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1.
Mutat Res ; 671(1-2): 93-9, 2009 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-19778542

RESUMO

Poly(ADP-ribose) polymerase-1 (PARP-1) is a base excision repair (BER) protein that binds to DNA single strand breaks (SSBs) and subsequently synthesizes and transfers poly(ADP-ribose) polymers to various nuclear proteins. Numerous biochemical studies have implicated PARP-1 as a modulator of BER; however, the role of PARP-1 in BER in living cells remains unclear partly due to lack of accurate quantitation of BER intermediates existing in cells. Since DT40 cells, chicken B lymphocytes, naturally lack PARP-2, DT40 cells allow for the investigation of the PARP-1 null phenotype without confounding by PARP-2. To test the hypothesis that PARP-1 is necessary for efficient BER during methylmethane sulfonate (MMS) exposure in vertebrate cells, intact DT40 cells and their isogenic PARP-1 null counterparts were challenged with different exposure scenarios for phenotypic characterization. With chronic exposure, PARP-1 null cells exhibited sensitivity to MMS but with an acute exposure did not accumulate base lesions or AP sites to a greater extent than wild-type cells. However, an increase in SSB content in PARP-1 null cell DNA, as indicated by glyoxal gel electrophoresis under neutral conditions, suggested the presence of BER intermediates. These data suggest that during exposure, PARP-1 impacts the stage of BER after excision of the deoxyribosephosphate moiety from the 5' end of DNA strand breaks by polymerase beta.


Assuntos
Quebras de DNA de Cadeia Simples , Metanossulfonato de Metila/toxicidade , Poli(ADP-Ribose) Polimerases/deficiência , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Galinhas , Humanos , Poli(ADP-Ribose) Polimerase-1 , Fatores de Transcrição/genética
2.
Genes (Basel) ; 10(4)2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31018584

RESUMO

Endonuclease VIII-like (NEIL) 1 and 3 proteins eliminate oxidative DNA base damage and psoralen DNA interstrand crosslinks through initiation of base excision repair. Current evidence points to a DNA replication associated repair function of NEIL1 and NEIL3, correlating with induced expression of the proteins in S/G2 phases of the cell cycle. However previous attempts to express and purify recombinant human NEIL3 in an active form have been challenging. In this study, both human NEIL1 and NEIL3 have been expressed and purified from E. coli, and the DNA glycosylase activity of these two proteins confirmed using single- and double-stranded DNA oligonucleotide substrates containing the oxidative bases, 5-hydroxyuracil, 8-oxoguanine and thymine glycol. To determine the biochemical role that NEIL1 and NEIL3 play during DNA replication, model replication fork substrates were designed containing the oxidized bases at one of three specific sites relative to the fork. Results indicate that whilst specificity for 5- hydroxyuracil and thymine glycol was observed, NEIL1 acts preferentially on double-stranded DNA, including the damage upstream to the replication fork, whereas NEIL3 preferentially excises oxidized bases from single stranded DNA and within open fork structures. Thus, NEIL1 and NEIL3 act in concert to remove oxidized bases from the replication fork.


Assuntos
DNA Glicosilases/metabolismo , Replicação do DNA , N-Glicosil Hidrolases/genética , Ciclo Celular , DNA/metabolismo , DNA de Cadeia Simples/metabolismo , Humanos , Modelos Genéticos , Timina/análogos & derivados , Timina/metabolismo , Uracila/análogos & derivados , Uracila/metabolismo
3.
DNA Repair (Amst) ; 5(1): 23-31, 2006 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-16290249

