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PubMed1985

Recognition of the structural distortions at the junctions between B and Z segments in negatively supercoiled DNA by osmium tetroxide.

It has been shown for the first time that conformational junction between contiguous right-handed B and left-handed Z segments can be recognized by a chemical probe. Plasmid pRW751 containing (dC-dG)13 and (dC-dG)16 blocks was treated with osmium tetroxide, pyridine (a reagent known to be single-strand selective) at physiological ionic conditions (0.1 and 0.2 M NaCl) and neutral pH. Mapping of the osmium binding sites by restriction enzyme digestion followed by nuclease S1 cleavage has revealed selective binding of osmium at, or near to, the end of the (dC-dG)n segments proximal to the 95 bp lac sequence. The junction of the shorter (dC-dG)13 segment was modified to a substantially greater extent than that of the longer segment. Partial inhibition of DNA cleavage by BamHI was observed at the restriction sites neighbouring to the both (dC-dG)n segments as a result of DNA modification by osmium tetroxide. The site-selective modification occurred only in supercoiled and not in relaxed molecules. Differences in the sensitivity of the B/Z junctions in pRW751 to the osmium tetroxide were explained by different structural features of these junctions.

Binding Sites↗

PubMed2005

Photochemoprevention of ultraviolet B signaling and photocarcinogenesis.

Exposure to solar radiation, particularly its ultraviolet (UV) B component, has a variety of harmful effects on human health. Some of these effects include sunburn cell formation, basal and squamous cell cancers, melanoma, cataracts, photoaging of the skin, and immune suppression. Amongst these various adverse effects of UV radiation, skin cancer is of the greatest concern. Over the years, changes in lifestyle has led to a significant increase in the amount of UV radiation that people receive, and this consequently has led to a surge in the incidence of skin cancer. The development of skin cancer is a complex multistage phenomenon involving three distinct stages exemplified by initiation, promotion and progression stages. Each of these stages is mediated via alterations in various cellular, biochemical, and molecular changes. Initiation, the first step in the carcinogenesis process is essentially an irreversible step in which genetic alterations occur in genes that ultimately leads to DNA modification and fixation of mutation. Tumor promotion is the essential process in cancer development involving clonal expansion of initiated cells giving rise to pre-malignant and then to malignant lesions, essentially by alterations in signal transduction pathways. Tumor progression involves the conversion of pre-malignant and malignant lesions into an invasive and potentially metastatic malignant tumor. All these processes for skin cancer development involve stimulation of DNA synthesis, DNA damage and proliferation, inflammation, immunosuppression, epidermal hyperplasia, cell cycle dysregulation, depletion of antioxidant defenses, impairment of signal transduction pathways, induction of cyclooxygenase, increase in prostaglandin synthesis, and induction of ornithine decarboxylase. Photochemoprevention has been appreciated as a viable approach to reduce the occurrence of skin cancer and in recent years, the use of agents, especially botanical antioxidants, present in the common diet and beverages consumed by human population have gained considerable attention as photochemopreventive agents for human use. Many such agents have also found a place in skin care products. Although this is more common in oriental countries, its popularity is significantly growing in western countries. In this article, we have summarized the available information of laboratory studies on UVB-mediated signaling that can be exploited as targets for photochemoprevention. We suggest that the use of skin care products supplemented with proven chemopreventive agents in conjunction with the use of sunscreens along with educational efforts may be an effective strategy for reducing UV-induced photodamage and skin cancer in humans. The mechanistic basis for the use of such products is discussed.

Chemoprevention↗

PubMed1999

Estrogen, DNA damage and mutations.

