RESUMO
The Saccharomyces cerevisiae RAD54 gene has critical roles in DNA double-strand break repair, homologous recombination, and gene targeting. Previous results show that the yeast gene enhances gene targeting when expressed in Arabidopsis thaliana. In this work we address the trans-species compatibility of Rad54 functions. We show that overexpression of yeast RAD54 in Arabidopsis enhances DNA damage resistance severalfold. Thus, the yeast gene is active in the Arabidopsis homologous-recombination repair system. Moreover, we have identified an A. thaliana ortholog of yeast RAD54, named AtRAD54. This gene, with close sequence similarity to RAD54, complements methylmethane sulfonate (MMS) sensitivity but not UV sensitivity or gene targeting defects of rad54Delta mutant yeast cells. Overexpression of AtRAD54 in Arabidopsis leads to enhanced resistance to DNA damage. This gene's assignment as a RAD54 ortholog is further supported by the interaction of AtRad54 with AtRad51 and the interactions between alien proteins (i.e., yeast Rad54 with AtRAD51 and yeast Rad51 with AtRad54) in a yeast two-hybrid experiment. These interactions hint at the molecular nature of this interkingdom complementation, although the stronger effect of the yeast Rad54 in plants than AtRad54 in yeast might be explained by an ability of the Rad54 protein to act alone, independently of its interaction with Rad51.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Sequência Conservada , Proteínas de Ligação a DNA/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/química , DNA Helicases , Reparo do DNA/efeitos da radiação , Enzimas Reparadoras do DNA , DNA Complementar , Proteínas de Ligação a DNA/química , Raios gama , Genes Fúngicos , Genes de Plantas , Teste de Complementação Genética , Dados de Sequência Molecular , Mutação/genética , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/efeitos da radiação , Homologia de Sequência de Aminoácidos , Técnicas do Sistema de Duplo-HíbridoRESUMO
The genome of plants, like that of other eukaryotes, is organized into chromatin, a compact structure that reduces the accessibility of DNA to machineries such as transcription, replication, and DNA recombination and repair. Plant genes, which contain the characteristic ATPase/helicase motifs of the chromatin remodeling Swi2/Snf2 family of proteins, have been thoroughly studied, but their role in homologous recombination or DNA repair has received limited attention. We have searched for homologs of the yeast RAD54 gene, whose role in recombination and repair and in chromatin remodeling is well established. Forty Arabidopsis SWI2/SNF2 genes were identified and the function of a selected group of 14 was analyzed. Mutant analysis and/or RNAi-mediated silencing showed that 11 of the 14 genes tested played a role in response to DNA damage. Two of the 14 genes were involved in homologous recombination between inverted repeats. The putative ortholog of RAD54 and close homologs of ERCC6/RAD26 were involved in DNA damage response, suggesting functional conservation across kingdoms. In addition, genes known for their role in development, such as PICKLE/GYMNOS and PIE1, or in silencing, such as DDM1, turned out to also be involved in DNA damage response. A comparison of ddm1 and met1 mutants suggests that DNA damage response is affected essentially by chromatin structure and that cytosine methylation is less critical. These results emphasize the broad involvement of the SWI2/SNF2 family, and thus of chromatin remodeling, in genome maintenance and the link between epigenetic and genetic processes.
Assuntos
Arabidopsis/genética , Genes de Plantas , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/genética , Montagem e Desmontagem da Cromatina/genética , Sequência Conservada , Dano ao DNA , DNA de Plantas/genética , DNA de Plantas/efeitos da radiação , Proteínas de Ligação a DNA/genética , Família Multigênica , Mutação , Fenótipo , Filogenia , Interferência de RNA , Tolerância a Radiação/genética , Recombinação Genética , Fatores de Transcrição/genéticaRESUMO
Gene targeting, which is homologous recombination-mediated integration of an extra-chromosomal DNA segment into a chromosomal target sequence, enables the precise disruption or replacement of any gene. Despite its value as a molecular genetic tool, gene targeting remains an inefficient technology in most species. We report that expression of the yeast RAD54 gene, a member of the SWI2/SNF2 chromatin remodeling gene family, enhances gene targeting in Arabidopsis by one to two orders of magnitude, from 10(-4) to 10(-3) in WT plants to 10(-2) to 10(-1). We show that integration events, detected with an assay based on the use of a fluorescent seed marker, are precise and germinally transmitted. These findings suggest that chromatin remodeling is rate-limiting for gene targeting in plants and improves the prospects for using gene targeting for the precise modification of plant genomes.