Replication protein A is required for meiotic recombination in Saccharomyces cerevisiae.
In Saccharomyces cerevisiae, meiotic recombination is initiated by transient DNA double-stranded breaks (DSBs). These DSBs undergo a 5' --> 3' resection to produce 3' single-stranded DNA ends that serve to channel DSBs into the RAD52 recombinational repair pathway. In vitro studies strongly suggest that several proteins of this pathway--Rad51, Rad52, Rad54, Rad55, Rad57, and replication protein A (RPA)--play a role in the strand exchange reaction. Here, we report a study of the meiotic phenotypes conferred by two missense mutations affecting the largest subunit of RPA, which are localized in the protein interaction domain (rfa1-t11) and in the DNA-binding domain (rfa1-t48). We find that both mutant diploids exhibit reduced sporulation efficiency, very poor spore viability, and a 10- to 100-fold decrease in meiotic recombination. Physical analyses indicate that both mutants form normal levels of meiosis-specific DSBs and that the broken ends are processed into 3'-OH single-stranded tails, indicating that the RPA complex present in these rfa1 mutants is functional in the initial steps of meiotic recombination. However, the 5' ends of the broken fragments undergo extensive resection, similar to what is observed in rad51, rad52, rad55, and rad57 mutants, indicating that these RPA mutants are defective in the repair of the Spo11-dependent DSBs that initiate homologous recombination during meiosis.
Soustelle
Christine
C
Institut Curie, Section de recherche, CNRS UMR144, 75248 Paris Cedex 05, France.
Vedel
Michèle
M
Kolodner
Richard
R
Nicolas
Alain
A
D003434:Crossing Over, Genetic / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000502:physiology*; D005785:Gene Conversion; D008540:Meiosis / Q000502:physiology*; D009154:Mutation; D011995:Recombination, Genetic; D051716:Replication Protein A; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000502:physiology*; D013172:Spores, Fungal / Q000502:physiology
D004268:DNA-Binding Proteins; D051716:Replication Protein A; D029701:Saccharomyces cerevisiae Proteins