Estrogen administration to rodents results in various types of DNA damage and ultimately leads to tumors in estrogen-responsive tissues. Yet these hormones have been classified as nonmutagenic, because they did not induce mutations in classical bacterial and mammalian mutation assays. In this review, we have discussed the induction by estrogens of DNA and chromosomal damage and of gene mutations, because the classical assays were designed to uncover mutations only at one specific locus and could not have detected other types of mutations or changes in other genes. Various types of estrogen-induced DNA damage include: (a) direct covalent binding of estrogen quinone metabolites to DNA; (b) enhancement of endogenous DNA adducts by chronic estrogen exposure of rodents; (c) free radical generation by metabolic redox cycling between quinone and hydroquinone forms of estrogens and free radical damage to DNA such as strand breakage, 8-hydroxylation of purine bases of DNA and lipid hydroperoxide-mediated DNA modification. Two different types of chromosomal damage have also been induced by estrogen in vivo and in cells in culture such as numerical chromosomal changes and also structural chromosomal aberrations. Gene mutations have been induced in several cell types in culture either by the parent estrogen or by reactive estrogen quinone metabolites. Furthermore, in estrogen-induced kidney tumors in hamsters, several mutations have been observed in the DNA polymerase beta gene mRNA. Estradiol also induces microsatellite instability in these kidney tumors and in premalignant kidney exposed to estradiol. Although this work is still ongoing, it can be concluded that estrogens are complete carcinogens capable of tumor initiation by mutation potentially in critical genes. The hormonal effects of estrogens may complete the development of tumors.

Animals↗

PubMed2001

The contribution of UDP-glucuronosyltransferase 1A9 on CYP1A2-mediated genotoxicity by aromatic and heterocyclic amines.

The importance of environmental and dietary arylamines, and heterocyclic amines in the etiology of human cancer is of growing interest. These pre-carcinogens are known to undergo bioactivation by cytochrome P450 (CYP)-directed oxidation, which then become substrates for the UDP-glucuronosyltransferases (UGTs). Thus, glucuronidation may contribute to the elimination of CYP-mediated reactive intermediate metabolites, preventing a toxic event. In this study, human UGTs were analyzed for their ability to modulate the mutagenic actions of N-hydroxy-arylamines formed by CYP1A2. Studies with recombinant human UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7 and UGT2B15 expressed in heterologous cell culture confirmed that UGT1A9 glucuronidated the mutagenic arylamines N-hydroxy-2-acetylaminofluorene (N-hydroxy-2AAF) and 2-hydroxyamino-1-methyl-6-phenylimidazo(4,5-b)pyridine (N-hydroxy-PhIP). To examine the mutagenic potential of these agents, a genotoxicity assay was employed using Salmonella typhimurium NM2009, a bacterial strain expressing the umuC SOS response gene fused to a beta-galactosidase reporter lacZ gene. DNA modification results in the induction of the umuC gene and subsequent enhancement of beta-galactosidase activity. Both N-hydroxy-2AAF and N-hydroxy-PhIP stimulated a dose-dependent increase in bacterial beta-galactosidase activity. In addition, the procarcinogens 2AAF and PhIP were efficiently bioactivated to bacterial mutagens when incubated with Escherichia coli membranes expressing CYP1A2 and NADPH reductase. CYP1A2 generated 2AAF- and PhIP-mediated DNA damage, but only the action of N-hydroxy-2AAF was blocked by expressed UGT1A9. These results indicate that UGT1A9 can control the outcome of a genotoxic response. The results also indicate that while a potential toxicant such as N-hydroxy-PhIP can serve as substrate for glucuronidation, its biological actions can exceed the capacity of the detoxification pathway to prevent the mutagenic episode.

2-Acetylaminofluorene↗

PubMed2005

Tissue-specific attenuation of endogenous DNA I-compounds in rats by carcinogen azoxymethane: possible role of dietary fish oil in colon cancer prevention.

I-compounds are bulky covalent DNA modifications that are derived from metabolic intermediates of nutrients. Some I-compounds may play protective roles against cancer, aging, and degenerative diseases. Many carcinogens and tumor promoters significantly reduce I-compound levels gradually during carcinogenesis. Colon cancer is the second leading cause of cancer death in the United States, whereas cancer of the small intestine is relatively rare. Here we have studied levels of I-compounds in DNA of colon and duodenum of male Sprague-Dawley rats treated with azoxymethane. The effects of dietary lipids (fish oil or corn oil) on colon and duodenal DNA I-compounds were also investigated. Rats fed a diet containing fish oil or corn oil were treated with 15 mg/kg azoxymethane. Animals were terminated 0, 6, 9, 12, or 24 hours after injection. I-compound levels were analyzed by the nuclease P1-enhanced (32)P-postlabeling assay. Rats treated with azoxymethane displayed lower levels of I-compounds in colon DNA compared with control groups (0 hour). However, I-compound levels in duodenal DNA were not diminished after azoxymethane treatment. Animals fed a fish oil diet showed higher levels of I-compounds in colonic DNA compared with corn oil groups (mean adduct levels for fish and corn oil groups were 13.35 and 10.69 in 10(9) nucleotides, respectively, P = 0.034). Taken together, these results support claims that fish oil, which contains a high level of omega-3 polyunsaturated fatty acids, may have potent chemopreventive effects on carcinogen-induced colon cancer. The fact that duodenal I-compounds were not diminished by azoxymethane treatment may have been due to the existence of tissue-specific factors protecting against carcinogenesis. In conclusion, our observations show that endogenous DNA adducts may serve not only as sensitive biomarkers in carcinogenesis and cancer prevention studies, but are also helpful to further our understanding of the chemopreventive properties of omega-3 fatty acids and mechanisms of carcinogenesis.

