RESUMEN
The SARS-CoV-2 helicase, non-structural protein 13 (Nsp13), plays an essential role in viral replication, translocating in the 5' â 3' direction as it unwinds double-stranded RNA/DNA. We investigated the impact of structurally distinct DNA lesions on DNA unwinding catalyzed by Nsp13. The selected lesions include two benzo[a]pyrene (B[a]P)-derived dG adducts, the UV-induced cyclobutane pyrimidine dimer (CPD), and the pyrimidine (6-4) pyrimidone (6-4PP) photolesion. The experimentally observed unwinding rate constants (kobs) and processivities (P) were examined. Relative to undamaged DNA, the kobs values were diminished by factors of up to ~15 for B[a]P adducts but only by factors of ~2-5 for photolesions. A minor-groove-oriented B[a]P adduct showed the smallest impact on P, which decreased by ~11% compared to unmodified DNA, while an intercalated one reduced P by ~67%. However, the photolesions showed a greater impact on the processivities; notably, the CPD, with the highest kobs value, exhibited the lowest P, which was reduced by ~90%. Our findings thus show that DNA unwinding efficiencies are lesion-dependent and most strongly inhibited by the CPD, leading to the conclusion that processivity is a better measure of DNA lesions' inhibitory effects than unwinding rate constants.
Asunto(s)
ADN Helicasas , SARS-CoV-2 , Proteínas no Estructurales Virales , SARS-CoV-2/metabolismo , Proteínas no Estructurales Virales/metabolismo , Proteínas no Estructurales Virales/química , ADN Helicasas/metabolismo , ADN Helicasas/química , ADN/metabolismo , ADN/química , Humanos , Daño del ADN , COVID-19/virología , Cinética , Metiltransferasas , ARN HelicasasRESUMEN
DNA helicase unwinding activity can be inhibited by small molecules and by covalently bound DNA lesions. Little is known about the relationships between the structural features of DNA lesions and their impact on unwinding rates and processivities. Employing E.coli RecQ helicase as a model system, and various conformationally defined DNA lesions, the unwinding rate constants kobs = kU + kD, and processivities P = (kU/(kU + kD) were determined (kU, unwinding rate constant; kD, helicase-DNA dissociation rate constant). The highest kobs values were observed in the case of intercalated benzo[a]pyrene (BP)-derived adenine adducts, while kobs values of guanine adducts with minor groove or base-displaced intercalated adduct conformations were ~10-20 times smaller. Full unwinding was observed in each case with the processivity P = 1.0 (100% unwinding). The kobs values of the non-bulky lesions T(6-4)T, CPD cyclobutane thymine dimers, and a guanine oxidation product, spiroiminodihydantoin (Sp), are up to 20 times greater than some of the bulky adduct values; their unwinding efficiencies are strongly inhibited with processivities P = 0.11 (CPD), 0.062 (T(6-4)T), and 0.63 (Sp). These latter observations can be accounted for by correlated decreases in unwinding rate constants and enhancements in the helicase DNA complex dissociation rate constants.
Asunto(s)
Escherichia coli , RecQ Helicasas , RecQ Helicasas/metabolismo , Escherichia coli/metabolismo , ADN/química , Relación Estructura-Actividad , Guanina/metabolismo , Aductos de ADN/metabolismoRESUMEN
The oxidatively generated genotoxic spiroiminodihydantoin (Sp) lesions are well-known substrates of the base excision repair (BER) pathway initiated by the bifunctional DNA glycosylase NEIL1. In this work, we reported that the excision kinetics of the single Sp lesions site-specifically embedded in the covalently closed circular DNA plasmids (contour length 2686 base pairs) by NEIL1 are biphasic under single-turnover conditions ([NEIL1] â« [SpDNApl]) in contrast with monophasic excision kinetics of the same lesions embedded in147-mer Sp-modified DNA duplexes. Under conditions of a large excess of plasmid DNA base pairs over NEIL1 molecules, the kinetics of excision of Sp lesions are biphasic in nature, exhibiting an initial burst phase, followed by a slower rate of formation of excision products The burst phase is associated with NEIL1-DNA plasmid complexes, while the slow kinetic phase is attributed to the dissociation of non-specific NEIL1-DNA complexes. The amplitude of the burst phase is limited because of the competing non-specific binding of NEIL1 to unmodified DNA sequences flanking the lesion. A numerical analysis of the incision kinetics yielded a value of φ â 0.03 for the fraction of NEIL1 encounters with plasmid molecules that result in the excision of the Sp lesion, and a characteristic dissociation time of non-specific NEIL1-DNA complexes (τ-ns â 8â s). The estimated average DNA translocation distance of NEIL1 is â¼80 base pairs. This estimate suggests that facilitated diffusion enhances the probability that NEIL1 can locate its substrate embedded in an excess of unmodified plasmid DNA nucleotides by a factor of â¼10.