RESUMO

Glycidaldehyde (GDA) is a bifunctional alkylating agent that has been shown to be mutagenic in vitro and carcinogenic in rodents. However, the molecular mechanism by which it exerts these effects is not established. GDA is capable of forming exocyclic hydroxymethyl-substituted etheno adducts on base residues in vitro. One of them, 7-(hydroxymethyl)-1,N6-ethenoadenine (7-hm-epsilonA), was identified as the principal adduct in mouse skin treated with GDA or a glycidyl ether. In this work, using defined oligonucleotides containing a site-specific 7-hm-epsilonA, the human and mouse alkylpurine-DNA-N-glycosylases (APNGs), responsible for the removal of the analogous 1,N6-ethenoadenine (epsilonA) adduct, are shown to recognize and excise 7-hm-epsilonA. Such an activity can be significantly modulated by both 5' neighboring and opposite sequence contexts. The efficiency of human or mouse APNG for excision of 7-hm-epsilonA is about half that, or similar to the excision of epsilonA, respectively. When human or mouse cell-free extracts were tested, however, the extent of 7-hm-epsilonA excision is dramatically lower than that for epsilonA, suggesting that, in the crude extracts, the APNG activities toward these two adducts are differentially affected. Using cell-free extracts from APNG deficient mice, this enzyme is shown to be the primary glycosylase excising 7-hm-epsilonA. A structural approach, using molecular modeling, was employed to examine how the structure of the 7-hm-epsilonA adduct affects DNA conformation, as compared to the epsilonA adduct. These novel substrate specificities could have both biological and structural implications.


Assuntos
Adenina/análogos & derivados , Adutos de DNA/metabolismo , DNA Glicosilases/metabolismo , Adenina/química , Adenina/metabolismo , Aldeídos/química , Aldeídos/metabolismo , Animais , Sistema Livre de Células , DNA Glicosilases/genética , Reparo do DNA/fisiologia , Compostos de Epóxi/química , Compostos de Epóxi/metabolismo , Humanos , Camundongos , Camundongos Knockout , Modelos Moleculares , Conformação de Ácido Nucleico , Ácidos Nucleicos Heteroduplexes/química
4.
Sci Rep ; 7(1): 17438, 2017 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-29234069

RESUMO

Interstrand cross-links (ICLs) are highly cytotoxic DNA lesions that block DNA replication and transcription by preventing strand separation. Previously, we demonstrated that the bacterial and human DNA glycosylases Nei and NEIL1 excise unhooked psoralen-derived ICLs in three-stranded DNA via hydrolysis of the glycosidic bond between the crosslinked base and deoxyribose sugar. Furthermore, NEIL3 from Xenopus laevis has been shown to cleave psoralen- and abasic site-induced ICLs in Xenopus egg extracts. Here we report that human NEIL3 cleaves psoralen-induced DNA-DNA cross-links in three-stranded and four-stranded DNA substrates to generate unhooked DNA fragments containing either an abasic site or a psoralen-thymine monoadduct. Furthermore, while Nei and NEIL1 also cleave a psoralen-induced four-stranded DNA substrate to generate two unhooked DNA duplexes with a nick, NEIL3 targets both DNA strands in the ICL without generating single-strand breaks. The DNA substrate specificities of these Nei-like enzymes imply the occurrence of long uninterrupted three- and four-stranded crosslinked DNA-DNA structures that may originate in vivo from DNA replication fork bypass of an ICL. In conclusion, the Nei-like DNA glycosylases unhook psoralen-derived ICLs in various DNA structures via a genuine repair mechanism in which complex DNA lesions can be removed without generation of highly toxic double-strand breaks.


Assuntos
Reagentes de Ligações Cruzadas/farmacologia , DNA Glicosilases/metabolismo , DNA/efeitos dos fármacos , DNA/metabolismo , Ficusina/farmacologia , N-Glicosil Hidrolases/metabolismo , Quebras de DNA , Desoxirribonuclease (Dímero de Pirimidina)/metabolismo , Escherichia coli , Proteínas de Escherichia coli/metabolismo , Humanos , Hidrólise , Conformação de Ácido Nucleico/efeitos dos fármacos
5.
FASEB J ; 19(2): 290-2, 2005 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15677345

