RESUMO
Members of the uracil-DNA glycosylase (UDG) enzyme family recognize and bind uracil, sequestering it within the binding site pocket and catalyzing the cleavage of the base from the deoxyribose, leaving an abasic site. The recognition and binding are passive and rely on innate dynamic motions of DNA wherein base pairs undergo thermally induced breakage and conformational fluctuations. Once the uracil breaks from its base pair, it can be recognized and bound by the enzyme, which then alters its conformation for sequestration and catalysis. Our results suggest that the thymine to uracil substitution, which differs only by a single methyl group, causes a destabilization of the duplex thermodynamics, which would lead to an increase in the population of the extrahelical state and increase the probability of uracil being recognized and excised from DNA by UDG. This destabilization is dependent on the identity of the nearest-neighbor base-pair stacks; a G·C nearest neighbor leads to thermal and enthalpic destabilization that is weaker that that seen with two A·T neighbors. In addition, uracil substitution yields a nearest-neighbor increase in the counterion uptake of the duplexes but decreases the level of immobilization of structural water for all substituted duplexes regardless of the neighbor identity or number of substitutions.
Assuntos
DNA/química , Timina/química , Uracila-DNA Glicosidase/química , Uracila/química , Pareamento de Bases , DNA/genética , Mutação , Conformação de Ácido Nucleico , Cloreto de Sódio/química , Termodinâmica , Água/químicaRESUMO
Wnt signaling is compromised early in the development of human colorectal cancer (CRC) due to truncating nonsense mutations in adenomatous polyposis coli (APC). CRC induced by chemical carcinogens, such as heterocyclic aromatic amines and azoxymethane, in mice also involves dysregulation of Wnt signaling but via activating missense mutations in the ß-catenin oncogene despite the fact that genetically modified mice harboring an inactive APC allele efficiently develop CRC. In contrast, activating mutations in ß-catenin are rarely observed in human CRC. Dysregulation of the Wnt signaling pathway by the two distinct mechanisms reveals insights into the etiology of human CRC. On the basis of calculations related to DNA adduct levels produced in mouse CRC models using mutagens, and the number of stem cells in the mouse colon, we show that two nonsense mutations required for biallelic disruption of APC are statistically unlikely to produce CRC in experiments using small numbers of mice. We calculate that an activating mutation in one allele near the critical GSK3ß phosphorylation site on ß-catenin is >105-times more likely to produce CRC by random mutagenesis due to chemicals than inactivating two alleles in APC, yet it does not occur in humans. Therefore, the mutagenesis mechanism in human CRC cannot be random. We explain that nonsense APC mutations predominate in human CRC because of deamination at 5-methylcytosine at CGA and CAG codons, coupled with the number of human colonic stem cells and lifespan. Our analyses, including a comparison of mutation type and age at CRC diagnosis in U.S. and Chinese patients, also indicate that APC mutations in CRC are not due to environmental mutagens that randomly damage DNA.
Assuntos
Polipose Adenomatosa do Colo/genética , Neoplasias do Colo/genética , Modelos Animais de Doenças , Mutagênese , Mutação , beta Catenina/genética , Polipose Adenomatosa do Colo/complicações , Animais , Neoplasias do Colo/complicações , Neoplasias do Colo/diagnóstico , HumanosRESUMO
The association between inflammation and the risk of colorectal cancer (CRC) is well documented in animal models and in humans, but the mechanistic role of inflammation in CRC is less well understood. To address this question, the induction of colon tumors was evaluated in (i) wild type (WT) and athymic BALB/c mice treated with the colon carcinogen azoxymethane (AOM) as a single agent, and (ii) in an inflammation model of colon cancer employing AOM and dextran sodium sulfate (DSS) in WT, athymic, TCRß(-/-) , TCRδ(-/-) and TCRß(-/-) TCRδ(-/-) C57Bl/6 mice. The athymic BALB/c mice treated with only AOM developed 90% fewer tumors than the WT mice. The difference in response was not due to metabolic activation of AOM or repair of DNA adducts. In the inflammation model using a standard sequential exposure to AOM followed by DSS treatment, the tumor incidence in WT mice was 58% with 7 adenomas and 6 adenocarcinomas. In contrast, the TCRß(-/-) , TCRδ(-/-) and TCRß(-/-) TCRδ(-/-) C57Bl/6 mice showed adenoma incidences of 10, 33, and 11%, respectively, and none of the immune compromised mice developed adenocarcinomas. When the DSS exposure was increased and the AOM lowered, no difference was observed between WT and TCRß(-/-) mice due to an increase in the incidence in the TCR null mice without concomitant increase in the WT mice. No tumors were observed in mice treated with AOM or DSS alone. © 2015 Wiley Periodicals, Inc.
