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1.
Nucleic Acids Res ; 37(7): 2116-25, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19223326

RESUMO

The 5-formyluracil (5-foU), a major mutagenic oxidative damage of thymine, is removed from DNA by Nth, Nei and MutM in Escherichia coli. However, DNA polymerases can also replicate past the 5-foU by incorporating C and G opposite the lesion, although the mechanism of correction of the incorporated bases is still unknown. In this study, using a borohydride-trapping assay, we identified a protein trapped by a 5-foU/C-containing oligonucleotide in an extract from E. coli mutM nth nei mutant. The protein was subsequently purified from the E. coli mutM nth nei mutant and was identified as KsgA, a 16S rRNA adenine methyltransferase. Recombinant KsgA also formed the trapped complex with 5-foU/C- and thymine glycol (Tg)/C-containing oligonucleotides. Furthermore, KsgA excised C opposite 5-foU, Tg and 5-hydroxymethyluracil (5-hmU) from duplex oligonucleotides via a beta-elimination reaction, whereas it could not remove the damaged base. In contrast, KsgA did not remove C opposite normal bases, 7,8-dihydro-8-oxoguanine and 2-hydroxyadenine. Finally, the introduction of the ksgA mutation increased spontaneous mutations in E. coli mutM mutY and nth nei mutants. These results demonstrate that KsgA has a novel DNA glycosylase/AP lyase activity for C mispaired with oxidized T that prevents the formation of mutations, which is in addition to its known rRNA adenine methyltransferase activity essential for ribosome biogenesis.


Assuntos
DNA Glicosilases/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Escherichia coli/enzimologia , Metiltransferases/metabolismo , Sequência de Aminoácidos , DNA Glicosilases/genética , DNA Glicosilases/isolamento & purificação , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/isolamento & purificação , DNA-Formamidopirimidina Glicosilase/química , DNA-Formamidopirimidina Glicosilase/genética , Desoxirribonuclease (Dímero de Pirimidina)/química , Desoxirribonuclease (Dímero de Pirimidina)/genética , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Metiltransferases/química , Metiltransferases/genética , Alinhamento de Sequência
2.
Mol Cell Biol ; 26(23): 9083-93, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16966376

RESUMO

Apurinic/apyrimidinic endonuclease 1, a key enzyme in repairing abasic sites in DNA, is an embryonic lethal in mice. We are examining its role in embryogenesis in zebra fish. Zebra fish contain two genomic copies (zfAPEX1a and zfAPEX1b) with identical coding sequences. zfAPEX1b lacks introns. Recombinant protein (ZAP1) is highly homologous with and has the same enzymatic properties as its human orthologue. ZAP1 is highly expressed throughout development. Embryos microinjected with morpholino oligonucleotide (MO) targeting the translation start site die at approximately the midblastula transition (MBT) without apoptosis. They are rescued with mRNA for human wild-type APEX1 but not for APEX1 encoding endonuclease-defective protein. Rescued embryos develop dysmorphic hearts, pericardial edema, few erythrocytes, small eyes, and abnormal notochords. Although the hearts in rescued embryos form defective loops ranging from no loop to one that is abnormally shaped, cardiac myosin (cmlc2) is present and contraction occurs. Embryos microinjected with MO targeting zfAPEX1a intron-exon junctions also pass the MBT with similar abnormalities. We conclude that AP endonuclease 1 is involved in both repairing DNA and regulating specific early stages of embryonic development.