RESUMO

OGG-1 DNA glycosylase (OGG-1) is an enzyme involved in DNA repair. It excises 7,8-dihydro-8-oxoguanine, which is formed by oxidative damage of guanine. We have investigated the role of OGG-1 in inflammation using three models of inflammation: endotoxic shock, diabetes, and contact hypersensitivity. We found that OGG-1(-/-) mice are resistant to endotoxin (lipopolysaccharide, LPS)-induced organ dysfunction, neutrophil infiltration and oxidative stress, when compared with the response seen in wild-type controls (OGG(+/+)). Furthermore, the deletion of the OGG-1 gene was associated with decreased serum cytokine and chemokine levels and prolonged survival after LPS treatment. Type I diabetes was induced by multiple low-dose streptozotocin treatment. OGG-1(-/-) mice were found to have significantly lower blood glucose levels and incidence of diabetes as compared with OGG-1(+/+) mice. Biochemical analysis of the pancreas showed that OGG-1(-/-) mice had greater insulin content, indicative of a greater beta-cell mass coupled with lower levels of the chemokine MIP-1alpha and Th1 cytokines IL-12 and TNF-alpha. Levels of protective Th2 cytokines, IL-4 and IL-10 were significantly higher in the pancreata of OGG-1(-/-) mice as compared with the levels measured in wild-type mice. In the contact hypersensitivity induced by oxazolone, the OGG-1(-/-) mice showed reduced neutrophil accumulation, chemokine, and Th1 and Th2 cytokine levels in the ear tissue. The current studies unveil a role for OGG-1 in the regulation of inflammation.


Assuntos
DNA Glicosilases/fisiologia , Animais , DNA Glicosilases/deficiência , DNA Glicosilases/genética , Dermatite de Contato/enzimologia , Dermatite de Contato/genética , Dermatite de Contato/metabolismo , Diabetes Mellitus Experimental/enzimologia , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Endotoxinas/efeitos adversos , Endotoxinas/metabolismo , Feminino , Inflamação/induzido quimicamente , Inflamação/enzimologia , Inflamação/genética , Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Genéticos , Choque Séptico/enzimologia , Choque Séptico/genética , Choque Séptico/metabolismo , Estreptozocina
6.
Nucleic Acids Res ; 32(1): 73-81, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-14704345

RESUMO

In nucleotide incision repair (NIR), an endonuclease nicks oxidatively damaged DNA in a DNA glycosylase-independent manner, providing the correct ends for DNA synthesis coupled to the repair of the remaining 5'-dangling modified nucleotide. This mechanistic feature is distinct from DNA glycosylase-mediated base excision repair. Here we report that Ape1, the major apurinic/apyrimidinic endonuclease in human cells, is the damage- specific endonuclease involved in NIR. We show that Ape1 incises DNA containing 5,6-dihydro-2'-deoxyuridine, 5,6-dihydrothymidine, 5-hydroxy-2'-deoxyuridine, alpha-2'-deoxyadenosine and alpha-thymidine adducts, generating 3'-hydroxyl and 5'-phosphate termini. The kinetic constants indicate that Ape1-catalysed NIR activity is highly efficient. The substrate specificity and protein conformation of Ape1 is modulated by MgCl2 concentrations, thus providing conditions under which NIR becomes a major activity in cell-free extracts. While the N-terminal region of Ape1 is not required for AP endonuclease function, we show that it regulates the NIR activity. The physiological relevance of the mammalian NIR pathway is discussed.


Assuntos
Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Nucleotídeos/metabolismo , Extratos Celulares , Dano ao DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/química , Células HeLa , Humanos , Cinética , Magnésio/farmacologia , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/metabolismo , Oxirredução , Conformação Proteica/efeitos dos fármacos , Especificidade por Substrato
7.
Cancer Res ; 63(22): 7699-703, 2003 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-14633693

RESUMO

Ethenobases are promutagenic DNA adducts formed by some environmental carcinogens and products of endogenous lipid peroxidation. Mutation spectra in tumors induced in mice by urethane or its metabolite vinyl carbamate (Vcar) are compatible with 1,N(6)-ethenoadenine (epsilonA) being an initiating lesion in the development of these tumors. As alkylpurine-DNA-N-glycosylase (APNG) releases epsilonA from DNA in vitro, wild-type and APNG-/- C57Bl/6J mice were treated with Vcar and levels of epsilonA and 3,N(4)-ethenocytosine (epsilonC), which is not a substrate of APNG, were analyzed in liver and lung DNA. At 6 h after the last dose, levels of epsilonA were 1.6-fold higher in DNA from APNG-/- mice and subsequently persisted at higher levels for longer than in DNA from wild-type animals, confirming that epsilonA is released by APNG in vivo. In contrast, approximately 14-fold lower levels of epsilonC were induced by Vcar, and the kinetics of formation and persistence of epsilonC was similar in the two mouse strains. The carcinogenicity of Vcar was compared in APNG-/- and wild-type suckling mice given a single dose of Vcar (30 or 150 nmol/g). After 1 year, only mice in the high-dose group developed hepatocellular carcinoma; however, the incidence was not higher in APNG-/- mice. Although higher levels and increased persistence of epsilonA was observed in hepatic DNA from APNG-/- mice at 150 nmol/g Vcar, apoptosis and cell proliferation levels were similar in both strains of mice. This may explain why differences in epsilonA formation/persistence observed here did not result in higher susceptibility of APNG-/- mice to hepatocarcinogenesis.


