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PubMed1986

The in vivo mutagenic frequency and specificity of O6-methylguanine in phi X174 replicative form DNA.

A bacteriophage phi X174-based site-specific mutagenesis system for the study of the in vivo mutagenic frequency and specificity of carcinogen-induced modification in DNA is presented. A (-)-strand primer containing O6-methylguanine in a specific site was hybridized to a single-stranded region in gene G of phi X gapped duplex DNA. The hybrid was enzymatically converted to replicative form DNA and was used to transform Escherichia coli cells. All gene G mutants generated by the modification were rescued by genetic complementation. An amber mutation in lysis gene E of the (+) strand of the replicative form DNA prevented lytic growth of wild-type phage derived from this strand. In each mutant-containing infective center produced from the transformed cells, gene G mutant phage were present in a 3:1 ratio compared to wild type. Thus, in vivo, O6-methylguanine in replicating phi X DNA has a mutagenic frequency of 75%. When repair of O6 methylguanine occurred, it was prereplicative. The mutations were due exclusively to the misincorporation of thymine.

Bacteriophage phi X 174↗
PubMed2002

alpha-L-ribo-configured locked nucleic acid (alpha-L-LNA): synthesis and properties.

The syntheses of monomeric nucleosides and 3'-O-phosphoramidite building blocks en route to alpha-L-ribo-configured locked nucleic acids (alpha-L-LNA), composed entirely of alpha-L-LNA monomers (alpha-L-ribo configuration) or of a mixture of alpha-L-LNA and DNA monomers (beta-D-ribo configuration), are described and the alpha-L-LNA oligomers are studied. Bicyclic 5-methylcytosin-1-yl and adenine-9-yl nucleoside derivatives have been prepared and the phosphoramidite approach has been used for the automated oligomerization leading to alpha-L-LNA oligomers. Binding studies revealed very efficient recognition of single-stranded DNA and RNA target oligonucleotide strands. Thus, stereoirregular alpha-L-LNA 11-mers containing a mixture of alpha-L-LNA monomers and DNA monomers ("mix-mer alpha-L-LNA") were shown to display DeltaT(m) values of +1 to +3 degrees C per modification toward DNA and +4 to +5 degrees C toward RNA when compared with the corresponding unmodified DNA x DNA and DNA x RNA reference duplexes. The corresponding DeltaT(m) values per modification for the stereoregular fully modified alpha-L-LNA were determined to be +4 degrees C (against DNA) and +5 degrees C (against RNA). 11-Mer alpha-L-LNAs (mix-mer alpha- L-LNA or fully modified alpha- L-LNA) were shown in vitro to be significantly stabilized toward 3'-exonucleolytic degradation. A duplex formed between RNA and either mix-mer alpha-L-LNA or fully modified alpha-L-LNA induced in vitro Escherichia coli RNase H-mediated cleavage, albeit very slow, of the RNA targets at high enzyme concentrations.

Circular Dichroism↗
PubMedDate not supplied

Role of the cytosine DNA-methyltransferase and p16INK4a genes in the development of mouse lung tumors.

CpG island methylation is an epigenetic modification of DNA associated with the silencing of gene transcription. The p16INK4a (p16) tumor suppressor gene is inactivated in human non-small cell lung cancers (NSCLCs) by either homozygous deletion or aberrant methylation. Inactivation of tumor suppressor genes by methylation has been linked in part to altered activity of the cytosine DNA-methyltransferase (DNA-MTase), the enzyme that catalyzes DNA methylation at CpG sites. The purpose of these studies was to define the role of DNA-MTase and p16 in the development of murine lung cancer. DNA-MTase activity was determined in alveolar type II and Clara cells from A/J and C3H mice that exhibit high and low susceptibility, respectively, for lung tumor formation. Increased DNA-MTase activity leading to an increase in overall DNA methylation was found only in alveolar type II cells, the target for murine adenocarcinomas. Both DNA-MTase and DNA methylation changes were detected 7 days after carcinogen exposure and, thus, were early events in neoplastic evolution. In addition, enzyme activity increased incrementally during lung cancer progression. Expression of p16 was detected in all primary lung tumors from A/J mice; however, levels of expression differed by up to 15-fold between tumors. The apparent low levels of expression seen in approximately half of the tumors was not attributed to methylation of the p16 gene. In contrast to the detection of p16 expression in primary tumors, this gene was deleted in four tumor-derived cell lines induced in the A/J mouse by NNK. The results from these studies indicate that the modulation of DNA-MTase activity was cell specific, segregated with susceptibility, and occurred early in neoplastic evolution. Thus, the marked increase in enzyme activity detected in alveolar type II cells after carcinogen treatment could be a major factor contributing to the high susceptibility for chemical-induced neoplasia associated with the A/J mouse strain. The inactivation of the p16 gene in murine cancers induced by NNK most likely arises as a late event via homozygous deletion.

