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/genéticaRESUMO
The THI4 gene of Saccharomyces cerevisiae encodes an enzyme of the thiamine biosynthetic pathway. The plant homolog thi1, from Arabidopsis thaliana, is also involved in thiamine biosynthesis; but was originally cloned due to its capacity to complement DNA repair deficient phenotypes in Escherichia coli. Here, the behavior of a thi4 disrupted strain was examined for increased sensitivity to treatment with the DNA damaging agents ultraviolet radiation (UV, 254 nm) and methyl methanesulfonate (MMS). Although the thi4 null mutant showed a similar level of survival as the wild-type strain, a higher frequency of respiratory mutants was induced by the two treatments. A similar phenotype was seen with wild-type strains expressing an antisense THI4 construct. Further analysis of respiratory mutants revealed that these were due to mutations of mitochondrial DNA (mtDNA) rather than nuclear DNA, consisting of rho-petite mutants. Moreover, the frequency of mutations was unaffected by the presence or absence of thiamine in the growth medium, and the defect leading to induction of petites in the thi4 mutant was corrected by expression of the Arabidopsis thi1 gene. Thus, Thi4 and its plant homolog appear to be dual functional proteins with roles in thiamine biosynthesis and mitochondrial DNA damage tolerance.
Assuntos
Proteínas de Arabidopsis , Dano ao DNA , Proteínas Fúngicas/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Tiamina/biossíntese , Arabidopsis/genética , Análise Mutacional de DNA , DNA Mitocondrial/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Corantes Fluorescentes , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/fisiologia , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Teste de Complementação Genética , Indóis/metabolismo , Metanossulfonato de Metila/farmacologia , Microscopia de Fluorescência , Mutagênese , Consumo de Oxigênio , Fenótipo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , RNA Antissenso/genética , RNA Antissenso/farmacologia , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Raios UltravioletaRESUMO
An Arabidopsis thaliana cDNA was isolated by complementation of the Escherichia coli mutant strain BW535 (xth, nfo, nth), which is defective in DNA base excision repair pathways. This cDNA partially complements the methyl methane sulfonate (MMS) sensitive phenotype of BW535. It also partially corrects the UV-sensitive phenotype of E. coli AB1886 (uvrA) and restores its ability to reactivate UV-irradiated lambda phage. It has an insert of ca. 1.3 kb with an open reading frame of 1047 bp (predicting a protein with a molecular mass of 36 kDa). This cDNA presents a high homology to a stress related gene from two species of Fusarium (sti35) and to genes whose products participate in the thiamine biosynthesis pathway, THI4, from Saccharomyces cerevisiae and nmt2 from Schizosaccharomyces pombe. The Arabidopsis predicted polypeptide has homology to several protein motifs: amino-terminal chloroplast transit peptide, dinucleotide binding site, DNA binding and bacterial DNA polymerases. The auxotrophy for thiamine in the yeast thi4::URA3 disruption strain is complemented by the Arabidopsis gene. Thus, the cloned gene, named thi1, is likely to function in the biosynthesis of thiamine in plants. The data presented in this work indicate that thi1 may also be involved in DNA damage tolerance in plant cells.