Asunto(s)
Daño del ADN , ADN Glicosilasas/metabolismo , Reparación del ADN , ADN/química , ADN Glicosilasas/genética , Células HeLa , Humanos , Oxidación-Reducción , Plásmidos/genéticaRESUMEN
The base and nucleotide excision repair pathways (BER and NER, respectively) are two major mechanisms that remove DNA lesions formed by the reactions of genotoxic intermediates with cellular DNA. It is generally believed that small non-bulky oxidatively generated DNA base modifications are removed by BER pathways, whereas DNA helix-distorting bulky lesions derived from the attack of chemical carcinogens or UV irradiation are repaired by the NER machinery. However, existing and growing experimental evidence indicates that oxidatively generated DNA lesions can be repaired by competitive BER and NER pathways in human cell extracts and intact human cells. Here, we focus on the interplay and competition of BER and NER pathways in excising oxidatively generated guanine lesions site-specifically positioned in plasmid DNA templates constructed by a gapped-vector technology. These experiments demonstrate a significant enhancement of the NER yields in covalently closed circular DNA plasmids (relative to the same, but linearized form of the same plasmid) harboring certain oxidatively generated guanine lesions. The interplay between the BER and NER pathways that remove oxidatively generated guanine lesions are reviewed and discussed in terms of competitive binding of the BER proteins and the DNA damage-sensing NER factor XPC-RAD23B to these lesions.
Asunto(s)
Reparación del ADN/fisiología , Guanina/metabolismo , Radicales Libres/química , Vectores Genéticos , Guanina/química , Humanos , Oxidación-Reducción , Plásmidos/genéticaRESUMEN
The base and nucleotide excision repair pathways (BER and NER, respectively) are two major mechanisms that remove DNA lesions formed by the reactions of genotoxic intermediates with cellular DNA. We have demonstrated earlier that the oxidatively generated guanine lesions spiroiminodihydantoin (Sp) and 5-guanidinohydantoin (Gh) are excised from double-stranded DNA by competing BER and NER in whole-cell extracts [Shafirovich, V., et al. (2016) J. Biol. Chem. 321, 5309-5319]. In this work we compared the NER and BER yields with single Gh or Sp lesions embedded at the same sites in covalently closed circular pUC19NN plasmid DNA (cccDNA) and in the same but linearized form (linDNA) of this plasmid. The kinetics of the Sp and Gh BER and NER incisions were monitored in HeLa cell extracts. The yield of NER products is â¼5 times greater in covalently closed circular DNA than in the linearized form, while the BER yield is smaller by â¼20-30% depending on the guanine lesion. Control BER experiments with 8-oxo-7,8-dihydroguanine (8-oxoG) show that the BER yield is increased by a factor of only 1.4 ± 0.2 in cccDNA relative to linDNA. These surprising differences in BER and NER activities are discussed in terms of the lack of termini in covalently closed circular DNA and the DNA lesion search dynamics of the NER DNA damage sensor XPC-RAD23B and the BER enzyme OGG1 that recognizes and excises 8-oxoG.
