DNA pairing and strand exchange by the Escherichia coli RecA and yeast Rad51 proteins without ATP hydrolysis: on the importance of not getting stuck

The bacterial RecA protein and the homologous Rad51 protein in eukaryotes both bind to single-stranded DNA (ssDNA), align it with a homologous duplex, and promote an extensive strand exchange between them. Both reactions have properties, including a tolerance of base analog substitutions that tend to eliminate major groove hydrogen bonding potential, that suggest a common molecular process underlies the DNA strand exchange promoted by RecA and Rad51. However, optimal conditions for the DNA pairing and DNA strand exchange reactions promoted by the RecA and Rad51 proteins in vitro are substantially different. When conditions are optimized independently for both proteins, RecA promotes DNA pairing reactions with short oligonucleotides at a faster rate than Rad51. For both proteins, conditions that improve DNA pairing can inhibit extensive DNA strand exchange reactions in the absence of ATP hydrolysis. Extensive strand exchange requires a spooling of duplex DNA into a recombinase-ssDNA complex, a process that can be halted by any interaction elsewhere on the same duplex that restricts free rotation of the duplex and/or complex, I.e. the reaction can get stuck. Optimization of an extensive DNA strand exchange without ATP hydrolysis requires conditions that decrease nonproductive interactions of recombinase-ssDNA complexes with the duplex DNA substrate.     

The rad51-K191R ATPase-defective mutant is impaired for presynaptic filament formation

The nucleoprotein filament formed by Rad51 polymerization on single-stranded DNA is essential for homologous pairing and strand exchange. ATP binding is required for Rad51 nucleoprotein filament formation and strand exchange, but ATP hydrolysis is not required for these functions in vitro. Previous studies have shown that a yeast strain expressing the rad51-K191R allele is sensitive to ionizing radiation, suggesting an important role for ATP hydrolysis in vivo. The recruitment of Rad51-K191R to double-strand breaks is defective in vivo, and this phenotype can be suppressed by elimination of the Srs2 helicase, an antagonist of Rad51 filament formation. The phenotype of the rad51-K191R strain is also suppressed by overexpression of Rad54. In vitro, the Rad51-K191R protein exhibits a slight decrease in binding to DNA, consistent with the defect in presynaptic filament formation. However, the rad51-K191R mutation is dominant in heterozygous diploids, indicating that the defect is not due simply to reduced affinity for DNA. We suggest the Rad51-K191R protein either forms an altered filament or is defective in turnover, resulting in a reduced pool of free protein available for DNA binding.     

Heterology tolerance and recognition of mismatched base pairs by human Rad51 protein

Human Rad51 (hRad51) promoted homology recognition and subsequent strand exchange are the key steps in human homologous recombination mediated repair of DNA double-strand breaks. However, it is still not clear how hRad51 deals with sequence heterology between the two homologous chromosomes in eukaryotic cells, which would lead to mismatched base pairs after strand exchange. Excessive tolerance of sequence heterology may compromise the fidelity of repair of DNA double-strand breaks. In this study, fluorescence resonance energy transfer (FRET) was used to monitor the heterology tolerance of human Rad51 mediated strand exchange reactions, in real time, by introducing either G-T or I-C mismatched base pairs between the two homologous DNA strands. The strand exchange reactions were much more sensitive to G-T than to I-C base pairs. These results imply that the recognition of homology and the tolerance of heterology by hRad51 may depend on the local structural motif adopted by the base pairs participating in strand exchange. AnhRad51 mutant protein (hRad51K133R), deficient in ATP hydrolysis, showed greater heterology tolerance to both types of mismatch base pairing, suggesting that ATPase activity may be important for maintenance of high fidelity homologous recombination DNA repair.     