Assuntos
Azoximetano/efeitos adversos , Neoplasias do Colo/epidemiologia , Neoplasias do Colo/patologia , Sulfato de Dextrana/administração & dosagem , Animais , Neoplasias do Colo/induzido quimicamente , Neoplasias do Colo/imunologia , Sulfato de Dextrana/farmacologia , Hospedeiro Imunocomprometido , Incidência , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Receptores de Antígenos de Linfócitos T/deficiênciaRESUMO
DNA glycosylases that remove alkylated and deaminated purine nucleobases are essential DNA repair enzymes that protect the genome, and at the same time confound cancer alkylation therapy, by excising cytotoxic N3-methyladenine bases formed by DNA-targeting anticancer compounds. The basis for glycosylase specificity towards N3- and N7-alkylpurines is believed to result from intrinsic instability of the modified bases and not from direct enzyme functional group chemistry. Here we present crystal structures of the recently discovered Bacillus cereus AlkD glycosylase in complex with DNAs containing alkylated, mismatched and abasic nucleotides. Unlike other glycosylases, AlkD captures the extrahelical lesion in a solvent-exposed orientation, providing an illustration for how hydrolysis of N3- and N7-alkylated bases may be facilitated by increased lifetime out of the DNA helix. The structures and supporting biochemical analysis of base flipping and catalysis reveal how the HEAT repeats of AlkD distort the DNA backbone to detect non-Watson-Crick base pairs without duplex intercalation.
Assuntos
Bacillus cereus/enzimologia , Dano ao DNA , DNA Glicosilases/metabolismo , Reparo do DNA/fisiologia , DNA/metabolismo , Alquilação , Sequência de Bases , Biocatálise , Cristalografia por Raios X , DNA/química , DNA/genética , Hidrólise , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Solventes/química , TermodinâmicaRESUMO
DNA in its simplest form is an ensemble of nucleic acids, water, and ions, and the conformation of DNA is dependent on the relative proportions of all three components. When DNA is covalently damaged by endogenous or exogenous reactive species, including those produced by some anticancer drugs, the ensemble undergoes localized changes that affect nucleic acid structure, thermodynamic stability, and the qualitative and quantative arrangement of associated cations and water molecules. Fortunately, the biological effects of low levels of DNA damage are successfully mitigated by a large number of proteins that efficiently recognize and repair DNA damage in the midst of a vast excess of canonical DNA. In this Account, we explore the impact of DNA modifications on the high resolution and dynamic structure of DNA, DNA stability, and the uptake of ions and water and explore how these changes may be sensed by proteins whose function is to initially locate DNA lesions. We discuss modifications on the nucleobases that are located in the major and minor grooves of DNA and include lesions that are observed in vivo, including oxidized bases, as well as some synthetic nucleobases that allow us to probe how the location and nature of different substituents affect the thermodynamics and structure of the DNA ensemble. It is demonstrated that disruption of a cation binding site in the major groove by modification of the N7-position on the purines, which is the major site for DNA alkylation, is enthalpically destabilizing. Accordingly, tethering a cationic charge in the major groove is enthalpically stabilizing. The combined structural and thermodynamic studies provide a detailed picture of how different DNA lesions affect the dynamics of DNA and how modified bases interact with their environment. Our work supports the hypothesis that there is a "thermodynamic signature" to DNA lesions that can be exploited in the initial search that requires differentiation between canonical DNA and DNA with a lesion. The differentiation between a lesion and a cognate lesion that is a substrate for a particular enzyme involves another layer of thermodynamic and kinetic factors.