Analysis of Variance↗

PubMed2005

Qualitatively predicting acetylation and methylation areas in DNA sequences.

Eukaryotic genomes are packaged by the wrapping of DNA around histone octamers to form nucleosomes. Nucleosome occupancy, acetylation, and methylation, which have a major impact on all nuclear processes involving DNA, have been recently mapped across the yeast genome using chromatin immunoprecipitation and DNA microarrays. However, this experimental protocol is laborious and expensive. Moreover, experimental methods often produce noisy results. In this paper, we introduce a computational approach to the qualitative prediction of nucleosome occupancy, acetylation, and methylation areas in DNA sequences. Our method uses support vector machines to discriminate between DNA areas with high and low relative occupancy, acetylation, or methylation, and rank k-gram features based on their support for these DNA modifications. Experimental results on the yeast genome reveal genetic area preferences of nucleosome occupancy, acetylation, and methylation that are consistent with previous studies. Supplementary files are available from http://www.jaist.ac.jp/~tran/nucleosome/.

Acetylation↗

PubMed1993

Sensitivity to tumor promotion of SENCAR and C57BL/6J mice correlates with oxidative events and DNA damage.

Significant differences in sensitivity to multistage carcinogenesis have been noted between mice that are sensitive (SENCAR) and resistant (C57BL/6J) to 12-O-tetradecanoylphorbol-13-acetate (TPA). However, the mechanism of this sensitivity has not yet been established. Recent studies from this laboratory have shown that TPA significantly enhances formation of hydrogen peroxide (H2O2) and oxidized DNA bases in SENCAR mouse skin, as it increases the infiltration of polymorphonuclear leukocytes (PMNs), as quantitated by myeloperoxidase (MPO). In the studies reported here, we compared SENCAR and C57BL/6J mice with respect to TPA-mediated edema, hyperplasia, PMN infiltration, oxidant formation and oxidative DNA damage in mouse skin. Topical application of two TPA doses (2x2-40 micrograms, 20 h apart) dose-dependently increased PMN infiltration and oxidant formation in both mouse strains, which was consistent with TPA-induced morphological alterations (edema and hyperplasia). However, at low TPA doses (2-4 micrograms), the increases over controls in the SENCAR mice were significantly greater (P < 0.01) than those in C57BL/6J mice. Comparison of the net values indicated that 4 micrograms TPA enhanced PMN infiltration (MPO units/cm2) and oxidant formation (nmol H2O2/cm2) in SENCAR mice by 7.7- and 11-fold respectively over those present in TPA-treated C57BL/6J mouse skin. At the same dose, TPA also significantly increased formation of thymidine glycol (dTG; 5.5-fold), 5-hydroxymethyl-2'-deoxyuridine (HMdU; 4.9-fold) and 8-hydroxyl-2-deoxyguanosine (8-OHdG; 11.4-fold) in SENCAR mouse epidermis. Then, the levels of all three declined. In C57BL/6J mice, there were virtually no increases at 4 micrograms TPA, but their levels gradually increased with higher TPA doses and reached maxima at 10 micrograms TPA for dTG (1.9-fold increase), at 20 micrograms TPA for 8-OHdG (6.0-fold), and at 30 micrograms TPA for HMdU (1.8-fold). We conclude that the TPA-mediated oxidative events and oxidative DNA modification by different doses of TPA correlate with the promoting potencies of those doses in both mouse strains. Therefore, they could be, at least in part, responsible for the strain-dependent sensitivity to tumor promotion.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed1987

Immunocytochemical detection of interaction products of cis-diamminedichloroplatinum(II) and cis-diammine(1,1-cyclobutanedicarboxylato)platinum(II) with DNA in rodent tissue sections.

