RESUMO
Abasic sites are one of the most common DNA lesions. All known abasic site repair mechanisms operate only when the damage is in double-stranded DNA. Here, we report the discovery of 5-hydroxymethylcytosine (5hmC) binding, ESC-specific (HMCES) as a sensor of abasic sites in single-stranded DNA. HMCES acts at replication forks, binds PCNA and single-stranded DNA, and generates a DNA-protein crosslink to shield abasic sites from error-prone processing. This unusual HMCES DNA-protein crosslink intermediate is resolved by proteasome-mediated degradation. Acting as a suicide enzyme, HMCES prevents translesion DNA synthesis and the action of endonucleases that would otherwise generate mutations and double-strand breaks. HMCES is evolutionarily conserved in all domains of life, and its biochemical properties are shared with its E. coli ortholog. Thus, HMCES is an ancient DNA lesion recognition protein that preserves genome integrity by promoting error-free repair of abasic sites in single-stranded DNA.
Assuntos
5-Metilcitosina/análogos & derivados , Reparo do DNA/fisiologia , DNA de Cadeia Simples/fisiologia , 5-Metilcitosina/metabolismo , Ácido Apurínico/metabolismo , DNA/metabolismo , Dano ao DNA/fisiologia , Replicação do DNA/fisiologia , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endonucleases , Escherichia coli/metabolismo , Polinucleotídeos/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismoRESUMO
Apurinic/apyrimidinic (AP) sites, or abasic sites, which are a common type of endogenous DNA damage, can forge interstrand DNA-DNA cross-links via reaction with the exocyclic amino group on a nearby 2Î-deoxyguanosine or 2Î-deoxyadenosine in the opposite strand. Here, we utilized a shuttle vector method to examine the efficiency and fidelity with which a reduced dG-AP cross-link-containing plasmid was replicated in cultured human cells. Our results showed that the cross-link constituted strong impediments to DNA replication in HEK293T cells, with the bypass efficiencies for the dG- and AP-containing strands being 40% and 20%, respectively. While depletion of polymerase (Pol) η did not perturb the bypass efficiency of the lesion, the bypass efficiency was markedly reduced (to 1-10%) in the isogenic cells deficient in Pol κ, Pol ι or Pol ζ, suggesting the mutual involvement of multiple translesion synthesis polymerases in bypassing the lesion. Additionally, replication of the cross-linked AP residue in HEK293T cells was moderately error-prone, inducing a total of â¼26% single-nucleobase substitutions at the lesion site, whereas replication past the cross-linked dG component occurred at a mutation frequency of â¼8%. Together, our results provided important insights into the effects of an AP-derived interstrand cross-link on the efficiency and accuracy of DNA replication in human cells.
Assuntos
Reparo do DNA , Replicação do DNA , Desoxiguanosina/metabolismo , Ácido Apurínico/metabolismo , DNA Polimerase Dirigida por DNA/fisiologia , Células HEK293 , HumanosRESUMO
Tyrosyl-DNA phosphodiesterase 1 (Tdp1) processes DNA 3'-end-blocking modifications, possesses DNA and RNA 3'-nucleosidase activity and is also able to hydrolyze an internal apurinic/apyrimidinic (AP) site and its synthetic analogs. The mechanism of Tdp1 interaction with DNA was analyzed using pre-steady state stopped-flow kinetics with tryptophan, 2-aminopurine and Förster resonance energy transfer fluorescence detection. Phosphorothioate or tetramethyl phosphoryl guanidine groups at the 3'-end of DNA have been used to prevent 3'-nucleosidase digestion by Tdp1. DNA binding and catalytic properties of Tdp1 and its mutants H493R (Tdp1 mutant SCAN1) and H263A have been compared. The data indicate that the initial step of Tdp1 interaction with DNA includes binding of Tdp1 to the DNA ends followed by the 3'-nucleosidase reaction. In the case of DNA containing AP site, three steps of fluorescence variation were detected that characterize (i) initial binding the enzyme to the termini of DNA, (ii) the conformational transitions of Tdp1 and (iii) search for and recognition of the AP-site in DNA, which leads to the formation of the catalytically active complex and to the AP-site cleavage reaction. Analysis of Tdp1 interaction with single- and double-stranded DNA substrates shows that the rates of the 3'-nucleosidase and AP-site cleavage reactions have similar values in the case of single-stranded DNA, whereas in double-stranded DNA, the cleavage of the AP-site proceeds two times faster than 3'-nucleosidase digestion. Therefore, the data show that the AP-site cleavage reaction is an essential function of Tdp1 which may comprise an independent of AP endonuclease 1 AP-site repair pathway.