Assuntos
Dano ao DNA , DNA/química , Termodinâmica , Pareamento de Bases , Sítios de Ligação , DNA/metabolismo , DNA Glicosilases/metabolismo , Reparo do DNA , Cinética , Conformação de Ácido Nucleico , ÁguaRESUMO
N3-methyladenine (3-mA), generated by the reaction of methylating agents with DNA, is considered a highly toxic but weakly mutagenic lesion. However, due to its intrinsic instability, some of the biological effects of the adduct can result from the formation of the corresponding depurination product [an apurinic (AP)-site]. Previously, we exploited Me-lex, i.e. {1-methyl-4-[1-methyl-4-(3-methoxysulfonylpropanamido)pyrrole-2-carboxamido]-pyrrole-2 carboxamido}propane, a minor groove equilibrium binder with selectivity for A/T rich sequences that efficiently reacts with DNA to afford 3-mA as the dominant product, to probe the biology of this lesion. Using human p53 cDNA as a target in a yeast system, a weak increase in mutagenicity was observed in the absence of Mag1 (3-methyladenine-DNA glycosylase 1, mag1), the enzyme devoted to remove 3-mA from DNA. Moreover, a significant increase in mutagenicity occurred in the absence of the enzymes involved in the repair of AP-sites (AP endonucleases 1 and 2, apn1apn2). Since methyl methanesulfonate (MMS) has been extensively used to explore the biological effects of 3-mA, even though it produces 3-mA in low relative yield, we compared the toxicity and mutagenicity induced by MMS and Me-lex in yeast. A mutagenesis reporter plasmid was damaged in vitro by MMS and then transformed into wild-type and Translesion Synthesis (TLS) Polζ (REV3) and Polη (RAD30) deficient strains. Furthermore, a mag1rad30 double mutant strain was constructed and transformed with the DNA plasmid damaged in vitro by Me-lex. The results confirm the important role of Polζ in the mutagenic bypass of MMS and Me-lex induced lesions, with Polη contributing only towards the bypass of Me-lex induced lesions, mainly in an error-free way. Previous and present results point towards the involvement of AP-sites, derived from the depurination of 3-mA, in the observed toxicity and mutagenicity.
Assuntos
Adenina/análogos & derivados , Metanossulfonato de Metila/toxicidade , Mutagênicos/toxicidade , Netropsina/análogos & derivados , Adenina/fisiologia , DNA Polimerase Dirigida por DNA/fisiologia , Humanos , Netropsina/toxicidade , Proteínas de Saccharomyces cerevisiae/fisiologiaRESUMO
A cationic 7-aminomethyl-7-deaza-2'-deoxyguanosine (7amG) was incorporated site-specifically into the self-complementary duplex d(G¹A²G³A4X5C6G7C8T9C¹°T¹¹C¹²)2 (X = 7amG). This construct placed two positively charged amines adjacent to the major groove edges of two symmetry-related guanines, providing a model for probing how cation binding in the major groove modulates the structure and stability of DNA. Molecular dynamics calculations restrained by nuclear magnetic resonance (NMR) data revealed that the tethered cationic amines were in plane with the modified base pairs. The tethered amines did not form salt bridges to the phosphodiester backbone. There was also no indication of the amines being capable of hydrogen bonding to flanking DNA bases. NMR spectroscopy as a function of temperature revealed that the X5 imino resonance remained sharp at 55 °C. Additionally, two 5'-neighboring base pairs, A4:T9 and G³:C¹°, were stabilized with respect to the exchange of their imino protons with solvent. The equilibrium constant for base pair opening at the A4:T9 base pair determined by magnetization transfer from water in the absence and presence of added ammonia base catalyst decreased for the modified duplex compared to that of the A4:T9 base pair in the unmodified duplex, which confirmed that the overall fraction of the A4:T9 base pair in the open state of the modified duplex decreased. This was also observed for the G³:C¹° base pair, where αK(op) for the G³:C¹° base pair in the modified duplex was 3.0 × 106 versus 4.1 × 106 for the same base pair in the unmodified duplex. In contrast, equilibrium constants for base pair opening at the X5:C8 and C6:G7 base pairs did not change at 15 °C. These results argue against the notion that electrostatic interactions with DNA are entirely entropic and suggest that major groove cations can stabilize DNA via enthalpic contributions to the free energy of duplex formation.
