RESUMEN
The N-(2-deoxy-d-erythro-pentofuranosyl)-urea DNA lesion forms following hydrolytic fragmentation of cis-5R,6S- and trans-5R,6R-dihydroxy-5,6-dihydrothymidine (thymine glycol, Tg) or from oxidation of 7,8-dihydro-8-oxo-deoxyguanosine (8-oxodG) and subsequent hydrolysis. It interconverts between α and ß deoxyribose anomers. Synthetic oligodeoxynucleotides containing this adduct are efficiently incised by unedited (K242) and edited (R242) forms of the hNEIL1 glycosylase. The structure of a complex between the active site unedited mutant CΔ100 P2G hNEIL1 (K242) glycosylase and double-stranded (ds) DNA containing a urea lesion reveals a pre-cleavage intermediate, in which the Gly2 N-terminal amine forms a conjugate with the deoxyribose C1' of the lesion, with the urea moiety remaining intact. This structure supports a proposed catalytic mechanism in which Glu3-mediated protonation of O4' facilitates attack at deoxyribose C1'. The deoxyribose is in the ring-opened configuration with the O4' oxygen protonated. The electron density of Lys242 suggests the 'residue 242-in conformation' associated with catalysis. This complex likely arises because the proton transfer steps involving Glu6 and Lys242 are hindered due to Glu6-mediated H-bonding with the Gly2 and the urea lesion. Consistent with crystallographic data, biochemical analyses show that the CΔ100 P2G hNEIL1 (K242) glycosylase exhibits a residual activity against urea-containing dsDNA.
Asunto(s)
ADN Glicosilasas , Reparación del ADN , Desoxirribosa , Urea , Desoxirribosa/química , ADN/química , Daño del ADN , ADN Glicosilasas/metabolismo , HumanosRESUMEN
Urea lesions in DNA arise from thymine glycol (Tg) or 8-oxo-dG; their genotoxicity is thought to arise in part due to their potential to accommodate the insertion of all four dNTPs during error-prone replication. Replication bypass with human DNA polymerase η (hPol η) confirmed that all four dNTPs were inserted opposite urea lesions but with purines exhibiting greater incorporation efficiency. X-ray crystal structures of ternary replication bypass complexes in the presence of Mg2+ ions with incoming dNTP analogs dAMPnPP, dCMPnPP, dGMPnPP, and dTMPnPP bound opposite urea lesions (hPol η·DNA·dNMPnPP complexes) revealed all were accommodated by hPol η. In each, the Watson-Crick face of the dNMPnPP was paired with the urea lesion, exploiting the ability of the amine and carbonyl groups of the urea to act as H-bond donors or acceptors, respectively. With incoming dAMPnPP or dGMPnPP, the distance between the imino nitrogen of urea and the N9 atoms of incoming dNMPnPP approximated the canonical distance of 9 Å in B-DNA. With incoming dCMPnPP or dTMPnPP, the corresponding distance of about 7 Å was less ideal. Improved base-stacking interactions were also observed with incoming purines vs pyrimidines. Nevertheless, in each instance, the α-phosphate of incoming dNMPnPPs was close to the 3'-hydroxyl group of the primer terminus, consistent with the catalysis of nucleotidyl transfer and the observation that all four nucleotides could be inserted opposite urea lesions. Preferential insertion of purines by hPol η may explain, in part, why the urea-directed spectrum of mutations arising from Tg vs 8-oxo-dG lesions differs.
Asunto(s)
Daño del ADN , ADN Polimerasa Dirigida por ADN , Humanos , 8-Hidroxi-2'-Desoxicoguanosina , ADN Polimerasa Dirigida por ADN/metabolismo , ADN/química , Replicación del ADN , Nucleótidos , Aductos de ADNRESUMEN
Aflatoxin B1 (AFB1) is a potent human liver carcinogen produced by certain molds, particularly Aspergillus flavus and Aspergillus parasiticus, which contaminate peanuts, corn, rice, cottonseed, and ground and tree nuts, principally in warm and humid climates. AFB1 undergoes bioactivation in the liver to produce AFB1-exo-8,9-epoxide, which forms the covalently bound cationic AFB1-N7-guanine (AFB1-N7-Gua) DNA adduct. This adduct is unstable and undergoes base-catalyzed opening of the guanine imidazolium ring to form two ring-opened diastereomeric 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)-9-hydroxy-aflatoxin B1 (AFB1-FapyGua) adducts. The AFB1 formamidopyrimidine (Fapy) adducts induce G â T transversion mutations and are likely responsible for the carcinogenic effects of AFB1. Quantitative liquid chromatography-mass spectrometry (LC-MS) methods have shown that AFB1-N7-Gua is eliminated in rodent and human urine, whereas ring-opened AFB1-FapyGua adducts persist in rodent liver. However, fresh frozen biopsy tissues are seldom available for biomonitoring AFB1 DNA adducts in humans, impeding research advances in this potent liver carcinogen. In contrast, formalin-fixed paraffin-embedded (FFPE) specimens used for histopathological analysis are often accessible for molecular studies. However, ensuring nucleic acid quality presents a challenge due to incomplete reversal of formalin-mediated DNA cross-links, which can preclude accurate quantitative measurements of DNA adducts. In this study, employing ion trap or high-resolution accurate Orbitrap mass spectrometry, we demonstrate that ring-opened AFB1-FapyGua adducts formed in AFB1-exposed newborn mice are stable to the formalin fixation and DNA de-cross-linking retrieval processes. The AFB1-FapyGua adducts can be detected at levels comparable to those in a match of fresh frozen liver. Orbitrap MS2 measurements can detect AFB1-FapyGua at a quantification limit of 4.0 adducts per 108 bases when only 0.8 µg of DNA is assayed on the column. Thus, our breakthrough DNA retrieval technology can be adapted to screen for AFB1 DNA adducts in FFPE human liver specimens from cohorts at risk of this potent liver carcinogen.