Assuntos
Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , DNA/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Ácido Apurínico/química , Ácido Apurínico/metabolismo , Sítios de Ligação/genética , DNA/química , DNA/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/química , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Transferência Ressonante de Energia de Fluorescência , Humanos , Hidrólise , Cinética , Mutação , Conformação de Ácido Nucleico , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/genética , Polinucleotídeos/química , Polinucleotídeos/metabolismo , Ligação Proteica , Especificidade por SubstratoRESUMO
DNA is rapidly cleaved under mild alkaline conditions at apyrimidinic/apurinic sites, but the half-life is several weeks in phosphate buffer (pH 7.5). However, abasic sites are â¼100-fold more reactive within nucleosome core particles (NCPs). Histone proteins catalyze the strand scission, and at superhelical location 1.5, the histone H4 tail is largely responsible for the accelerated cleavage. The rate constant for strand scission at an abasic site is enhanced further in a nucleosome core particle when it is part of a bistranded lesion containing a proximal strand break. Cleavage of this form results in a highly deleterious double-strand break. This acceleration is dependent upon the position of the abasic lesion in the NCP and its structure. The enhancement in cleavage rate at an apurinic/apyrimidinic site rapidly drops off as the distance between the strand break and abasic site increases and is negligible once the two forms of damage are separated by 7 bp. However, the enhancement of the rate of double-strand break formation increases when the size of the gap is increased from one to two nucleotides. In contrast, the cleavage rate enhancement at 2-deoxyribonolactone within bistranded lesions is more modest, and it is similar in free DNA and nucleosome core particles. We postulate that the enhanced rate of double-strand break formation at bistranded lesions containing apurinic/apyrimidinic sites within nucleosome core particles is a general phenomenon and is due to increased DNA flexibility.
Assuntos
Ácido Apurínico/química , Quebras de DNA de Cadeia Dupla , DNA/química , Nucleossomos/química , Ácido Apurínico/metabolismo , DNA/genética , DNA/metabolismo , Reparo do DNA , DNA de Cadeia Simples , Histonas/química , Histonas/metabolismo , Modelos Químicos , Modelos Moleculares , Estrutura Molecular , Conformação de Ácido Nucleico , Nucleossomos/genética , Nucleossomos/metabolismo , Domínios ProteicosRESUMO
Apurinic/apyrimidinic (AP) sites are constantly formed in cellular DNA due to instability of the glycosidic bond, particularly at purines and various oxidized, alkylated, or otherwise damaged nucleobases. AP sites are also generated by DNA glycosylases that initiate DNA base excision repair. These lesions represent a significant block to DNA replication and are extremely mutagenic. Some DNA glycosylases possess AP lyase activities that nick the DNA strand at the deoxyribose moiety via a ß- or ß,δ-elimination reaction. Various amines can incise AP sites via a similar mechanism, but this non-enzymatic cleavage typically requires high reagent concentrations. Herein, we describe a new class of small molecules that function at low micromolar concentrations as both ß- and ß,δ-elimination catalysts at AP sites. Structure-activity relationships have established several characteristics that appear to be necessary for the formation of an iminium ion intermediate that self-catalyzes the elimination at the deoxyribose ring.
Assuntos
Clivagem do DNA , Dano ao DNA , Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , DNA/genética , Ácido Apurínico/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Biocatálise , DNA/metabolismoRESUMO
Tyrosyl-DNA phosphodiesterase 1 (Tdp1) promotes catalytic scission of a phosphodiester bond between the 3'-end of DNA and the hydroxyl group of a tyrosine residue, as well as cleaving off a variety of other 3'-terminal phosphate-linked DNA substituents. We have shown recently that Tdp1 can initiate an apurinic/apyrimidinic (AP) site repair pathway that is independent from the one mediated by AP endonuclease 1 (APE1). Until recently, there was no method available of tracking the AP-site cleaving activity of Tdp1 by real-time fluorescence assay. In the present study we demonstrate a highly specific real-time detection of the AP-site cleaving activity of Tdp1 which allows one to distinguish it from the activity of APE1 by using a short hairpin oligonucleotide with a 1,12-dodecanediol loop, a 5'-fluorophore, and a 3'-quencher. Specific phosphodiesterase activity of Tdp1, which is usually able to remove quencher from the 3'-end of DNA, was suppressed in our approach by introducing a noncleavable phosphate group mimic between the 3'-end and the quencher. As a nondigestible 3'-phosphate analogue, we have used a new uncharged tetramethyl phosphoryl guanidine (Tmg) group, which is resistant to 3'-phosphodiesterase cleavage.
