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 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.