Adenocarcinoma↗
PubMedDate not supplied

Thymo-adrenals reaction in pre-pubertal and pubertal Wistar rats in response to acute administration of calcitonin.

Pre- and post-pubertal Wistar rats were injected i.m. with a single dose equivalent to 1 UI of Calcitonin (Calsyn-50 Rorer, Swiss). Its effects upon the thymus and adrenals were assessed after 1, 3 and respectively, 8 days since injection. The results pointed to differences of reaction of the thymus and adrenals dependent on rat ages and on the period of hormone persistence in the organism. The effects in mature rats are characterized by a stress state, reflected in adrenal reaction, but without impact upon the thymus in point of biochemical parameters. In rats of that age, modifications in both organs are much reduced after 8 days since calcitonin injection. In pubertal rats, adrenal activity is temperate, and thymus registers modification of DNA, of oxygen consumption and of nitrogen aminic after 3 days since injection. This modification is maintained for 8 days.

Adrenal Glands↗
PubMed1981

Temporal patterns of covalent DNA adducts in rat liver after single and multiple doses of aflatoxin B1.

We examined patterns of covalent modifications of DNA produced in rat liver after exposure to single and multiple doses of aflatoxin B1. The principal product, previously identified as 2,3-dihydro-3-hydroxy(N7-guanyl) aflatoxin B1, was removed rapidly from liver DNA in vivo after a 0.6-mg/kg dose was administered i.p. to male Fischer rats. This lesion had an apparent half-life of 7.5 hr. Similar kinetics of disappearance was seen for two other aflatoxin adducts, one of which was previously identified as an N7-guanine adduct of aflatoxin P1. The kinetics of formation and disappearance differed for two other products believed to be produced by scission of the imidazole ring of the 7-substituted guanine moiety of the principal adduct in the DNA molecule. These adducts were removed slowly, if at all, during the 72-hr period studied. Approximately 20% of the principal N7 adduct initially formed was converted to these products in 24 hr, at which time they were the predominant lesions in DNA. Administration of multiple doses of aflatoxin B1, using a regimen shown to produce a high incidence of hepatocellular carcinoma, caused accumulation of these persistent products in liver DNA over a 14-day period.

Aflatoxins↗
PubMedDate not supplied

Epigenetic silencing mediated by CpG island methylation: potential as a therapeutic target and as a biomarker.

Many genes become transcriptionally silenced during the development of cancer. As well as affecting disease progression, gene silencing has the potential to influence drug resistance and clinical outcome following therapy. In addition to silencing due to gene mutations, covalent epigenetic modifications such as DNA hypermethylation and histone post-translational modifications are associated with transcriptional inactivation of many genes and are an important early event during carcinogenesis and tumour development. Aberrant methylation of CpG islands in promoters is associated with transcriptional inactivation of genes involved in all aspects of tumour development. Genes involved in key DNA damage response pathways, such as cell cycle control, apoptosis signalling and DNA repair, can frequently become methylated and epigenetically silenced in tumours. This may lead to differences in intrinsic sensitivity of tumours to chemotherapy, depending on the specific function of the gene inactivated. Furthermore, it is proposed that chemotherapy itself can exert a selective pressure on epigenetically silenced drug sensitivity genes present in subpopulations of cells, leading to acquired chemoresistance. Since the DNA sequence of epigenetically inactivated genes are not mutated but rather subject to reversible modifications via DNA methyltransferases (DNMTs) or histone modification, it is possible to reverse silencing using small molecule inhibitors. Such compounds show anti-tumour activity and can increase the sensitivity of drug resistant preclinical tumour models. Clinical trials of epigenetic therapies are now underway. Epigenetic profiling, using DNA methylation and histone analysis, will provide guidance on optimisation of these therapies with conventional chemotherapy and will help identify patient populations who may particularly benefit from such approaches.