Assuntos
Ácido Apurínico/metabolismo , Bioensaio/métodos , Oligonucleotídeos/química , Diester Fosfórico Hidrolases/metabolismo , Polinucleotídeos/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Corantes Fluorescentes/química , Cinética , Microscopia de Fluorescência , Mutação , Oligonucleotídeos/metabolismo , Diester Fosfórico Hidrolases/análise , Diester Fosfórico Hidrolases/genética , Especificidade por SubstratoRESUMO
The mechanism of hydrolysis of the apurinic/apyrimidinic (AP) site and its synthetic analogs by using tyrosyl-DNA phosphodiesterase 1 (Tdp1) was analyzed. Tdp1 catalyzes the cleavage of AP site and the synthetic analog of the AP site, 3-hydroxy-2(hydroxymethyl)-tetrahydrofuran (THF), in DNA by hydrolysis of the phosphodiester bond between the substituent and 5' adjacent phosphate. The product of Tdp1 cleavage in the case of the AP site is unstable and is hydrolyzed with the formation of 3'- and 5'-margin phosphates. The following repair demands the ordered action of polynucleotide kinase phosphorylase, with XRCC1, DNA polymerase ß, and DNA ligase. In the case of THF, Tdp1 generates break with the 5'-THF and the 3'-phosphate termini. Tdp1 is also able to effectively cleave non-nucleotide insertions in DNA, decanediol and diethyleneglycol moieties by the same mechanism as in the case of THF cleavage. The efficiency of Tdp1 catalyzed hydrolysis of AP-site analog correlates with the DNA helix distortion induced by the substituent. The following repair of 5'-THF and other AP-site analogs can be processed by the long-patch base excision repair pathway.
Assuntos
Ácido Apurínico/metabolismo , Reparo do DNA , DNA/metabolismo , Furanos/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Polinucleotídeos/metabolismo , Ácido Apurínico/análogos & derivados , DNA/química , DNA Polimerase beta/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Hidrólise , Conformação de Ácido Nucleico , Fosfatos/metabolismo , Polinucleotídeo 5'-Hidroxiquinase/metabolismo , Transdução de Sinais , Especificidade por Substrato , Proteína 1 Complementadora Cruzada de Reparo de Raio-XRESUMO
A unique capability of Endonuclease IV in discrimination of mismatches neighboring a natural abasic site in DNA strands has been demonstrated, which enables genotyping of SNPs with high discrimination factors and differentiation of as low as 0.1-0.01% of target DNA strands from a large background of single-base different interfering strands.
Assuntos
Ácido Apurínico/química , Técnicas Biossensoriais , DNA/genética , Desoxirribonuclease IV (Fago T4-Induzido)/metabolismo , Polimorfismo de Nucleotídeo Único/genética , Polinucleotídeos/química , Ácido Apurínico/metabolismo , Sítios de Ligação , DNA/química , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Genótipo , Humanos , Cinética , Polinucleotídeos/metabolismo , Especificidade por SubstratoRESUMO
The goal of the present work was to determine the impact of N3-methyladenine (3-mA), an important lesion generated by many environmental agents and anticancer drugs, on in vivo DNA replication and in vitro RNA transcription. Due to 3-mA chemical instability, the stable isostere 3-methyl-3-deazaadenine (3-m-c(3)A) was site specifically positioned into an oligodeoxynucleotide. The oligomer was, then incorporated into a vector system that is rapidly converted to ssDNA inside yeast cells and requires DNA replication opposite the lesion for plasmid clonal selection. For control purposes, an adenine or a stable apurinic/apyrimidinic (AP)-lesion was placed at the same site. The presence of each lesion in the oligonucleotide was confirmed by MALDI-TOF analysis. Plasmids were then transfected into yeast cells. While the AP-site dramatically reduced plasmid replication in all strains, the 3-m-c(3)A had a slight effect in the rad30 background which significantly increased only in a rev3rad30 background. Considering TLS events opposite 3-m-c(3)A, the lack of Polη was associated with a substantial increase in AT>GC transitions (p=0.0011), while in the absence of Polζ only events derived from an error free bypass were detected. The 3-m-c(3)A also did not affect in vitro transcription, while the AP-site was a strong block to T7 RNA progression when located in the transcribed strand. We conclude that, in these experimental systems, 3-m-c(3)A is efficiently bypassed by replication in vivo and by transcription in vitro.