Asunto(s)
Aflatoxina B1 , Aductos de ADN , Ratones , Humanos , Animales , Aflatoxina B1/química , Adhesión en Parafina , ADN/metabolismo , Carcinógenos/metabolismo , Espectrometría de Masas , Guanina , FormaldehídoRESUMEN
The genotoxic 3-(2-deoxy-ß-D-erythro-pentofuranosyl)pyrimido[1,2-α]purin-10(3H)-one (M1dG) DNA lesion arises from endogenous exposures to base propenals generated by oxidative damage and from exposures to malondialdehyde (MDA), produced by lipid peroxidation. Once formed, M1dG may oxidize, in vivo, to 3-(2-deoxy-ß-D-erythropentofuranosyl)-pyrimido[1,2-f]purine-6,10(3H,5H)-dione (6-oxo-M1dG). The latter blocks DNA replication and is a substrate for error-prone mutagenic bypass by the Y-family DNA polymerase hpol η. To examine structural consequences of 6-oxo-M1dG damage in DNA, we conducted NMR studies of 6-oxo-M1dG incorporated site-specifically into 5' -d(C1A2T3X4A5T6G7A8C9G10C11T12)-3':5'-d(A13G14C15G16T17C18A19T20C21A22T23G24)-3' (X = 6-oxo-M1dG). NMR spectra afforded detailed resonance assignments. Chemical shift analyses revealed that nucleobase C21, complementary to 6-oxo-M1dG, was deshielded compared with the unmodified duplex. Sequential NOEs between 6-oxo-M1dG and A5 were disrupted, as well as NOEs between T20 and C21 in the complementary strand. The structure of the 6-oxo-M1dG modified DNA duplex was refined by using molecular dynamics (rMD) calculations restrained by NOE data. It revealed that 6-oxo-M1dG intercalated into the duplex and remained in the anti-conformation about the glycosyl bond. The complementary cytosine C21 extruded into the major groove, accommodating the intercalated 6-oxo-M1dG. The 6-oxo-M1dG H7 and H8 protons faced toward the major groove, while the 6-oxo-M1dG imidazole proton H2 faced into the major groove. Structural perturbations to dsDNA were limited to the 6-oxo-M1dG damaged base pair and the flanking T3:A22 and A5:T20 base pairs. Both neighboring base pairs remained within the Watson-Crick hydrogen bonding contact. The 6-oxo-M1dG did not stack well with the 5'-neighboring base pair T3:A22 but showed improved stacking with the 3'-neighboring base pair A5:T20. Overall, the base-displaced intercalated structure was consistent with thermal destabilization of the 6-oxo-M1dG damaged DNA duplex; thermal melting temperature data showed a 15 °C decrease in Tm compared to the unmodified duplex. The structural consequences of 6-oxo-M1dG formation in DNA are evaluated in the context of the chemical biology of this lesion.