Asunto(s)
ADN/metabolismo , Guanina/metabolismo , Nucleótidos/metabolismo , ADN/química , Reparación del ADN , Guanina/química , Células HeLa , Humanos , Conformación de Ácido Nucleico , Nucleótidos/química , Oxidación-Reducción , PlásmidosRESUMEN
The Nucleotide Excision Repair (NER) mechanism removes a wide spectrum of structurally different lesions that critically depend on the binding of the DNA damage sensing NER factor XPC-RAD23B (XPC) to the lesions. The bulky mutagenic benzo[a]pyrene diol epoxide metabolite-derived cis- and trans-B[a]P-dG lesions (G*) adopt base-displaced intercalative (cis) or minor groove (trans) conformations in fully paired DNA duplexes with the canonical C opposite G* (G*:C duplexes). While XPC has a high affinity for binding to these DNA lesions in fully complementary double-stranded DNA, we show here that deleting only the C in the complementary strand opposite the lesion G* embedded in 50-mer duplexes, fully abrogates XPC binding. Accurate values of XPC dissociation constants (KD) were determined by employing an excess of unmodified DNA as a competitor; this approach eliminated the binding and accumulation of multiple XPC molecules to the same DNA duplexes, a phenomenon that prevented the accurate estimation of XPC binding affinities in previous studies. Surprisingly, a detailed comparison of XPC dissociation constants KD of unmodified and lesion-containing G*:Del complexes, showed that the KD values were -2.5-3.6 times greater in the case of G*:Del than in the unmodified G:Del and fully base-paired G:C duplexes. The origins of this unexpected XPC lesion avoidance effect is attributed to the intercalation of the bulky, planar B[a]P aromatic ring system between adjacent DNA bases that thermodynamically stabilize the G*:Del duplexes. The strong lesion-base stacking interactions associated with the absence of the partner base, prevent the DNA structural distortions needed for the binding of the BHD2 and BHD3 ß-hairpins of XPC to the deletion duplexes, thus accounting for the loss of XPC binding and the known NER-resistance of G*:Del duplexes.
Asunto(s)
7,8-Dihidro-7,8-dihidroxibenzo(a)pireno 9,10-óxido/metabolismo , Aductos de ADN/metabolismo , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , 7,8-Dihidro-7,8-dihidroxibenzo(a)pireno 9,10-óxido/química , ADN/química , ADN/metabolismo , Aductos de ADN/química , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/química , Humanos , Cinética , Simulación de Dinámica Molecular , Conformación de Ácido Nucleico , Conformación Proteica , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Especificidad por SustratoRESUMEN
The excision of DNA lesions by human nucleotide excision repair (NER) has been extensively studied in human cell extracts. Employing DNA duplexes with fewer than 200 bp containing a single bulky, benzo[a]pyrene-derived guanine lesion (B[a]P-dG), the NER yields are typically on the order of â¼5-10%, or less. Remarkably, the NER yield is enhanced by a factor of â¼6 when the B[a]P-dG lesion is embedded in a covalently closed circular pUC19NN plasmid (contour length of 2686 bp) rather than in the same plasmid linearized by a restriction enzyme with the B[a]P-dG adduct positioned at the 945th nucleotide counted from the 5'-end of the linearized DNA molecules. Furthermore, the NER yield in the circular pUC19NN plasmid is â¼9 times greater than in a short 147-mer DNA duplex with the B[a]P-dG adduct positioned in the middle. Although the NER factors responsible for these differences were not explicitly identified here, we hypothesize that the initial DNA damage sensor XPC-RAD23B is a likely candidate; it is known to search for DNA lesions by a constrained one-dimensional search mechanism [Cheon, N. Y., et al. (2019) Nucleic Acids Res. 47, 8337-8347], and our results are consistent with the notion that it dissociates more readily from the blunt ends than from the inner regions of linear DNA duplexes, thus accounting for the remarkable enhancement in NER yields associated with the single B[a]P-dG adduct embedded in covalently closed circular plasmids.