Assuntos
Adenina/análogos & derivados , Adutos de DNA/metabolismo , Replicação do DNA , Saccharomyces cerevisiae/genética , Transcrição Gênica , Adenina/metabolismo , Ácido Apurínico/metabolismo , DNA Fúngico/metabolismo , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , RNA Polimerases Dirigidas por DNA/biossíntese , RNA Polimerases Dirigidas por DNA/genética , Técnicas de Inativação de Genes , Genes Reporter , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Virais/biossíntese , Proteínas Virais/genéticaRESUMO
Apurinic/apyrimidinic (AP) sites are ubiquitous DNA lesions that are highly mutagenic and cytotoxic if not repaired. In addition, clusters of two or more abasic lesions within one to two turns of DNA, a hallmark of ionizing radiation, are repaired much less efficiently and thus present greater mutagenic potential. Abasic sites are chemically labile, but naked DNA containing them undergoes strand scission slowly with a half-life on the order of weeks. We find that independently generated AP sites within nucleosome core particles are highly destabilized, with strand scission occurring â¼60-fold more rapidly than in naked DNA. The majority of core particles containing single AP lesions accumulate DNA-protein cross-links, which persist following strand scission. The N-terminal region of histone protein H4 contributes significantly to DNA-protein cross-links and strand scission when AP sites are produced approximately 1.5 helical turns from the nucleosome dyad, which is a known hot spot for nucleosomal DNA damage. Reaction rates for AP sites at two positions within this region differ by â¼4-fold. However, the strand scission of the slowest reacting AP site is accelerated when it is part of a repair resistant bistranded lesion composed of two AP sites, resulting in rapid formation of double strand breaks in high yields. Multiple lysine residues within a single H4 protein catalyze double strand cleavage through a mechanism believed to involve a templating effect. These results show that AP sites within the nucleosome produce significant amounts of DNA-protein cross-links and generate double strand breaks, the most deleterious form of DNA damage.
Assuntos
Dano ao DNA , DNA/metabolismo , Nucleossomos/metabolismo , Proteínas/metabolismo , Ácido Apurínico/química , Ácido Apurínico/genética , Ácido Apurínico/metabolismo , Sequência de Bases , Reagentes de Ligações Cruzadas/química , Reagentes de Ligações Cruzadas/metabolismo , DNA/química , DNA/genética , Reparo do DNA , Eletroforese em Gel de Poliacrilamida , Histonas/química , Histonas/genética , Histonas/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Conformação de Ácido Nucleico , Nucleossomos/genética , Polinucleotídeos/química , Polinucleotídeos/genética , Polinucleotídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas/química , Proteínas/genéticaRESUMO
An isothermal signal amplification technique for specific DNA sequences, known as cycling probe technology (CPT), has enabled rapid acquisition of genomic information. Here we report an analogous technique for the detection of an activated transcription factor, a transcription element-binding assay with fluorescent amplification by apurinic/apyrimidinic (AP) site lysis cycle (TEFAL). This simple amplification assay can detect activated transcription factors by using a unique nucleic acid probe containing a consensus binding sequence and an AP site, which enables the CPT reaction with AP endonuclease. In this article, we demonstrate that this method detects the functional CLOCK/BMAL1 heterodimer via the TEFAL probe containing the E-box consensus sequence to which the CLOCK/BMAL1 heterodimer binds. Using TEFAL combined with immunoassays, we measured oscillations in the amount of CLOCK/BMAL1 heterodimer in serum-stimulated HeLa cells. Furthermore, we succeeded in measuring the circadian accumulation of the functional CLOCK/BMAL1 heterodimer in human buccal mucosa cells. TEFAL contributes greatly to the study of transcription factor activation in mammalian tissues and cell extracts and is a powerful tool for less invasive investigation of human circadian rhythms.
