ATP-dependent and ATP-independent roles for the Rad54 chromatin remodeling enzyme during recombinational repair of a DNA double strand break

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-single-stranded 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.     

Functions of the Snf2/Swi2 family Rad54 motor protein in homologous recombination

Homologous recombination is a central pathway to maintain genomic stability and is involved in the repair of DNA damage and replication fork support, as well as accurate chromosome segregation during meiosis. Rad54 is a dsDNA-dependent ATPase of the Snf2/Swi2 family of SF2 helicases, although Rad54 lacks classical helicase activity and cannot carry out the strand displacement reactions typical for DNA helicases. Rad54 is a potent and processive motor protein that translocates on dsDNA, potentially executing several functions in recombinational DNA repair. Rad54 acts in concert with Rad51, the central protein of recombination that performs the key reactions of homology search and DNA strand invasion. Here, we will review the role of the Rad54 protein in homologous recombination with an emphasis on mechanistic studies with the yeast and human enzymes. We will discuss how these results relate to in vivo functions of Rad54 during homologous recombination in somatic cells and during meiosis. This article is part of a Special Issue entitled: Snf2/Swi2 ATPase structure and function.     

Tailed duplex DNA is the preferred substrate for Rad51 protein-mediated homologous pairing

The repair of potentially lethal DNA double-stranded breaks (DSBs) by homologous recombination requires processing of the broken DNA into a resected DNA duplex with a protruding 3'-single-stranded DNA (ssDNA) tail. Accordingly, the canonical models for DSB repair require invasion of an intact homologous DNA template by the 3'-end of the ssDNA, a characteristic that the bacterial pairing protein RecA possesses. Unexpectedly, we find that for the eukaryotic homolog, Rad51 protein, the 5'-end of ssDNA is more invasive than the 3'-end. This pairing bias is unaffected by Rad52, Rad54 or Rad55-57 proteins. However, further investigation reveals that, in contrast to RecA protein, the preferred DNA substrate for Rad51 protein is not ssDNA but rather dsDNA with ssDNA tails. This important distinction permits the Rad51 proteins to promote DNA strand invasion using either 3'- or 5'-ends with similar efficiency.     

Rad54 protein is targeted to pairing loci by the Rad51 nucleoprotein filament

Rad51 and Rad54 proteins are important for the repair of double-stranded DNA (dsDNA) breaks by homologous recombination in eukaryotes. Rad51 assembles on single-stranded DNA (ssDNA) to form a helical nucleoprotein filament that performs homologous pairing with dsDNA; Rad54 stimulates this pairing substantially. Here, we demonstrate that Rad54 acts in concert with the mature Rad51-ssDNA filament. Enhancement of DNA pairing by Rad54 is greatest at an equimolar ratio relative to Rad51 within the filament. Reciprocally, the Rad51-ssDNA filament enhances both the dsDNA-dependent ATPase and the dsDNA unwinding activities of Rad54. We conclude that Rad54 participates in the DNA homology search as a component of the Rad51-nucleoprotein filament and that the filament delivers Rad54 to the dsDNA pairing locus, thereby linking the unwinding of potential target DNA with the homology search process.     

Analysis of mouse Rad54 expression and its implications for homologous recombination

Homologous recombination is one of the major pathways for repair of DNA double-strand breaks (DSBs). Important proteins in this pathway are Rad51 and Rad54. Rad51 forms a nucleoprotein filament on single-stranded DNA (ssDNA) that mediates pairing with and strand invasion of homologous duplex DNA with the assist of Rad54. We estimated that the nucleus of a mouse embryonic stem (ES) cells contains on average 4.7x10(5) Rad51 and 2.4x10(5) Rad54 molecules. Furthermore, we showed that the amount of Rad54 was subject to cell cycle regulation. We discuss our results with respect to two models that describe how Rad54 stimulates Rad51-mediated DNA strand invasion. The models differ in whether Rad54 functions locally or globally. In the first model, Rad54 acts in cis relative to the site of strand invasion. Rad54 coats the Rad51 nucleoprotein filament in stoichiometric amounts and binds to the target duplex DNA at the site that is homologous to the ssDNA in the Rad51 nucleoprotein filament. Subsequently, it promotes duplex DNA unwinding. In the second model, Rad54 acts in trans relative to the site of strand invasion. Rad54 binds duplex DNA distant from the site that will be unwound. Translocation of Rad54 along the duplex DNA increases superhelical stress thereby promoting duplex DNA unwinding.     