Assuntos
Carcinógenos/toxicidade , DNA Glicosilases/deficiência , Reparo do DNA/fisiologia , DNA/efeitos dos fármacos , DNA/metabolismo , Desoxiadenosinas/metabolismo , Uretana/análogos & derivados , Uretana/toxicidade , Animais , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Dano ao DNA , DNA Glicosilases/genética , Desoxiadenosinas/biossíntese , Feminino , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
8.
Mutat Res ; 531(1-2): 165-75, 2003 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-14637253

RESUMO

The generation of mouse models of base excision repair deficiency has resulted in a re-examination of the cellular defence mechanisms that exist to counteract oxidative base damage. Contrary to exhibiting various detrimental effects of the gene disruption, the different strains of DNA-N-glycosylase deficient mice have proved to be remarkably resilient to the loss of the major activities that catalyse the removal of oxidised bases from DNA. Indeed, with a few exceptions, there is little evidence for the accumulation of oxidised bases in tissues and organs of the glycosylase knockout mice, even in older animals. This is highly suggestive of hitherto unknown backup mechanisms for dealing with the removal of oxidative base damage from genomic DNA. Results from both a genomics-based approach and biochemical analyses of cell free extracts from DNA glycosylase knockout mice have indicated that this is so and there is increasing evidence of several novel DNA glycosylase/AP lyases in mammalian cells that are capable of acting on oxidised bases in vitro. This, in parallel with other repair mechanisms involving mismatch repair, the Cockayne syndrome B protein and the efficient and accurate bypass of replication blocking lesions by a battery of translesion DNA polymerases, may explain the lack of severe phenotype observed for the DNA glycosylase deficient mice discussed in this article.


Assuntos
DNA Glicosilases/deficiência , Animais , Dano ao DNA , DNA Glicosilases/metabolismo , Camundongos , Camundongos Knockout , Modelos Animais , Estresse Oxidativo
9.
Exp Cell Res ; 313(16): 3604-15, 2007 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-17765223

RESUMO

The use of gene mutation/knock-out strategies in mouse embryonic stem (ES) cells has revolutionized the study of gene function in ES cells and embryonic development. However, the construction of vectors for homologous recombination strategies requires considerable expertise and time. We describe two novel vectors that can generate site specific knock-out or EGFP knock-in ES cells within 6 weeks from construct design to identification of positive ES cell clones. As proof-of-principle, we have utilized the knock-out targeting vector to modify the NEIL2 locus in ES cells. In addition, using the knock-in vector, we have inserted EGFP downstream of the 5T4 oncofetal antigen promoter in ES cells (5T4-GFP ES cells). Undifferentiated 5T4-GFP ES cells lack EGFP and maintain expression of the pluripotent markers OCT-4 and NANOG. Upon differentiation, EGFP expression is increased in 5T4-GFP ES cells and this correlates with 5T4 transcript expression of the unmodified allele, loss of Nanog and Oct-4 transcripts and upregulation of differentiation-associated transcripts. Furthermore, we demonstrate that fluorescent activated cell sorting of 5T4-GFP ES cells allows isolation of pluripotent or differentiated cells from a heterogeneous population. These vectors provide researchers with a rapid method of modifying specific ES cell genes to study cellular differentiation and embryonic development.


