RESUMO
We have previously shown that recombination between 400-bp substrates containing only 4-bp differences, when present in an inverted repeat orientation, is suppressed by >20-fold in wild-type strains of S. cerevisiae. Among the genes involved in this suppression were three genes involved in mismatch repair--MSH2, MSH3, and MSH6--and one in nucleotide excision repair, RAD1. We now report the involvement of these genes in interchromosomal recombination occurring via crossovers using these same short substrates. In these experiments, recombination was stimulated by a double-strand break generated by the HO endonuclease and can occur between completely identical (homologous) substrates or between nonidentical (homeologous) substrates. In addition, a unique feature of this system is that recombining DNA strands can be given a choice of either type of substrate. We find that interchromosomal crossover recombination with these short substrates is severely inhibited in the absence of MSH2, MSH3, or RAD1 and is relatively insensitive to the presence of mismatches. We propose that crossover recombination with these short substrates requires the products of MSH2, MSH3, and RAD1 and that these proteins have functions in recombination in addition to the removal of terminal nonhomology. We further propose that the observed insensitivity to homeology is a result of the difference in recombinational mechanism and/or the timing of the observed recombination events. These results are in contrast with those obtained using longer substrates and may be particularly relevant to recombination events between the abundant short repeated sequences that characterize the genomes of higher eukaryotes.
Assuntos
Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Genes Fúngicos , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Pareamento Incorreto de Bases , Sequência de Bases , Cromossomos Fúngicos/genética , Troca Genética , Enzimas Reparadoras do DNA , DNA Fúngico/química , DNA Fúngico/genética , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Íntrons , Modelos Genéticos , Proteína 2 Homóloga a MutS/genética , Proteína 2 Homóloga a MutS/metabolismo , Proteína 3 Homóloga a MutS , Recombinação Genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade por SubstratoRESUMO
A TRP5-based reversion system that allows the rates of all possible base pair substitutions to be measured when the TRP5 locus is in both orientations relative to a defined origin of replication has been developed. This system should be useful for a wide variety of mutation and repair studies in yeast.