Assuntos
DNA/química , Modelos Moleculares , Nucleosídeo Q/análogos & derivados , Oligodesoxirribonucleotídeos/química , Cinética , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular , Conformação de Ácido Nucleico , Desnaturação de Ácido Nucleico , Nucleosídeo Q/química , Motivos de Nucleotídeos , Oligodesoxirribonucleotídeos/síntese química , TermodinâmicaRESUMO
Derivatives of methyl 3-(1-methyl-5-(1-methyl-5-(propylcarbamoyl)-1H-pyrrol-3-ylcarbamoyl)-1H-pyrrol-3-ylamino)-3-oxopropane-1-sulfonate (1), a peptide-based DNA minor groove binding methylating agent, were synthesized and characterized. In all cases, the N-terminus was appended with an O-methyl sulfonate ester, while the C-terminus group was varied with nonpolar and polar side chains. In addition, the number of pyrrole rings was varied from 2 (dipeptide) to 3 (tripeptide). The ability of the different analogues to efficiently generate N3-methyladenine was demonstrated as was their selectivity for minor groove (N3-methyladenine) versus major groove (N7-methylguanine) methylation. Induced circular dichroism studies were used to measure the DNA equilibrium binding properties of the stable sulfone analogues; the tripeptide binds with affinity that is >10-fold higher than that of the dipeptide. The toxicities of the compounds were evaluated in alkA/tag glycosylase mutant E. coli and in human WT glioma cells and in cells overexpressing and under-expressing N-methylpurine-DNA glycosylase, which excises N3-methyladenine from DNA. The results show that equilibrium binding correlates with the levels of N3-methyladenine produced and cellular toxicity. The toxicity of 1 was inversely related to the expression of MPG in both the bacterial and mammalian cell lines. The enhanced toxicity parallels the reduced activation of PARP and the diminished rate of formation of aldehyde reactive sites observed in the MPG knockdown cells. It is proposed that unrepaired N3-methyladenine is toxic due to its ability to directly block DNA polymerization.
Assuntos
Alquilantes/síntese química , DNA/química , Adenina/análogos & derivados , Adenina/química , Alquilantes/química , Alquilantes/toxicidade , Animais , Bovinos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , DNA/metabolismo , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , DNA Glicosilases/química , DNA Glicosilases/metabolismo , Metilação de DNA , Escherichia coli/enzimologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Humanos , Peptídeos/química , Peptídeos/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , TermodinâmicaRESUMO
The oxidation of DNA resulting from reactive oxygen species generated during aerobic respiration is a major cause of genetic damage that, if not repaired, can lead to mutations and potentially an increase in the incidence of cancer and aging. A major oxidation product generated in cells is 8-oxoguanine (oxoG), which is removed from the nucleotide pool by the enzymatic hydrolysis of 8-oxo-2'-deoxyguanosine triphosphate and from genomic DNA by 8-oxoguanine-DNA glycosylase. Finding and repairing oxoG in the midst of a large excess of unmodified DNA requires a combination of rapid scanning of the DNA for the lesion followed by specific excision of the damaged base. The repair of oxoG involves flipping the lesion out of the DNA stack and into the active site of the 8-oxoguanine-DNA glycosylase. This would suggest that thermodynamic stability, in terms of the rate for local denaturation, could play a role in lesion recognition. While prior X-ray crystal and NMR structures show that DNA with oxoG lesions appears virtually identical to the corresponding unmodified duplex, thermodynamic studies indicate that oxoG has a destabilizing influence. Our studies show that oxoG destabilizes DNA (ΔΔG of 2-8 kcal mol(-1) over a 16-116 mM NaCl range) due to a significant reduction in the enthalpy term. The presence of oxoG has a profound effect on the level and nature of DNA hydration indicating that the environment around an oxoGâ¢C is fundamentally different than that found at Gâ¢C. The temperature-dependent imino proton NMR spectrum of oxoG modified DNA confirms the destabilization of the oxoGâ¢C pairing and those base pairs that are 5' of the lesion. The instability of the oxoG modification is attributed to changes in the hydrophilicity of the base and its impact on major groove cation binding.
