Specific negative effects resulting from elevated levels of the recombinational repair protein Rad54p in Saccharomyces cerevisiae.

Abstract

RAD54 is an important gene in the RAD52 group that controls recombinational repair of DNA damage in Saccharomyces cerevisiae. Rad54p is a DNA-dependent ATPase and shares seven conserved sequence motifs with proteins of the Swi2p/Snf2p family. Genetic analysis of mutations in motif IA, the putative ATP-binding fold of Rad54p, demonstrated the functional importance of this motif. Overexpression of these mutant proteins resulted in strong, dominant-negative effects on cell survival. High levels of full-length wild-type Rad54p or specific parts of Rad54p also resulted in negative effects, dependent on the ploidy of the host cell. This differential effect was not under a/alpha mating-type control. Deletion of the RAD54 gene led to a small but significant increase in the mutation rate. However, the negative overexpression effects in haploid cells could not be explained by an accumulation of (recessive) lethal mutations. All negative overexpression effects were found to be enhanced under genotoxic stress. We suggest that the negative overexpression effects are the result of unbalanced protein-protein interactions, indicating that Rad54p is involved in multiple interactions, dependent on the physiological situation. Diploid wild-type cells contained an estimated 7000 Rad54p molecules/cell, whereas haploid cells about 3500/cell. Rad54p levels were highest in actively growing cells compared to stationary phase cells. Rad54 protein levels were found to be elevated after DNA damage.