Animals↗
PubMed1999

Evidence of horizontal transfer of the EcoO109I restriction-modification gene to Escherichia coli chromosomal DNA.

A DNA fragment carrying the genes coding for EcoO109I endonuclease and EcoO109I methylase, which recognize the nucleotide sequence 5'-(A/G)GGNCC(C/T)-3', was cloned from the chromosomal DNA of Escherichia coli H709c. The EcoO109I restriction-modification (R-M) system was found to be inserted between the int and psu genes from satellite bacteriophage P4, which were lysogenized in the chromosome at the P4 phage attachment site of the corresponding leuX gene observed in E. coli K-12 chromosomal DNA. The sid gene of the prophage was inactivated by insertion of one copy of IS21. These findings may shed light on the horizontal transfer and stable maintenance of the R-M system.

Amino Acid Sequence↗
PubMedDate not supplied

[Weakening of bacteriophage lambda EcoK DNA restriction in the presence of plasmid pKM101 ard+. I. General characteristics and genetic localization].

The host-controlled K-restriction of unmodified phage lambda is ten to hundred-fold alleviated in the E. coli K12 strain, carring plasmid pKM101 of N-incompatibility group. By restriction mapping Tn5 insertion in pKM101, which reduced pKM101-mediated alleviation of K-restriction, was shown to by located within BglII-B-fragment approximately 9 kb anticlockwise from the EcoRI-site of pKM101. We have termed the gene(s) promoting the alleviation of K-restriction ARD (Alleviation of Restriction of DNA). It was shown that (i) plasmid pKM101-mediated alleviation of K-restriction did not depend on bacterial genes LexA, RecBC, umuC and plasmid gene muc; (ii) ard gene did not mediate EcoK type modification of DNA and did not enhance the modification activity of EcoK system in a way similar to that observed with RAL gene of phage lambda. Action of Ard gene of plasmid pKM101 is highly specific: alleviation of restriction of DNA lambda takes place only in K-strains of E. coli and is practically absent in B-strains and also in E. coli strains which have restricting enzymes of 11 type, EcoRI and EcoRIII.

Bacteriophage lambda↗
PubMed1986

Inhibition of DNA polymerase activity by methyl methanesulfonate.

Methyl methanesulfonate (MMS) inhibits both thymidine incorporation into DNA in mitogen-activated human lymphocytes and deoxythymidine triphosphate incorporation into template DNA by DNA polymerase-alpha in a cell-free system. When MMS-modified DNA was used as the template for DNA synthesis utilizing unmodified DNA polymerase-alpha, nucleotide incorporation into template DNA was not inhibited. When unmodified DNA was used as the template for DNA synthesis utilizing MMS-modified DNA polymerase-alpha, nucleotide incorporation was differentially inhibited dependent on the MMS concentration. An analysis of the kinetics of DNA polymerase-alpha inhibition showed that incorporation of all 4 deoxynucleoside triphosphates into DNA template was noncompetitively inhibited by MMS, which is consistent with nonspecific MMS modification of the enzyme. These data indicate that MMS modification of DNA polymerase-alpha alone is sufficient to inhibit the incorporation of deoxynucleoside triphosphates into template DNA in vitro. The data further indicate that alkylation of both DNA polymerase-alpha and DNA template synergistically increases inhibition of DNA synthesis.

Alkylation↗
PubMed2000

Anti-mutagenic structural modification by fluorine-substitution in highly mutagenic 4-methylquinoline derivatives.