Asunto(s)
Reparación del ADN , ADN Circular/química , ADN Circular/genética , Guanina , Plásmidos/genética , Secuencia de BasesRESUMEN
The interplay between nucleotide excision repair (NER) and base excision repair (BER) of nonbulky, oxidatively generated DNA lesions has long been a subject of significant interest. The hydantoin oxidation products of 8-oxoguanine, spiroiminodihydantoin (Sp) and 5-guanidinohydantoin (Gh), are substrates of both BER and NER in HeLa cell extracts and human cells [Shafirovich, V., et al. (2019) Chem. Res. Toxicol. 32, 753-761]. The primary factor that recognizes DNA lesions is the DNA damage-sensing factor XPC-RAD23B (XPC), while the glycosylase NEIL1 is known to remove Gh and Sp lesions from double-stranded DNA. It is shown here that in aqueous solutions containing nanomolar concentrations of proteins, XPC and NEIL1 compete for binding to 147-mer oligonucleotide duplexes that contain single Gh or Sp lesions under conditions of [protein] â« [DNA], thus inhibiting the rate of BER catalyzed by NEIL1. The non-covalently bound NEIL1 molecules can be displaced by XPC at concentration ratios R = [XPC]/[NEIL1] > 0.2, while full displacement of NEIL1 is observed at R ≥ 0.5. In the absence of XPC and under single-turnover conditions, only the burst phase is observable. However, with a progressive increase in the XPC concentration, the amplitude of the burst phase decreases gradually, and a slower time-dependent phase of incision product formation manifests itself with rate constants of 3.0 × 10-3 s-1 (Gh) and 0.90 × 10-3 s-1 (Sp). These slow kinetics are attributed to the dissociation of XPC-DNA complexes that allow for the rebinding of NEIL1 to the temporarily exposed Gh or Sp lesions, and the incisions observed under these steady-state conditions.
Asunto(s)
ADN Glicosilasas/metabolismo , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Hidantoínas/metabolismo , Unión Competitiva , ADN/efectos de los fármacos , Reparación del ADN , Humanos , Hidantoínas/farmacología , Conformación Molecular , Oxidación-ReducciónRESUMEN
Purine 5',8-cyclo-2'-deoxynucleosides (cPu) are tandem-type lesions observed among the DNA purine modifications and identified in mammalian cellular DNA in vivo. These lesions can be present in two diasteroisomeric forms, 5'R and 5'S, for each 2'-deoxyadenosine and 2'-deoxyguanosine moiety. They are generated exclusively by hydroxyl radical attack to 2'-deoxyribose units generating C5' radicals, followed by cyclization with the C8 position of the purine base. This review describes the main recent achievements in the preparation of the cPu molecular library for analytical and DNA synthesis applications for the studies of the enzymatic recognition and repair mechanisms, their impact on transcription and genetic instability, quantitative determination of the levels of lesions in various types of cells and animal model systems, and relationships between the levels of lesions and human health, disease, and aging, as well as the defining of the detection limits and quantification protocols.
Asunto(s)
Daño del ADN , Purinas/química , Purinas/metabolismo , Animales , Reparación del ADN , Humanos , Oligonucleótidos/síntesis química , Oligonucleótidos/química , Especies Reactivas de Oxígeno/metabolismo , Bibliotecas de Moléculas PequeñasRESUMEN
The interchange between different repair mechanisms in human cells has long been a subject of interest. Here, we provide a direct demonstration that the oxidatively generated guanine lesions spiroiminodihydantoin (Sp) and 5-guanidinohydantoin (Gh) embedded in double-stranded DNA are substrates of both base excision repair (BER) and nucleotide excision repair (NER) mechanisms in intact human cells. Site-specifically modified, 32P-internally labeled double-stranded DNA substrates were transfected into fibroblasts or HeLa cells, and the BER and/or NER mono- and dual incision products were quantitatively recovered after 2-8 h incubation periods and lysis of the cells. DNA duplexes bearing single benzo[ a]pyrene-derived guanine adduct were employed as positive controls of NER. The NER activities, but not the BER activities, were abolished in XPA-/- cells, while the BER yields were strongly reduced in NEIL1-/- cells. Co-transfecting different concentrations of analogous DNA sequences bearing the BER substrates 5-hydroxyuracil diminish the BER yields of Sp lesions and enhance the yields of NER products. These results are consistent with a model based on the local availability of BER and NER factors in human cells and their competitive binding to the same Sp or Gh BER/NER substrates.