Assuntos
Antígenos de Neoplasias/genética , Células-Tronco Embrionárias/metabolismo , Vetores Genéticos , Proteínas de Fluorescência Verde/metabolismo , Regiões Promotoras Genéticas/genética , Recombinação Genética , Animais , Sequência de Bases , Biomarcadores/metabolismo , Diferenciação Celular , Linhagem Celular , Células Clonais , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/enzimologia , Camundongos , Dados de Sequência Molecular , Timidina Quinase/metabolismo
10.
J Biol Chem ; 277(52): 50487-90, 2002 Dec 27.
Artigo em Inglês | MEDLINE | ID: mdl-12401779

RESUMO

In mammalian cells, thymine glycols and other oxidized pyrimidines such as 5,6-dihydrouracil are removed from DNA by the NTH1 protein, a bifunctional DNA-N-glycosylase. However, mNTH1 knock-out mice in common with other DNA glycosylase-deficient mice do not show any severe abnormalities associated with accumulation of DNA damage and mutations. In the present study we used an in vitro repair system to investigate the mechanism for the removal of 5,6-dihydrouracil from DNA by mNTH1-deficient cell-free extracts derived from testes of mNTH1 knock-out mice. We found that these extracts are able to support the removal of 5,6-dihydrouracil from DNA at about 20% of the efficiency of normal extracts. Furthermore, we also found that single-nucleotide patch base excision repair is the major pathway for removal of 5,6-dihydrouracil in mNTH1-deficient cell extracts, suggesting the involvement of other DNA glycosylase(s) in the removal of oxidized pyrimidines.


Assuntos
Reparo do DNA , Desoxirribonuclease (Dímero de Pirimidina) , Endodesoxirribonucleases/genética , Proteínas de Escherichia coli , Uracila/análogos & derivados , Uracila/metabolismo , Animais , Sequência de Bases , Sistema Livre de Células , Dano ao DNA , DNA Glicosilases , Endodesoxirribonucleases/deficiência , Endodesoxirribonucleases/metabolismo , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Mutação , N-Glicosil Hidrolases/deficiência , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/metabolismo , Proteínas Recombinantes/metabolismo
11.
J Biol Chem ; 277(30): 26987-93, 2002 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-12016206

RESUMO

The promutagenic and genotoxic exocyclic DNA adduct 1,N(2)-ethenoguanine (1,N(2)-epsilonG) is a major product formed in DNA exposed to lipid peroxidation-derived aldehydes in vitro. Here, we report that two structurally unrelated proteins, the Escherichia coli mismatch-specific uracil-DNA glycosylase (MUG) and the human alkylpurine-DNA-N-glycosylase (ANPG), can release 1,N(2)-epsilonG from defined oligonucleotides containing a single modified base. A comparison of the kinetic constants of the reaction indicates that the MUG protein removes the 1,N(2)-epsilonG lesion more efficiently (k(cat)/K(m) = 0.95 x 10(-3) min(-1) nm(-1)) than the ANPG protein (k(cat)/K(m) = 0.1 x 10(-3) min(-1) nm(-1)). Additionally, while the nonconserved, N-terminal 73 amino acids of the ANPG protein are not required for activity on 1,N(6)-ethenoadenine, hypoxanthine, or N-methylpurines, we show that they are essential for 1,N(2)-epsilonG-DNA glycosylase activity. Both the MUG and ANPG proteins preferentially excise 1,N(2)-epsilonG when it is opposite dC; however, unlike MUG, ANPG is unable to excise 1,N(2)-epsilonG when it is opposite dG. Using cell-free extracts from genetically modified E. coli and murine embryonic fibroblasts lacking MUG and mANPG activity, respectively, we show that the incision of the 1,N(2)-epsilonG-containing duplex oligonucleotide has an absolute requirement for MUG or ANPG. Taken together these observations suggest a possible role for these proteins in counteracting the genotoxic effects of 1,N(2)-epsilonG residues in vivo.