Rad52 promotes postinvasion steps of meiotic double-strand-break repair

During DNA double-strand-break (DSB) repair by recombination, the broken chromosome uses a homologous chromosome as a repair template. Early steps of recombination are well characterized: DSB ends assemble filaments of RecA-family proteins that catalyze homologous pairing and strand-invasion reactions. By contrast, the postinvasion steps of recombination are poorly characterized. Rad52 plays an essential role during early steps of recombination by mediating assembly of a RecA homolog, Rad51, into nucleoprotein filaments. The meiosis-specific RecA-homolog Dmc1 does not show this dependence, however. By exploiting the Rad52 independence of Dmc1, we reveal that Rad52 promotes postinvasion steps of both crossover and noncrossover pathways of meiotic recombination in Saccharomyces cerevisiae. This activity resides in the N-terminal region of Rad52, which can anneal complementary DNA strands, and is independent of its Rad51-assembly function. Our findings show that Rad52 functions in temporally and biochemically distinct reactions and suggest a general annealing mechanism for reuniting DSB ends during recombination.     

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.     

Multiple interactions with the Rad51 recombinase govern the homologous recombination function of Rad54

In eukaryotes, Rad51 and Rad54 functionally cooperate to mediate homologous recombination and the repair of damaged chromosomes by recombination. Rad51, the eukaryotic counterpart of the bacterial RecA recombinase, forms filaments on single-stranded DNA that are capable of pairing the bound DNA with a homologous double-stranded donor to yield joint molecules. Rad54 enhances the homologous DNA pairing reaction, and this stimulatory effect involves a physical interaction with Rad51. Correspondingly, the ability of Rad54 to hydrolyze ATP and introduce superhelical tension into covalently closed circular plasmid DNA is stimulated by Rad51. By controlled proteolysis, we show that the amino-terminal region of yeast Rad54 is rather unstructured. Truncation mutations that delete the N-terminal 113 or 129 amino acid residues of Rad54 attenuate or ablate physical and functional interactions with Rad51 under physiological ionic strength, respectively. Surprisingly, under less stringent conditions, the Rad54 Delta129 protein can interact with Rad51 in affinity pull-down and functional assays. These results highlight the functional importance of the N-terminal Rad51 interaction domain of Rad54 and reveal that Rad54 contacts Rad51 through separable epitopes.     

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.     

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 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.     

Structural basis for octameric ring formation and DNA interaction of the human homologous-pairing protein Dmc1

The human Dmc1 protein, a RecA/Rad51 homolog, is a meiosis-specific DNA recombinase that catalyzes homologous pairing. RecA and Rad51 form helical filaments, while Dmc1 forms an octameric ring. In the present study, we crystallized the full-length human Dmc1 protein and solved the structure of the Dmc1 octameric ring. The monomeric structure of the Dmc1 protein closely resembled those of the human and archaeal Rad51 proteins. In addition to the polymerization motif that was previously identified in the Rad51 proteins, we found another hydrogen bonding interaction at the polymer interface, which could explain why Dmc1 forms stable octameric rings instead of helical filaments. Mutagenesis studies identified the inner and outer basic patches that are important for homologous pairing. The inner patch binds both single-stranded and double-stranded DNAs, while the outer one binds single-stranded DNA. Based on these results, we propose a model for the interaction of the Dmc1 rings with DNA.     

A RecA homologue in Ustilago maydis that is distinct and evolutionarily distant from Rad51 actively promotes DNA pairing reactions in the absence of auxiliary factors

