Assembly of RecA-like recombinases: distinct roles for mediator proteins in mitosis and meiosis.

Members of the RecA family of recombinases from bacteriophage T4, Escherichia coli, yeast, and higher eukaryotes function in recombination as higher-order oligomers assembled on tracts of single-strand DNA (ssDNA). Biochemical studies have shown that assembly of recombinase involves accessory factors. These studies have identified a class of proteins, called recombination mediator proteins, that act by promoting assembly of recombinase on ssDNA tracts that are bound by ssDNA-binding protein (ssb). In the absence of mediators, ssb inhibits recombination reactions by competing with recombinase for DNA-binding sites. Here we briefly review mediated recombinase assembly and present results of new in vivo experiments. Immuno-double-staining experiments in Saccharomyces cerevisiae suggest that Rad51, the eukaryotic recombinase, can assemble at or near sites containing ssb (replication protein A, RPA) during the response to DNA damage, consistent with a need for mediator activity. Correspondingly, mediator gene mutants display defects in Rad51 assembly after DNA damage and during meiosis, although the requirements for assembly are distinct in the two cases. In meiosis, both Rad52 and Rad55/57 are required, whereas either Rad52 or Rad55/57 is sufficient to promote assembly of Rad51 in irradiated mitotic cells. Rad52 promotes normal amounts of Rad51 assembly in the absence of Rad55 at 30 degrees C but not 20 degrees C, accounting for the cold sensitivity of rad55 null mutants. Finally, we show that assembly of Rad51 is induced by radiation during S phase but not during G(1), consistent with the role of Rad51 in repairing the spontaneous damage that occurs during DNA replication.

Rad52 associates with RPA and functions with rad55 and rad57 to assemble meiotic recombination complexes.

We show that the Saccharomyces cerevisiae recombination protein Rad52 and the single-strand DNA-binding protein RPA assemble into cytologically detectable subnuclear complexes (foci) during meiotic recombination. Immunostaining shows extensive colocalization of Rad52 and RPA and more limited colocalization of Rad52 with the strand exchange protein Rad51. Rad52 and RPA foci are distinct from those formed by Rad51, and its meiosis-specific relative Dmc1, in that they are also detected in meiosis during replication. In addition, RPA foci are observed during mitotic S phase. Double-strand breaks (DSBs) promote formation of RPA, Rad52, and Rad51 foci. Mutants that lack Spo11, a protein required for DSB formation, are defective in focus formation, and this defect is suppressed by ionizing radiation in a dose-dependent manner. DSBs are not sufficient for the appearance of Rad51 foci; Rad52, Rad55, and Rad57 are also required supporting a model in which these three proteins promote meiotic recombination by promoting the assembly of strand exchange complexes.