Assuntos
Adutos de DNA , DNA Glicosilases , Escherichia coli/metabolismo , Guanina/análogos & derivados , Guanina/farmacologia , N-Glicosil Hidrolases/metabolismo , Animais , Sistema Livre de Células , Reparo do DNA , Humanos , Cinética , Camundongos , Modelos Químicos , Oligonucleotídeos/farmacologia , Ligação Proteica , Especificidade por Substrato , Uracila-DNA Glicosidase
12.
J Biol Chem ; 278(36): 33701-7, 2003 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-12819227

RESUMO

Mitochondrial DNA is constantly exposed to high levels of endogenously produced reactive oxygen species, resulting in elevated levels of oxidative damaged DNA bases. A large spectrum of DNA base alterations can be detected after oxidative stress, and many of these are highly mutagenic. Thus, an efficient repair of these is necessary for survival. Some of the DNA repair pathways involved have been characterized, but others are not yet determined. A DNA repair activity for thymine glycol and other oxidized pyrimidines has been described in mammalian mitochondria, but the nature of the glycosylases involved in this pathway remains unclear. The generation of mouse strains lacking murine thymine glycol-DNA glycosylase (mNTH1) and/or murine 8-oxoguanine-DNA glycosylase (mOGG1), the two major DNA N-glycosylase/apurinic/apyrimidinic (AP) lyases involved in the repair of oxidative base damage in the nucleus, has provided very useful biological model systems for the study of the function of these and other glycosylases in mitochondrial DNA repair. In this study, mouse liver mitochondrial extracts were generated from mNTH1-, mOGG1-, and [mNTH1, mOGG1]-deficient mice to ascertain the role of each of these glycosylases in the repair of oxidized pyrimidine base damage. We also characterized for the first time the incision of various modified bases in mitochondrial extracts from a double-knock-out [mNTH1, mOGG1]-deficient mouse. We show that mNTH1 is responsible for the repair of thymine glycols in mitochondrial DNA, whereas other glycosylase/AP lyases also participate in removing other oxidized pyrimidines, such as 5-hydroxycytosine and 5-hydroxyuracil. We did not detect a backup glycosylase or glycosylase/AP lyase activity for thymine glycol in the mitochondrial mouse extracts.


Assuntos
Desoxirribonuclease (Dímero de Pirimidina) , Proteínas de Escherichia coli , Mitocôndrias Hepáticas/metabolismo , N-Glicosil Hidrolases/fisiologia , Oxigênio/metabolismo , Pirimidinas/química , Animais , Sequência de Bases , Western Blotting , Boroidretos/farmacologia , Núcleo Celular/metabolismo , Reparo do DNA , DNA-Formamidopirimidina Glicosilase , Eletroforese em Gel de Poliacrilamida , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/fisiologia , Fígado/metabolismo , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , N-Glicosil Hidrolases/genética , Oligonucleotídeos/química , Estresse Oxidativo , Espécies Reativas de Oxigênio , Uracila/química
13.
Biochem Biophys Res Commun ; 319(1): 240-6, 2004 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-15158468

RESUMO

The base excision repair (BER) pathway plays a key role in protecting the genome from endogenous DNA damage. Current methods to measure BER activities are indirect and cumbersome. Here, we introduce a direct method to assay DNA excision repair that is suitable for automation and industrial use, based on the fluorescence quenching mechanism of molecular beacons. We designed a single-stranded DNA oligonucleotide labelled with a 5'-fluorescein (F) and a 3'-Dabcyl (D) in which the fluorophore, F, is held in close proximity to the quencher, D, by the stem-loop structure design of the oligonucleotide. Following removal of the modified base or incision of the oligonucleotide, the fluorophore is separated from the quencher and fluorescence can be detected as a function of time. Several modified beacons have been used to validate the assay on both cell-free extracts and purified proteins. We have further developed the method to analyze BER in cultured cells. As described, the molecular beacon-based assay can be applied to all DNA modifications processed by DNA excision/incision repair pathways. Possible applications of the assay are discussed, including high-throughput real-time DNA repair measurements both in vitro and in living cells.


Assuntos
Dano ao DNA , Reparo do DNA , Técnicas Genéticas , Animais , Sistema Livre de Células , DNA/química , DNA Glicosilases/química , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Células HeLa , Humanos , Camundongos , Células NIH 3T3 , Conformação de Ácido Nucleico , Oligonucleotídeos/química , Oxigênio/metabolismo , Saccharomyces cerevisiae/metabolismo , Espectrometria de Fluorescência , Fatores de Tempo
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