RESUMO
In budding yeast the DNA helicase Mph1 prevents genome rearrangements during ectopic homologous recombination (HR) by suppressing the formation of crossovers (COs). Here we show that during ectopic HR repair, the anti-CO function of Mph1 is intricately associated with the mismatch repair (MMR) factor, MutSalpha. In particular, during HR repair using a completely homologous substrate, we reveal an MMR-independent function of MutSalpha in generating COs that is specifically antagonized by Mph1, but not Sgs1. In contrast, both Mph1 and MutSalpha are required to efficiently suppress COs in the presence of a homeologous substrate. Mph1 acts redundantly with Sgs1 in this respect since mph1Delta sgs1Delta double mutant cells pheno-copy MutSalpha mutants and completely fail to discriminate homologous and homeologous sequences during HR repair. However, this defect of mph1Delta sgs1Delta cells is not due to an inability to carry out MMR but rather is accompanied by elevated levels of gene conversion (GC) and bi-directional GC tracts specifically in non-crossover products. Models describing how Mph1, MutSalpha and Sgs1 act in concert to suppress genome rearrangements during ectopic HR repair are discussed.
Assuntos
RNA Helicases DEAD-box/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Proteína 2 Homóloga a MutS/metabolismo , Recombinação Genética , Proteínas de Saccharomyces cerevisiae/metabolismo , RNA Helicases DEAD-box/genética , Reparo de Erro de Pareamento de DNA , Proteínas de Ligação a DNA/genética , Conversão Gênica , Proteína 2 Homóloga a MutS/genética , Mutação , RecQ Helicases/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
To date, the majority of studies of bacterial gene expression have been carried out on large communities, as techniques for analysis of expression in individual cells have not been available. Recent developments now allow us to use reporter genes to monitor gene expression in individual bacterial cells. Conventional reporters are not suitable for studies of living single cells. However, variants of GFP have proved to be ideal for the study of development, cell biology, and pathogenesis and are now the reporters of choice for microbial studies. In combination with techniques such as DFI and IVET and the use of flow cytometry and advanced fluorescence microscopy, the latest generation of GFP reporters allows the investigation of gene expression in individual bacterial cells within particular environments. These studies promise to bring a new level of understanding to the fields of bacterial pathogenesis and environmental microbiology.