We have previously shown that fluorine-substitution at position 3 of quinoline deprived this molecule of mutagenicity, possibly due to interference with the yield of its metabolically activated form, the 1,4-hydrated 2,3-epoxide (enamine epoxide), which is directly responsible for the mutagenic modification of DNA. To further explore the possibility of a method for anti-mutagenic modification of mutagens by fluorine-substitution, 4-methylquinoline (4-MeQ), the most mutagenic form of all the quinoline derivatives examined so far, was used as a target in the present study. Five mono- and di-fluorinated derivatives of 4-MeQ, 2-fluoro-4-methylquinoline (2-F-4-MeQ), 6-F-4-MeQ, 7-F-4-MeQ, 2,6-difluoro-4-methylquinoline (2, 6-diF-4-MeQ), and 2,7-diF-4-MeQ, were subjected to analysis of their structure-mutagenicity relationships. The 2-fluorinated derivatives (2-F-4-MeQ, 2,6-diF-4-MeQ, and 2,7-diF-4-MeQ) were all non-mutagenic in the Ames test. 7-F-4-MeQ was as highly mutagenic as, and 6-F-4-MeQ was less mutagenic than non-fluorinated 4-MeQ. Metabolic studies were also conducted with 4-MeQ, 2-F-4-MeQ, 6-F-4-MeQ, and 7-F-4-MeQ, using a liver microsomal enzyme fraction prepared from the 3-methylcholanthrene-treated rat. The HPLC analytical data showed that, although the metabolic patterns (hydroxylation at 4-methyl group as a main metabolic pathway and 3-hydroxylation as a minor pathway) of these four F-MeQs were similar to one another, only the 3-hydroxy metabolite of 2-F-4-MeQ was not produced under the present experimental conditions employed. These results suggest that fluorine-substitution at position 2 of 4-MeQ inhibited the formation of the enamine epoxide in the pyridine moiety and deprived this molecule of mutagenicity as in the case of quinoline.

Animals↗
PubMed1992

Purification and biochemical characterization of the EcaI DNA methyltransferase.

The EcaI GGTNACC-specific DNA-adenine modification methyltransferase has been purified to apparent homogeneity. The active form of the DNA methyltransferase is a single polypeptide. The enzyme has a pH optimum at pH 8.0 and a temperature optimum at 25 degrees C. EcaI DNA methyltransferase transfers one methyl group to the adenine of the recognition site in a single binding event. The Km was 170 nM for DNA and 1.8 microM for the methyl donor S-adenosylmethionine. Methylated DNA is a competitive inhibitor with respect to DNA (Ki = 3.5 nM). The other product of the DNA-methylation reaction, S-adenosylhomocysteine was found to be a competitive inhibitor with respect to S-adenosylmethionine (Ki = 2.7 microM). The S-adenosylmethionine analog sinefungin was shown to be a very strong inhibitor (Ki = 3.5 nM) of the DNA methyltransferase reaction.

Adenosine↗
PubMed1986

Formation of cyclic nucleic acid adducts from some simple alpha, beta-unsaturated carbonyl compounds and cyclic nitrosamines.