Asunto(s)
Aductos de ADN , ADN , ADN/química , Purinas/química , Daño del ADN , Conformación Molecular , Protones , Conformación de Ácido Nucleico , Desoxiguanosina/químicaRESUMEN
O6-Methyl-2'-deoxyguanosine (O6-MeG) is one of the most common DNA lesions and arises as a consequence of both xenobiotic carcinogens and endogenous methylation by S-adenosylmethionine. O6-MeG frequently causes G-to-A mutations during DNA replication due to the misincorporation of dTTP and continued DNA synthesis. Efforts to detect DNA adducts such as O6-MeG, and to understand their impacts on DNA structure and function, have motivated the creation of nucleoside analogs with altered base moieties to afford a more favorable interaction with the adduct as compared to the unmodified nucleotide. Such analogs directed at O6-MeG include benzimidazolinone and benzimidazole nucleotides, as well as their extended π surface analogs naphthimidazolinone and napthimidazole derivatives. These analogs form a more stable pair with O6-MeG than with G, most likely due to a combination of H-bonding and stacking. While extending the π surface of the analogs enhances their performance as adduct-directed probes, the precise origins of the increased affinity between the synthetic analogs and O6-MeG remain unclear. To better understand relevant conformational and pairing properties, we used X-ray crystallography and analyzed the structures of the DNA duplexes with naphthimidazolinone inserted opposite G or O6-MeG. The structures reveal a complex interaction of the analog found either in an anti orientation and stacked inside the duplex, either above or below G or O6-MeG, or in a syn orientation and paired opposite G with formation of a single H-bond. The experimental structural data are consistent with the stabilizing effect of the synthetic analog observed in UV melting experiments and calculations and moreover reveal that the origin of these observations appears to be superior stacking between O6-MeG and the extended π system of the synthetic probe.
Asunto(s)
Aductos de ADN , Nucleósidos , Bencimidazoles , Carcinógenos , ADN/química , Desoxiguanosina/análogos & derivados , Conformación de Ácido Nucleico , Nucleósidos/química , Nucleótidos , S-Adenosilmetionina , XenobióticosRESUMEN
Abasic (AP) sites are one of the most common forms of DNA damage. The deoxyribose ring of AP sites undergoes anomerization between α and ß configurations, via an electrophilic aldehyde intermediate. In sequences where an adenine residue is located on the opposing strand and offset 1 nt to the 3' side of the AP site, the nucleophilic N6-dA amino group can react with the AP aldehyde residue to form an interstrand cross-link (ICL). Here, we present an experimentally determined structure of the dA-AP ICL by NMR spectroscopy. The ICL was constructed in the oligodeoxynucleotide 5'-d(T1A2T3G4T5C6T7A8A9G10T11T12C13A14T15C16T17A18)-3':5'-d(T19A20G21A22T23G24A25A26C27X28T29A30G31A32C33A34T35A36)-3' (X=AP site), with the dA-AP ICL forming between A8 and X28. The NMR spectra indicated an ordered structure for the cross-linked DNA duplex and afforded detailed spectroscopic resonance assignments. Structural refinement, using molecular dynamics calculations restrained by NOE data (rMD), revealed the structure of the ICL. In the dA-AP ICL, the 2'-deoxyribosyl ring of the AP site was ring-closed and in the ß configuration. Juxtapositioning the N6-dA amino group and the aldehydic C1 of the AP site within bonding distance while simultaneously maintaining two flanking unpaired A9 and T29 bases stacked within the DNA is accomplished by the unwinding of the DNA at the ICL. The structural data is discussed in the context of recent studies describing the replication-dependent unhooking of the dA-AP ICL by the base excision repair glycosylase NEIL3.
Asunto(s)
Adenina/química , Aldehídos/química , Reactivos de Enlaces Cruzados/química , Daño del ADN , ADN/química , Reparación del ADN , Humanos , Conformación de Ácido NucleicoRESUMEN
Dietary exposure to aflatoxins is a significant risk factor in the development of hepatocellular carcinomas. Following bioactivation by microsomal P450s, the reaction of aflatoxin B1 (AFB1) with guanine (Gua) in DNA leads to the formation of stable, imidazole ring-opened 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)-9-hydroxyaflatoxin B1 (AFB1-FapyGua) adducts. In contrast to most base modifications that result in destabilization of the DNA duplex, the AFB1-FapyGua adduct increases the thermal stability of DNA via 5'-interface intercalation and base-stacking interactions. Although it was anticipated that this stabilization might make these lesions difficult to repair relative to helix distorting modifications, prior studies have shown that both the nucleotide and base excision repair pathways participate in the removal of the AFB1-FapyGua adduct. Specifically for base excision repair, we previously showed that the DNA glycosylase NEIL1 excises AFB1-FapyGua and catalyzes strand scission in both synthetic oligodeoxynucleotides and liver DNA of exposed mice. Since it is anticipated that error-prone replication bypass of unrepaired AFB1-FapyGua adducts contributes to cellular transformation and carcinogenesis, the structural and thermodynamic parameters that modulate the efficiencies of these repair pathways are of considerable interest. We hypothesized that the DNA sequence context in which the AFB1-FapyGua adduct is formed might modulate duplex stability and consequently alter the efficiencies of NEIL1-initiated repair. To address this hypothesis, site-specific AFB1-FapyGua adducts were synthesized in three sequence contexts, with the 5' neighbor nucleotide being varied. DNA structural stability analyses were conducted using UV absorbance- and NMR-based melting experiments. These data revealed differentials in thermal stabilities associated with the 5'-neighbor base pair. Single turnover kinetic analyses using the NEIL1 glycosylase demonstrated corresponding sequence-dependent differences in the repair of this adduct, such that there was an inverse correlation between the stabilization of the duplex and the efficiency of NEIL1-mediated catalysis.