Rad54: the Swiss Army knife of homologous recombination?

Homologous recombination (HR) is a ubiquitous cellular pathway that mediates transfer of genetic information between homologous or near homologous (homeologous) DNA sequences. During meiosis it ensures proper chromosome segregation in the first division. Moreover, HR is critical for the tolerance and repair of DNA damage, as well as in the recovery of stalled and broken replication forks. Together these functions preserve genomic stability and assure high fidelity transmission of the genetic material in the mitotic and meiotic cell divisions. This review will focus on the Rad54 protein, a member of the Snf2-family of SF2 helicases, which translocates on dsDNA but does not display strand displacement activity typical for a helicase. A wealth of genetic, cytological, biochemical and structural data suggests that Rad54 is a core factor of HR, possibly acting at multiple stages during HR in concert with the central homologous pairing protein Rad51.     

Rad54 dissociates homologous recombination intermediates by branch migration

Double-strand DNA breaks (DSBs) cause cell death and genome instability. Homologous recombination is a major DSB repair pathway that operates by forming joint molecules with homologous DNA sequences, which are used as templates to achieve accurate repair. In eukaryotes, Rad51 protein (RecA homolog) searches for homologous sequences and catalyzes the formation of joint molecules (D-loops). Once joint molecules have been formed, DNA polymerase extends the 3' single-stranded DNA tails of the broken chromosome, restoring the lost information. How joint molecules subsequently dissociate is unknown. We reconstituted DSB repair in vitro using purified human homologous recombination proteins and DNA polymerase eta. We found that Rad54 protein, owing to its ATP-dependent branch-migration activity, can cause dissociation of joint molecules. These results suggest a previously uncharacterized mechanism of DSB repair in which Rad54 branch-migration activity plays an important role.     

A novel function of Rad54 protein. Stabilization of the Rad51 nucleoprotein filament

Homologous recombination is important for the repair of double-stranded DNA breaks in all organisms. Rad51 and Rad54 proteins are two key components of the homologous recombination machinery in eukaryotes. In vitro, Rad51 protein assembles with single-stranded DNA to form the helical nucleoprotein filament that promotes DNA strand exchange, a basic step of homologous recombination. Rad54 protein interacts with this Rad51 nucleoprotein filament and stimulates its DNA pairing activity, suggesting that Rad54 protein is a component of the nucleoprotein complex involved in the DNA homology search. Here, using physical criteria, we demonstrate directly the formation of Rad54-Rad51-DNA nucleoprotein co-complexes that contain equimolar amounts of each protein. The binding of Rad54 protein significantly stabilizes the Rad51 nucleoprotein filament formed on either single-stranded DNA or double-stranded DNA. The Rad54-stabilized nucleoprotein filament is more competent in DNA strand exchange and acts over a broader range of solution conditions. Thus, the co-assembly of an interacting partner with the Rad51 nucleoprotein filament represents a novel means of stabilizing the biochemical entity central to homologous recombination, and reveals a new function of Rad54 protein.     

Xrcc3 is recruited to DNA double strand breaks early and independent of Rad51

Rad51-mediated homologous recombination (HR) is essential for maintenance of genome integrity. The Xrcc3 protein functions in HR DNA repair, and studies suggest it has multiple roles at different stages in this pathway. Defects in vertebrate XRCC3 result in elevated levels of spontaneous and DNA damage-induced chromosomal abnormalities, as well as increased sensitivity to DNA damaging agents. Formation of DNA damaged-induced nuclear Rad51 foci requires Xrcc3 and the other Rad51 paralog proteins (Rad51B, Rad51C, Rad51D, Xrcc2), thus supporting a model in which an early function of Xrcc3 involves promoting assembly of active Rad51 repair complexes. However, it is not known whether Xrcc3 or other Rad51 paralog proteins accumulate at DNA breaks, and if they do whether their stable association with breaks requires Rad51. Here we report for the first time that Xrcc3 forms distinct foci in human cells and that nuclear Xrcc3 begins to localize at sites of DNA damage within 10 min after radiation treatment. RNAi-mediated knock down of Rad51 has no effect on the DNA damage-induced localization of Xrcc3 to DNA breaks. Our data are consistent with a model in which Xrcc3 associates directly with DNA breaks independent of Rad51, and subsequently facilitates formation of the Rad51 nucleoprotein filament.     