Assuntos
Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Coração/embriologia , Hematopoese/fisiologia , Peixe-Zebra/embriologia , Sequência de Aminoácidos , Animais , Sequência Conservada , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/química , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/isolamento & purificação , Embrião não Mamífero , Dosagem de Genes , Coração/anatomia & histologia , Hematopoese/genética , Histocitoquímica , Humanos , Hibridização In Situ , Cinética , Microinjeções , Dados de Sequência Molecular , Mutação , Oligonucleotídeos Antissenso/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Sítio de Iniciação de Transcrição
3.
Mol Pharmacol ; 73(3): 669-77, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18042731

RESUMO

Human apurinic/apyrimidinic endonuclease (Ape1) plays an important role by processing the >10,000 highly toxic abasic sites generated in the genome of each cell every day. Ape1 has recently emerged as a target for inhibition, in that its overexpression in tumors has been linked with poor response to both radiation and chemotherapy and lower overall patient survival. Inhibition of Ape1 using siRNA or the expression of a dominant-negative form of the protein has been shown to sensitize cells to DNA-damaging agents, including various chemotherapeutic agents. However, potent small-molecule inhibitors of Ape1 remain to be found. To this end, we screened Ape1 against the NCI Diversity Set of small molecules and discovered aromatic nitroso, carboxylate, sulfonamide, and arylstibonic acid compounds with micromolar affinities for the protein. A further screen of a 37-compound arylstibonic acid sublibrary identified ligands with IC(50) values in the range of 4 to 300 nM. The negatively charged stibonic acids act by a partial-mixed mode and probably serve as DNA phosphate mimics. These compounds provide a useful scaffold for development of chemotherapeutic agents against Ape1.


Assuntos
Antimônio/farmacologia , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/antagonistas & inibidores , Endonucleases/antagonistas & inibidores , Antimônio/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , DNA/análise , DNA/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/isolamento & purificação , Humanos , Concentração Inibidora 50 , Cinética , Ligantes , Modelos Químicos , Osteossarcoma/tratamento farmacológico , Osteossarcoma/enzimologia , Osteossarcoma/genética , Osteossarcoma/patologia , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/isolamento & purificação , Eletricidade Estática , Relação Estrutura-Atividade , Especificidade por Substrato
4.
Biochem Biophys Res Commun ; 368(1): 68-73, 2008 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-18206643

RESUMO

Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/ref-1) is a multifunctional protein involved both in DNA base excision repair and redox regulation. In this study we evaluated the protective role of Tat-mediated APE1/ref-1 transduction on the tumor necrosis factor (TNF)-alpha-activated endothelial activation in cultured human umbilical vein endothelial cells. To construct Tat-APE1/ref-1 fusion protein, human full length of APE1/ref-1 was fused with Tat-protein transduction domain. Purified Tat-APE1/ref-1 fusion protein efficiently transduced cultured endothelial cells in a dose-dependent manner and reached maximum expression at 1h after incubation. Transduced Tat-APE1/ref-1 showed inhibitory activity on the TNF-alpha-induced monocyte adhesion and vascular cell adhesion molecule-1 expression in cultured endothelial cells. These results suggest Tat-APE1/ref-1 might be useful to reduce vascular endothelial activation or vascular inflammatory disorders.


Assuntos
DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Produtos do Gene tat/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Adesão Celular , Células Cultivadas , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/isolamento & purificação , Células Endoteliais/citologia , Produtos do Gene tat/genética , Produtos do Gene tat/isolamento & purificação , Humanos , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismo
5.
Nucleic Acids Res ; 34(7): 2067-76, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16617147

RESUMO

Abasic (AP)-endonuclease (APE) is responsible for repair of AP sites, and single-strand DNA breaks with 3' blocking groups that are generated either spontaneously or during repair of damaged or abnormal bases via the DNA base excision repair (BER) pathway in both nucleus and mitochondria. Mammalian cells express only one nuclear APE, 36 kDa APE1, which is essential for survival. Mammalian mitochondrial (mt) BER enzymes other than mtAPE have been characterized. In order to identify and characterize mtAPE, we purified the APE activity from beef liver mitochondria to near homogeneity, and showed that the mtAPE which has 3-fold higher specific activity relative to APE1 is derived from the latter with deletion of 33 N-terminal residues which contain the nuclear localization signal. The mtAPE-sized product could be generated by incubating 35S-labeled APE1 with crude mitochondrial extract, but not with cytosolic or nuclear extract, suggesting that cleavage of APE1 by a specific mitochondria-associated N-terminal peptidase is a prerequisite for mitochondrial import. The low abundance of mtAPE, particularly in cultured cells might be the reason for its earlier lack of detection by western analysis.