Assuntos
Fatores de Transcrição ARNTL/análise , Proteínas CLOCK/análise , Imunoensaio/métodos , Sondas de Ácido Nucleico/química , Ácido Apurínico/metabolismo , Ritmo Circadiano , Dimerização , Células HeLa , Humanos , Polinucleotídeos/metabolismo , Ligação Proteica , Interferência de RNARESUMO
Apurinic/apyrimidinic (AP or abasic) sites are common DNA lesions that arise from spontaneous depurination or by base excision repair (BER) of modified bases. Accumulation of impaired AP sites could lead to increased genomic instability that in turn could lead to a more malignant phenotypic behavior of tumors. We, therefore, evaluated the effects of AP sites on survival in resected non-small cell lung cancer (NSCLC) patients. Resected tumor specimens from 99 patients with NSCLC who underwent surgical resection were collected. The enzyme-linked immunosorbent assay was applied to measure the levels of AP sites in tumor DNA. The median number of AP sites per 10(5) nucleotides was 12.4 for all the study subjects. Patients with low levels of AP site had significantly longer survival time compared with ones with medium or high levels of AP site (log-rank test: P=0.015). In Cox regression analysis, patients with medium or high levels of AP sites had over twofold increased hazard of death. In addition, we found a statistically significant correlation between levels of AP sites and age (rho=0.560, P<0.001). The results of this study demonstrated that levels of AP sites could predict survival in resected NSCLC patients. We postulate that an intact BER mechanism may reduce the accumulation of oxidative DNA damage that are thought to contribute to the tumor's malignant potential and therefore the risk of death.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Dano ao DNA , DNA de Neoplasias/genética , Neoplasias Pulmonares/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Ácido Apurínico/análise , Ácido Apurínico/metabolismo , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/cirurgia , DNA de Neoplasias/metabolismo , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/estatística & dados numéricos , Feminino , Humanos , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/cirurgia , Masculino , Pessoa de Meia-Idade , Polinucleotídeos/análise , Polinucleotídeos/metabolismo , Modelos de Riscos ProporcionaisRESUMO
Many oxidative DNA lesions are handled well by base excision repair (BER), but some types may be problematic. Recent work indicates that 2-deoxyribonolactone (dL) is such a lesion by forming stable, covalent cross-links between the abasic residue and DNA repair proteins with lyase activity. In the case of DNA polymerase beta, the reaction is potentiated by incision of dL by Ape1, the major mammalian AP endonuclease. When repair is prevented, polymerase beta is the most reactive cross-linking protein in whole-cell extracts. Cross-linking with dL is largely avoided by processing the damage through the "long-patch" (multinucleotide) BER pathway. However, if excess damage leads to the accumulation of unrepaired oxidative lesions in DNA, there may be a danger of polymerase beta-mediated cross-link formation. Understanding how cells respond to such complex damage is an important issue. In addition to its role in defending against DNA damage caused by exogenous agents, Ape1 protein is essential for coping with the endogenous DNA damage in human cells grown in culture. Suppression of Ape1 using RNA-interference technology causes arrest of cell proliferation and activation of apoptosis in various cell types, correlated with the accumulation of unrepaired abasic DNA damage. Notably, all these effects are reversed by expression of the unrelated protein Apn1 of S. cerevisiae, which shares only the enzymatic repair function with Ape1 (AP endonuclease).
Assuntos
Dano ao DNA , Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Animais , Apoptose , Ácido Apurínico/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/deficiência , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Humanos , Açúcares Ácidos/química , Açúcares Ácidos/metabolismoRESUMO
Dibenzo[a,l]pyrene (DB[a,l]P) is the most potent carcinogenic polycyclic aromatic hydrocarbon and has been identified in the environment. Comparative tumorigenicity studies in mouse skin and rat mammary gland indicate that DB[a,l]P is slightly more potent than DB[a,l]P-11,12-dihydrodiol and much more potent than (+/-)-syn-DB[a,l]P-11,12-dihydrodiol-13,14-epoxide {(+/-)-syn-DB[a,l]PDE} and (+/-)-anti-DB[a,l]PDE. We report here the identification and quantification of the depurinating adducts formed in mouse skin treated with DB[a,l]P, DB[a,l]P-11,12-dihydrodiol, (+/-)-syn-DB[a,l]PDE, or (+/-)-anti-DB[a,l]PDE and rat mammary gland treated with DB[a,l]P. The biologically formed adducts were compared with standard adducts by their retention times on HPLC and their spectra obtained by fluorescence line-narrowing spectroscopy at low temperature. In mouse skin treated with DB[a,l]P, depurinating adducts comprised 99% of the total adducts. Most of the depurinating adducts were formed by one-electron oxidation, with 63% at Ade and 12% at Gua. The remainder were formed by the diol epoxide, with 18% at Ade and 6% at Gua. When mouse skin was treated with DB[a,l]P-11,12-dihydrodiol, depurinating adducts comprised 80% of the total, and the predominant one was with Ade (69%). Treatment of skin with (+/-)-syn-DB[a,l]PDE resulted in 32% depurinating adducts, primarily at Ade (25%), whereas treatment with (+/-)-anti-DB[a,l]PDE produced 97% stable adducts. The formation of depurinating adducts following treatment of rat mammary gland with DB[a,l]P resulted in approximately 98% depurinating adducts, with the major adducts formed by one-electron oxidation. Only one depurinating diol epoxide adduct was formed. Tumorigenicity, mutations, and DNA adduct data suggest that depurinating Ade adducts play a major role in the initiation of tumors by DB[a,l]P.