Calf thymus DNA was modified in vitro by cis-diamminedichloroplatinum(II) (cisDDP), complexed with methylated bovine serum albumin and used to immunize rabbits. The anti-cisDDP-DNA antiserum obtained was applied in a double peroxidase-antiperoxidase staining procedure to localize cisDDP-DNA and cis-diammine(1,1-cyclobutanedicarboxylato)platinum(II) (CBDCA)-DNA interaction products in cryostat tissue sections of mice and rats. Rats received cisDDP (0-10 mg/kg) and were killed after 24 h. Mice received cisDDP (0-15 mg/kg) or CBDCA (200 mg/kg), and were killed after 2 h-162 days. For each time-dose combination two mice or one rat were used; agents were given i.p. Specific nuclear staining was observed in all tissues examined from cisDDP- or CBDCA-treated animals. No significant nuclear staining could be observed in tissue sections from control rats and mice. The extent of staining after cisDDP was dose and time dependent. The lowest dose of cisDDP after which specific nuclear staining could be detected varied from tissue to tissue [e.g., 0.1 mg/kg, pancreas (mouse); 0.5 mg/kg, liver, kidney (mouse, rat)]. The longest time interval after a single dose of 6 mg/kg cisDDP in which adducts could be visualized also depended on the tissue and varied between 9 days (spleen, testis) and 162 days (kidney). The staining intensity in liver and kidney, measured microdensitometrically, decreased relatively fast in the first days after treatment, but much slower thereafter. In the kidney, cisDDP-induced DNA modification showed regional variation: inner cortex greater than outer cortex greater than medulla (rat) and cortex greater than medulla (mouse). In the mouse kidney, a small subpopulation of tubular cells in close association with the renal corpuscles showed a remarkably high staining intensity after both cisDDP and CBDCA administration. Tissues that showed clear cisDDP-induced histological alterations (kidney, pancreas, testis, and duodenum) also showed moderate to high levels of cisDDP-DNA interaction products. A correlation between cell damage (measured histologically) and cisDDP-DNA binding within one tissue type was demonstrated in the rat inner renal cortex, the murine renal cortex, and in duodenal epithelial cells of both mice and rats.

Animals↗

PubMedDate not supplied

[Dibromoethylacetate - a new agent for fixing the unwound regions of DNA. Denaturation maps of mitochondrial DNA from the rat liver].

On the basis of the reaction of dibromoethylacetate with adenine and cytosine at the uncoiled regions of DNA it was possible to fix these molten regions (the degree of denaturation was about 16%) for rat liver mitochondrial DNA. Modification of all accessible adenine and cytosine residues in 0.14 M Na-acetate, pH 5.65, was completed at 70-80 degrees C in 15-20 min. Using the computer orientation for the set of fixed BamHI-fragments and the linear molecules of full length the denaturation map of mtDNA was constructed, the GC-content of the molten regions was about 28%. The dibromoethylacetate is a perspective agent for screening DNA with a low content of destabilizing regions (for instance, DNA of malignant cells).

Adenine↗

PubMed2004

Complete genomic sequence of bacteriophage B3, a Mu-like phage of Pseudomonas aeruginosa.

Bacteriophage B3 is a transposable phage of Pseudomonas aeruginosa. In this report, we present the complete DNA sequence and annotation of the B3 genome. DNA sequence analysis revealed that the B3 genome is 38,439 bp long with a G+C content of 63.3%. The genome contains 59 proposed open reading frames (ORFs) organized into at least three operons. Of these ORFs, the predicted proteins from 41 ORFs (68%) display significant similarity to other phage or bacterial proteins. Many of the predicted B3 proteins are homologous to those encoded by the early genes and head genes of Mu and Mu-like prophages found in sequenced bacterial genomes. Only two of the predicted B3 tail proteins are homologous to other well-characterized phage tail proteins; however, several Mu-like prophages and transposable phage D3112 encode approximately 10 highly similar proteins in their predicted tail gene regions. Comparison of the B3 genomic organization with that of Mu revealed evidence of multiple genetic rearrangements, the most notable being the inversion of the proposed B3 immunity/early gene region, the loss of Mu-like tail genes, and an extreme leftward shift of the B3 DNA modification gene cluster. These differences illustrate and support the widely held view that tailed phages are genetic mosaics arising by the exchange of functional modules within a diverse genetic pool.

