these observations, we suggest that formation of a UmuD'C-RecA*-DNA quaternary complex protects the UmuD'C proteins from proteolytic degradation and as a consequence helps to promote the switch from error-free to error-prone mechanisms of DNA repair.

Bacterial Proteins↗
PubMed1993

In vivo evidence that UV-induced C-->T mutations at dipyrimidine sites could result from the replicative bypass of cis-syn cyclobutane dimers or their deamination products.

The major mutations induced by UV light are C-->T transitions at dipyrimidines and arise from the incorporation of A opposite the C of dipyrimidine photoproducts. The incorporation of A has most often been explained by the known preference of a polymerase to do so opposite noninstructional DNA damage such as an abasic site (A rule). There are also mechanisms that suppose, however, that cis-syn dipyrimidine photodimers are instructional. In one such mechanism (tautomer bypass), the incorporation of A is directed by the tautomer of a C of a dimer that is equivalent in base-pairing properties to U [Person et al. (1974) Genetics 78, 1035-1049]. In another mechanism (deamination bypass), the incorporation of A is directed by a U of a dimer that results from the deamination of the C of a dimer [Taylor & O'Day (1990) Biochemistry 29, 1624-1632]. The viability of these mechanisms was tested by obtaining the mutation spectrum of a TU dimer in Escherichia coli by application of a standard method for site-directed mutagenesis. To this end, a 41-mer containing a site-specific TU dimer was constructed via ligation of a dimer-containing decamer that was produced by triplet-sensitized irradiation and used to prime DNA synthesis on a uracil-containing (+) strand of an M13 clone containing a double mismatch opposite the dimer. The reaction mixture was used to transfect a uracil glycosylase proficient, photoproduct repair deficient E. coli host, and all progeny phage weakly hybridizing to the parental (+) or (-) strands were sequenced. Under non-SOS conditions the TU dimer almost completely blocked replication, while under SOS conditions it directed the incorporation of two As with much higher specificity (96%) than would an abasic site. The implications of these results to the mechanism of the UV-induced TC-->TT mutation, and by extension to the CT-->TT, CC-->TC, CC-->CT, and the tandem CC-->TT mutations, are discussed.

Amination↗
PubMed1996

Genetic evidence for an activator required for induction of colicin-like bacteriocin 28b production in Serratia marcescens by DNA-damaging agents.

Bacteriocin 28b production is induced by mitomycin in wild-type Serratia marcescens 2170 but not in Escherichia coli harboring the bacteriocin 28b structural gene (bss). Studies with a bss-lacZ transcriptional fusion showed that mitomycin increased the level of bss gene transcription in S. marcescens but not in the E. coli background. A S. marcescens Tn5 insertion mutant was obtained (S. marcescens 2170 reg::Tn5) whose bacteriocin 28b production and bss gene transcription were not increased by mitomycin treatment. Cloning and DNA sequencing of the mutated region showed that the Tn5 insertion was flanked by an SOS box sequence and three genes that are probably cotranscribed (regA, regB, and regC). These three genes had homology to phage holins, phage lysozymes, and the Ogr transcriptional activator of P2 and related bacteriophages, respectively. Recombinant plasmid containing this wild-type DNA region complemented the reg::Tn5 regulatory mutant. A transcriptional fusion between a 157-bp DNA fragment, containing the apparent SOS box upstream of the regA gene, and the cat gene showed increased chloramphenicol acetyltransferase activity upon mitomycin treatment. Upstream of the bss gene, a sequence similar to the consensus sequence proposed to bind Ogr protein was found, but no sequence similar to an SOS box was detected. Our results suggest that transcriptional induction of bacteriocin 28b upon mitomycin treatment is mediated by the regC gene whose own transcription would be LexA dependent.

Amino Acid Sequence↗
PubMed1989

General principles of DNA repair in microorganisms and implications for future research.