ATP-dependent and independent functions of Rad54 in genome maintenance

Rad54, a member of the SWI/SNF protein family of DNA-dependent ATPases, repairs DNA double-strand breaks (DSBs) through homologous recombination. Here we demonstrate that Rad54 is required for the timely accumulation of the homologous recombination proteins Rad51 and Brca2 at DSBs. Because replication protein A and Nbs1 accumulation is not affected by Rad54 depletion, Rad54 is downstream of DSB resection. Rad54-mediated Rad51 accumulation does not require Rad54's ATPase activity. Thus, our experiments demonstrate that SWI/SNF proteins may have functions independent of their ATPase activity. However, quantitative real-time analysis of Rad54 focus formation indicates that Rad54's ATPase activity is required for the disassociation of Rad54 from DNA and Rad54 turnover at DSBs. Although the non-DNA-bound fraction of Rad54 reversibly interacts with a focus, independent of its ATPase status, the DNA-bound fraction is immobilized in the absence of ATP hydrolysis by Rad54. Finally, we show that ATP hydrolysis by Rad54 is required for the redistribution of DSB repair sites within the nucleus.     

Rad52 and Rad59 exhibit both overlapping and distinct functions

Homologous recombination is an important pathway for the repair of DNA double-strand breaks (DSBs). In the yeast Saccharomyces cerevisiae, Rad52 is a central recombination protein, whereas its paralogue, Rad59, plays a more subtle role in homologous recombination. Both proteins can mediate annealing of complementary single-stranded DNA in vitro, but only Rad52 interacts with replication protein A and the Rad51 recombinase. We have studied the functional overlap between Rad52 and Rad59 in living cells using chimeras of the two proteins and site-directed mutagenesis. We find that Rad52 and Rad59 have both overlapping as well as separate functions in DSB repair. Importantly, the N-terminus of Rad52 possesses functions not supplied by Rad59, which may account for its central role in homologous recombination.     

Mouse Rad54 affects DNA conformation and DNA-damage-induced Rad51 foci formation

Error-free repair by homologous recombination of DNA double-strand breaks induced by ionizing radiation (IR) requires the Rad52 group proteins, including Rad51 and Rad54, in the yeast Saccharomyces cerevisiae [1]. The formation of a 'joint' molecule between the damaged DNA and the homologous repair template is a key step in recombination mediated by Rad51 and stimulated by Rad54 [2] [3] [4] [5]. Mammalian homologs of Rad51 and Rad54 have been identified [2] [3] [6]. Here, we demonstrate that mouse Rad54 (mRad54) formed IR-induced nuclear foci that colocalized with mRad51. Interaction between mRad51 and mRad54 was induced by genotoxic stress, but only when lesions that required mRad54 for their repair were formed. Interestingly, mRad54 was essential for the formation of IR-induced mRad51 foci. Rad54 belongs to the SWI2/SNF2 protein family, members of which modulate protein-DNA interactions in an ATP-driven manner [7]. Results of a topological assay suggested that purified human Rad54 (hRad54) protein can unwind double-stranded (ds) DNA at the expense of ATP hydrolysis. Unwinding of the homologous repair template could promote the formation or stabilization of hRad51-mediated joint molecules. Rad54 appears to be required downstream of other Rad52 group proteins, such as Rad52 and the Rad55-Rad57 heterodimer, that assist Rad51 in interacting with the broken DNA [2] [3] [4].     