Asunto(s)
Aflatoxina B1/metabolismo , Aductos de ADN/metabolismo , ADN Glicosilasas/metabolismo , ADN/metabolismo , Guanina/metabolismo , Pirimidinas/metabolismo , Aflatoxina B1/química , Secuencia de Bases , Biocatálisis , ADN/química , Aductos de ADN/química , ADN Glicosilasas/química , Guanina/química , Humanos , Estructura Molecular , Pirimidinas/químicaRESUMEN
Global distribution of hepatocellular carcinomas (HCCs) is dominated by its incidence in developing countries, accounting for >700,000 estimated deaths per year, with dietary exposures to aflatoxin (AFB1) and subsequent DNA adduct formation being a significant driver. Genetic variants that increase individual susceptibility to AFB1-induced HCCs are poorly understood. Herein, it is shown that the DNA base excision repair (BER) enzyme, DNA glycosylase NEIL1, efficiently recognizes and excises the highly mutagenic imidazole ring-opened AFB1-deoxyguanosine adduct (AFB1-Fapy-dG). Consistent with this in vitro result, newborn mice injected with AFB1 show significant increases in the levels of AFB1-Fapy-dG in Neil1-/- vs. wild-type liver DNA. Further, Neil1-/- mice are highly susceptible to AFB1-induced HCCs relative to WT controls, with both the frequency and average size of hepatocellular carcinomas being elevated in Neil1-/- The magnitude of this effect in Neil1-/- mice is greater than that previously measured in Xeroderma pigmentosum complementation group A (XPA) mice that are deficient in nucleotide excision repair (NER). Given that several human polymorphic variants of NEIL1 are catalytically inactive for their DNA glycosylase activity, these deficiencies may increase susceptibility to AFB1-associated HCCs.
Asunto(s)
Aflatoxinas/toxicidad , Carcinoma Hepatocelular/prevención & control , Aductos de ADN/efectos de los fármacos , ADN Glicosilasas/fisiología , Neoplasias Hepáticas Experimentales/prevención & control , Sustancias Protectoras/farmacología , Animales , Carcinoma Hepatocelular/inducido químicamente , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Femenino , Neoplasias Hepáticas Experimentales/inducido químicamente , Neoplasias Hepáticas Experimentales/metabolismo , Neoplasias Hepáticas Experimentales/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Venenos/toxicidadRESUMEN
Routine dietary consumption of foods that contain aflatoxins is the second leading cause of environmental carcinogenesis worldwide. Aflatoxin-driven mutagenesis is initiated through metabolic activation of aflatoxin B1 (AFB1) to its epoxide form that reacts with N7 guanine in DNA. The resulting AFB1-N7-dG adduct undergoes either spontaneous depurination or imidazole-ring opening yielding formamidopyrimidine AFB1 (AFB1-Fapy-dG). Because this latter adduct is known to persist in human tissues and contributes to the high frequency G-to-T mutation signature associated with many hepatocellular carcinomas, we sought to establish the identity of the polymerase(s) involved in processing this lesion. Although our previous biochemical analyses demonstrated the ability of polymerase ζ (pol ζ) to incorporate an A opposite AFB1-Fapy-dG and extend from this mismatch, biological evidence supporting a unique role for this polymerase in cellular tolerance following aflatoxin exposure has not been established. Following challenge with AFB1, survival of mouse cells deficient in pol ζ (Rev3L-/-) was significantly reduced relative to Rev3L+/- cells or Rev3L-/- cells complemented through expression of the wild-type human REV3L. Furthermore, cell-cycle progression of Rev3L-/- mouse embryo fibroblasts was arrested in late S/G2 following AFB1 exposure. These Rev3L-/- cells showed an increase in replication-dependent formation of γ-H2AX foci, micronuclei, and chromosomal aberrations (chromatid breaks and radials) relative to Rev3L+/- cells. These data suggest that pol ζ is essential for processing AFB1-induced DNA adducts and that, in its absence, cells do not have an efficient backup polymerase or a repair/tolerance mechanism facilitating survival.