The analysis of DNA repair and mutagenesis in organisms that have high resolution genetic and molecular biological systems has led, and will continue to lead, to major advances in our understanding of these processes. It is striking that almost all of the major insights highlighted in this chapter have been gained within the last 10 years and, in fact, several of the discoveries described here occurred during the actual writing of this report. It is worth reflecting that in the mid 1970s there was a widely held view that the essential elements of DNA repair in E. coli were well understood and, at that time, a considerable number of investigators shifted their focus to systems that did not offer the high resolution genetic and molecular biologic tools of E. coli and S. cerevisiae. The remarkable progress achieved over the past ten years underscores the limitations of that viewpoint. It is difficult, and probably unwise, to pinpoint specific issues in DNA repair in E. coli and S. cerevisiae as special targets for future research since so much remains to be learned. Most of the striking discoveries outlined in this chapter have come from skilled investigators using the full range of high resolution genetic, molecular biologic, and biochemical tools to address fundamental problems in DNA repair and mutagenesis. In most cases, it would have been impossible to have predicted the critical discoveries that have been made. For example, UV and chemical mutagenesis require the induction of a pair of genes, mediated by DNA-damage-stimulated proteolytic cleavage of a repressor and then the subsequent activation of one of these gene products by a mechanistically related proteolytic cleavage. In 1975 an hypothesis proposing such a mechanism for UV and chemical mutagenesis would have been dismissed as being utterly fanciful. Yet this is the current reality. As in the cases of the other findings detailed here, the key insights came not from the testing of fanciful hypotheses but rather from systematic scientific studies of fundamental problems. Funding in this area should then support inquiries of fundamental issues in DNA repair and mutagenesis by investigators who are skilled at using the full range of sophisticated experimental tools available for these organisms. It is worth noting that supporting this type of research will shed light not only on mechanisms of DNA repair and mutagenesis but also on more fundamental issues in the biology of living organisms.(ABSTRACT TRUNCATED AT 400 WORDS)

Adaptation, Physiological↗
PubMed1992

Isolation of DNA damage-inducible promoters in Escherichia coli: regulation of polB (dinA), dinG, and dinH by LexA repressor.

A new genetic screening method has been developed to isolate Escherichia coli promoters which are components of the SOS regulon. Plasmids containing the regulatory regions of polB (dinA) and two new loci, dinG and dinH, were characterized. Galactokinase gene fusion experiments indicated that transcription of these genes is inducible by treatment with mitomycin and conforms to a classical model of SOS regulation involving simple LexA repression. Mapping studies using the E. coli DNA library of Kohara et al. (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987) revealed that dinG and dinH are located at 17.8 and 19.8 min on the chromosome, respectively. The nucleotide sequence of the dinH regulatory region contains a segment which is very similar to previously characterized binding sites for LexA protein. An asymmetric, noncanonical 20-bp LexA operator in the cloned dinG promoter region was identified. Additional experiments have revealed that the nucleotide sequence of the gene immediately downstream of the DNA damage-inducible polB locus encodes a polypeptide which has extensive sequence homology to several known and putative DNA and RNA helicase proteins. This gene, which is not regulated by the LexA repressor, has been designated hepA. The predicted amino acid sequence of the product of hepA contains several highly conserved sequence motifs that are also found in enzymes such as the RecQ and UvrB proteins of E. coli and the Rad3 protein of Saccharomyces cerevisiae.

Amino Acid Sequence↗
PubMed1988

Excision repair of thymine glycols, urea residues, and apurinic sites in Escherichia coli.

The genetic requirements for the excision repair of thymine glycols, urea residues, and apurinic (AP) sites were examined by measuring the survival in Escherichia coli mutants of phi X174 replicative form (RF) I transfecting DNA containing selectively introduced lesions. phi X RF I DNA containing thymine glycols was inactivated at a greater rate in mutants deficient in endonuclease III (nth) than in wild-type hosts, suggesting that endonuclease III is involved in the repair of thymine glycols in vivo. phi X RF I DNA containing thymine glycols was also inactivated at a greater rate in mutants that were deficient in both exonuclease III and endonuclease IV (xth nfo) than in wild-type hosts, suggesting that a class II AP endonuclease is required for the in vivo processing of thymine glycols. phi X duplex-transfecting DNA containing urea residues or AP sites was inactivated at a greater rate in xth nfo double mutants than in wild-type, but not single-mutant, hosts, suggesting that exonuclease III or endonuclease IV is required for the repair of these damages and that either activity can substitute for the other. These data are in agreement with the known in vitro substrate specificities of endonuclease III, exonuclease III, and endonuclease IV.