Assuntos
DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Mitocôndrias/enzimologia , Proteínas Mitocondriais/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/isolamento & purificação , Estabilidade Enzimática , Humanos , Cinética , Camundongos , Mitocôndrias Hepáticas/enzimologia , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/isolamento & purificação , Dados de Sequência Molecular , Peptídeo Hidrolases/metabolismo , Proteínas Recombinantes/metabolismo , Deleção de Sequência
6.
DNA Repair (Amst) ; 5(12): 1439-48, 2006 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-16982218

RESUMO

DNA glycosylases/AP lyases initiate repair of oxidized bases in the genomes of all organisms by excising these lesions and then cleaving the DNA strand at the resulting abasic (AP) sites and generate 3' phospho alpha,beta-unsaturated aldehyde (3' PUA) or 3' phosphate (3' P) terminus. In Escherichia coli, the AP-endonucleases (APEs) hydrolyze both 3' blocking groups (3' PUA and 3' P) to generate the 3'-OH termini needed for repair synthesis. In mammalian cells, the previously characterized DNA glycosylases, NTH1 and OGG1, produce 3' PUA, which is removed by the only AP-endonuclease, APE1. However, APE1 is barely active in removing 3' phosphate generated by the recently discovered mammalian DNA glycosylases NEIL1 and NEIL2. We showed earlier that the 3' phosphate generated by NEIL1 is efficiently removed by polynucleotide kinase (PNK) and not APE1. Here we show that the NEIL2-initiated repair of 5-hydroxyuracil (5-OHU) similarly requires PNK. We have also observed stable interaction between NEIL2 and other BER proteins DNA polymerase beta (Pol beta), DNA ligase IIIalpha (Lig IIIalpha) and XRCC1. In spite of their limited sequence homology, NEIL1 and NEIL2 interact with the same domains of Pol beta and Lig IIIalpha. Surprisingly, while the catalytically dispensable C-terminal region of NEIL1 is the common interacting domain, the essential N-terminal segment of NEIL2 is involved in analogous interaction. The BER proteins including NEIL2, PNK, Pol beta, Lig IIIalpha and XRCC1 (but not APE1) could be isolated as a complex from human cells, competent for repair of 5-OHU in plasmid DNA.


Assuntos
DNA Glicosilases/metabolismo , Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , DNA/metabolismo , Polinucleotídeo 5'-Hidroxiquinase/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , DNA Glicosilases/isolamento & purificação , DNA Ligase Dependente de ATP , DNA Ligases/isolamento & purificação , DNA Ligases/metabolismo , DNA Polimerase beta/isolamento & purificação , DNA Polimerase beta/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/isolamento & purificação , Humanos , Complexos Multiproteicos , Plasmídeos/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose , Polinucleotídeo 5'-Hidroxiquinase/isolamento & purificação , Estrutura Terciária de Proteína , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Transfecção , Técnicas do Sistema de Duplo-Híbrido , Proteínas de Xenopus
7.
DNA Repair (Amst) ; 4(6): 655-70, 2005 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-15907773

