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1.
Gene ; 208(2): 207-13, 1998 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-9524267

RESUMO

The human gene XPB, defective in xeroderma pigmentosum patients complementation group B, encodes a DNA helicase involved in several DNA metabolic pathways, including DNA repair and transcription. The high conservation of this gene has allowed the cloning of homologs in various species, such as mouse, yeast and Drosophila. Not much information on the molecular basis of nucleotide excision repair in plants is available, but these organisms may have similar mechanisms to other eukaryotes. A homolog of XPB was isolated in Arabidopsis thaliana by using polymerase chain reaction (PCR) with degenerate oligonucleotides based on protein domains which are conserved among several species. Screening of an Arabidopsis cDNA library led to the identification and isolation of a cDNA clone with 2670 bp encoding a predicted protein of 767 amino acids, denoted araXPB. Genomic analysis indicated that this is a nuclear single copy gene in plant cells. Northern blot with the cDNA probe revealed a major transcript which migrated at approx. 2,800 b, in agreement with the size of the cDNA isolated. The araXPB protein shares approximately 50% identical and 70% conserved amino acids with the yeast and human homologs. The plant protein maintains all the functional domains found in the other proteins, including nuclear localization signal, DNA-binding domain and helicase motifs, suggesting that it might also act as part of the RNA transcription apparatus, as well as nucleotide excision repair in plant cells.


Assuntos
Arabidopsis/genética , Arabidopsis/metabolismo , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Sequência de Aminoácidos , Animais , Clonagem Molecular , Sequência Conservada , DNA Helicases/química , DNA Complementar , DNA de Plantas/genética , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/química , Drosophila/genética , Genes de Plantas , Humanos , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Saccharomyces cerevisiae/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Xeroderma Pigmentoso/genética
2.
Free Radic Res ; 21(2): 75-83, 1994 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-7921166

RESUMO

To elucidate the mechanisms of mutagenesis by singlet oxygen DNA damage in mammalian cells, a SV40-derived single-stranded shuttle vector was exposed to the water soluble endoperoxide 3,3'-(1,4-naphthylidene) dipropionate (NDPO2). The damaged vector was transfected into monkey COS7 cells and the plasmid progeny exhibited up to 10 fold increase on the mutation frequency in the supF target gene, when compared to untreated vector. The sequence in the supF locus of such mutants revealed that singlet oxygen-induced mutagenesis in single-stranded vector is significantly different from spontaneous mutagenesis. Among the base substitutions, most of the mutations involved deoxyguanosines, being G to T transversions the predominant type of change. The data indicate that mutagenesis by singlet oxygen in mammalian cells may be generated by an error prone bypass of damaged deoxyguanosines at the template DNA.


Assuntos
Dano ao DNA/efeitos dos fármacos , Vetores Genéticos , Guanina , Mutagênese , Oxigênio/farmacologia , Timina , Animais , Linhagem Celular , Escherichia coli/genética , Haplorrinos , Plasmídeos , Oxigênio Singlete , Transfecção
3.
Nucleic Acids Res ; 20(16): 4319-23, 1992 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-1324479

RESUMO

In order to characterize the molecular nature of singlet oxygen (1O2) induced mutations in mammalian cells, a SV40-based shuttle vector (pi SVPC13) was treated with singlet oxygen arising from the thermal decomposition of the water-soluble endoperoxide of 3,3'-(1,4-naphthylidene) dipropionate (NDPO2). After the passage of damaged plasmid through monkey COS7 cells, the vector was shuffled into E. coli cells, allowing the screening of supF mutants. The mutation spectrum analysis shows that single and multiple base substitutions arose in 82.5% of the mutants, the others being rearrangements. The distribution of mutations within the supF gene is not random and some hotspots are evident. Most of the point mutations (98.4%) involve G:C base pairs and G:C to T:A transversion was the most frequent mutation (50.8%), followed by G:C to C:G transversion (32.8%). These results indicate that mutagenesis in mammalian cells, mediated by 1O2-induced DNA damage, is targeted selectively at guanine residues.


Assuntos
DNA Viral/efeitos dos fármacos , Oxigênio/toxicidade , Fotoquímica , RNA de Transferência/genética , Animais , Sequência de Bases , Linhagem Celular , Vetores Genéticos/genética , Haplorrinos , Dados de Sequência Molecular , Mutagênese/genética , Testes de Mutagenicidade , Plasmídeos/genética , Vírus 40 dos Símios/genética , Oxigênio Singlete
4.
Nucleic Acids Res ; 20(10): 2465-9, 1992 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-1375992

RESUMO

In vitro DNA synthesis on single stranded templates damaged by singlet oxygen was investigated in the supF tRNA gene sequence, using several DNA polymerases. Singlet oxygen was generated by the thermal decomposition of the water soluble with the endoperoxide of disodium 3,3'-(1,4-naphthylidene) dipropionate (NDPO2). The data demonstrated that damage at deoxyguanosine residues interrupts DNA polymerization. Modified T7 phage and Thermus aquaticus DNA polymerases were found to synthesize DNA fragments which terminated opposite deoxyguanosine, while T4 phage DNA polymerase and avian myeloblast virus reverse transcriptase were blocked one nucleotide 3' to deoxyguanosine positions on the template. DNA polymerase I (Klenow fragment) from Escherichia coli was inhibited at both positions, before and at the putative damaged sites. The blocking lesions, induced by 5 mM NDPO2, were estimated to be approximately 1.5 per 260 nucleotides, corresponding to 2% of deoxyguanosines. The distribution of lesions in the supF gene did not reveal any specific sequence context which showed distinct susceptibility to the attack of singlet oxygen.


Assuntos
Reparo do DNA/efeitos dos fármacos , DNA de Cadeia Simples/efeitos dos fármacos , Desoxiguanosina/metabolismo , Naftóis/farmacologia , Oxigênio/farmacologia , Proteínas de Bactérias/genética , Sequência de Bases , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Escherichia coli/genética , Dados de Sequência Molecular , Inibidores da Síntese de Ácido Nucleico , Fotoquímica , Plasmídeos/genética , RNA de Transferência/genética , DNA Polimerase Dirigida por RNA/metabolismo , Inibidores da Transcriptase Reversa , Oxigênio Singlete
5.
Photochem Photobiol ; 55(1): 39-45, 1992 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-1318549

RESUMO

The effects of singlet oxygen (1O2), generated by the thermal decomposition of water soluble NDPO2 (endoperoxide of the disodium 3,3'-(1,4-naphthylidene) dipropionate), on a single-stranded shuttle vector were analysed. 1O2 induces a much higher level of breaks in the phosphodiester backbone of single-stranded than double-stranded DNA. This may be due to a higher accessibility of guanine residue, primarily damaged by 1O2. The damaged vector was transfected into monkey COS7 cells where single-stranded DNA was converted to the double-stranded replicative form DNA. After 3 days, extrachromosomal DNA was extracted and the plasmids rescued in E. coli to study mutagenesis. There is a significant increase in mutation frequency of damaged single-stranded DNA in comparison to untreated DNA. It is concluded that 1O2 induces breaks in the backbone of single-stranded DNA and that the 1O2-damaged molecules are mutated after passage through mammalian cells.


Assuntos
Dano ao DNA , DNA de Cadeia Simples/efeitos da radiação , Oxigênio/farmacologia , Vírus 40 dos Símios/genética , Transfecção , Animais , Linhagem Celular , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , DNA de Cadeia Simples/genética , Escherichia coli/genética , Vetores Genéticos , Mutagênese , Fotoquímica , Oxigênio Singlete
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