Coliphages↗
PubMed2000

Identification of mucAB-like homologs on two IncT plasmids, R394 and Rts-1.

Recent phylogenetic analysis of the superfamily of lesion-replicating DNA polymerases suggest that they can be broadly divided into four sub-groups comprised of UmuC-like, DinB-like, Rev1-like and Rad30-like proteins. The UmuC-like sub-family is best characterized at the genetic level and sequence analysis of eleven umu orthologs, residing on bacterial chromosomes or on self-transmissible R-plasmids allows further subdivision into five sub-groups (UmuDC, MucAB, ImpAB, RumAB and RulAB) based on amino acid sequence conservation. Some of these orthologs are apparently inactive in situ, but may promote increased mutagenesis and survival when subcloned and expressed from high-copy number plasmids. We were, therefore, interested in devising an assay that would identify umuC-like genes in situ in the absence of a functional assay. To this end, degenerate primers directed towards conserved amino acid regions within the UmuC-like sub-family of DNA polymerases were designed and used to identify mucAB-like operons on the IncT plasmids, R394 and Rts-1.Interestingly, DNA sequence analysis of an approximately 7kb region of R394 identified two LexA-regulated genes immediately downstream of mucAB((R394)) that are similar to the chromosomally-encoded Escherichia coli tus gene and the IncI plasmid-encoded impC gene, respectively. Analysis of the R394 and Rts-1 mucB genes revealed that both contain insertions which result in the expression of a truncated inactive MucB protein. While R394 was unable to restore mutagenesis functions to a DeltaumuDC E. coli strain, Rts-1 surprisingly promoted significant levels of MMS-induced SOS mutagenesis, raising the possibility that Rts-1 encodes another, yet unidentified, umu-like homolog.

Amino Acid Sequence↗
PubMed1989

Volatile nitrosamine levels and genotoxicity of food samples from high-risk areas for nasopharyngeal carcinoma before and after nitrosation.

Traditional life-style, especially food habits, infection by Epstein-Barr virus (EBV) and genetic factors, have been associated with an increased risk of nasopharyngeal carcinoma (NPC). N-Nitroso compounds and other carcinogens either present in food or formed endogenously, as well as food constituents that activate EBV, have been suspected as etiological factors in NPC pathogenesis. For their characterization preserved food items, frequently consumed in NPC endemic areas in Tunisia, South China and Greenland, were sampled and screened for the presence of mutagens and volatile nitrosamines before and after nitrosation. Aqueous extracts as well as 2 organic extracts of the samples were assayed for genotoxicity in 2 Salmonella typhimurium strains and the SOS chromotest. The same extracts had previously been analyzed for volatile nitrosamines and for EBV-activating substances in Raji cells. In our study, 13 out of 16 food samples showed a weak, directly-acting genotoxicity in the SOS chromotest in at least one of the extracts, but only one sample from Greenland was found to be weakly mutagenic in Salmonella TA 98. Chemical nitrosation for 9 out of 15 samples of aqueous food extracts increased the genotoxic effect in the SOS chromotest. Levels of volatile nitrosamines were also elevated for 12 out of 15 samples; highest levels of N-nitrosodimethylamine were found in hard salted and dried fish from China (1,200 micrograms/kg) and highest N-nitrosopyrrolidine levels in a Tunisian spice (3,840 micrograms/kg). In non-nitrosated aqueous food extracts, the level of volatile nitrosamines and genotoxic activities were not correlated with the EBV-inducing activity of the same samples. After chemical nitrosation, EBV-inducing activity was decreased or showed no change and was not correlated with increases in either the genotoxicity or the nitrosamine levels. Our results suggest that EBV-activating compounds belong to a different class of substances. However, there was an association between the changes in genotoxicity and nitrosamine levels due to nitrosation.