RESUMO

The Saccharomyces cerevisiae mutant strain YW778, which lacks apurinic/apyrimidinic (AP) endonuclease and 3'-diesterase DNA repair activities, displays high levels of spontaneous mutations and hypersensitivities to several DNA damaging agents. We searched a cDNA library derived from the nematode Caenorhabditis elegans for gene products that would rescue the DNA repair defects of this yeast mutant. We isolated two genes, apn-1 and exo-3, encoding proteins that have not been previously characterized. Both APN-1 and EXO-3 share significant identity with the functionally established Escherichia coli AP endonucleases, endonuclease IV and exonuclease III, respectively. Strain YW778 expressing either apn-1 or exo-3 shows parental levels of spontaneous mutations, as well as resistance to DNA damaging agents that produce AP sites and DNA single strand breaks with blocked 3'-ends. Using an in vitro assay, we show that the apn-1 and exo-3 genes independently express AP endonuclease activity in the yeast mutant. We further characterize the EXO-3 protein and three of its mutated variants E68A, D190A, and H279A. The E68A variant retains both AP endonuclease and 3'-diesterase repair activities in vitro, yet severely lacks the ability to protect strain YW778 from spontaneous and drug-induced DNA lesions, suggesting that this variant E68A may possess a defect that interferes with the repair process in vivo. In contrast, D190A and H279A are completely devoid of DNA repair activities and fail to rescue the genetic instability of strain YW778. Our data strongly suggest that EXO-3 and APN-1 are enzymes possessing intrinsic AP endonuclease and 3'-diesterase activities.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimologia , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Teste de Complementação Genética , Proteínas de Helminto/metabolismo , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Animais , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/isolamento & purificação , Reparo do DNA , DNA Bacteriano , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/química , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/isolamento & purificação , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/isolamento & purificação , Endodesoxirribonucleases/metabolismo , Escherichia coli/genética , Expressão Gênica , Biblioteca Gênica , Vetores Genéticos , Glutationa Transferase/metabolismo , Proteínas de Helminto/genética , Proteínas de Helminto/isolamento & purificação , Dados de Sequência Molecular , Mutagênese Insercional , Plasmídeos/genética , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos
8.
Methods Enzymol ; 408: 33-48, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16793361

RESUMO

NEIL1 and NEIL2 were newly discovered as mammalian orthologs of Escherichia coli Nei and Fpg, oxidized base-specific DNA glycosylases. These are distinct from previously characterized OGG1 and NTH1, the other two glycosylases for repairing oxidatively damaged bases in mammalian cells, in regards to reaction mechanism. Recombinant human NEIL1 and NEIL2 were purified from E. coli and biochemically characterized. Some damaged bases are common substrates for both groups of enzymes. However, in contrast to the lack of activity of NTH1 and OGG1 for substrate lesions in single-stranded DNA, the NEILs have unique preference for bubble or single-stranded DNA substrates, suggesting their preferential involvement in repairing transcribed or replicating DNA sequences.


Assuntos
Dano ao DNA , DNA Glicosilases/metabolismo , Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos) , DNA/metabolismo , DNA Glicosilases/genética , DNA Glicosilases/isolamento & purificação , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/isolamento & purificação , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Genoma Humano , Humanos , Oxirredução , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
9.
Nucleic Acids Res ; 32(12): 3531-6, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15247342

RESUMO

DNA strand breaks containing 3'-phosphoglycolate (3'-PG) ends are the major lesions induced by ionizing radiation. The repair of this lesion is not completely understood and several activities are thought to be involved in processing of 3'-PG ends. In this study we examined activities in human whole cell extracts (WCE) responsible for removal of 3'-PG. Using a radiolabelled oligonucleotide containing a single nucleotide gap with internal 5'-phosphate and 3'-PG ends, we demonstrate that the major 3'-PG activity in human WCE is Mg2+ dependent and that this activity co-purifies with AP endonuclease 1 (APE1) over phosphocellulose and gel filtration chromatography. Furthermore, immunodepletion of APE1 from active gel filtration fractions using APE1 specific antibodies reveals that the major activity against 3'-PG in human WCE is APE1.