Assuntos
DNA/química , Guanina/análogos & derivados , Pareamento de Bases , Calorimetria , Guanina/química , Ressonância Magnética Nuclear Biomolecular , Oligonucleotídeos/química , Cloreto de Sódio/química , Espectrofotometria Ultravioleta , Temperatura , Água/químicaRESUMO
Oxidation of DNA due to exposure to reactive oxygen species is a major source of DNA damage. One of the oxidation lesions formed, 5-hydroxy-2'-deoxycytidine, has been shown to miscode by some replicative DNA polymerases but not by error prone polymerases capable of translesion synthesis. The 5-hydroxy-2'-deoxycytidine lesion is repaired by DNA glycosylases that require the 5-hydroxycytidine base to be extrahelical so it can enter into the enzyme's active site where it is excised off the DNA backbone to afford an abasic site. The thermodynamic and nuclear magnetic resonance results presented here describe the effect of a 5-hydroxy-2'-deoxycytidine·2'-deoxyguanosine base pair on the stability of two different DNA duplexes. The results demonstrate that the lesion is highly destabilizing and that the energy barrier for the unstacking of 5-hydroxy-2'-deoxycytidine from the DNA duplex may be low. This could provide a thermodynamic mode of adduct identification by DNA glycosylases that requires the lesion to be extrahelical.
Assuntos
Dano ao DNA , Desoxicitidina/análogos & derivados , Desoxiguanosina/química , Sítios de Ligação , Dicroísmo Circular , DNA Glicosilases/química , DNA Glicosilases/metabolismo , Reparo do DNA , Desoxicitidina/química , Desoxicitidina/metabolismo , Desoxiguanosina/metabolismo , Ressonância Magnética Nuclear Biomolecular , Conformação de Ácido Nucleico , TermodinâmicaRESUMO
The repair of abasic sites that arise in DNA from hydrolytic depurination/depyrimidination of the nitrogenous bases from the sugar-phosphate backbone and the action of DNA glycosylases on deaminated, oxidized, and alkylated bases are critical to cell survival. Apurinic/apyrimidinic endonuclease-1/redox effector factor-1 (APE-1; aka APE1/ref-1) is responsible for the initial removal of abasic lesions as part of the base excision repair pathway. Deletion of APE-1 activity is embryonic lethal in animals and is lethal in cells. Potential inhibitors of the repair function of APE-1 were identified based upon molecular modeling of the crystal structure of the APE-1 protein. We describe the characterization of several unique nanomolar inhibitors using two complementary biochemical screens. The most active molecules all contain a 2-methyl-4-amino-6,7-dioxolo-quinoline structure that is predicted from the modeling to anchor the compounds in the endonuclease site of the protein. The mechanism of action of the selected compounds was probed by fluorescence and competition studies, which indicate, in a specific case, direct interaction between the inhibitor and the active site of the protein. It is demonstrated that the inhibitors induce time-dependent increases in the accumulation of abasic sites in cells at levels that correlate with their potency to inhibit APE-1 endonuclease excision. The inhibitor molecules also potentiate by 5-fold the toxicity of a DNA methylating agent that creates abasic sites. The molecules represent a new class of APE-1 inhibitors that can be used to probe the biology of this critical enzyme and to sensitize resistant tumor cells to the cytotoxicity of clinically used DNA damaging anticancer drugs.