Carcinoma↗
PubMed2004

AtHKT1 facilitates Na+ homeostasis and K+ nutrition in planta.

Genetic and physiological data establish that Arabidopsis AtHKT1 facilitates Na(+) homeostasis in planta and by this function modulates K(+) nutrient status. Mutations that disrupt AtHKT1 function suppress NaCl sensitivity of sos1-1 and sos2-2, as well as of sos3-1 seedlings grown in vitro and plants grown in controlled environmental conditions. hkt1 suppression of sos3-1 NaCl sensitivity is linked to higher Na(+) content in the shoot and lower content of the ion in the root, reducing the Na(+) imbalance between these organs that is caused by sos3-1. AtHKT1 transgene expression, driven by its innate promoter, increases NaCl but not LiCl or KCl sensitivity of wild-type (Col-0 gl1) or of sos3-1 seedlings. NaCl sensitivity induced by AtHKT1 transgene expression is linked to a lower K(+) to Na(+) ratio in the root. However, hkt1 mutations increase NaCl sensitivity of both seedlings in vitro and plants grown in controlled environmental conditions, which is correlated with a lower K(+) to Na(+) ratio in the shoot. These results establish that AtHKT1 is a focal determinant of Na(+) homeostasis in planta, as either positive or negative modulation of its function disturbs ion status that is manifested as salt sensitivity. K(+)-deficient growth of sos1-1, sos2-2, and sos3-1 seedlings is suppressed completely by hkt1-1. AtHKT1 transgene expression exacerbates K(+) deficiency of sos3-1 or wild-type seedlings. Together, these results indicate that AtHKT1 controls Na(+) homeostasis in planta and through this function regulates K(+) nutrient status.

Arabidopsis↗
PubMed1999

Mutations affecting the ability of the Escherichia coli UmuD' protein to participate in SOS mutagenesis.

The products of the SOS-regulated umuDC operon are required for most UV and chemical mutagenesis in Escherichia coli, a process that results from a translesion synthesis mechanism. The UmuD protein is activated for its role in mutagenesis by a RecA-facilitated autodigestion that removes the N-terminal 24 amino acids. A previous genetic screen for nonmutable umuD mutants had resulted in the isolation of a set of missense mutants that produced UmuD proteins that were deficient in RecA-mediated cleavage (J. R. Battista, T. Ohta, T. Nohmi, W. Sun, and G. C. Walker, Proc. Natl. Acad. Sci. USA 87:7190-7194, 1990). To identify elements of the UmuD' protein necessary for its role in translesion synthesis, we began with umuD', a modified form of the umuD gene that directly encodes the UmuD' protein, and obtained missense umuD' mutants deficient in UV and methyl methanesulfonate mutagenesis. The D39G, L40R, and T51I mutations affect residues located at the UmuD'2 homodimer interface and interfere with homodimer formation in vivo. The D75A mutation affects a highly conserved residue located at one end of the central strand in a three-stranded beta-sheet and appears to interfere with UmuD'2 homodimer formation indirectly by affecting the structure of the UmuD' monomer. When expressed from a multicopy plasmid, the L40R umuD' mutant gene exhibited a dominant negative effect on a chromosomal umuD+ gene with respect to UV mutagenesis, suggesting that the mutation has an effect on UmuD' function that goes beyond its impairment of homodimer formation. The G129D mutation affects a highly conserved residue that lies at the end of the long C-terminal beta-strand and results in a mutant UmuD' protein that exhibits a strongly dominant negative effect on UV mutagenesis in a umuD+ strain. The A30V and E35K mutations alter residues in the N-terminal arms of the UmuD'2 homodimer, which are mobile in solution.

Bacterial Proteins↗
PubMedDate not supplied

Mechanisms of mutagenesis in mammalian cells. Application to human thyroid tumours.