Asunto(s)
Carcinoma Hepatocelular/genética , Proteínas de Unión al ADN/genética , ADN Polimerasa Dirigida por ADN/genética , Neoplasias Hepáticas/genética , Aflatoxina B1/análogos & derivados , Aflatoxina B1/genética , Aflatoxina B1/toxicidad , Aflatoxinas/toxicidad , Animales , Carcinoma Hepatocelular/inducido químicamente , Carcinoma Hepatocelular/patología , Supervivencia Celular/efectos de los fármacos , Aberraciones Cromosómicas/efectos de los fármacos , Citidina/análogos & derivados , Citidina/genética , Citidina/toxicidad , Aductos de ADN/efectos de los fármacos , Aductos de ADN/genética , Daño del ADN/efectos de los fármacos , Reparación del ADN/genética , ADN Polimerasa Dirigida por ADN/química , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Neoplasias Hepáticas/inducido químicamente , Neoplasias Hepáticas/patología , Ratones , Mutagénesis/efectos de los fármacos , Mutagénesis/genética , MutaciónRESUMEN
The most common lesion in DNA occurring due to clinical treatment with Temozolomide or cellular exposures to other methylating agents is 7-methylguanine (N7-Me-dG). It can undergo a secondary reaction to form N6-(2-deoxy-d-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5- N-methylformamidopyrimidine (MeFapy-dG). MeFapy-dG undergoes epimerization in DNA to produce either α or ß deoxyribose anomers. Additionally, conformational rotation around the formyl bond, C5- N5 bond, and glycosidic bond may occur. To characterize and quantitate the mixture of these isomers in DNA, a 13C-MeFapy-dG lesion, in which the CH3 group of the MeFapy-dG was isotopically labeled, was incorporated into the trimer 5'-TXT-3' and the dodecamer 5'-CATXATGACGCT-3' (X = 13C-MeFapy-dG). NMR spectroscopy of both the trimer and dodecamer revealed that the MeFapy-dG lesion exists in single strand DNA as ten configurationally and conformationally discrete species, eight of which may be unequivocally assigned. In the duplex dodecamer, the MeFapy-dG lesion exists as six configurationally and conformationally discrete species. Analyses of NMR data in the single strand trimer confirm that for each deoxyribose anomer, atropisomerism occurs around the C5- N5 bond to produce R a and S a atropisomers. Each atropisomer exhibits geometrical isomerism about the formyl bond yielding E and Z conformations. 1H NMR experiments allow the relative abundances of the species to be determined. For the single strand trimer, the α and ß anomers exist in a 3:7 ratio, favoring the ß anomer. For the ß anomer, with respect to the C5- N5 bond, the R a and S a atropisomers are equally populated. However, the Z geometrical isomer of the formyl moiety is preferred. For the α anomer, the E- S a isomer is present at 12%, whereas all other isomers are present at 5-7%. DNA processing enzymes may differentially recognize different isomers of the MeFapy-dG lesion. Moreover, DNA sequence-specific differences in the populations of configurational and conformational species may modulate biological responses to the MeFapy-dG lesion.
Asunto(s)
Aductos de ADN/toxicidad , ADN/efectos de los fármacos , Espectroscopía de Resonancia Magnética con Carbono-13/métodos , Cromatografía Líquida de Alta Presión/métodos , ADN/química , Daño del ADN , Reparación del ADN , Replicación del ADN , Electroforesis Capilar/métodos , Isomerismo , Conformación de Ácido Nucleico , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodosRESUMEN
The environmental pollutant 3-nitrobenzanthrone produces bulky aminobenzanthrone (ABA) DNA adducts with both guanine and adenine nucleobases. A major product occurs at the C8 position of guanine (C8-dG-ABA). These adducts present a strong block to replicative polymerases but, remarkably, can be bypassed in a largely error-free manner by the human Y-family polymeraseâ η (hPol η). Here, we report the crystal structure of a ternary Polâ DNAâ dCTP complex between a C8-dG-ABA-containing template:primer duplex and hPol η. The complex was captured at the insertion stage and provides crucial insight into the mechanism of error-free bypass of this bulky lesion. Specifically, bypass involves accommodation of the ABA moiety inside a hydrophobic cleft to the side of the enzyme active site and formation of an intra-nucleotide hydrogen bond between the phosphate and ABA amino moiety, allowing the adducted guanine to form a standard Watson-Crick pair with the incoming dCTP.
Asunto(s)
Benzo(a)Antracenos/metabolismo , Aductos de ADN/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Desoxiguanosina/análogos & derivados , Benzo(a)Antracenos/química , Aductos de ADN/química , ADN Polimerasa Dirigida por ADN/química , Desoxiguanosina/química , Desoxiguanosina/metabolismo , Humanos , Modelos Moleculares , Estructura MolecularRESUMEN
2-Amino-3-methylimidazo[4,5-f]quinolone (IQ), a heterocyclic amine found in cooked meats, undergoes bioactivation to a nitrenium ion, which alkylates guanines at both the C8-dG and N2-dG positions. The conformation of a site-specific N2-dG-IQ adduct in an oligodeoxynucleotide duplex containing the iterated CG repeat restriction site of the NarI endonuclease has been determined. The IQ moiety intercalates, with the IQ H4a and CH3 protons facing the minor groove, and the IQ H7a, H8a and H9a protons facing the major groove. The adducted dG maintains the anti-conformation about the glycosyl bond. The complementary dC is extruded into the major groove. The duplex maintains its thermal stability, which is attributed to stacking between the IQ moiety and the 5'- and 3'-neighboring base pairs. This conformation is compared to that of the C8-dG-IQ adduct in the same sequence, which also formed a 'base-displaced intercalated' conformation. However, the C8-dG-IQ adopted the syn conformation placing the Watson-Crick edge of the modified dG into the major groove. In addition, the C8-dG-IQ adduct was oriented with the IQ CH3 group and H4a and H5a facing the major groove. These differences may lead to differential processing during DNA repair and replication.