Assuntos
DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Glicolatos/metabolismo , Extratos Celulares/química , Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/isolamento & purificação , Células HeLa , Humanos , Oligonucleotídeos/metabolismo , Testes de Precipitina
10.
DNA Repair (Amst) ; 3(11): 1483-92, 2004 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-15380104

RESUMO

E. coli nucleoside diphosphate kinase (EcoNDK) is an important cellular enzyme required to maintain balanced nucleotide pools in the cells. Recently, it was reported that EcoNDK is also a multifunctional base excision repair enzyme, possessing uracil-DNA glycosylase (UDG) and AP-DNA processing activities. We investigated for the presence of such activities in M. tuberculosis NDK (MtuNDK), which shares 45.2% identity, and 52.6% similarity with EcoNDK. In contrast to the robust uracil excision activity reported for EcoNDK, MtuNDK preparation exhibited very poor excision of uracil from DNA. However, this activity was undetectable when MtuNDK was purified from an ung(-) strain of E. coli, or when the assays were performed in the presence of extremely low amounts of a highly specific proteinaceous inhibitor, Ugi which forms an extremely tight complex with the host Ung (UDG), showing that MtuNDK preparation was contaminated with UDG. Reinvestigation of uracil processing activity of EcoNDK, showed that even this protein lacked UDG activity. All preparations of NDK were shown to be active by their autophosphorylation activity. Ugi neither displayed a physical interaction with EcoNDK nor did it affect autophosphorylation of NDKs. Further, neither of the NDK preparations processed the AP-DNA generated by UDG treatment of the uracil containing DNA duplexes. However, partially purified preparations of NDK did process such DNA substrates.


Assuntos
DNA Bacteriano/química , DNA Bacteriano/metabolismo , Escherichia coli/enzimologia , Mycobacterium tuberculosis/enzimologia , Núcleosídeo-Difosfato Quinase/metabolismo , Uracila/química , Sequência de Bases , Dano ao DNA , DNA Glicosilases/genética , DNA Glicosilases/isolamento & purificação , DNA Glicosilases/metabolismo , Reparo do DNA , DNA Bacteriano/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/isolamento & purificação , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Mycobacterium tuberculosis/genética , Núcleosídeo-Difosfato Quinase/genética , Núcleosídeo-Difosfato Quinase/isolamento & purificação , Uracila-DNA Glicosidase
11.
DNA Repair (Amst) ; 35: 27-36, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26439176

RESUMO

Clustered DNA damage is a unique characteristic of radiation-induced DNA damage and the formation of these sites poses a serious challenge to the cell's repair machinery. Within a cell DNA is compacted, with nucleosomes being the first order of higher level structure. However, few data are reported on the efficiency of clustered-lesion processing within nucleosomal DNA templates. Here, we show retardation of cleavage of a single AP site by purified APE1 when contained in nucleosomal DNA, compared to cleavage of an AP site in non-nucleosomal DNA. This retardation seen in nucleosomal DNA was alleviated by incubation with CHO-K1 nuclear extract. When clustered DNA damage sites containing bistranded AP sites were present in nucleosomal DNA, efficient cleavage of the AP sites was observed after treatment with nuclear extract. The resultant DSB formation led to DNA dissociating from the histone core and nucleosomal dispersion. Clustered damaged sites containing bistranded AP site/8-oxoG residues showed no retardation of cleavage of the AP site but retardation of 8-oxoG excision, compared to isolated lesions, thus DSB formation was not seen. An increased understanding of processing of clustered DNA damage in a nucleosomal environment may lead to new strategies to enhance the cytotoxic effects of radiotherapeutics.


Assuntos
Clivagem do DNA , Dano ao DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/química , Nucleossomos/química , Animais , Células CHO , Extratos Celulares/química , Núcleo Celular/enzimologia , Cricetulus , DNA Glicosilases/química , Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/isolamento & purificação , Guanina/análogos & derivados , Guanina/química , Humanos , Moldes Genéticos
12.
Methods Mol Biol ; 257: 115-24, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-14770001

RESUMO

We describe a basic, fast, and reliable technique to isolate and characterize ribonucleoprotein (RNP) using antibody to a constituent protein. The antibody serves to immunopurify RNP from total cells or nuclear and cytoplasmic cell fractions under conditions that promote RNP integrity. The presence of other RNP proteins as well as transcripts can then be analyzed by Western blotting and reverse transcription polymerase chain reaction, respectively. RNase treatment before immunopurification can be used to assess the dependence of protein-protein interactions on RNA. We also describe a modification using beta-mercaptoethanol that facilitates analyzing proteins that comigrate with antibody light or heavy chains.