To determine the structures of the DNA adducts of two cyclic nitrosamines, N-nitrosopyrrolidine and N-nitrosomorpholine, the model compounds, alpha-acetoxy-N-nitrosopyrrolidine, 4-(carbethoxynitrosamino)butanal and 2-(carbethoxynitrosamino)ethoxyacetaldehyde, were allowed to react with deoxyguanosine in the presence of porcine liver esterase or base. These model compounds are stable precursors of intermediates formed upon metabolic alpha-hydroxylation of N-nitrosopyrrolidine and N-nitrosomorpholine. The major adducts formed in these reactions were isolated and characterized, on the basis of ultraviolet absorption, mass spectrometry, proton nuclear magnetic resonance and chromatographic properties, as structurally unique 1,N2-cyclic deoxyguanosine adducts. Reaction of crotonaldehyde and glyoxal with deoxyguanosine at 37 degrees C, pH 7, also led to the formation of 1,N2-cyclic deoxyguanosine adducts identical to those formed from the model compounds for alpha-hydroxylation of N-nitrosopyrrolidine and N-nitrosomorpholine, respectively. The 1,N2-cyclic deoxyguanosine adducts were also formed in DNA, upon incubation with N-nitrosopyrrolidine and rat liver microsomes. Acrolein, the simplest alpha, beta-unsaturated carbonyl compound, reacted readily with deoxyguanosine to form three major adducts. These adducts were characterized as cyclic 1,N2-propanodeoxyguanosine adducts resulting from Michael addition of acrolein to the 1- and N2-positions of deoxyguanosine followed by ring closure. One of these adducts, as well as the corresponding crotonaldehyde adduct, was formed in calf thymus DNA upon reaction with acrolein or crotonaldehyde under physiological conditions. The level of modification of DNA by acrolein was considerably higher than that by either crotonaldehyde or N-nitrosopyrrolidine.

Acetaldehyde↗
PubMed1996

Cloning and analysis of the genes encoding the type IIS restriction-modification system HphI from Haemophilus parahaemolyticus.

The genomic region encoding the type IIS restriction-modification (R-M) system HphI (enzymes recognizing the asymmetric sequence 5'-GGTGA-3'/5'-TCACC-3') from Haemophilus parahaemolyticus were cloned into Escherichia coli and sequenced. Sequence analysis of the R-M HphI system revealed three adjacent genes aligned in the same orientation: a cytosine 5 methyltransferase (gene hphIMC), an adenine N6 methyltransferase (hphIMA) and the HphI restriction endonuclease (gene hphIR). Either methyltransferase is capable of protecting plasmid DNA in vivo against the action of the cognate restriction endonuclease. hphIMA methylation renders plasmid DNA resistant to R.Hindill at overlapping sites, suggesting that the adenine methyltransferase modifies the 3'-terminal A residue on the GGTGA strand. Strong homology was found between the N-terminal part of the m6A methyltransferasease and an unidentified reading frame interrupted by an incomplete gaIE gene of Neisseria meningitidis. The HphI R-M genes are flanked by a copy of a 56 bp direct nucleotide repeat on each side. Similar sequences have also been identified in the non-coding regions of H.influenzae Rd DNA. Possible involvement of the repeat sequences in the mobility of the HphI R-M system is discussed.

Amino Acid Sequence↗
PubMed1987

[Addressed modification of the promoter region of DNA in a complex with RNA polymerase by alkylating oligonucleotide derivatives].

Addressed chemical modification of double-stranded DNA unwound with the specific protein has been demonstrated. 4(N-2-chloroethyl-N-methylamino)benzyl-5'-phosphoamides of oligonucleotides d(CG)rC, d(ATCG)rC, d(AATCG)rC, which can serve as primers in the RNA polymerase-catalyzed transcription, alkylated A2 promoter region of pSK-A2 plasmid in its "open" complex with E. coli RNA polymerase.

Alkylating Agents↗
PubMed1990

NMR-studies of carcinogen reactions with DNA: ethylene dibromide and aflatoxin B1.

Two examples are described of the use of NMR spectroscopy to study the modification of DNA structure by carcinogens. The reaction of ethylene dibromide involves initial conjugation with glutathione, catalysed by glutathione S-transferase. Reaction of this adduct with DNA occurs at N7 of guanine. Through the use of stereospecifically 1,2-dideuteriated ethylene dibromide, the mechanism of reaction has been shown to involve an odd number, i.e. three, of SN2 inversions. Correlation spectra (COSY) were employed to analyse reaction stereochemistry. The relative configuration of the deuterium atoms in the products was initially assigned by 1H nuclear Overhauser effect (NOE) difference spectra and then confirmed by an independent synthesis of stereospecifically dideuteriated glutathione-guanine adducts. The second example involves reaction of the epoxide of aflatoxin B1 with DNA to form covalent adducts at N7 of guanine. Adduct formation was found to enhance duplex stability. Chemical shift changes for aflatoxin protons in the covalent adduct when compared with those for aflatoxin B1 non-covalently associated with DNA suggest that covalently linked aflatoxin is intercalated. NOEs confirm that the aflatoxin moiety is intercalated and show that it is on the 5' side of the guanine. This geometry leads to d(ATCGAT)2 forming an adduct in which only one chain has been modified by aflatoxin, while d(ATGCAT)2 forms a complex in which both chains have been modified.