Base Sequence↗

PubMed2003

Chromatin compaction by human MeCP2. Assembly of novel secondary chromatin structures in the absence of DNA methylation.

MeCP2 is a transcriptional repressor that contains an N-terminal methylated DNA-binding domain, a central transcription regulation domain, and a C-terminal domain of unknown function. Whereas current models of MeCP2 function evoke localized recruitment of histone deacetylases to specific methylated regions of the genome, it is unclear whether MeCP2 requires DNA methylation to bind to chromatin or whether MeCP2 binding influences chromatin structure in the absence of other proteins. To address these issues, we have characterized the complexes formed between MeCP2 and biochemically defined nucleosomal arrays. At molar ratios near 1 MeCP2/nucleosome, unmethylated nucleosomal arrays formed both extensively condensed ellipsoidal particles and oligomeric suprastructures. Furthermore, MeCP2-mediated chromatin compaction occurred in the absence of monovalent or divalent cations, in distinct contrast to all other known chromatin-condensing proteins. Analysis of specific missense and nonsense MeCP2 mutants indicated that the ability to condense chromatin resides in region(s) of the protein other than the methylated DNA-binding domain. These data demonstrate that MeCP2 assembles novel secondary chromatin structures independent of DNA modification and suggest that the ability of MeCP2 to silence chromatin may be related in part to its effects on large-scale chromatin organization.

Chromatin↗

PubMed1987

Expression of the cloned coliphage T3 S-adenosylmethionine hydrolase gene inhibits DNA methylation and polyamine biosynthesis in Escherichia coli.

We have developed a new research tool for the study of S-adenosylmethionine (AdoMet) metabolism by cloning the coliphage T3 AdoMet hydrolase (AdoMetase; EC 3.3.1.2) gene into the M13mp8 expression vector. The recombinant bacteriophage clones expressed an AdoMetase activity in Escherichia coli like that found in T3-infected cells. High levels of AdoMetase expression impaired AdoMet-mediated activities such as dam and dcm methylase-directed DNA modifications and the synthesis of spermidine from putrescine. Expression vectors containing the cloned AdoMetase gene thus provide an alternate approach to the use of chemical inhibitors or mutants defective in AdoMet biosynthesis to probe the effect of AdoMet limitation.

Cloning, Molecular↗

PubMed1994

Gene activation studied by immunological methods.

Gene activation can be studied at several levels: transcription (mRNA), translation (proteins), or phenotypical alterations (functional activity or morphology). These levels can be studied in situ or biochemically by the use of specific probes for normal or altered DNA, mRNA, or proteins. Immunological probes are potent tools for studies of alterations induced by xenobiotics in target organs. When the effects of xenobiotics are studied in whole tissue, the cellular heterogeneity of the organ must be taken into account. For this reason, combined in situ and biochemical techniques are necessary. Antibodies to normal or altered cellular constituents are used for identification, quantitation, and cellular localization of proteins and modified DNA. Many xenobiotics alter gene activation by interactions with DNA. After activation, 2-acetylaminofluorene (AAF) forms DNA adducts, which can be identified immunologically. Combined with bromodeoxyuridine (BrdU) pulse labeling, techniques have been developed to demonstrate reduced adduct concentrations in proliferating cells and preneoplastic foci in the livers of AAF-fed rats. Carcinogen-induced DNA modifications are implicated as a major mechanism of altered gene activation in neoplasia, leading to phenotypical alterations. Also, cellular differentiation may be affected by xenobiotics. Differentiation-associated markers can be used for studies of gene activation. In mouse skin, the keratins K1 and K10 are only expressed in suprabasal, differentiating cells. BrdU pulse chase experiments combined with double immunofluorescence have revealed that K1 and K10 are sequentially turned on 18 to 24 hr after DNA synthesis and are followed by suprabasal migration. After a single application of the tumor promotor 12-O-tetradecanoylphorbol-13-acetate (TPA), cell migration starts directly after mitosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Blotting, Western↗

PubMed2003

Incorporation of reporter molecule-labeled nucleotides by DNA polymerases. II. High-density labeling of natural DNA.

