Characterization of Mei5-Sae3, a mediator of the Saccharomyces cerevisiae recombinase Dmc1.

摘要

Homologous recombination, the exchange of genetic material between allelic sequences, is an essential process in the repair of damaged DNA and in the progression of meiosis. DNA strand exchange, the molecular process that forms the basis for homologous recombination, is catalyzed by recombinases, which promote strand exchange by forming helical filaments on single-stranded DNA. The proper regulation of recombinase activity by single-stranded DNA binding proteins, mediators, and other recombinase accessory factors is essential for genome integrity; dysfunction of regulation leading to either hyperrecombination or hyporecombination is potentially oncogenic. In this study, homologous recombination is studied in the context of yeast meiotic repair. During meiosis, double-strand DNA breaks are induced; the repair of these breaks through homologous recombination creates physical linkages between chromosomes that are essential for proper segregation during meiosis. In Saccharomyces cerevisiae, Dmc1, a homologue of the bacterial recombinase RecA, is a meiosis-specific recombinase whose activity is essential for homologous recombination during meiosis. Recently, the protein complex Mei5-Sae3 was identified as an accessory factor for Dmc1 and was shown to co-associate with Dmc1 on meiotic chromosomes. Mei5-Sae3 shares sequence homology with the Schizosaccharomyces pombe protein complex Sfr1-Swi5, which has been shown to stimulate the recombinational activity of Dmc1. In this study, Mei5-Sae3 has been purified and its ability to bind to DNA, as well as its preference for single-stranded DNA, have been described. Furthermore, the ability of Mei5-Sae3 to overcome the inhibition imposed on Dmc1 strand assimilation activity and DNA binding by RPA, the yeast single-stranded DNA binding protein, has been demonstrated, indicating that Mei5-Sae3 functions as a mediator for Dmc1. Finally, a direct interaction between Mei5-Sae3 and RPA has been demonstrated.;Additionally, an allele of Dmc1 (Dmc1-I282T) has been tested for its ability to act as a suppressor of the meiotic defects of mei5 yeast. While expression of DMC1-1282T failed to rescue the spore viability, meiotic progression, or meiotic repair defects of mei5 mutants, it did partially rescue Dmc1 focus formation.;The insights and implications of the biochemical activities and RPA interaction of Mei5-Sae3, as well as those of the partial rescue of focus formation by DMC1-1282T, are discussed.