Assuntos
DNA Liase (Sítios Apurínicos ou Apirimidínicos)/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Sequência de Bases , Domínio Catalítico , Linhagem Celular Tumoral , DNA/genética , DNA/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/química , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/toxicidade , Humanos , Simulação de Acoplamento Molecular , Oxirredução/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/toxicidadeRESUMO
Understanding the origins of mutations in tumor suppressor genes and oncogenes associated with cancers in different tissues is critical to the development of potential prevention strategies. Analysis of >10,000 nonsense mutations in 63 tumor suppressor genes based on the ratio of the number of nonsense mutations per codon type is reported for each gene. The ratio for Câ¢GâTâ¢A nonsense mutations at Arg CGA codons to the number of CGA codons in all cancers is 23 (3088 total nonsense mutations for 134 CGA codons in the 63 suppressor genes). The ratio for this codon, which is attributed to hydrolytic deamination of 5-methylcytosine at CpG sites based on the sequence context, is 6-fold higher than the next highest ratio that involves a Câ¢GâTâ¢A transition at Trp TGG codons. Câ¢GâAâ¢T transversions at Glu, Ser, Tyr, Gly and Cys codons account for 25 % of the total nonsense mutations but the mutation per codon ratio for these codons is 1.0. Analysis of the bases 5' of the mutated CGA codons in the 63 tumor suppressor genes in all cancers shows a preference of 5'-G > C â¼ T â¼ A, which is not indicative of a role for enzymatic deamination by deaminases. Overall Câ¢GâTâ¢A mutations account for 61 % of all of the nonsense mutations in the collection of tumor suppressor genes. It is demonstrated that the ratio of Câ¢GâTâ¢A deamination-associated nonsense mutations at CGA codons (hydrolytic deamination) to the number of frame shift insertion/deletion mutations (i.e., replication based) for 5 major tumor suppressors genes are very similar in 3 different tissues that undergo a wide range of stem cell divisions. Therefore, the frequency of deamination mutations parallels the number of stem cell replications. This may reflect the generation of more solvent accessible single-stranded DNA regions during polymerization that are kinetically more prone to deamination.
Assuntos
Códon sem Sentido , Códon/química , Genes Supressores de Tumor , Neoplasias/genética , Códon/metabolismo , Bases de Dados Factuais , Regulação Neoplásica da Expressão Gênica , Humanos , Taxa de Mutação , Neoplasias/patologiaRESUMO
We recently demonstrated that Polzeta and Rev1 contribute to alleviate the lethal effects of Me-lex, which selectively generates 3-methyladenine, by error prone lesion bypass. In order to determine the role of Poleta in the biological fate of Me-lex induced lesions, the RAD30 (Poleta) gene was deleted in the yIG397 parental strain and in its rev3 (Polzeta) derivative, and the strains transformed with plasmid DNA damaged in vitro by Me-lex. While deletion of RAD30 increased the toxicity of Me-lex, the impact on mutagenicity varied depending on the concentration of Me-lex induced DNA damage and the overall TLS capacity of the cells. For the first time the Me-lex induced mutation spectrum in rad30 strain was determined and compared with the spectrum previously determined in WT strain. Overall, the two mutation spectra were not significantly different. The effect on mutation frequency and the features of the Me-lex induced mutation spectra were suggestive of error prone (significant decrease of mutation frequency and significant decrease of AT>TA at a mutation hotspot in rad30 vs RAD30) but also error free (significant increase of AT>GC in rad30 vs RAD30) Poleta dependent bypass of lesions. In summary, our previous results with Polzeta and Rev1 mutants, the present results with Poleta, and the known physical and functional interactions among TLS proteins, lead us to propose that the bypass of Me-lex induced lesions is a multi-DNA polymerases process that is mostly effective when all three yeast TLS polymerases are present.
Assuntos
Adenina/análogos & derivados , Dano ao DNA , DNA Fúngico/efeitos dos fármacos , DNA Polimerase Dirigida por DNA/fisiologia , Mutagênese , Netropsina/análogos & derivados , Saccharomyces cerevisiae/efeitos dos fármacos , Adenina/toxicidade , DNA Fúngico/genética , Deleção de Genes , Mutação/genética , Netropsina/toxicidade , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/fisiologia , Proteína Supressora de Tumor p53/genéticaRESUMO
The replacement of the 7-N atom on guanine (G) with a C-H to give 7-deazaguanine (c(7)G) alters the electronic properties of the heterocyclic base and eliminates a potential major groove cation binding site, which affects the organization of salts and water in the major groove. This has a destabilizing effect on DNA. We report herein the characterization of DNA oligomers containing 7-(aminomethyl)-7-deazaguanine (1) residues using a variety of spectroscopic and thermodynamic approaches. 1 is an intramolecular model for the major groove binding of cations and basic amino acid residues to G. In contrast to c(7)G, the tethering of a cation in the major groove using 1 affords DNA that is as, or more, stable than the corresponding unmodified DNA. The stabilization is associated with the folding enthalpy and hydration.