Rad54 Phosphorylation Promotes Homologous Recombination by Balancing Rad54 Mobility and DNA Binding

The repair of DNA double-strand breaks by homologous recombination is of crucial importance for maintaining genomic stability. Two major players during this repair pathway are Rad51 and Rad54. Previously, it was shown that Rad54 exists as a monomer or oligomer when bound to DNA and drives the displacement of Rad51 by translocating along the DNA. Moreover, phosphorylation of Rad54 was reported to stimulate this clearance of Rad51 from DNA. However, it is currently unclear how phosphorylation of Rad54 modulates its molecular-structural function and how it affects the activity of monomeric or oligomeric Rad54 during the removal of Rad51. To examine the impact of Rad54 phosphorylation on a molecular-structural level, we applied molecular dynamics simulations of Rad54 monomers and hexamers in the absence or presence of DNA. Our results suggest that 1) phosphorylation of Rad54 stabilizes the monomeric form by reducing the interlobe movement of Rad54 monomers and might therefore facilitate multimer formation around DNA and 2) phosphorylation of Rad54 in a higher-order hexamer reduces its binding strength to DNA, which is a requirement for efficient mobility on DNA. To further address the relationship between the mobility of Rad54 and its phosphorylation state, we performed fluorescence recovery after photobleaching experiments in living cells, which expressed different versions of the Rad54 protein. Here, we could measure that the phosphomimetic version of Rad54 was highly mobile on DNA, whereas a nonphosphorylatable mutant displayed a mobility defect. Taken together, these data show that the phosphorylation of Rad54 is a critical event in balancing the DNA binding strength and mobility of Rad54 and might therefore provide optimal conditions for DNA translocation and subsequent removal of Rad51 during homologous recombination repair.     

Rad54 protein possesses chromatin-remodeling activity stimulated by the Rad51-ssDNA nucleoprotein filament

In Saccharomyces cerevisiae, the Rad54 protein participates in the recombinational repair of double-strand DNA breaks together with the Rad51, Rad52, Rad55 and Rad57 proteins. In vitro, Rad54 interacts with Rad51 and stimulates DNA strand exchange promoted by Rad51 protein. Rad54 is a SWI2/SNF2-related protein that possesses double-stranded DNA-dependent ATPase activity and changes DNA topology in an ATP hydrolysis-dependent manner. Here we show that Rad54 catalyzes bidirectional nucleosome redistribution by sliding nucleosomes along DNA. Nucleosome redistribution is greatly stimulated by the Rad51 nucleoprotein filament but does not require the presence of homologous single-stranded DNA within the filament. On the basis of these data, we propose that Rad54 facilitates chromatin remodeling and, perhaps more generally, protein clearing at the homology search step of genetic recombination.     

Recombinational repair in yeast: functional interactions between Rad51 and Rad54 proteins.

Rad51p is a eukaryotic homolog of RecA, the central homologous pairing and strand exchange protein in Escherichia coli. Rad54p belongs to the Swi2p/Snf2p family of DNA-stimulated ATPases. Both proteins are also important members of the RAD52 group which controls recombinational DNA damage repair of double-strand breaks and other DNA lesions in Saccharomyces cerevisiae. Here we demonstrate by genetic, molecular and biochemical criteria that Rad51 and Rad54 proteins interact. Strikingly, overexpression of Rad54p can functionally suppress the UV and methyl methanesulfonate sensitivity caused by a deletion of the RAD51 gene. However, no suppression was observed for the defects of rad51 cells in the repair of gamma-ray-induced DNA damage, mating type switching or spontaneous hetero-allelic recombination. This suppression is genetically dependent on the presence of two other members of the recombinational repair group, RAD55 and RAD57. Our data provide compelling evidence that Rad51 and Rad54 proteins interact in vivo and that this interaction is functionally important for recombinational DNA damage repair. As both proteins are conserved throughout evolution from yeasts to humans, a similar protein-protein interaction may be expected in other organisms.     

The chromatin remodeler p400 ATPase facilitates Rad51-mediated repair of DNA double-strand breaks

DNA damage signaling and repair take place in a chromatin context. Consequently, chromatin-modifying enzymes, including adenosine triphosphate–dependent chromatin remodeling enzymes, play an important role in the management of DNA double-strand breaks (DSBs). Here, we show that the p400 ATPase is required for DNA repair by homologous recombination (HR). Indeed, although p400 is not required for DNA damage signaling, DNA DSB repair is defective in the absence of p400. We demonstrate that p400 is important for HR-dependent processes, such as recruitment of Rad51 to DSB (a key component of HR), homology-directed repair, and survival after DNA damage. Strikingly, p400 and Rad51 are present in the same complex and both favor chromatin remodeling around DSBs. Altogether, our data provide a direct molecular link between Rad51 and a chromatin remodeling enzyme involved in chromatin decompaction around DNA DSBs.     