Mutations are defined as stable and irreversible modifications of the normal genetic message due to small changes in the number or type of bases, or to large modifications of the genome such as deletions, insertions or chromosome rearrangements. These lesions are due to either polymerase errors during normal DNA replication or unrepaired DNA lesions, which will give rise to mutations through a mutagenic pathway. The molecular process leading to mutagenesis depends largely on the type of DNA lesions. Base modifications, such as 8-oxo-guanine or thymine glycol, both induced by ionizing radiations (IR), are readily replicated leading to direct mutations, usually base-pair substitutions. The 8-oxo-G gives rise predominantly to G to T transversions, the type of mutations found in ras or p53 gene from IR-induced tumors. Bulky adducts produced by chemical carcinogens or UV-irradiation are usually repaired by the nucleotide excision repair (NER) pathway which is able to detect structural distortion in the normal double-strand DNA backbone. These lesions represent a blockage to DNA and RNA polymerases as well as some signal for p53 accumulation in the damaged cell. In the absence of repair, these lesions could be eventually replicated owing to the induction of specific proteins at least in bacteria during the SOS process. The precise nature of the error-prone replication across an unexcised DNA lesion in the template is not fully understood in detailed biochemical terms, in mammalian cells. IR basically produce a very large number of DNA lesions from unique base modifications to single- or double-strand breaks and even complex DNA lesions due to the passage of very high energy particles or to a local re-emission of numerous radicals. The breakage of the double-helix is a difficult lesion to repair. Either it will result in cell death or, after an incorrect recombinational pathway, it will induce frameshifts, large deletions or chromosomal rearrangements. Most of the IR-induced mutations are recessive ones, requiring therefore a second genetic event in order to exhibit any harmful effect and a long latency period before the development of a radiation-induced tumor. The fact that IR essentially induced deletions and chromosomal translocations renders very difficult the use of the p53 gene as a marker for mutation analysis. In agreement with the type of lesions induced by IR, it is interesting to point out that the presence has been observed, in a vast majority of radiation-induced papillary thyroid carcinomas (PTC), of an activated ret proto-oncogene originated by the fusion of the tyrosine kinase 3' domain of this gene with the 5' domain of four different genes. These ret chimeric genes which are due to intra- or inter-chromosomal translocations, were called RET/PTC1 to PTC5. The RET/PTC rearrangements were found in PTC from children contaminated by the Chernobyl fall-out as well as in tumours from patients with a history of therapeutic external radiation, with a frequency of 60-84%. This frequency was only 15% in 'spontaneous' PTC. The type of ret chimeric gene predominantly originated by the accidental or therapeutic IR was different. Indeed, PTC1 was present in 75% of the tumours linked to a therapeutic radiation and PTC3 in 75% of the Chernobyl ones. The other forms of RET/PTC were observed in only a minority of the post-Chernobyl PTC (< 20%). The difference in the frequency of PTC1 and PTC3 in both types of PTC, is statistically significant (P < 10(-5), Fischer's exact test). In two of the post-therapeutic radiation PTC, RET/PTC1 and PTC3 were simultaneously present. A PTC1 gene was also observed in 45% of the adenomas appearing after therapeutic radiation. The long-period of latency between exposure to IR and the appearance of thyroid tumours is probably due to the conversion of a heterozygote genotype of IR-induced mutations to a homozygote one. It will be interesting to use this time lag in accidental or therapeutic-irradiated p

DNA Damage↗
PubMed1996

Comparing the presence, potency, and potential hazard of genotoxins extracted from a broad range of industrial effluents.