The modification of nucleic acids using nucleotides linked to detectable reporter or functional groups is an important experimental tool in modern molecular biology. This enhances DNA or RNA detection as well as expanding the catalytic repertoire of nucleic acids. Here we present the evaluation of a broad range of modified deoxyribonucleoside 5'-triphosphates (dNTPs) covering all four naturally occurring nucleobases for potential use in DNA modification. A total of 30 modified dNTPs with either fluorescent or non-fluorescent reporter group attachments were systematically evaluated individually and in combinations for high-density incorporation using different model and natural DNA templates. Furthermore, we show a side-by-side comparison of the incorporation efficiencies of a family A (Taq) and B (Vent(R) exo-) type DNA polymerase using the differently modified dNTP substrates. Our results show superior performance by a family B-type DNA polymerase, Vent(R) exo-, which is able to fully synthesize a 300 bp DNA product when all natural dNTPs are completely replaced by their biotin-labeled dNTP analogs. Moreover, we present systematic testing of various combinations of fluorescent dye-modified dNTPs enabling the simultaneous labeling of DNA with up to four differently modified dNTPs.

Base Sequence↗

PubMedDate not supplied

Selective telomere activation and the control of antigen gene expression in trypanosomes.

African trypanosomes escape the immune defence of their mammalian host by changing their antigenic surface coat. Antigenic variation occurs through differential gene activation: only one antigen gene is transcribed at a time, among a large collection of specific sequences. This transcription always takes place in a telomere, but it seems that different telomeres can be used alternatively as the gene expression site. Since the trypanosome genome is made up of numerous chromosomes, it would appear that a highly selective process allows the activation of only one telomere at a time. This process seems linked to the differential inactivation of a peculiar telomeric DNA modification system. Two mechanisms allow antigen genes to be expressed. First, a gene copy can be inserted in the expression site by replacing the formerly expressed gene. This is due to gene conversion, whose extent can vary considerably, according to the degree of homology between the recombining partners. The second mechanism involves the activation of another telomere along with deactivation of the telomere containing the previously expressed gene. This form of activation can occur without apparent DNA rearrangement. The alternate use of these mechanisms leads to rapid changes in the antigen gene repertoire, due to gain and loss of different sequences, and to alteration of their activation rate.

Animals↗

PubMed2000

Morphological transformation by 8-hydroxy-2'-deoxyguanosine in Syrian hamster embryo (SHE) cells.

8-Hydroxy-2'-deoxyguanosine (OH8dG) is one of the most prevalent oxidative DNA modifications found in eukaryotic cells. Previous studies have suggested an association between OH8dG formation and carcinogenesis. However, it is unclear whether OH8dG formation results in the necessary genotoxic events for cancer development. In the present study, the formation of OH8dG and its ability to transform Syrian hamster embryo (SHE) cells was examined. Methylene blue, a photosensitizer that in the presence of light can generate singlet oxygen by a type II mechanism, was used to produce oxidative DNA damage (predominantly OH8dG) in SHE cells. Photoactivated methylene blue produced a dose-dependent increase in OH8dG as well as a dose-dependent increase in morphological transformation in SHE cells. SHE cells transfected with DNA that contained increasing concentrations of OH8dG displayed a dose-dependent increase in morphological transformation. Treatment with beta-carotene (a singlet oxygen quencher) inhibited both the formation of OH8dG and the induction of morphological transformation in photoactivated methylene blue-treated SHE cells. These results suggest that formation of OH8dG can induce morphological transformation and provide further support for a role of OH8dG formation in the carcinogenesis process.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed2002

A new regulatory DNA motif of the gamma subclass Proteobacteria: identification of the LexA protein binding site of the plant pathogen Xylella fastidiosa.