Assuntos
Pareamento de Bases , Citosina , DNA/química , Desoxiguanosina/química , Guanina , Nucleosídeo Q/análogos & derivados , Oligodesoxirribonucleotídeos/química , Sequência de Bases , DNA/genética , Nucleosídeo Q/química , Oligodesoxirribonucleotídeos/genética , Temperatura , TermodinâmicaRESUMO
The relative toxicity and mutagenicity of Me-lex, which selectively generates 3-methyladenine (3-MeA), is dependent on the nature of the DNA repair background. Base excision repair (BER)-defective S. cerevisiae strains mag1 and apn1apn2 were both significantly more sensitive to Me-lex toxicity, but only the latter is significantly more prone to Me-lex-induced mutagenesis. To examine the contribution of translesion synthesis (TLS) DNA polymerases in the bypass of Me-lex-induced lesions, the REV3 and REV1 genes were independently deleted in the parental yeast strain and in different DNA repair-deficient derivatives: the nucleotide excision repair (NER)-deficient rad14, and the BER-deficient mag1 or apn1apn2 strains. The strains contained an integrated ADE2 reporter gene under control of the transcription factor p53. A centromeric yeast expression vector containing the wild-type p53 cDNA was treated in vitro with increasing concentrations of Me-lex and transformed into the different yeast strains. The toxicity of Me-lex-induced lesions was evaluated based on the plasmid transformation efficiency compared to the untreated vector, while Me-lex mutagenicity was assessed using the p53 reporter assay. In the present study, we demonstrate that disruption of Polzeta (through deletion of its catalytic subunit coded by REV3) or Rev1 (by REV1 deletion) increased Me-lex lethality and decreased Me-lex mutagenicity in both the NER-defective (rad14) and BER-defective (mag1; apn1apn2) strains. Therefore, Polzeta and Rev1 contribute to resistance of the lethal effects of Me-lex-induced lesions (3-MeA and derived AP sites) by bypassing lesions and fixing some mutations.
Assuntos
Adenina/análogos & derivados , Antimutagênicos/farmacologia , Metilação de DNA/efeitos dos fármacos , Mutagênicos/toxicidade , Netropsina/análogos & derivados , Nucleotidiltransferases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Adenina/química , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , DNA Glicosilases/genética , DNA Glicosilases/metabolismo , Reparo do DNA/fisiologia , Enzimas Reparadoras do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Netropsina/toxicidade , Nucleotidiltransferases/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteína Supressora de Tumor p53RESUMO
We have investigated the mutagenicity induced at the Hprt locus in Chinese hamster ovary (CHO) cells treated with increasing concentrations of Me-lex, a minor groove selective methylating agent that efficiently generates more than 90-95% of 3-MeA DNA adducts. Me-lex treatment was cytotoxic but weakly mutagenic, resulting in up to 7-fold induction above background in the Hprt mutation frequency. The molecular nature of 43 Hprt mutations induced by Me-lex was determined by sequence analysis of the Hprt cDNA and genomic analysis of the gene locus. Base pair substitutions represented about 25% of Me-lex induced mutations. The mutation spectrum revealed a high percentage of genomic deletions (51%) comprising single/multiple exon(s) and even the loss of the complete locus. When the distribution of mutations among different classes was considered, the difference between the spontaneous and Me-lex induced CHO spectra was statistically significant (p<0.012), indicating that the sites where mutations occurred were Me-lex specific. Based upon these results we hypothesize that a large proportion of mutations may result from the processing of 3-MeA, the main adduct induced by Me-lex, within A/T rich sequences in non-coding regions of the Hprt gene. The processing of these lesions by DNA polymerases could result in recombination and genomic deletions, thus representing a severe threat for genome integrity.
Assuntos
Adenina/análogos & derivados , Deleção de Sequência , Adenina/metabolismo , Adenina/farmacologia , Animais , Sequência de Bases , Células CHO , Cricetinae , Cricetulus , Metilação de DNA , Hipoxantina Fosforribosiltransferase/genética , MutagênicosRESUMO
The incorporation of 7-deazaguanine modifications into DNA is frequently used to probe protein recognition of H-bonding information in the major groove of DNA. While it is generally assumed that 7-deazaguanine forms a normal Watson-Crick base pair with cytosine, detailed thermodynamic and structural analyses of this modification have not been reported. The replacement of the 7-N atom on guanine with a C-H, alters the electronic properties of the heterocycle and eliminates a major groove cation-binding site that could affect the organization of salts and water in the major groove. We report herein the characterization of synthetic DNA oligomers containing 7-deazaguanine using a variety of complementary approaches: UV thermal melting, differential scanning calorimetry (DSC), circular dichroism (CD), chemical probing and NMR. The results indicate that the incorporation of a 7-deazaguanine modification has a significant effect on the dynamic structure of the DNA at the flanking residue. This appears to be mediated by changes in hydration and cation organization.