We examined the genotoxicity of dichloromethane extracts from 50 final effluent samples collected from 42 industries, including pulp and paper, chemical manufacturing, metal refining, metal surface treatment, and municipal waste water treatment. Effluents were initially fractionated into dissolved substances, and substances adsorbed to suspended particulate matter. Acid/base partitioning was used to further fractionate aqueous extracts. Genotoxicity of extracts was found to be related to sample type, industry type, metabolic activation status, and extract fluorescence (380 nm excitation, 430 nm emission). S9 metabolic activation reduced genotoxic potency in over 90% of the extracts examined. Expression of potency values per equivalent unit of original sample revealed that effluent particulate matter is, on average, almost four orders of magnitude more potent than aqueous filtrates. Suspended particulate matter from organic and inorganic chemical production, petroleum and metal refining, and from metal surface treatment facilities, provided extracts that were significantly more genotoxic than those from sewage treatment and pulp and paper facilities. Aqueous filtrates from inorganic and organic chemical production, metal refining, and surface treatment facilities were significantly more genotoxic than those emitted by aluminum and petroleum refineries. Overall, the results suggest that pulp and paper mills emit mostly soluble genotoxins, while petroleum and aluminum refineries emit predominantly particle-associated genotoxins. Although some extracts elicited a strong SOS response, the potency of the extractable residues was low when compared to highly potent pure substances such as benzo(alpha) pyrene. On average, a mg of dichloromethane-extractable residue has an SOS genotoxicity equivalent to 0.1-1.0 microg of benzo(alpha) pyrene. Predicted Ames mutagenic potency values corresponded reasonably well with industrial waste mutagenic potency values corresponded reasonably well with industrial waste mutagenic potency values published by other researchers. Genotoxic loading values were calculated to quantify the total daily genotoxic emission and potential hazard of each industry. Highest loadings were from sewage treatment, pulp and paper, and metal refining facilities. Highest loading values were the SOS genotoxic equivalent of over 30 kg of benzo(alpha)pyrene per day. The ultimate hazard of genotoxic emissions is not known. Actual hazard assessment is complicated by a poor understanding of the postemission behavior of genotoxins. Exposure of downstream biota is likely substantial.

Analysis of Variance↗
PubMed1996

Sorption of organic genotoxins to particulate matter in industrial effluents.

In an earlier work [White PA et al. (1996): Environ Mol Mutagen 27:116-139] we examined the genotoxicity of dichloromethane extracts from a variety of industrial effluent samples. in this companion work, we used the SOS Chromotest to investigate the sorption of the extracted genotoxins to effluent suspended particulate matter. The affinity of the genotoxins for particulate matter is expressed as a genotoxicity sorption partition coefficient (Kd-genotox). The results indicate that industries known for their emission of combustion by-products, such as polycyclic aromatic hydrocarbons, often have high Kd-genotox values (>/= 10(6). These include metal refining and founding industries as well metal surface treatment facilities. In contrast, Kd-genotox values for pulp and paper mills and sewage treatment facilities are several orders of magnitude lower (</= 10(4)). In several cases the calculated Kd-genotox values are in agreement with the Kow values of genotoxic substances isolated from genotoxic industrial waste samples studied by other researchers. The sorption partition coefficient, in conjunction with concentration of available particulate matter, was used to determine the percent of organic genotoxins adsorbed to effluent suspended particulate matter. Values range from 2.3% to 99.8%. High values (>70%) were obtained for metal surface treatment and inorganic and organic chemical production facilities. Low values (>30%) were obtained for sewage treatment facilities and pulp and paper mills. The results also demonstrate the effect of variations in the concentration of available particulate matter on the genotoxicity of both aqueous and particulate extracts. The results suggest that the sorptive properties of the particulate matter itself are reduced when the concentration of particulate matter is very high (>1,000 mg per 1). The use of sorption partition information in inferring the physical-chemical nature of the putative genotoxins and the implications of the results for assessing the hazard posed to aquatic biota by industrial genotoxins are discussed.

Adsorption↗
PubMed1997

Activation and detoxification of dinitropyrenes by cytosol and microsomes from Aroclor-pretreated rats in the Ames and umu assays.