Asunto(s)
Aminoquinolinas/química , Bencimidazoles/química , Aductos de ADN/química , Secuencia de Bases , ADN/química , Desoxirribonucleasas de Localización Especificada Tipo II , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos/química , ProtonesRESUMEN
5-Hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) form during active demethylation of 5-methylcytosine (5mC) and are implicated in epigenetic regulation of the genome. They are differentially processed by thymine DNA glycosylase (TDG), an enzyme involved in active demethylation of 5mC. Three modified Dickerson-Drew dodecamer (DDD) sequences, amenable to crystallographic and spectroscopic analyses and containing the 5'-CG-3' sequence associated with genomic cytosine methylation, containing 5hmC, 5fC, or 5caC placed site-specifically into the 5'-T(8)X(9)G(10)-3' sequence of the DDD, were compared. The presence of 5caC at the X(9) base increased the stability of the DDD, whereas 5hmC or 5fC did not. Both 5hmC and 5fC increased imino proton exchange rates and calculated rate constants for base pair opening at the neighboring base pair A(5):T(8), whereas 5caC did not. At the oxidized base pair G(4):X(9), 5fC exhibited an increase in the imino proton exchange rate and the calculated kop. In all cases, minimal effects to imino proton exchange rates occurred at the neighboring base pair C(3):G(10). No evidence was observed for imino tautomerization, accompanied by wobble base pairing, for 5hmC, 5fC, or 5caC when positioned at base pair G(4):X(9); each favored Watson-Crick base pairing. However, both 5fC and 5caC exhibited intranucleobase hydrogen bonding between their formyl or carboxyl oxygens, respectively, and the adjacent cytosine N(4) exocyclic amines. The lesion-specific differences observed in the DDD may be implicated in recognition of 5hmC, 5fC, or 5caC in DNA by TDG. However, they do not correlate with differential excision of 5hmC, 5fC, or 5caC by TDG, which may be mediated by differences in transition states of the enzyme-bound complexes.
Asunto(s)
Citosina/análogos & derivados , ADN/química , Oligonucleótidos/química , 5-Metilcitosina/análogos & derivados , Citosina/química , Timina ADN Glicosilasa/químicaRESUMEN
Hepatocellular carcinomas (HCCs) are the third leading cause of cancer deaths worldwide. The highest rates of early onset HCCs occur in geographical regions with high aflatoxin B1 (AFB1) exposure, concomitant with hepatitis B infection. Although the carcinogenic basis of AFB1 has been ascribed to its mutagenic effects, the mutagenic property of the primary AFB1-DNA adduct, AFB1-N7-Gua, in mammalian cells has not been studied extensively. Taking advantage of the ability to create vectors containing a site-specific DNA adduct, the mutagenic potential was determined in primate cells. This adduct was highly mutagenic following replication in COS-7 cells, with a mutation frequency of 45%. The spectrum of mutations was predominantly G to T base substitutions, a result that is consistent with previous mutation data derived from aflatoxin-associated HCCs. To assess which DNA polymerases (pol) might contribute to the mutational outcome, in vitro replication studies were performed. Unexpectedly, replicative pol δ and the error-prone translesion synthesis pol ζ were able to accurately bypass AFB1-N7-Gua. In contrast, replication bypass using pol κ was shown to occur with low fidelity and could account for the commonly detected G to T transversions.
Asunto(s)
Aflatoxina B1/toxicidad , Carcinoma Hepatocelular/genética , Aductos de ADN/genética , Replicación del ADN , Neoplasias Hepáticas/genética , Aflatoxina B1/genética , Aflatoxina B1/metabolismo , Animales , Células COS , Carcinoma Hepatocelular/inducido químicamente , Chlorocebus aethiops , Aductos de ADN/metabolismo , Daño del ADN , Reparación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Humanos , Neoplasias Hepáticas/inducido químicamente , Mutación PuntualRESUMEN
N(6)-(2-Deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine (MeFapy-dG) arises from N7-methylation of deoxyguanosine followed by imidazole ring opening. The lesion has been reported to persist in animal tissues. Previous in vitro replication bypass investigations of the MeFapy-dG adduct revealed predominant insertion of C opposite the lesion, dependent on the identity of the DNA polymerase (Pol) and the local sequence context. Here we report crystal structures of ternary Pol·DNA·dNTP complexes between MeFapy-dG-adducted DNA template:primer duplexes and the Y-family polymerases human Pol η and P2 Pol IV (Dpo4) from Sulfolobus solfataricus. The structures of the hPol η and Dpo4 complexes at the insertion and extension stages, respectively, are representative of error-free replication, with MeFapy-dG in the anti conformation and forming Watson-Crick pairs with dCTP or dC.