Assuntos
DNA/química , Desoxicitidina/química , Desoxiguanosina/análogos & derivados , Pareamento de Bases , Varredura Diferencial de Calorimetria , Cátions/química , Dicroísmo Circular , Citosina/química , Desoxiguanosina/química , Ligação de Hidrogênio , Ressonância Magnética Nuclear Biomolecular , Conformação de Ácido Nucleico , Espectrofotometria Ultravioleta , Eletricidade Estática , Temperatura , Água/químicaRESUMO
The etiology of glioblastoma multiforme (GBM), the most serious form of brain cancer, remains obscure, although it has been proposed that cancer risk is a function of random polymerase errors that occur during stem cell division and the resulting mutations in oncogenes and tumor suppressor genes. Analysis of the 8 genes (PTEN, TP53, EGFR, PIK3R1, PIK3CA, NF1, RB1, IDH1) that are mutated in at least 5% of GBM tumors indicates a non-random mutation pattern that reflects a significant role for hydrolytic deamination at CpG sites. The formation of activating mutations in some genes, e.g., IDH1, where a very limited set of mutations are oncogenic, statistically cannot involve random mutagenesis due to polymerase errors that occur during each stem cell replication. Comparison of the in vitro misincorporation tendencies of three replicative polymerases and the "random" mutation pattern in a subset of genes indicates non-polymerase based pathways are involved. Analysis of the mutation patterns shows that chemical deamination that occurs at a slow rate at each CpG is favored over random polymerase errors by a factor of more than 10 million. Therefore, if a truncating nonsense mutation in a tumor suppressor, or an activating missense mutation in an oncogene, can occur due to a C > T base substitution at a CpG sequence, it is highly favored over other mutation pathways.
RESUMO
Site-specific insertion of 5-(3-aminopropyl)-2'-deoxyuridine (Z3dU) and 7-deaza-dG into the Dickerson-Drew dodecamers 5'-d(C (1)G (2)C (3)G (4)A (5)A (6)T (7)T (8)C (9) Z (10)C (11)G (12))-3'.5'-d(C (13)G (14)C (15)G (16)A (17)A (18)T (19)T (20)C (21) Z (22)C (23)G (24))-3' (named DDD (Z10)) and 5'-d(C (1)G (2)C (3)G (4)A (5)A (6)T (7) X (8)C (9) Z (10)C (11)G (12))-3'.5'-d(C (13)G (14)C (15)G (16)A (17)A (18)T (19) X (20)C (21) Z (22)C (23)G (24))-3' (named DDD (2+Z10)) (X = Z3dU; Z = 7-deaza-dG) suggests a mechanism underlying the formation of interstrand N+2 DNA cross-links by nitrogen mustards, e.g., melphalan and mechlorethamine. Analysis of the DDD (2+Z10) duplex reveals that the tethered cations at base pairs A (5).X (20) and X (8).A (17) extend within the major groove in the 3'-direction, toward conserved Mg (2+) binding sites located adjacent to N+2 base pairs C (3).Z (22) and Z (10).C (15). Bridging waters located between the tethered amines and either Z (10) or Z (22) O (6) stabilize the tethered cations and allow interactions with the N + 2 base pairs without DNA bending. Incorporation of 7-deaza-dG into the DDD (2+Z10) duplex weakens but does not eliminate electrostatic interactions between tethered amines and Z (10) O (6) and Z (22) O (6). The results suggest a mechanism by which tethered N7-dG aziridinium ions, the active species involved in formation of interstrand 5'-GNC-3' cross-links by nitrogen mustards, modify the electrostatics of the major groove and position the aziridinium ions proximate to the major groove edge of the N+2 C.G base pair, facilitating interstrand cross-linking.