1,3-, 1,6-, and 1,8-Dinitropyrene (1,3-, 1,6-, and 1,8-DNP) are direct-acting mutagens in that they do not require an exogenous source of enzymes for activation to mutagens in the Ames assay. However, the addition of mammalian S9 preparations, or the microsomal and cytosolic compartments comprising S9, modulate the mutagenic response of these DNPs. In this study, we compared the mutagenic response of these DNPs in the presence of cytosol and microsomal fractions from the liver of Aroclor-pretreated (AR) and control rats, in the Ames mutagenicity assay and umu gene induction assay. 1,3- and 1,8-DNP were deactivated to a greater extent by microsomes from AR-induced and control rats than was 1,6-DNP, in both the umu and Ames assays. In the Ames assay, S9 was more potent in deactivating the DNP than an equivalent concentration of microsomes from the same S9 preparation. Also, S9 from AR-pretreated rats deactivated the isomers to a greater extent than S9 from control rats. In contrast to the constant deactivation of all the isomers in the two assays catalyzed by microsomes and S9, the response with cytosol from AR-pretreated rats differed with respect to the three isomers in the Ames and umu assays. When cytosol from AR-treated rats was added, the mutagenicity of 1,3- and 1,6-DNP, but not 1,8-DNP, was significantly (P < 0.05) increased in the Ames assay while the mutagenicity of the three DNPs was increased in the umu assay. Also, a biphasic response was observed in the umu assay with 1,6- and 1,8-DNP, in that AR-cytosol enhanced the mutagenicity at low protein concentrations (5-50 micrograms protein/reaction) but abrogated the response at higher protein concentrations. The effect of cytosol from control rats depended on the isomer tested; 1,3-DNP was activated above the background level in both assays (nearly towfold) while 1,6-DNP and 1,8-DNP were only activated at low protein concentrations in the umu assay. In the Ames assay, cytosol from AR-pretreated rats did not alter the mutagenic response with 1,8-DNP, while control cytosol significantly (P < 0.05) deactivated 1,8-DNP at all substrate concentrations tested. In summary, this study showed that the mutagenicity of 1,3-DNP was similar in the two assays but the responses with 1,6- and 1,8-DNP differed in the two assays. These isomeric differences could be due to the varying metabolic pathways of the three DNPs as well as the detectable end points of the two assays.

Animals↗
PubMed2000

Oxidative mutagenesis in Escherichia coli strains lacking ROS-scavenging enzymes and/or 8-oxoguanine defenses.

Escherichia coli strains with different combinations of null mutations in the katG, katE, sodA, sodB, fpg, and mutY genes were constructed to compare their spontaneous mutation frequencies and sensitivities to various oxidants with those of bacteria solely deficient in catalase (katG katE) or cytosolic superoxide dismutase (sodA sodB) and the parental strain possessing a full complement of these enzymes. The MutY DNA glycosylase represented the major protection against the mutagenic consequences of processes associated with normal aerobic metabolism. Spontaneous mutagenesis in MutY-lacking bacteria was not influenced by the absence of (A)BC excinuclease or the presence of MucAB proteins, a result consistent with 8-oxoguanine being a principal premutational lesion. In contrast, catalase and SOD represented the major protection against the genotoxic consequences of bursts of oxidative stress caused by reactive-oxygen-generating compounds. Therefore, only bacteria simultaneously defective in both katG and katE or sodA and sodB genes were hypersensitive with respect to mutability by peroxide and superoxide, respectively. These data suggest that oxidative lesions other than 8-oxoguanine contribute to mutagenesis by hydrogen peroxide and redox-cycling chemicals.

DNA Repair↗
PubMed2002

The "tale" of UmuD and its role in SOS mutagenesis.

Recently, the Escherichia coli umuD and umuC genes have been shown to encode E. coli's fifth DNA polymerase, pol V (consisting of a heterotrimer of UmuD'(2)C). The main function of pol V appears to be the bypass of DNA lesions that would otherwise block replication by pols I-IV. This process is error-prone and leads to a striking increase in mutations at sites of DNA damage. While the enzymatic properties of pol V are now only beginning to be fully appreciated, a great deal is known about how E. coli regulates the intracellular levels of the Umu proteins so that the lesion-bypassing activity of pol V is available to help cells survive the deleterious consequences of DNA damage, yet keeps any unwarranted activity on undamaged templates to a minimum. Our review summarizes the multiple restrictions imposed upon pol V, so as to limit its activity in vivo and, in particular, highlights the pivotal role that the N-terminal tail of UmuD plays in regulating SOS mutagenesis.

ATP-Dependent Proteases↗