Aflatoxin B1↗
PubMed2000

Modification of natural, double-helical DNA by antitumor cis- and trans-[Cl(2)(Me(2)SO(4))(4)Ru] in cell-free media.

Modifications of natural DNA in cell-free media by the antitumor ruthenium compounds cis- and trans-[Cl(2)(Me(2)SO(4))(4)Ru] were studied by various biochemical and biophysical methods. These methods included: binding studies by means of flameless atomic absorption spectrophotometry, mapping of DNA adducts by means of transcription assay, use of ethidium bromide as a fluorescent probe of DNA adducts of metal complexes, an interstrand cross-linking assay employing gel electrophoresis under denaturing conditions, measurements of DNA unwinding by gel electrophoresis, differential pulse polarographic analysis of DNA conformation, and analysis of liquid crystalline dispersions of DNA by circular dichroism. The results indicated that both ruthenium compounds irreversibly coordinated to DNA; the rate of binding of the cis isomer was considerably lower than that of the trans isomer. The DNA-binding mode of trans-[Cl(2)(Me(2)SO(4))(4)Ru] included formation of bifunctional adducts such as intrastrand cross-links between neighboring purine residues and a small amount ( approximately 1%) of interstrand cross-links. cis-[Cl(2)(Me(2)SO(4))(4)Ru] formed mainly monofunctional lesions on natural DNA. Both ruthenium isomers induced conformational alterations of non-denaturational character in DNA, the trans compound being more effective. In addition, DNA adducts of trans-[Cl(2)(Me(2)SO(4))(4)Ru] were capable of inhibiting RNA synthesis by DNA-dependent RNA polymerases, while the adducts of the cis isomer were not. Thus, several features of the DNA-binding mode of trans-[Cl(2)(Me(2)SO(4))(4)Ru] were similar to those of antitumor cis-diamminedichloroplatinum (II), which may be relevant to the biological effects of this antitumor ruthenium drug. On the other hand, the different DNA-binding mode of cis-[Cl(2)(Me(2)SO(4))(4)Ru] was consistent with its less pronounced biological effects.

Antineoplastic Agents↗
PubMed2000

Type I restriction systems: sophisticated molecular machines (a legacy of Bertani and Weigle).

Restriction enzymes are well known as reagents widely used by molecular biologists for genetic manipulation and analysis, but these reagents represent only one class (type II) of a wider range of enzymes that recognize specific nucleotide sequences in DNA molecules and detect the provenance of the DNA on the basis of specific modifications to their target sequence. Type I restriction and modification (R-M) systems are complex; a single multifunctional enzyme can respond to the modification state of its target sequence with the alternative activities of modification or restriction. In the absence of DNA modification, a type I R-M enzyme behaves like a molecular motor, translocating vast stretches of DNA towards itself before eventually breaking the DNA molecule. These sophisticated enzymes are the focus of this review, which will emphasize those aspects that give insights into more general problems of molecular and microbial biology. Current molecular experiments explore target recognition, intramolecular communication, and enzyme activities, including DNA translocation. Type I R-M systems are notable for their ability to evolve new specificities, even in laboratory cultures. This observation raises the important question of how bacteria protect their chromosomes from destruction by newly acquired restriction specifities. Recent experiments demonstrate proteolytic mechanisms by which cells avoid DNA breakage by a type I R-M system whenever their chromosomal DNA acquires unmodified target sequences. Finally, the review will reflect the present impact of genomic sequences on a field that has previously derived information almost exclusively from the analysis of bacteria commonly studied in the laboratory.

Amino Acid Sequence↗

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