Assuntos
Ácido Apurínico/metabolismo , Benzopirenos/toxicidade , Carcinógenos/toxicidade , Adutos de DNA/metabolismo , Glândulas Mamárias Animais/efeitos dos fármacos , Pele/efeitos dos fármacos , Animais , Ácido Apurínico/química , Benzopirenos/metabolismo , Carcinógenos/metabolismo , DNA/química , DNA/metabolismo , Adutos de DNA/análise , Feminino , Glândulas Mamárias Animais/química , Glândulas Mamárias Animais/metabolismo , Camundongos , Ratos , Ratos Sprague-Dawley , Pele/química , Pele/metabolismoRESUMO
The stable adducts of dibenzo[a,l]pyrene (DB[a,l]P) formed by rat liver microsomes in vitro were previously quantified, whereas the depurinating adducts were both identified and quantified [Li, et al. (1995) Biochemistry 34, 8043]. In this article, we report the identification and quantification of the stable DNA adducts obtained from DB[a,l]P and DB[a,l]P-11,12-dihydrodiol activated by rat liver microsomes and from reaction of (+/-)-anti-DB[a,l]P-11,12-dihydrodiol-13,14-epoxide (DB[a,l]PDE) and (+/-)-syn-DB[a,l]PDE with DNA in vitro. In addition, the stable DNA adducts were identified and quantified following treatment of mouse skin with DB[a,l]P, DB[a,l]P-11,12-dihydrodiol, (+/-)-anti-DB[a,l]PDE, or (+/-)-syn-DB[a,l]PDE in vivo and treatment of rat mammary gland with DB[a,l]P in vivo. The DNA adducts were analyzed by the (32)P-postlabeling method, and the major adducts were identified by comparison with standards. The six stable adducts of DB[a,l]P formed by rat liver microsomes in vitro were either guanine or adenine adducts of anti-DB[a,l]PDE or syn-DB[a,l]PDE. About 43% of the detected stable adducts from microsomes were with guanine and 44% were with adenine. The pattern of adducts formed from DB[a,l]P-11,12-dihydrodiol with microsomes was very similar to that from DB[a,l]P. Reaction of (+/-)-anti-DB[a,l]PDE with DNA in vitro formed higher levels of stable adducts (55% from guanine and 39% from adenine) than (+/-)-syn-DB[a,l]PDE did (about 44% with guanine and 47% with adenine). In mouse skin treated with DB[a,l]P, 1% of the total adducts detected were stable adducts, comprised of 51% guanine adducts and 46% from adenine; with DB[a,l]P-11,12-dihydrodiol, 54% of the total were stable adducts, with a pattern of adducts similar to those formed from DB[a,l]P. Treatment of mouse skin with (+/-)-syn-DB[a,l]PDE formed 68% stable adducts, mostly at guanine. With (+/-)-anti-DB[a,l]PDE, mouse skin contained almost exclusively (97%) stable adducts: 61% guanine adducts and 33% adenine adducts. In rat mammary gland treated with DB[a,l]P, 2% of the total adducts were stable, with 42% guanine adducts and 55% adenine adducts. Approximately equal to or greater amounts of stable guanine adducts were formed in all systems, except for rat mammary gland. In contrast, the majority of depurinating adducts were adenine adducts. The carcinogenic potencies of these compounds in mouse skin, published earlier, do not qualitatively or quantitatively correlate with stable adducts, but rather with depurinating adducts.