Asunto(s)
Guanina/metabolismo , Células Cultivadas , Reparación del ADN , Fibroblastos/metabolismo , Guanina/química , Células HeLa , Humanos , Cinética , Estructura Molecular , Oxidación-ReducciónRESUMEN
The mechanistic aspects of one-electron oxidation of G-quadruplexes in the basket (Na+ ions) and hybrid (K+ ions) conformations were investigated by transient absorption laser kinetic spectroscopy and HPLC detection of the 8-oxo-7,8-dihydroguanine (8-oxoG) oxidation product. The photo-induced one-electron abstraction from G-quadruplexes was initiated by sulfate radical anions (SO4 Ë- ) derived from the photolysis of persulfate ions by 308 nm excimer laser pulses. In neutral aqueous solutions (pH 7.0), the transient absorbance of neutral guanine radicals, G(-H)Ë, is observed following the complete decay of SO4 Ë- radicals (~10 µs after the actinic laser flash). In both basket and hybrid conformations, the G(-H)Ë decay is biphasic with one component decaying with a lifetime of ~0.1 ms, and the other with a lifetime of 20-30 ms. The fast decay component (~0.1 ms) in G-quadruplexes is correlated with the formation of 8-oxoG lesions. We propose that in G-quadruplexes, G(-H)Ë radicals retain radical cation character by sharing the N1-proton with the O6 -atom of G in the [GË+ : G] Hoogsteen base pair; this [G(-H)Ë: H+ G â GË+ : G] leads to the hydration of GË+ radical cation within the millisecond time domain, and is followed by the formation of the 8-oxoG lesions.
Asunto(s)
G-Cuádruplex , Guanina/análogos & derivados , Modelos Químicos , Telómero , Guanina/química , Humanos , Cinética , Oxidación-ReducciónRESUMEN
The nonbulky 5',8-cyclopurine DNA lesions (cP) and the bulky, benzo[ a]pyrene diol epoxide-derived stereoisomeric cis- and trans- N2-guanine adducts (BPDE-dG) are good substrates of the human nucleotide excision repair (NER) mechanism. These DNA lesions were embedded at the In or Out rotational settings near the dyad axis in nucleosome core particles reconstituted either with native histones extracted from HeLa cells (HeLa-NCP) or with recombinant histones (Rec-NCP). The cP lesions are completely resistant to NER in human HeLa cell extracts. The BPDE-dG adducts are also NER-resistant in Rec-NCPs but are good substrates of NER in HeLa-NCPs. The four BPDE-dG adduct samples are excised with different efficiencies in free DNA, but in HeLa-NCPs, the efficiencies are reduced by a common factor of 2.2 ± 0.2 relative to the NER efficiencies in free DNA. The NER response of the BPDE-dG adducts in HeLa-NCPs is not directly correlated with the observed differences in the thermodynamic destabilization of HeLa-NCPs, the Förster resonance energy transfer values, or hydroxyl radical footprint patterns and is weakly dependent on the rotational settings. These and other observations suggest that NER is initiated by the binding of the DNA damage-sensing NER factor XPC-RAD23B to a transiently opened BPDE-modified DNA sequence that corresponds to the known footprint of XPC-DNA-RAD23B complexes (≥30 bp). These observations are consistent with the hypothesis that post-translational modifications and the dimensions and properties of the DNA lesions are the major factors that have an impact on the dynamics and initiation of NER in nucleosomes.
Asunto(s)
Aductos de ADN/química , Daño del ADN , Reparación del ADN , ADN/química , Nucleosomas/química , Purinas/química , Aductos de ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Células HeLa , Humanos , Nucleosomas/genéticaRESUMEN
The most common, oxidatively generated lesion in cellular DNA is 8-oxo-7,8-dihydroguanine, which can be oxidized further to yield highly mutagenic spiroiminodihydantoin (Sp) and 5-guanidinohydantoin (Gh) in DNA. In human cell-free extracts, both lesions can be excised by base excision repair and global genomic nucleotide excision repair. However, it is not known if these lesions can be removed by transcription-coupled DNA repair (TCR), a pathway that clears lesions from DNA that impede RNA synthesis. To determine if Sp or Gh impedes transcription, which could make each a viable substrate for TCR, either an Sp or a Gh lesion was positioned on the transcribed strand of DNA under the control of a promoter that supports transcription by human RNA polymerase II. These constructs were incubated in HeLa nuclear extracts that contained active RNA polymerase II, and the resulting transcripts were resolved by denaturing polyacrylamide gel electrophoresis. The structurally rigid Sp strongly blocks transcription elongation, permitting 1.6 ± 0.5% nominal lesion bypass. In contrast, the conformationally flexible Gh poses less of a block to human RNAPII, allowing 9 ± 2% bypass. Furthermore, fractional lesion bypass for Sp and Gh is minimally affected by glycosylase activity found in the HeLa nuclear extract. These data specifically suggest that both Sp and Gh may well be susceptible to TCR because each poses a significant block to human RNA polymerase II progression. A more general principle is also proposed: Conformational flexibility may be an important structural feature of DNA lesions that enhances their transcriptional bypass.
