RESUMO
Human GEN1 and yeast Yen1 are endonucleases with the ability to cleave Holliday junctions (HJs), which are proposed intermediates in recombination. In vivo, GEN1 and Yen1 function secondarily to Mus81, which has weak activity on intact HJs. We show that the genetic relationship is reversed in Drosophila, with Gen mutants having more severe defects than mus81 mutants. In vitro, DmGen, like HsGEN1, efficiently cleaves HJs, 5Î flaps, splayed arms, and replication fork structures. We find that the cleavage rates for 5Î flaps are significantly higher than those for HJs for both DmGen and HsGEN1, even in vast excess of enzyme over substrate. Kinetic studies suggest that the difference in cleavage rates results from a slow, rate-limiting conformational change prior to HJ cleavage: formation of a productive dimer on the HJ. Despite the stark difference in vivo that Drosophila uses Gen over Mus81 and humans use MUS81 over GEN1, we find the in vitro activities of DmGen and HsGEN1 to be strikingly similar. These findings suggest that simpler branched structures may be more important substrates for Gen orthologs in vivo, and highlight the utility of using the Drosophila model system to further understand these enzymes.
Assuntos
Dano ao DNA , Reparo do DNA , DNA Cruciforme/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Endonucleases/metabolismo , Resolvases de Junção Holliday/metabolismo , Animais , Sequência de Bases , Citoplasma/metabolismo , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Embrião não Mamífero/metabolismo , Humanos , Modelos Biológicos , Mutação/genética , Multimerização Proteica , Transporte Proteico , Proteínas de Schizosaccharomyces pombe/metabolismo , Especificidade por SubstratoAssuntos
Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Troca Genética , DNA Helicases/genética , DNA Helicases/metabolismo , DNA Cruciforme/genética , Proteínas de Ligação a DNA/genética , Desoxirribonucleases/genética , Endonucleases/genética , Meiose/genética , Recombinação Genética , Animais , HumanosRESUMO
The soybean aphid (Aphis glycines Matsumura) is an important soybean [Glycine max (L.) Merr.] pest in North America. The dominant aphid resistance gene Rag1 was previously mapped from the cultivar 'Dowling' to a 12 cM marker interval on soybean chromosome 7 (formerly linkage group M). The development of additional genetic markers mapping closer to Rag1 was needed to accurately position the gene to improve the effectiveness of marker-assisted selection (MAS) and to eventually clone it. The objectives of this study were to identify single nucleotide polymorphisms (SNPs) near Rag1 and to position these SNPs relative to Rag1. To generate a fine map of the Rag1 interval, 824 BC(4)F(2) and 1,000 BC(4)F(3) plants segregating for the gene were screened with markers flanking Rag1. Plants with recombination events close to the gene were tested with SNPs identified in previous studies along with new SNPs identified from the preliminary Williams 82 draft soybean genome shotgun sequence using direct re-sequencing and gene-scanning melt-curve analysis. Progeny of these recombinant plants were evaluated for aphid resistance. These efforts resulted in the mapping of Rag1 between the two SNP markers 46169.7 and 21A, which corresponds to a physical distance on the Williams 82 8x draft assembly (Glyma1.01) of 115 kilobase pair (kb). Several candidate genes for Rag1 are present within the 115-kb interval. The markers identified in this study that are closely linked to Rag1 will be a useful resource in MAS for this important aphid resistance gene.
Assuntos
Afídeos/fisiologia , Mapeamento Cromossômico , Genes RAG-1/genética , Glycine max , Imunidade Inata/genética , Doenças das Plantas , Animais , Produtos Agrícolas/genética , Produtos Agrícolas/imunologia , Produtos Agrícolas/parasitologia , Ligação Genética , Marcadores Genéticos , Imunidade Inata/imunologia , Doenças das Plantas/imunologia , Doenças das Plantas/parasitologia , Polimorfismo de Nucleotídeo Único , Recombinação Genética , Glycine max/genética , Glycine max/imunologia , Glycine max/parasitologiaRESUMO
The recognition of DNA double-strand breaks (DSBs) using a phospho-specific antibody to the histone 2A variant has become the gold standard assay for DNA damage detection. Here we report on the development of the first monoclonal antibody to the phospho-specific form of Drosophila H2AV and characterize the specificity of this antibody to programmed DSBs in oocytes and rereplication sites in endocycling cells by immunofluorescence assays and to DSBs resulting from irradiation in both cell culture and whole tissue by Western blot assays. These studies show that the antibody derived in the study is highly specific for this modification that occurs at DSB sites, and therefore will be a new useful tool within the Drosophila community for the study of DNA damage response, DSB repair, meiotic recombination and chemical agents that cause DNA damage.