Assuntos
Benzopirenos/toxicidade , Carcinógenos/toxicidade , Adutos de DNA/metabolismo , Glândulas Mamárias Animais/efeitos dos fármacos , Pele/efeitos dos fármacos , Animais , Ácido Apurínico/química , Ácido Apurínico/metabolismo , Benzopirenos/química , Benzopirenos/metabolismo , Carcinógenos/química , Carcinógenos/metabolismo , Adutos de DNA/análise , Dano ao DNA , Feminino , Glândulas Mamárias Animais/química , Glândulas Mamárias Animais/metabolismo , Camundongos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Radioisótopos de Fósforo , Ratos , Ratos Sprague-Dawley , Pele/química , Pele/metabolismoRESUMO
Abasic lesions are unable to form Watson-Crick hydrogen bonds with nucleotides. Nonetheless, polymerase and repair enzymes distinguish between various oxidized abasic lesions, as well as from nonoxidized abasic sites (AP). The C2-AP lesion is produced when DNA is exposed to gamma-radiolysis. Its effects on polymerases and repair enzymes are unknown. A recently reported method for the chemical synthesis of oligonucleotides containing C2-AP at a defined site was utilized for studying the activity of Klenow exo(-) and repair enzymes on templates containing the lesion. The C2-AP lesion has a similar effect on Klenow exo(-) as do AP and C4-AP sites. Deoxyadenosine is preferentially incorporated opposite C2-AP, but extension of the primer past the lesion is strongly blocked. C2-AP is incised less efficiently by exonuclease III and endonuclease IV than are other abasic lesions. Furthermore, although a Schiff base between C2-AP and endonuclease III can be chemically trapped, the location of the 3'-phosphate alpha with respect to the aldehyde prevents beta-elimination associated with the lyase activity of type I base excision repair enzymes. The interactions of the C2'-oxidized abasic site with Klenow exo(-) and repair enzymes suggest that the lesion will be mutagenic and that it will be removed by strand displacement synthesis and flap endonuclease processing via a long patch repair mechanism.
Assuntos
Ácido Apurínico/metabolismo , Reparo do DNA , Replicação do DNA , DNA Bacteriano/metabolismo , Polinucleotídeos/metabolismo , Ácido Apurínico/síntese química , Ácido Apurínico/genética , DNA Polimerase I/química , DNA Polimerase I/metabolismo , Primers do DNA/síntese química , Primers do DNA/metabolismo , DNA Bacteriano/síntese química , DNA Bacteriano/genética , Desoxirribonuclease IV (Fago T4-Induzido)/química , Desoxirribonuclease IV (Fago T4-Induzido)/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Exodesoxirribonucleases/química , Exodesoxirribonucleases/metabolismo , Ácidos Nucleicos Heteroduplexes/síntese química , Ácidos Nucleicos Heteroduplexes/genética , Ácidos Nucleicos Heteroduplexes/metabolismo , Hibridização de Ácido Nucleico , Oligonucleotídeos/síntese química , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Oxirredução , Polinucleotídeos/síntese química , Polinucleotídeos/genética , Bases de Schiff/química , Bases de Schiff/metabolismo , Moldes GenéticosRESUMO
Ionizing radiation induces clustered DNA damage sites which have been shown to challenge the repair mechanism(s) of the cell. Evidence demonstrating that base excision repair is compromised during the repair of an abasic (AP) site present within a clustered damage site is presented. Simple bistranded clustered damage sites, comprised of either an AP-site and 8-oxoG or two AP-sites, one or five bases 3' or 5' to each other, were synthesized in oligonucleotides, and repair was carried out in xrs5 nuclear extracts. The rate of repair of an AP-site when present opposite 8-oxoG is reduced by up to 2-fold relative to that when an AP-site is present as an isolated lesion. The mechanism of repair of the AP-site shows asymmetry, depending on its position relative to 8-oxoG on the opposite strand. The AP-site is rejoined by short-patch base excision repair when the lesions are 5' to each other, whereas when the lesions are 3' to one another, rejoining of the AP-site occurs by both long-patch and short-patch repair processes. The major stalling of repair occurs at the DNA ligase step. 8-OxoG and an AP-site present within a cluster are processed sequentially, limiting the formation of double-strand breaks to <4%. In contrast, when two AP-sites are contained within the clustered DNA damage site, both AP-sites are incised simultaneously, giving rise to double-strand breaks. This study provides new insight into understanding the processes that lead to the biological consequences of radiation-induced DNA damage and ultimately tumorigenesis.