Escherichia coli LexA protein is the repressor of a gene network whose members are directly involved in the repair of damaged DNA and in the survival of bacterial cells until DNA lesions have been eliminated. The lexA gene is widely present in bacteria, although the sequences of only three LexA-binding sites are known: Gram-positive, alpha Proteobacteria and some members of gamma Proteobacteria represented by E. coli. Taking advantage of the fact that the genome sequence of the plant-pathogenic bacterium Xylella fastidiosa has been determined, its lexA gene has been cloned and overexpressed in E. coli to purify its product. After demonstration that X. fastidiosa lexA and recA genes are co-transcribed, gel mobility shift assays and directed mutagenesis experiments using the promoter of the lexA-recA transcriptional unit demonstrated that the X. fastidiosa LexA protein specifically binds the imperfect palindrome TTAGN(6)TACTA. This is the first LexA binding sequence identified in the gamma Proteobacteria differing from the E. coli-like LexA box. Although a computational search has revealed the presence of TTAGN(6)TACTA-like motifs upstream of X. fastidiosa genes other than lexA, X. fastidiosa LexA only binds the promoter of one of them, XF2313, encoding a putative DNA-modification methylase. Moreover, X. fastidiosa LexA protein does not bind any of the other genes whose homologues are regulated by the LexA repressor in E. coli (uvrA, uvrB, ssb, ruvAB, ftsK, dinG, recN and ybfE). RT-PCR quantitative analysis has also demonstrated that lexA-recA and XF2313 genes, as well as the X. fastidiosa genes which are homologues to those of E. coli belonging to the LexA regulon, with the exception of ssb, are DNA damage-inducible in X. fastidiosa.

Bacterial Proteins↗

PubMed1998

Heritable and cancer risks of exposures to anticancer drugs: inter-species comparisons of covalent deoxyribonucleic acid-binding agents.

In the past years, several methodologies were developed for potency ranking of genotoxic carcinogens and germ cell mutagens. In this paper, we analyzed six sub-classes of covalent deoxyribonucleic acid (DNA) binding antineoplastic drugs comprising a total of 37 chemicals and, in addition, four alkyl-epoxides, using four approaches for the ranking of genotoxic agents on a potency scale: the EPA/IARC genetic activity profile (GAP) database, the ICPEMC agent score system, and the analysis of qualitative and quantitative structure-activity and activity-activity relationships (SARs, AARs) between types of DNA modifications and genotoxic endpoints. Considerations of SARs and AARs focused entirely on in vivo data for mutagenicity in male germ cells (mouse, Drosophila), carcinogenicity (TD50s) and acute toxicity (LD50s) in rodents, whereas the former two approaches combined the entire database on in vivo and in vitro mutagenicity tests. The analysis shows that the understanding and prediction of rank positions of individual genotoxic agents requires information on their mechanism of action. Based on SARs and AARs, the covalent DNA binding antineoplastic drugs can be divided into three categories. Category 1 comprises mono-functional alkylating agents that primarily react with N7 and N3 moieties of purines in DNA. Efficient DNA repair is the major protective mechanism for their low and often not measurable genotoxic effects in repair-competent germ cells, and the need of high exposure doses for tumor induction in rodents. Due to cell type related differences in the efficiency of DNA repair, a strong target cell specificity in various species regarding the potency of these agents for adverse effects is found. Three of the four evaluation systems rank category 1 agents lower than those of the other two categories. Category 2 type mutagens produce O-alkyl adducts in DNA in addition to N-alkyl adducts. In general, certain O-alkyl DNA adducts appear to be slowly repaired, or even not at all, which make this kind of agents potent carcinogens and germ cell mutagens. Especially the inefficient repair of O-alkyl-pyrimidines causes the high mutational response of cells to these agents. Agents of this category give high potency scores in all four expert systems. The major determinant for the high rank positions on any scale of genotoxic of category 3 agents is their ability to induce primarily structural chromosomal changes. These agents are able to cross-link DNA. Their high intrinsic genotoxic potency appears to be related to the number of DNA cross-links per target dose unit they can induce. A confounding factor among category 3 agents is that often the genotoxic endpoints occur close to or at toxic levels, and that the width of the mutagenic dose range, i.e., the dose area between the lowest observed effect level and the LD50, is smaller (usually no more than 1 logarithmic unit) than for chemicals of the other two categories. For all three categories of genotoxic agents, strong correlations are observed between their carcinogenic potency, acute toxicity and germ cell specificity.

Animals↗