Assuntos
Fracionamento Celular/métodos , Imunoglobulina G/metabolismo , RNA Mensageiro/metabolismo , Ribonucleoproteínas/isolamento & purificação , Animais , Western Blotting/métodos , Células COS , Núcleo Celular , Células Cultivadas , Chlorocebus aethiops , Citoplasma , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/imunologia , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/isolamento & purificação , Humanos , Imunoglobulina G/imunologia , Mercaptoetanol/metabolismo , Complexo Proteico Nuclear de Ligação ao Cap/imunologia , Complexo Proteico Nuclear de Ligação ao Cap/isolamento & purificação , Ligação Proteica , RNA Mensageiro/genética , Proteínas de Ligação a RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribonucleases/farmacologia , Ribonucleoproteínas/imunologia , Fatores de Transcrição/imunologia , Fatores de Transcrição/isolamento & purificação
13.
Methods Mol Biol ; 928: 161-74, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22956141

RESUMO

Human apurinic/apyrimidinic endonuclease-1 (APE-1) is essential for base excision repair and plays a major role in DNA repair and maintaining genomic stability. Cancer cells treated with conventional DNA-damaging agents develop resistance due in part to upregulation of enzymes involved in DNA repair. It is hypothesized that inhibiting DNA repair machinery should sensitize the cells to DNA-damaging agents. Previously, it has been shown that APE-1 is implicated in drug resistance and cancer progression. Therefore, APE-1 inhibitors are being sought after for their synergistic properties with various chemotherapeutics agents. Screening of several compound libraries and optimization of known inhibitors of APE-1 endonuclease activity have been accelerated by the use of high-throughput screening. Nevertheless, potential inhibitors must be tested in other counterscreens to validate their selectivity for APE-1. Here, we describe in-depth protocols for APE-1 purification and development of assays specific for APE-1 endonuclease activity.


Assuntos
DNA Liase (Sítios Apurínicos ou Apirimidínicos)/isolamento & purificação , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Ensaios Enzimáticos/métodos
14.
Antioxid Redox Signal ; 14(8): 1387-401, 2011 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-20874257

RESUMO

APE1 is a multifunctional protein possessing DNA repair and redox activation of transcription factors. Blocking these functions leads to apoptosis, antiangiogenesis, cell-growth inhibition, and other effects, depending on which function is blocked. Because a selective inhibitor of the APE redox function has potential as a novel anticancer therapeutic, new analogues of E3330 were synthesized. Mass spectrometry was used to characterize the interactions of the analogues (RN8-51, 10-52, and 7-60) with APE1. RN10-52 and RN7-60 were found to react rapidly with APE1, forming covalent adducts, whereas RN8-51 reacted reversibly. Median inhibitory concentration (IC(50) values of all three compounds were significantly lower than that of E3330. EMSA, transactivation assays, and endothelial tube growth-inhibition analysis demonstrated the specificity of E3330 and its analogues in blocking the APE1 redox function and demonstrated that the analogues had up to a sixfold greater effect than did E3330. Studies using cancer cell lines demonstrated that E3330 and one analogue, RN8-51, decreased the cell line growth with little apoptosis, whereas the third, RN7-60, caused a dramatic effect. RN8-51 shows particular promise for further anticancer therapeutic development. This progress in synthesizing and isolating biologically active novel E3330 analogues that effectively inhibit the APE1 redox function validates the utility of further translational anticancer therapeutic development.