Asunto(s)
Guanidinas/farmacología , Guanosina/análogos & derivados , Hidantoínas/farmacología , ARN Polimerasa II/antagonistas & inhibidores , Compuestos de Espiro/farmacología , Elongación de la Transcripción Genética/efectos de los fármacos , Daño del ADN , Reparación del ADN , Guanidinas/síntesis química , Guanidinas/química , Guanosina/síntesis química , Guanosina/química , Guanosina/farmacología , Células HeLa , Humanos , Hidantoínas/síntesis química , Hidantoínas/química , Conformación Molecular , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Compuestos de Espiro/síntesis química , Compuestos de Espiro/química , Relación Estructura-ActividadRESUMEN
It is generally believed that the mammalian nucleotide excision repair pathway removes DNA helix-distorting bulky DNA lesions, while small non-bulky lesions are repaired by base excision repair (BER). However, recent work demonstrates that the oxidativly generated guanine oxidation products, spiroimininodihydantoin (Sp), 5-guanidinohydantoin (Gh), and certain intrastrand cross-linked lesions, are good substrates of NER and BER pathways that compete with one another in human cell extracts. The oxidation of guanine by peroxynitrite is known to generate 5-guanidino-4-nitroimidazole (NIm) which is structurally similar to Gh, except that the 4-nitro group in NIm is replaced by a keto group in Gh. However, unlike Gh, NIm is an excellent substrate of BER, but not of NER. These and other related results are reviewed and discussed in this article.
Asunto(s)
Daño del ADN , ADN Glicosilasas/metabolismo , Reparación del ADN , ADN Forma B/química , Estrés Oxidativo , ADN Forma B/metabolismo , Guanidinas , Humanos , Hidantoínas , Nitroimidazoles , Oxidación-Reducción , Especificidad por SustratoRESUMEN
Oxidatively generated guanine radicals in DNA can undergo various nucleophilic reactions including the formation of C8-guanine cross-links with adjacent or nearby N3-thymines in DNA in the presence of O2. These G[8-3]T lesions have been identified in the DNA of human cells exposed to oxidative stress, and are most likely genotoxic if not removed by cellular defence mechanisms. The abilities of several representative polymerases to bypass the G[8-3]T lesions in two different sequence contexts, G*T* and G*CT*, were assessed in vitro. The polymerase BF (bacillus fragment) from Bacillus stearothermophilus, the Y-family archaeal polymerases Dpo4 from Sulfolobus sulfataricus P2, and human DNA pol κ and pol η were selected for the study. The A-family polymerase BF was strongly blocked, while relatively weak translesion synthesis was observed in the case of Y-family polymerases Dpo4 and pol κ. Primer extension catalyzed by pol η was also partially stalled at various positions at or near the G[8-3]T cross-linked bases, but a significant and distributive primer extension was observed beyond the sites of the lesions with the efficiency being consistently greater in the case of G*CT* than in the case of G*T* lesions. The results obtained with pol η are compared with translesion synthesis past other intrastrand cross-linked lesions with previously published results of others that include the isomeric G[8-5m]T lesions generated by ionizing radiation, the cis-syn cyclobutane pyrimidine dimer and the 6-4 photoproduct generated by UV irradiation, and the Pt-G*G* lesions derived from the reactions of the chemotherapeutic agent cisplatin with DNA.