Asunto(s)
Daño del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Desoxiguanosina/química , Pirimidinas/química , Sulfolobus solfataricus/enzimología , Modelos MolecularesRESUMEN
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.
Asunto(s)
Daño del ADN , ADN/química , Termodinámica , Emparejamiento Base , Sitios de Unión , ADN/metabolismo , ADN Glicosilasas/metabolismo , Reparación del ADN , Cinética , Conformación de Ácido Nucleico , AguaRESUMEN
Aflatoxin B(1) (AFB(1)), a mycotoxin produced by Aspergillus flavus, is oxidized by cytochrome P450 enzymes to aflatoxin B(1)-8,9-epoxide, which alkylates DNA at N7-dG. Under basic conditions, this N7-dG adduct rearranges to yield the trans-8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)-9-hydroxy aflatoxin B(1) (AFB(1)−FAPY) adduct. The AFB(1)−FAPY adduct exhibits geometrical isomerism involving the formamide moiety. NMR analyses of duplex oligodeoxynucleotides containing the 5'-XA-3', 5'-XC-3', 5'-XT-3', and 5'-XY-3' sequences (X = AFB(1)−FAPY; Y = 7-deaza-dG)demonstrate that the equilibrium between E and Z isomers is controlled by major groove hydrogen bonding interactions.Structural analysis of the adduct in the 5'-XA-3' sequence indicates the preference of the E isomer of the formamide group,attributed to formation of a hydrogen bond between the formyl oxygen and the N(6) exocyclic amino group of the 3'-neighboradenine. While the 5'-XA-3' sequence exhibits the E isomer, the 5'-XC-3' sequence exhibits a 7:3 E:Z ratio at equilibrium at 283K. The E isomer is favored by a hydrogen bond between the formyl oxygen and the N(4)-dC exocyclic amino group of the 3'-neighbor cytosine. The 5'-XT-3' and 5'-XY-3' sequences cannot form such a hydrogen bond between the formyl oxygen and the 3'-neighbor T or Y, respectively, and in these sequence contexts the Z isomer is favored. Additional equilibria between α and ß anomers and the potential to exhibit atropisomers about the C5−N(5) bond do not depend upon sequence. In each of the four DNA sequences, the AFB(1)−FAPY adduct maintains the ß deoxyribose configuration. Each of these four sequences feature the atropisomer of the AFB(1) moiety that is intercalated above the 5'-face of the damaged guanine. This enforces the Ra axialc onformation for the C5−N(5) bond.
Asunto(s)
Aflatoxina B1/análogos & derivados , Aflatoxina B1/química , Citidina/análogos & derivados , ADN/química , Secuencia de Bases , Citidina/química , Espectroscopía de Resonancia Magnética , EstereoisomerismoRESUMEN
The conformation of an N(2)-dG adduct arising from the heterocyclic amine 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), a potent food mutagen, was determined in 5'-d(C(1)T(2)C(3)X(4)G(5)C(6)G(7)C(8)C(9)A(10)T(11)C(12))-3':5'-d(G(13)A(14)T(15)G(16)G(17)C(18)G(19)C(20)C(21)G(22)A(23)G(24))-3'; X = N(2)-dG-IQ, in which the modified nucleotide X(4) corresponds to G(1) in the 5'-d(G(1)G(2)CG(3)CC)-3' NarI restriction endonuclease site. Circular dichroism (CD) revealed blue shifts relative to the unmodified duplex, consistent with adduct-induced twisting, and a hypochromic effect for the IQ absorbance in the near UV region. NMR revealed that the N(2)-dG-IQ adduct adopted a base-displaced intercalated conformation in which the modified guanine remained in the anti conformation about the glycosidic bond, the IQ moiety intercalated into the duplex, and the complementary base C(21) was displaced into the major groove. The processing of the N(2)-dG-IQ lesion by hpol η is sequence-dependent; when placed at the reiterated G(3) position, but not at the G(1) position, this lesion exhibits a propensity for frameshift replication [Choi, J. Y., et al. (2006) J. Biol. Chem., 281, 25297-25306]. The structure of the N(2)-dG-IQ adduct at the nonreiterated G(1) position was compared to that of the same adduct placed at the G(3) position [Stavros, K. M., et al. (2014) Nucleic Acids Res., 42, 3450-3463]. CD indicted minimal spectral differences between the G(1) vs G(3) N(2)-dG-IQ adducts. NMR indicated that the N(2)-dG-IQ adduct exhibited similar base-displaced intercalated conformations at both the G(1) and G(3) positions. This result differed as compared to the corresponding C8-dG-IQ adducts placed at the same positions. The C8-dG-IQ adduct adopted a minor groove conformation when placed at position G(1) but a base-displaced intercalated conformation when placed at position G(3) in the NarI sequence. The present studies suggest that differences in lesion bypass by hpol η may be mediated by differences in the 3'-flanking sequences, perhaps modulating the ability to accommodate transient strand slippage intermediates.
