ATP-dependent and ATP-independent Roles for the Rad54 Chromatin Remodeling Enzyme during Recombinational Repair of a DNA Double Strand Break

Abstract

The efficient and accurate repair of DNA double strand breaks (DSBs) is critical to cell survival, and defects in this process can lead to genome instability and cancers. In eukaryotes, the Rad52 group of proteins dictates the repair of DSBs by the error-free process of homologous recombination (HR). A critical step in eukaryotic HR is the formation of the initial Rad51-singlestranded DNA presynaptic nucleoprotein filament. This presynaptic filament participates in a homology search process that leads to the formation of a DNA joint molecule and recombinational repair of the DSB. Recently, we showed that the Rad54 protein functions as a mediator of Rad51 binding to single-stranded DNA, and here, we find that this activity does not require ATP hydrolysis. We also identify a novel Rad54-dependent chromatin remodeling event that occurs in vivo during the DNA strand invasion step of HR. This ATP-dependent remodeling activity of Rad54 appears to control subsequent steps in the HR process. Double strand breaks (DSBs)1 are a common form of DNA damage, resulting from a variety of environmental insults including ionizing radiation and chemical attack. The most common cause of DSBs is internal reactive oxygen species, accounting for thousands of breaks per cell per day. The inability to repair these breaks leads to genomic instability. Misrepair of these breaks can result in deletions, insertions, and translocations. In higher eukaryotes, improper DSB repair can lead to tumorigenesis. It is therefore of vital importance that cells repair these breaks accurately and faithfully.Several pathways have evolved for the repair of DSBs. In yeast, the predominant pathway is thought to be homologous recombination (1, 2). Homologous recombination (HR) provides an "error-free" method for repairing DSBs as it utilizes homologous DNA sequences as a template to repair the lesion with no loss of genetic information. The genes in the RAD52 epistasis group, required for HR, are highly conserved from yeast to man (2-4), highlighting the importance of this molecular pathway.Studies in yeast have suggested a sequence of molecular events that occur following the formation of a DSB (2, 4, 5). Nucleolytic end processing leads to resection of the 5Ј ends of DNA that flank the break, generating long stretches of 3Ј single-stranded DNA. Rad51p binds the single-stranded DNA, forming right-handed helical nucleoprotein filaments. In vitro, Rad52p (6), Rad54p (7), and a Rad55p/Rad57p heterodimer (8) mediate this early step by overcoming the inhibitory effects of the heterotrimeric single-stranded DNA-binding protein, RPA.In vivo, these same Rad proteins are also required for optimal recruitment of Rad51p to DNA surrounding a single DSB in yeast (7, 9). The Rad51-nucleoprotein filament is believed to search the genome for homologous sequences, resulting in the formation of a heteroduplex "joint molecule" (10). Joint molecule formation is followed by extension of the incoming strand by DNA polymerases and branch migration. The end result is the repair of...

Synergistic action of the Saccharomyces cerevisiae homologous recombination factors Rad54 and Rad51 in chromatin remodeling

“…Interestingly, Rad54 dissociates Rad51 from duplex DNA via its DNA translocase activity, a function believed to be important for the intracellular recycling of Rad51 and possibly for freeing the primer end in the D-loop structure to initiate the DNA synthesis reaction [30]. Rad54 facilitates the assembly and maintenance of the Rad51 presynaptic filament as well [31,32], and the interactions of Rad54 and Rad51 are germane for their mutual targeting to a site-specific DSB in cells [31,33,34]. The Rad51 presynaptic filament stabilization role of Rad54 does not require its ATPase activity [32,34].…”

“…Rad54 facilitates the assembly and maintenance of the Rad51 presynaptic filament as well [31,32], and the interactions of Rad54 and Rad51 are germane for their mutual targeting to a site-specific DSB in cells [31,33,34]. The Rad51 presynaptic filament stabilization role of Rad54 does not require its ATPase activity [32,34]. Thus, Rad54 plays a major role in regulating the activities of Rad51.…”

Chromatin dynamics coupled to DNA repair

“…113 In an early stage of HR, Rad54 helps to recruit Rad51 to recipient DNA and to stabilize Rad51 presynaptic filaments, irrespective of its ATPase activity. 112 Rad51 stimulates Rad54 to remodel nucleosomes in vitro and in vivo in an ATPase-dependent manner. 112 response.…”

“…112 Rad51 stimulates Rad54 to remodel nucleosomes in vitro and in vivo in an ATPase-dependent manner. 112 response. 79,94,128 HDACs act late on in the DNA-repair process reducing the amount of histone acetylation once repair has been completed.…”

“…108 Rad54 is a member of the Swi2/Snf2 family that binds singlestranded DNA and facilitates the chromatin remodeling that occurs during the strand invasion of recipient DNA to the intact homologous chromosome or sister chromatid. 112 This protein acts as a key mediator during early and late stages of HR repair. 113 In an early stage of HR, Rad54 helps to recruit Rad51 to recipient DNA and to stabilize Rad51 presynaptic filaments, irrespective of its ATPase activity.…”