Two RecA homologues have been identified to date in Ustilago maydis. One is orthologous to Rad51 while the other, Rec2, is structurally quite divergent and evolutionarily distant. DNA repair and recombination proficiency in U. maydis requires both Rec2 and Rad51. Here we have examined biochemical activities of Rec2 protein purified after overexpression of the cloned gene. Rec2 requires DNA as a cofactor to hydrolyze ATP and depends on ATP to promote homologous pairing and DNA strand exchange. ATPgammaS was found to substitute for ATP in all pairing reactions examined. With superhelical DNA and a homologous single-stranded oligonucleotide as substrates, Rec2 actively promoted formation and dissociation of D-loops. When an RNA oligonucleotide was substituted it was found that R-loops could also be formed and utilized as primer/template for limited DNA synthesis. In DNA strand exchange reactions using oligonucleotides, we found that Rec2 exhibited a pairing bias that is opposite that of RecA. Single-stranded oligonucleotides were activated for DNA strand exchange when attached as tails protruding from a duplex sequence due to enhanced binding of Rec2. The results indicate that Rec2 is competent, and in certain ways even better than Rad51, in the ability to provide the fundamental DNA pairing activity necessary for recombinational repair. We propose that the emerging paradigm for homologous recombination featuring Rad51 as the essential catalytic component for strand exchange may not be universal in eukaryotes.     

The Essential Functions of Human Rad51 Are Independent of ATP Hydrolysis

Genetic recombination and the repair of double-strand DNA breaks in Saccharomyces cerevisiae require Rad51, a homologue of the Escherichia coli RecA protein. In vitro, Rad51 binds DNA to form an extended nucleoprotein filament and catalyzes the ATP-dependent exchange of DNA between molecules with homologous sequences. Vertebrate Rad51 is essential for cell proliferation. Using site-directed mutagenesis of highly conserved residues of human Rad51 (hRad51) and gene targeting of the RAD51 locus in chicken DT40 cells, we examined the importance of Rad51's highly conserved ATP-binding domain. Mutant hRad51 incapable of ATP hydrolysis (hRad51K-133R) binds DNA less efficiently than the wild type but catalyzes strand exchange between homologous DNAs. hRad51 does not need to hydrolyze ATP to allow vertebrate cell proliferation, form nuclear foci, or repair radiation-induced DNA damage. However, cells expressing hRad51K-133R show greatly reduced targeted integration frequencies. These findings show that ATP hydrolysis is involved in DNA binding by hRad51 and suggest that the extent of DNA complexed with hRad51 in nucleoprotein influences the efficiency of recombination.     

The meiosis-specific recombinase hDmc1 forms ring structures and interacts with hRad51

Eukaryotic cells encode two homologs of Escherichia coli RecA protein, Rad51 and Dmc1, which are required for meiotic recombination. Rad51, like E.coli RecA, forms helical nucleoprotein filaments that promote joint molecule and heteroduplex DNA formation. Electron microscopy reveals that the human meiosis-specific recombinase Dmc1 forms ring structures that bind single-stranded (ss) and double-stranded (ds) DNA. The protein binds preferentially to ssDNA tails and gaps in duplex DNA. hDmc1-ssDNA complexes exhibit an irregular, often compacted structure, and promote strand-transfer reactions with homologous duplex DNA. hDmc1 binds duplex DNA with reduced affinity to form nucleoprotein complexes. In contrast to helical RecA/Rad51 filaments, however, Dmc1 filaments are composed of a linear array of stacked protein rings. Consistent with the requirement for two recombinases in meiotic recombination, hDmc1 interacts directly with hRad51.     

Rad54 protein exerts diverse modes of ATPase activity on duplex DNA partially and fully covered with Rad51 protein

Rad54 protein is a Snf2-like ATPase with a specialized function in the recombinational repair of DNA damage. Rad54 is thought to stimulate the search of homology via formation of a specific complex with the presynaptic Rad51 filament on single-stranded DNA. Herein, we address the interaction of Rad54 with Rad51 filaments on double-stranded (ds) DNA, an intermediate in DNA strand exchange with unclear functional significance. We show that Saccharomyces cerevisiae Rad54 exerts distinct modes of ATPase activity on partially and fully saturated filaments of Rad51 protein on dsDNA. The highest ATPase activity is observed on dsDNA containing short patches of yeast Rad51 filaments resulting in a 6-fold increase compared with protein-free DNA. This enhanced ATPase mode of yeast Rad54 can also be elicited by partial filaments of human Rad51 protein but to a lesser extent. In contrast, the interaction of Rad54 protein with duplex DNA fully covered with Rad51 is entirely species-specific. When yeast Rad51 fully covers dsDNA, Rad54 protein hydrolyzes ATP in a reduced mode at 60-80% of its rate on protein-free DNA. Instead, saturated filaments with human Rad51 fail to support the yeast Rad54 ATPase. We suggest that the interaction of Rad54 with dsDNA-Rad51 complexes is of functional importance in homologous recombination.     