Asunto(s)
ADN/química , Desoxiguanosina/análogos & derivados , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Sustancias Intercalantes/química , Espectroscopía de Resonancia Magnética , Quinolinas/química , Dicroismo Circular , ADN/metabolismo , Desoxiguanosina/química , Conformación Molecular , Conformación de Ácido Nucleico , Protones , Espectrofotometría UltravioletaRESUMEN
3-Nitrobenzanthrone (3-NBA), an environmental mutagen found in diesel exhaust and a suspected carcinogen, undergoes metabolic reduction followed by reaction with DNA to form aminobenzanthrone (ABA) adducts, with the major alkylation product being N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone (C8-dG-ABA). Site-specific synthesis of the C8-dG-ABA adduct in the oligodeoxynucleotide 5'-d(GTGCXTGTTTGT)-3':5'-d(ACAAACACGCAC)-3'; X = C8-dG-ABA adduct, including codons 272-275 of the p53 gene, has allowed for investigation into the structural and thermodynamic properties of this adduct. The conformation of the C8-dG-ABA adduct was determined using NMR spectroscopy and was refined using molecular dynamics (MD) calculations restrained by experimentally determined interproton distance restraints obtained from NOE experiments. The refined structure revealed that the C8-dG-ABA adduct formed a base-displaced intercalated conformation. The adducted guanine was shifted into the syn conformation about the glycosidic bond. The 5'- and 3'-neighboring base pairs remained intact. While this facilitated π-stacking interactions between the ABA moiety and neighboring bases, the thermal melting temperature (Tm) of the adduct-containing duplex showed a decrease of 11 °C as compared to the corresponding unmodified oligodeoxynucleotide duplex. Overall, in this sequence, the base-displaced intercalated conformation of the C8-dG-ABA lesion bears similarity to structures of other arylamine C8-dG adducts. However, in this sequence, the base-displaced intercalated conformation for the C8-dG-ABA adduct differs from the conformation of the N(2)-dG-ABA adduct reported by de los Santos and co-workers, in which it is oriented in the minor groove toward the 5' end of the duplex, with the modified guanine remaining in the anti conformation about the glyosidic torsion angle, and the complementary base remaining within the duplex. The results are discussed in relationship to differences between the C8-dG-ABA and N(2)-dG-ABA adducts with respect to susceptibility to nucleotide excision repair (NER).
Asunto(s)
Benzo(a)Antracenos/química , Aductos de ADN/química , Desoxiguanosina/química , Simulación de Dinámica Molecular , Espectroscopía de Resonancia Magnética , Conformación MolecularRESUMEN
The 2'-deoxynucleoside containing the synthetic base 1-[(2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)-tetrahydrofuran-2-yl)-1H-perimidin-2(3H)-one] (dPer) recognizes in DNA the O(6)-benzyl-2'-deoxyguanosine nucleoside (O(6)-Bn-dG), formed by exposure to N-benzylmethylnitrosamine. Herein, we show how dPer distinguishes between O(6)-Bn-dG and dG in DNA. The structure of the modified Dickerson-Drew dodecamer (DDD) in which guanine at position G(4) has been replaced by O(6)-Bn-dG and cytosine C(9) has been replaced with dPer to form the modified O(6)-Bn-dG:dPer (DDD-XY) duplex [5'-d(C(1)G(2)C(3)X(4)A(5)A(6)T(7)T(8)Y(9)G(10)C(11)G(12))-3']2 (X = O(6)-Bn-dG, Y = dPer) reveals that dPer intercalates into the duplex and adopts the syn conformation about the glycosyl bond. This provides a binding pocket that allows the benzyl group of O(6)-Bn-dG to intercalate between Per and thymine of the 3'-neighbor A:T base pair. Nuclear magnetic resonance data suggest that a similar intercalative recognition mechanism applies in this sequence in solution. However, in solution, the benzyl ring of O(6)-Bn-dG undergoes rotation on the nuclear magnetic resonance time scale. In contrast, the structure of the modified DDD in which cytosine at position C(9) is replaced with dPer to form the dG:dPer (DDD-GY) [5'-d(C(1)G(2)C(3)G(4)A(5)A(6)T(7)T(8)Y(9)G(10)C(11)G(12))-3']2 duplex (Y = dPer) reveals that dPer adopts the anti conformation about the glycosyl bond and forms a less stable wobble pairing interaction with guanine.