RESUMO
To test the DNA double-strand break (DSB) repair activities present in Drosophila early embryos, we have analyzed the circularization of a microinjected linear plasmid. In order to study repair by homologous recombination, the linear plasmid was injected with an homologous fragment encompassing the break. After extraction from embryos, repair products were analyzed directly by PCR and after their cloning into bacteria. We demonstrate, in addition to the repair by homologous recombination, the presence of an efficient end-joining activity in embryos. Plasmid circularization by end-joining was accompanied by short deletions frequently associated with non-random insertions. Most importantly, pre-irradiation of embryos specifically enhanced the accurate repair by homologous recombination. Such a stimulation is described for the first time in the context of a whole higher organism.
Assuntos
Reparo do DNA/genética , Reparo do DNA/efeitos da radiação , DNA/genética , Drosophila/embriologia , Embrião não Mamífero/efeitos da radiação , Raios gama , Animais , Sequência de Bases , DNA/administração & dosagem , DNA/metabolismo , DNA/efeitos da radiação , Análise Mutacional de DNA , DNA Circular/genética , DNA Circular/metabolismo , DNA Circular/efeitos da radiação , Drosophila/enzimologia , Drosophila/genética , Drosophila/efeitos da radiação , Embrião não Mamífero/enzimologia , Embrião não Mamífero/metabolismo , Microinjeções , Modelos Genéticos , Dados de Sequência Molecular , Mutação/genética , Mutação/efeitos da radiação , Plasmídeos/genética , Plasmídeos/metabolismo , Plasmídeos/efeitos da radiação , Reação em Cadeia da Polimerase , Recombinação Genética/genética , Recombinação Genética/efeitos da radiação , Homologia de Sequência do Ácido NucleicoRESUMO
We previously reported evidence that the so-called reactivity level, a peculiar cellular state of oocytes that regulates the frequency of transposition of I factor, a LINE element-like retrotransposon, might be one manifestation of a DNA repair system. In this article, we report data showing that the reactivity level is correlated with the frequency of crossing over, at least on the X chromosome and on the pericentromeric region of the third chromosome. Moreover, a check for X-chromosome losses and recessive lethals produced after gamma irradiation in flies with different reactivity levels, but common genetic backgrounds, brings more precise evidence for the relationship between reactivity levels and DNA repair. Those results support the existence of a repair-recombination system whose efficiency is modulated by endogenous and environmental factors. The implications of this biological system in connecting genomic variability and environment may shed new lights on adaptative mechanisms. We propose to call it VAMOS for variability modulation system.