Asunto(s)
ADN Polimerasa III/metabolismo , ADN/genética , ADN/metabolismo , Guanina/química , Timina/química , Catálisis , ADN/química , Daño del ADN , ADN Polimerasa III/química , Reparación del ADN , Replicación del ADN , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/metabolismo , Guanina/metabolismo , Humanos , Estructura Molecular , Timina/metabolismoRESUMEN
The well known biomarker of oxidative stress, 8-oxo-7,8-dihydroguanine, is more susceptible to further oxidation than the parent guanine base and can be oxidatively transformed to the genotoxic spiroiminodihydantoin (Sp) and 5-guanidinohydantoin (Gh) lesions. Incubation of 135-mer duplexes with single Sp or Gh lesions in human cell extracts yields a characteristic nucleotide excision repair (NER)-induced ladder of short dual incision oligonucleotide fragments in addition to base excision repair (BER) incision products. The ladders were not observed when NER was inhibited either by mouse monoclonal antibody (5F12) to human XPA or in XPC(-/-) fibroblast cell extracts. However, normal NER activity appeared when the XPC(-/-) cell extracts were complemented with XPC-RAD23B proteins. The Sp and Gh lesions are excellent substrates of both BER and NER. In contrast, 5-guanidino-4-nitroimidazole, a product of the oxidation of guanine in DNA by peroxynitrite, is an excellent substrate of BER only. In the case of mouse embryonic fibroblasts, BER of the Sp lesion is strongly reduced in NEIL1(-/-) relative to NEIL1(+/+) extracts. In summary, in human cell extracts, BER and NER activities co-exist and excise Gh and Sp DNA lesions, suggesting that the relative NER/BER product ratios may depend on competitive BER and NER protein binding to these lesions.
Asunto(s)
Reparación del ADN , Guanina/análogos & derivados , Estrés Oxidativo , Animales , Línea Celular , Células , ADN Glicosilasas/genética , ADN Glicosilasas/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Guanina/metabolismo , Guanina/toxicidad , Células HeLa , Humanos , RatonesRESUMEN
In nucleosomes, the access of DNA lesions to nucleotide excision repair is hindered by histone proteins. However, evidence that the nature of the DNA lesions may play a role in facilitating access is emerging, but these phenomena are not well-understood. We have used molecular dynamics simulations to elucidate the structural, dynamic, and energetic properties of the R and S 5'-8-cyclo-2'-dG and the (+)-cis-anti-B[a]P-dG lesions in a nucleosome. Our results show that the (+)-cis-anti-B[a]P-dG adduct is more dynamic and more destabilizing than the smaller and more constrained 5',8-cyclo-2'-dG lesions, suggesting more facile access to the more bulky (+)-cis-anti-B[a]P-dG lesion.
Asunto(s)
Reparación del ADN , Nucleosomas/química , ADN/química , Aductos de ADN/química , Simulación de Dinámica Molecular , Resonancia Magnética Nuclear Biomolecular , Conformación de Ácido Nucleico , TermodinámicaRESUMEN
Oxidatively generated guanine radical cations in DNA can undergo various nucleophilic reactions including the formation of C8-guanine cross-links with adjacent or nearby N3-thymines in DNA in the presence of O2. The G*[C8-N3]T* lesions have been identified in the DNA of human cells exposed to oxidative stress, and are most likely genotoxic if not removed by cellular defense mechanisms. It has been shown that the G*[C8-N3]T* lesions are substrates of nucleotide excision repair in human cell extracts. Cleavage at the sites of the lesions was also observed but not further investigated (Ding et al. (2012) Nucleic Acids Res. 40, 2506-2517). Using a panel of eukaryotic and prokaryotic bifunctional DNA glycosylases/lyases (NEIL1, Nei, Fpg, Nth, and NTH1) and apurinic/apyrimidinic (AP) endonucleases (Apn1, APE1, and Nfo), the analysis of cleavage fragments by PAGE and MALDI-TOF/MS show that the G*[C8-N3]T* lesions in 17-mer duplexes are incised on either side of G*, that none of the recovered cleavage fragments contain G*, and that T* is converted to a normal T in the 3'-fragment cleavage products. The abilities of the DNA glycosylases to incise the DNA strand adjacent to G*, while this base is initially cross-linked with T*, is a surprising observation and an indication of the versatility of these base excision repair proteins.