Analyses of the yeast Rad51 recombinase A265V mutant reveal different in vivo roles of Swi2-like factors

The Saccharomyces cerevisiae Swi2-like factors Rad54 and Rdh54 play multifaceted roles in homologous recombination via their DNA translocase activity. Aside from promoting Rad51-mediated DNA strand invasion of a partner chromatid, Rad54 and Rdh54 can remove Rad51 from duplex DNA for intracellular recycling. Although the in vitro properties of the two proteins are similar, differences between the phenotypes of the null allele mutants suggest that they play different roles in vivo. Through the isolation of a novel RAD51 allele encoding a protein with reduced affinity for DNA, we provide evidence that Rad54 and Rdh54 have different in vivo interactions with Rad51. The mutant Rad51 forms a complex on duplex DNA that is more susceptible to dissociation by Rdh54. This Rad51 variant distinguishes the in vivo functions of Rad54 and Rdh54, leading to the conclusion that two translocases remove Rad51 from different substrates in vivo. Additionally, we show that a third Swi2-like factor, Uls1, contributes toward Rad51 clearance from chromatin in the absence of Rad54 and Rdh54, and define a hierarchy of action of the Swi2-like translocases for chromosome damage repair.     

Rad54p is a chromatin remodeling enzyme required for heteroduplex DNA joint formation with chromatin

In eukaryotic cells, the repair of DNA double-strand breaks by homologous recombination requires a RecA-like recombinase, Rad51p, and a Swi2p/Snf2p-like ATPase, Rad54p. Here we find that yeast Rad51p and Rad54p support robust homologous pairing between single-stranded DNA and a chromatin donor. In contrast, bacterial RecA is incapable of catalyzing homologous pairing with a chromatin donor. We also show that Rad54p possesses many of the biochemical properties of bona fide ATP-dependent chromatin-remodeling enzymes, such as ySWI/SNF. Rad54p can enhance the accessibility of DNA within nucleosomal arrays, but it does not seem to disrupt nucleosome positioning. Taken together, our results indicate that Rad54p is a chromatin-remodeling enzyme that promotes homologous DNA pairing events within the context of chromatin.     

INO80-dependent chromatin remodeling regulates early and late stages of mitotic homologous recombination

Chromatin remodeling is emerging as a critical regulator of DNA repair factor access to DNA damage, and optimum accessibility of these factors is a major determinant of DNA repair outcome. Hence, chromatin remodeling is likely to play a key role in genome stabilization and tumor suppression. We previously showed that nucleosome eviction near double-strand breaks (DSBs) in yeast is regulated by the INO80 nucleosome remodeling complex and is defective in mutants lacking the Arp8 subunit of INO80. In the absence of homologous donor sequences, RPA recruitment to a DSB appeared normal in arp8Δ, but Rad51 recruitment was defective. We now show that the early strand invasion step of homologous recombination (HR) is markedly delayed in an arp8Δ haploid, but there is only a minor defect in haploid HR efficiency (MAT switching). In an arp8Δ diploid, interhomolog DSB repair by HR shows a modest defect that is partially suppressed by overexpression of Rad51 or its mediator, Rad52. In wild type cells, DSB repair typically results in gene conversion, and most gene conversion tracts are continuous, reflecting efficient mismatch repair of heteroduplex DNA. In contrast, arp8Δ gene conversion tracts are longer and frequently discontinuous, indicating defects in late stages of HR. Interestingly, when a homologous donor sequence is present, Rad51 is recruited normally to a DSB in arp8Δ, but its transfer to the donor is delayed, and this correlates with defective displacement of donor nucleosomes. We propose that retained nucleosomes at donors destabilize heteroduplex DNA or impair mismatch recognition, reflected in delayed strand invasion and altered conversion tracts.