Yeast Rad54 promotes Rad51-dependent homologous DNA pairing via ATP hydrolysis-driven change in DNA double helix conformation.

Saccharomyces cerevisiae RAD54 gene functions in the formation of heteroduplex DNA, a key intermediate in recombination processes. Rad54 is monomeric in solution, but forms a dimer/oligomer on DNA. Rad54 dimer/oligomer alters the conformation of the DNA double helix in an ATP-dependent manner, as revealed by a change in the DNA linking number in a topoisomerase I-linked reaction. DNA conformational alteration does not occur in the presence of non-hydrolyzable ATP analogues, nor when mutant rad54 proteins defective in ATP hydrolysis replace Rad54. Accordingly, the Rad54 ATPase activity is shown to be required for biological function in vivo and for promoting Rad51-mediated homologous DNA pairing in vitro. Taken together, the results are consistent with a model in which a Rad54 dimer/oligomer promotes nascent heteroduplex joint formation via a specific interaction with Rad51 protein and an ability to transiently unwind duplex DNA.     

Identification of the subunit-subunit interface of Xenopus Rad51.1 protein: similarity to RecA

Rad51, like its prokaryotic homolog RecA, forms a helical filament for homologous DNA recombination and recombinational DNA repair. Comparison of the three-dimensional structures of human Rad51 and Escherichia coli RecA indicated that the tyrosine residue at position 191 in human Rad51 lies at the centre of a putative subunit-subunit contact interface. We inserted a tryptophan residue as a fluorescent probe at the corresponding position in Xenopus Rad51.1 and found that its fluorescence depended upon the protein concentration, indicating that the residue is truly in the subunit-subunit interface. We also found that 3 M urea, which promoted the dissociation of Rad51 filament without complete unfolding of the protein, exposed the tryptophan residue to solvent. The fluorescence was not modified by binding to DNA and only slightly modified by ATP, indicating that the same site is used for formation of the active ATP-Rad51-DNA filament. The slight changes in fluorescence caused by ATP and ADP suggest that the subunit-subunit contact is altered, leading to the elongation of the filament by these nucleotides, as with the RecA filament. Thus, Rad51 forms filaments by subunit-subunit contact much like RecA does.     

Roles of ATP binding and ATP hydrolysis in human Rad51 recombinase function

The Rad51 recombinase polymerizes on ssDNA to yield a right-handed nucleoprotein filament, called the presynaptic filament, that can search for homology in duplex DNA and pair the recombining DNA molecules to form a DNA joint. ATP is needed for presynaptic filament assembly and homologous DNA pairing, but the roles of ATP binding and ATP hydrolysis in the overall reaction scheme have not yet been clearly defined. To address this issue, we have constructed two mutants of hRad51, hRad51 K133A and hRad51 K133R, expressed these mutant variants in Escherichia coli, and purified them to near homogeneity. Both hRad51 mutant variants are greatly attenuated for ATPase activity, but hRad51 K133R retains the ability to protect DNA from restriction enzyme digest and induce topological changes in duplex DNA in an ATP-dependent manner, whereas the hRad51 K133A variant is inactive. With biochemical means, we show that the presynaptic filament becomes greatly stabilized when ATP hydrolysis is prevented, leading to an enhanced ability of the presynaptic filament to catalyze homologous pairing. These results help form the basis for understanding the functions of ATP binding and ATP hydrolysis in hRad51-mediated recombination reactions.     