Asunto(s)
Reparación del ADN , ADN/química , Oligonucleótidos/química , Secuencia de Bases , ADN/metabolismo , ADN Glicosilasas/metabolismo , Células HeLa , Humanos , Datos de Secuencia Molecular , Oligonucleótidos/metabolismo , Oxidación-ReducciónRESUMEN
A direct method has been developed for the in vitro synthesis of stable DNA-protein cross-links (DPC's) between guanine and amino acids (lysine and arginine). This method employs the combination of guanine neutral radicals, G(-H)Ë, and side-chain C-centered amino acid radicals. The latter were generated indirectly after first causing the selective photoionization of 2-aminopurine (2AP) embedded in the oligonucleotide, 5'-d(CC[2AP]TCGCTACC), by intense nanosecond 308 nm excimer laser pulses. The 2AP radical cation deprotonates rapidly to form the 2AP(-H)Ë neutral radical which, in turn, oxidizes the nearby guanine to form the neutral guanine G(-H)Ë radical, as described previously (Shafirovich et al., J. Phys. Chem. B, 2001, 105, 8431). In parallel, the hydrated electrons, generated by the photoionization of 2AP, are scavenged by nitrous oxide to generate hydroxyl radicals. In the presence of a large excess of the amino acids, the hydroxyl radicals oxidize the latter to produce C-centered amino acid radicals that combine with the G(-H)Ë radicals to form the guanine-amino acid cross-linked oligonucleotide product. Analogous products were generated by photoionizing the free nucleoside, 2',3',5'-tri-O-acetylguanosine, (tri-O-Ac-Guo), using intense nanosecond 266 nm Nd:YAG laser pulse irradiation. The guanine-amino acid cross-links thus produced site-specifically positioned either in oligonucleotides, or in the free nucleoside tri-O-Ac-Guo were isolated by HPLC methods and identified by high resolution LC-TOF/MS and LC-MS/MS methods. The possibility that analogous guanine-amino acid cross-linked products could be formed in vivo using single hit radical generation mechanisms during oxidative stress is discussed.
Asunto(s)
Aminoácidos/química , Reactivos de Enlaces Cruzados/síntesis química , Guanina/química , Reactivos de Enlaces Cruzados/química , Radicales Libres/química , Estructura MolecularRESUMEN
The mechanistic aspects of hydration of guanine radical cations, G(â¢+) in double- and single-stranded oligonucleotides were investigated by direct time-resolved spectroscopic monitoring methods. The G(â¢+) radical one-electron oxidation products were generated by SO4(â¢-) radical anions derived from the photolysis of S2O8(2-) anions by 308 nm laser pulses. In neutral aqueous solutions (pH 7.0), after the complete decay of SO4(â¢-) radicals (â¼5 µs after the actinic laser flash) the transient absorbance of neutral guanine radicals, G(-H)(â¢) with maximum at 312 nm, is dominant. The kinetics of decay of G(-H)(â¢) radicals depend strongly on the DNA secondary structure. In double-stranded DNA, the G(-H)(â¢) decay is biphasic with one component decaying with a lifetime of â¼2.2 ms and the other with a lifetime of â¼0.18 s. By contrast, in single-stranded DNA the G(-H)(â¢) radicals decay monophasically with a â¼ 0.28 s lifetime. The ms decay component in double-stranded DNA is correlated with the enhancement of 8-oxo-7,8-dihydroguanine (8-oxoG) yields which are â¼7 greater than in single-stranded DNA. In double-stranded DNA, it is proposed that the G(-H)(â¢) radicals retain radical cation character by sharing the N1-proton with the N3-site of C in the [G(â¢+):C] base pair. This [G(-H)(â¢):H(+)C â G(â¢+):C] equilibrium allows for the hydration of G(â¢+) followed by formation of 8-oxoG. By contrast, in single-stranded DNA, deprotonation of G(â¢+) and the irreversible escape of the proton into the aqueous phase competes more effectively with the hydration mechanism, thus diminishing the yield of 8-oxoG, as observed experimentally.