Spontaneous and double-strand break-induced recombination, and gene conversion tract lengths, are differentially affected by overexpression of wild-type or ATPase-defective yeast Rad54

Rad54 plays key roles in homologous recombination (HR) and double-strand break (DSB) repair in yeast, along with Rad51, Rad52, Rad55 and Rad57. Rad54 belongs to the Swi2/Snf2 family of DNA-stimulated ATPases. Rad51 nucleoprotein filaments catalyze DNA strand exchange and Rad54 augments this activity of Rad51. Mutations in the Rad54 ATPase domain (ATPase(–)) impair Rad54 function in vitro, sensitize yeast to killing by methylmethane sulfonate and reduce spontaneous gene conversion. We found that overexpression of ATPase(–) Rad54 reduced spontaneous direct repeat gene conversion and increased both spontaneous direct repeat deletion and spontaneous allelic conversion. Overexpression of ATPase(–) Rad54 decreased DSB-induced allelic conversion, but increased chromosome loss and DSB-dependent lethality. Thus, ATP hydrolysis by Rad54 contributes to genome stability by promoting high-fidelity DSB repair and suppressing spontaneous deletions. Overexpression of wild-type Rad54 did not alter DSB-induced HR levels, but conversion tract lengths were reduced. Interestingly, ATPase(–) Rad54 decreased overall HR levels and increased tract lengths. These tract length changes provide new in vivo evidence that Rad54 functions in the post-synaptic phase during recombinational repair of DSBs.