Abstract
The Saccharomyces cerevisiae Rdh54 protein is a member of the Swi2/Snf2 family of DNA translocases required for meiotic and mitotic recombination and DNA repair. Rdh54 interacts with the general recombinases Rad51 and Dmc1 and promotes D-loop formation with either recombinase. Rdh54 also mediates the removal of Rad51 from undamaged chromatin in mitotic cells, which prevents formation of nonrecombinogenic complexes that can otherwise become toxic for cell growth. To determine which of the mitotic roles of Rdh54 are dependent on Rad51 complex formation, we finely mapped the Rad51 interaction domain in Rdh54, generated N-terminal truncation variants, and characterized their attributes biochemically and in cells. Here, we provide evidence suggesting that the N-terminal region of Rdh54 is not necessary for the response to the DNA-damaging agent methyl methanesulfonate. However, truncation variants missing 75-200 residues at the N terminus are sensitive to Rad51 overexpression. Interestingly, a hybrid protein containing the N-terminal region of Rad54, responsible for Rad51 interaction, fused to the Swi2/Snf2 core of Rdh54 is able to effectively complement the sensitivity to both methyl methanesulfonate and excess Rad51 in rdh54 null cells. Altogether, these results reveal a distinction between damage sensitivity and Rad51 removal with regard to Rdh54 interaction with Rad51.
Highlights
Rdh54 is a motor protein that is able to dissociate Rad51 recombinase from chromatin
We provide evidence that the N-terminal region of Rdh54 is required to overcome the toxic effect of Rad51 overexpression but is dispensable for the repair of DNA damaged by methyl methanesulfonate (MMS)
Generation of rdh54 N-terminal Truncation Mutant Constructs—Biochemical studies have shown that Rdh54 interacts with the Rad51 recombinase and that the binding domain resides within the N terminus (Fig. 1) [15]
Summary
Rdh is a motor protein that is able to dissociate Rad recombinase from chromatin. Rad and Rdh, members of the Swi2/ Snf family of DNA translocases, have been shown to enhance the efficiency of Rad51-mediated D-loop formation These translocases are capable of displacing Rad from dsDNA, an attribute that is likely important for clearing Rad from the nascent D-loop to facilitate repair DNA synthesis and for preventing the nonspecific accumulation of Rad on bulk chromatin [5, 6]. In vitro studies have shown that the Rad51-binding domain of Rdh resides within its N terminus and suggested that complex formation is required for promoting efficient D-loop reaction, for its chromatin-remodeling activity, and for Rad removal from dsDNA [8, 15].
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