RESUMEN
BACKGROUND: Efficient mechanisms for rejoining of DNA double-strand breaks (DSBs) are vital because misrepair of such lesions leads to mutation, aneuploidy and loss of cell viability. DSB repair is mediated by proteins acting in two major pathways, called homologous recombination and nonhomologous end-joining. Repair efficiency is also modulated by other processes such as sister chromatid cohesion, nucleosome remodeling and DNA damage checkpoints. The total number of genes influencing DSB repair efficiency is unknown. RESULTS: To identify new yeast genes affecting DSB repair, genes linked to gamma radiation resistance in previous genome-wide surveys were tested for their impact on repair of site-specific DSBs generated by in vivo expression of EcoRI endonuclease. Eight members of the RAD52 group of DNA repair genes (RAD50, RAD51, RAD52, RAD54, RAD55, RAD57, MRE11 and XRS2) and 73 additional genes were found to be required for efficient repair of EcoRI-induced DSBs in screens utilizing both MATa and MATα deletion strain libraries. Most mutants were also sensitive to the clastogenic chemicals MMS and bleomycin. Several of the non-RAD52 group genes have previously been linked to DNA repair and over half of the genes affect nuclear processes. Many proteins encoded by the protective genes have previously been shown to associate physically with each other and with known DNA repair proteins in high-throughput proteomics studies. A majority of the proteins (64%) share sequence similarity with human proteins, suggesting that they serve similar functions. CONCLUSIONS: We have used a genetic screening approach to detect new genes required for efficient repair of DSBs in Saccharomyces cerevisiae. The findings have spotlighted new genes that are critical for maintenance of genome integrity and are therefore of greatest concern for their potential impact when the corresponding gene orthologs and homologs are inactivated or polymorphic in human cells.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN/genética , Genómica , Saccharomyces cerevisiae/genética , Animales , Antineoplásicos/farmacología , Bleomicina/farmacología , Roturas del ADN de Doble Cadena/efectos de los fármacos , Roturas del ADN de Doble Cadena/efectos de la radiación , Reparación del ADN/efectos de los fármacos , Reparación del ADN/efectos de la radiación , Rayos gamma , Genes de Plantas/genética , Humanos , Metilmetanosulfonato/farmacología , Ratones , Ratas , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/efectos de la radiaciónRESUMEN
Chemical-based methods have been developed for transformation of DNA into log-phase cells of the budding yeast Saccharomyces cerevisiae with high efficiency. Transformation of early stationary-phase cells, e.g. cells grown in overnight liquid cultures or as colonies on plates, is less efficient than log-phase cells but is simpler and more adaptable to high-throughput projects. In this study we have tested different approaches for transformation of early stationary-phase cell cultures and identified a method utilizing polyethylene glycol (PEG), lithium acetate and dimethyl sulphoxide (DMSO) as the most efficient. Plasmid DNA transformations using this method could be improved modestly by allowing cells to recover from the chemical treatment in rich broth before plating to selective media. Strong increases in transformation efficiencies were observed when cells were treated briefly with dithiothreitol (DTT). Tests using several different yeast strain backgrounds indicated that DTT treatment could enhance transformation efficiencies by up to 40-fold. Evaluation of multiple parameters affecting the efficiency of the method led to development of an optimized protocol achieving > 50 000 transformants/µg DNA in most backgrounds tested.