Assuntos
Antineoplásicos/farmacologia , Benzoquinonas/farmacologia , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Propionatos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Antineoplásicos/síntese química , Antineoplásicos/química , Benzoquinonas/síntese química , Benzoquinonas/química , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Clonagem Molecular , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/isolamento & purificação , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Modelos Moleculares , Estrutura Molecular , Organização e Administração , Oxirredução/efeitos dos fármacos , Propionatos/síntese química , Propionatos/química , Relação Estrutura-Atividade
15.
J Virol ; 80(10): 4847-57, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16641276

RESUMO

We show here that the African swine fever virus (ASFV) protein pE296R, predicted to be a class II apurinic/apyrimidinic (AP) endonuclease, possesses endonucleolytic activity specific for AP sites. Biochemical characterization of the purified recombinant enzyme indicated that the K(m) and catalytic efficiency values for the endonucleolytic reaction are in the range of those reported for Escherichia coli endonuclease IV (endo IV) and human Ape1. In addition to endonuclease activity, the ASFV enzyme has a proofreading 3'-->5' exonuclease activity that is considerably more efficient in the elimination of a mismatch than in that of a correctly paired base. The three-dimensional structure predicted for the pE296R protein underscores the structural similarities between endo IV and the viral protein, supporting a common mechanism for the cleavage reaction. During infection, the protein is expressed at early times and accumulates at later times. The early enzyme is localized in the nucleus and the cytoplasm, while the late protein is found only in the cytoplasm. ASFV carries two other proteins, DNA polymerase X and ligase, that, together with the viral AP endonuclease, could act as a viral base excision repair system to protect the virus genome in the highly oxidative environment of the swine macrophage, the virus host cell. Using an ASFV deletion mutant lacking the E296R gene, we have determined that the viral endonuclease is required for virus growth in macrophages but not in Vero cells. This finding supports the existence of a viral reparative system to maintain virus viability in the infected macrophage.


Assuntos
Vírus da Febre Suína Africana/enzimologia , Vírus da Febre Suína Africana/crescimento & desenvolvimento , Reparo do DNA/fisiologia , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/fisiologia , Macrófagos/enzimologia , Macrófagos/virologia , Vírus da Febre Suína Africana/patogenicidade , Sequência de Aminoácidos , Animais , Domínio Catalítico/genética , Chlorocebus aethiops , Sequência Conservada , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/isolamento & purificação , Desoxirribonuclease IV (Fago T4-Induzido)/genética , Proteínas de Escherichia coli/genética , Dados de Sequência Molecular , Mutação , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Suínos , Células Vero
16.
J Biol Chem ; 278(47): 46994-7001, 2003 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-12966083

RESUMO

Recent crystallographic studies reveal loops in human AP endonuclease 1 (APE1) that interact with the major and minor grooves of DNA containing apurinic/apyrimidinic (AP) sites. These loops are postulated to stabilize the DNA helix and the flipped out AP residue. The loop alpha8 interacts with the major groove on the 3' side of the AP site. To determine the essentiality of the amino acids that constitute the alpha8 loop, we created a mutant library containing random nucleotides at codons 222-229 that, in wild-type APE1, specify the sequence NPKGNKKN. Upon expression of the library (2 x 10(6) different clones) in Escherichia coli and multiple rounds of selection with the alkylating agent methyl-methane sulfonate (MMS), we obtained approximately 2 x 10(5) active mutants that complemented the MMS sensitivity of AP endonuclease-deficient E. coli. DNA sequencing showed that active mutants tolerated amino acid substitutions at all eight randomized positions. Basic and uncharged polar amino acids together comprised the majority of substitutions, reflecting the positively charged, polar character of the wild-type loop. Asn-222, Asn-226, and Asn-229 exhibited the least mutability, consistent with x-ray data showing that each asparagine contacts a DNA phosphate. Substitutions at residues 226-229, located nearer to the AP site, that reduced basicity or hydrogen bonding potential, increased Km 2- to 6-fold and decreased AP site binding; substitutions at residues 222-225 exhibited lesser effects. This initial mutational analysis of the alpha8 loop supports and extends the conclusion of crystallographic studies that the loop is important for binding of AP.DNA and AP site incision.