New insights into the mechanism of homologous recombination in yeast

Genome stability is of primary importance for the survival and proper functioning of all organisms. Double-strand breaks (DSBs) arise spontaneously during growth, or can be created by external insults. Repair of DSBs by homologous recombination provides an efficient and fruitful pathway to restore chromosomal integrity. Exciting new work in yeast has lately provided insights into this complex process. Many of the proteins involved in recombination have been isolated and the details of the repair mechanism are now being unraveled at the molecular level. In this review, we focus on recent studies which dissect the recombinational repair of a single broken chromosome. After DSB formation, a decision is made regarding the mechanism of repair (recombination or non-homologous end-joining). This decision is under genetic control. Once committed to the recombination pathway, the broken chromosomal ends are resected by a still unclear mechanism in which the DNA damage checkpoint protein Rad24 participates. At this stage several proteins are recruited to the broken ends, including Rad51p, Rad52p, Rad55p, Rad57p, and possibly Rad54p. A genomic search for homology ensues, followed by strand invasion, promoted by the Rad51 filament with the participation of Rad55p, Rad57p and Rad54p. DNA synthesis then takes place, restoring the resected ends. Crossing-over formation depends on the length of the homologous recombining sequences, and is usually counteracted by the activity of the mismatch repair system. Given the conservation of the repair mechanisms and genes throughout evolution, these studies have profound implications for other eukaryotic organisms.     

Fission yeast rad51 and dmc1, two efficient DNA recombinases forming helical nucleoprotein filaments

Homologous recombination is important for the repair of double-strand breaks during meiosis. Eukaryotic cells require two homologs of Escherichia coli RecA protein, Rad51 and Dmc1, for meiotic recombination. To date, it is not clear, at the biochemical level, why two homologs of RecA are necessary during meiosis. To gain insight into this, we purified Schizosaccharomyces pombe Rad51 and Dmc1 to homogeneity. Purified Rad51 and Dmc1 form homo-oligomers, bind single-stranded DNA preferentially, and exhibit DNA-stimulated ATPase activity. Both Rad51 and Dmc1 promote the renaturation of complementary single-stranded DNA. Importantly, Rad51 and Dmc1 proteins catalyze ATP-dependent strand exchange reactions with homologous duplex DNA. Electron microscopy reveals that both S. pombe Rad51 and Dmc1 form nucleoprotein filaments. Rad51 formed helical nucleoprotein filaments on single-stranded DNA, whereas Dmc1 was found in two forms, as helical filaments and also as stacked rings. These results demonstrate that Rad51 and Dmc1 are both efficient recombinases in lower eukaryotes and reveal closer functional and structural similarities between the meiotic recombinase Dmc1 and Rad51. The DNA strand exchange activity of both Rad51 and Dmc1 is most likely critical for proper meiotic DNA double-strand break repair in lower eukaryotes.     

Rad54, a Swi2/Snf2-like recombinational repair protein,disassembles Rad51:dsDNA filaments

Rad54 protein is a member of the Swi2/Snf2-like family of DNA-dependent/stimulated ATPases that dissociate and remodel protein complexes on dsDNA. Rad54 functions in the recombinational DNA repair (RAD52) pathway. Here we show that Rad54 protein dissociates Rad51 from nucleoprotein filaments formed on dsDNA. Addition of Rad54 protein overcomes inhibition of DNA strand exchange by Rad51 protein bound to substrate dsDNA. Species preference in the Rad51 dissociation and DNA strand exchange assays underlines the importance of specific Rad54-Rad51 protein interactions. Rad51 protein is unable to release dsDNA upon ATP hydrolysis, leaving it stuck on the heteroduplex DNA product after DNA strand exchange. We suggest that Rad54 protein is involved in the turnover of Rad51-dsDNA filaments.