Assuntos
Ácido Apurínico/química , Núcleo Celular/química , Dano ao DNA , Reparo do DNA , DNA/química , Guanosina/análogos & derivados , Animais , Antígenos Nucleares/genética , Ácido Apurínico/metabolismo , Células CHO , Extratos Celulares/química , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cricetinae , DNA/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/química , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Proteínas de Ligação a DNA/genética , Guanosina/química , Humanos , Autoantígeno Ku , Oligonucleotídeos/síntese químicaRESUMO
BACKGROUND: DNA with two sites of damage in close proximity might not be repaired as efficiently as DNA with a single damage site. MATERIALS AND METHODS: To study this hypothesis, we utilized short oligodeoxyribonucleotides with a stable adduct 7 or 16 nucleotides (nt) downstream from an apurinic (AP) site. Repair by extracts of human breast epithelial MCF-10A1 cells was assayed by quantifying the incorporation of [alpha-32P]dTTP. RESULTS: The level of repair of an oligodeoxyribonucleotide with an AP site 7 nt from a stable adduct was comparable to that of the oligodeoxyribonucleotide with only an AP site. A decrease in overall repair of oligodeoxyribonucleotides containing an AP site and a stable adduct was observed if these lesions were 16 nt apart compared to the presence of only an AP site. CONCLUSION: The ability of human breast MCF-10A1 cells to repair DNA adducts and AP sites is affected by other near-by lesions.
Assuntos
Ácido Apurínico/genética , Extratos Celulares/farmacologia , Adutos de DNA/genética , Dano ao DNA , Reparo do DNA/fisiologia , Oligodesoxirribonucleotídeos/genética , Ácido Apurínico/metabolismo , Sequência de Bases , Mama/química , Mama/citologia , Linhagem Celular , Adutos de DNA/metabolismo , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Desoxirribonucleotídeos/genética , Desoxirribonucleotídeos/metabolismo , Células Epiteliais/química , Humanos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/metabolismo , Fator de Transcrição AP-1/deficiência , Fator de Transcrição AP-1/genética , Proteína de Xeroderma Pigmentoso Grupo ARESUMO
The human ribosomal protein S3 (hS3) possesses multifunctional activities that are involved in both protein translation, as well as the ability of cleaving apurinic/apyrimidinic (AP) DNA via a beta-elimination reaction. We recently showed that hS3 also has a surprising binding affinity for an 7,8-dihydro-8-oxoguanine (8-oxoG) residue embedded in a 5' end labeled 37mer DNA oligonucleotide. To understand the interaction of hS3 and DNA templates containing 8-oxoG, we carried out real-time analysis using surface plasmon resonance (SPR). Notably, hS3 was found to have an apparent three orders of magnitude higher binding affinity (KD) for 8-oxoG than the human N-glycosylase/AP lyase base excision repair (BER) enzyme OGG1. An even more dramatic five orders of magnitude higher binding affinity for AP DNA was found for hS3 as opposed to hOGG1. These results suggest that ribosomal protein hS3 may have a multifunctional role that may also affect functions associated with DNA base excision repair transactions.
Assuntos
Ácido Apurínico/metabolismo , Reparo do DNA , Guanina/análogos & derivados , Guanina/metabolismo , Polinucleotídeos/metabolismo , Proteínas Ribossômicas/metabolismo , Sítios de Ligação , DNA Glicosilases/metabolismo , Humanos , Ligação Proteica , Ressonância de Plasmônio de SuperfícieRESUMO
Base excision repair (BER) is one of the major pathways for repair of simple DNA base lesions and is carried out through a series of coordinated reactions relying on several different enzymatic activities and accessory proteins. Imbalance of BER activities has been reported to be linked to genetic instability and cancer. To experimentally address the mechanisms orchestrating BER, we monitored both the overall rate and the rate-limiting steps in the repair in cell-free extracts of five different endogenously occurring DNA lesions (abasic site, uracil, 8-oxoguanine, hypoxanthine and 5,6-dihydrouracil) and the effect of addition of rate-limiting BER components on the rate and co-ordination of BER reactions. We find that several mechanisms including regulation of DNA glycosylase turnover and involvement of poly(ADP-ribose) polymerase participate in synchronization of the repair events. We also find that repair of different DNA lesions involves different mechanisms for optimizing repair rates without accumulation of intermediates. Repair of some lesions such as 8-oxoguanine is regulated by glycosylase turnover and progress without substantial accumulation of repair intermediates. However, during repair of the apurinic/apyrimidinic (AP) sites or 5,6-dihydrouracil, poly(ADP-ribose) polymerase plays an important role in the coordination of the rates of repair reactions.