Assuntos
DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , DNA/metabolismo , Mutação , Sequência de Aminoácidos , Substituição de Aminoácidos , Ácido Apurínico , Sítios de Ligação , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/isolamento & purificação , Humanos , Hidrólise , Cinética , Biblioteca de Peptídeos , Polinucleotídeos , Ligação Proteica
17.
Carcinogenesis ; 1(3): 263-70, 1980 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22283008

RESUMO

The endonuclease (AP endonuclease) specific for apurinic/apyrimidinic sites (AP sites) has been purified and characterized from normal human autopsy liver. Three chromatographically distinct species of AP endonuclease were resolved by phosphocellulose chromatography. The species had molecular weights ranging from 24,000 to 31,000. Species 3 was significantly more thermostable than was either species 1 or species 2. Species 2 had an apparent Km of 400 nM AP sites whereas species 3 had an apparent Km of 2000 nM AP sites. All of the species required Mg2+ for activity. The concentration of Mg2+ which resulted in half maximal velocity was 5 mM for species 3 but was only 1.2 mM for species 1. The observed differences in physical and catalytic properties of the three species suggest they are isozymes. Incision of damaged DNA by the three putative isozymes was restricted to AP sites and resulted in the production of 3'hydroxyl and 5' phosphorylated termini.


Assuntos
Dano ao DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , DNA/metabolismo , Fígado/enzimologia , Autopsia , Celulose/análogos & derivados , Celulose/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/isolamento & purificação , Humanos , Concentração de Íons de Hidrogênio , Isoenzimas , Cinética , Magnésio/metabolismo , Peso Molecular , Especificidade por Substrato
18.
Br J Cancer ; 91(6): 1166-73, 2004 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-15316562

RESUMO

Apurinic/apyrimidinic endonuclease (APE) is a key enzyme involved in DNA base excision repair (BER) that is often expressed at elevated levels in human cancers. Pancreatic cancer cells treated with the nucleoside analogue gemcitabine (2', 2'-difluoro-2'deoxycytidine) showed increases in APE/redox effector factor (ref-1) protein levels (approximately two-fold for Panc-1 and six-fold for MiaPaCa-2), with corresponding increases in endonuclease activity. These results suggested that the activation of APE/ref-1 might be an adaptive response that contributes to gemcitabine resistance by facilitating BER. To test this hypothesis, we examined the effects of disrupting APE/ref-1 using antisense on gemcitabine toxicity. Antisense oligonucleotides decreased protein levels three-fold in MiaPaCa-2 and five-fold in Panc-1 in comparison to controls, associated with reduced endonuclease activity. Combination treatments with antisense oligonucleotides and gemcitabine partially suppressed the increase in APE/ref-1 activity seen in cells exposed to gemcitabine alone. While clonogenic assays showed only slight decreases in colony formation in cells treated with either antisense oligonucleotides or gemcitabine alone, the combination with APE/ref-1 antisense resulted in a 2-log enhancement of gemcitabine toxicity in Panc-1 cells. Overall these findings suggest that APE/ref-1 plays a significant role in gemcitabine resistance in some pancreatic cancer cells, and support the further investigation of novel treatments that target this protein.


Assuntos
Antimetabólitos Antineoplásicos/toxicidade , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Desoxicitidina/análogos & derivados , Desoxicitidina/toxicidade , Oligonucleotídeos Antissenso/farmacologia , Transporte Biológico , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/efeitos dos fármacos , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/isolamento & purificação , Resistencia a Medicamentos Antineoplásicos , Citometria de Fluxo , Humanos , Cinética , Oligonucleotídeos Antissenso/farmacocinética , Neoplasias Pancreáticas , Transfecção , Gencitabina
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