DNA binding, annealing, and strand exchange activities of Brh2 protein from Ustilago maydis

Brh2 is the Ustilago maydis ortholog of the BRCA2 tumor suppressor. It functions in repair of DNA by homologous recombination by controlling the action of Rad51. A critical aspect in the control appears to be the recruitment of Rad51 to single-stranded DNA regions exposed as lesions after damage or following a disturbance in DNA synthesis. In previous experimentation, Brh2 was shown to nucleate formation of the Rad51 nucleoprotein filament that becomes the active element in promoting homologous pairing and DNA strand exchange. Nucleation was found to be initiated at junctions of double-stranded and single-stranded DNA. Here we investigated the DNA binding specificity of Brh2 in more detail using oligonucleotide substrates. We observed that Brh2 prefers partially duplex structures with single-stranded branches, flaps, or D-loops. We found also that Brh2 has an inherent ability to promote DNA annealing and strand exchange reactions on free as well as RPA-coated substrates. Unlike Rad51, Brh2 was able to promote DNA strand exchange when preincubated with double-stranded DNA. These findings raise the notion that Brh2 may have roles in homologous recombination beyond the previously established Rad51 mediator activity.     

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.     

Meiotic Recombination: An Affair of Two Recombinases

In E. coli, homologous recombination is catalyzed by the RecA recombinase. Two RecA-like factors, Rad51 and Dmc1, are found in eukaryotes. Whereas Rad51 is needed for homologous recombination reactions in both mitotic and meiotic cells, the role of Dmc1 is restricted to meiosis. Recent work has shown that, like RecA and Rad51, Dmc1 mediates the homologous DNA pairing strand exchange reaction via a filamentous intermediate assembled on single-stranded DNA. Emerging evidence suggests that the tumor suppressor BRCA2 functions in the assembly of nucleoprotein filaments of Rad51 and Dmc1. The manner in which Rad51 and Dmc1 functionally cooperate in meiotic recombination remains to be determined.     

Structure of REC2, a recombinational repair gene of Ustilago maydis, and its function in homologous recombination between plasmid and chromosomal sequences.

Mutation in the REC2 gene of Ustilago maydis leads to defects in DNA repair, recombination, and meiosis. Analysis of the primary sequence of the Rec2 protein reveals a region with significant homology to bacterial RecA protein and to the yeast recombination proteins Dmc1, Rad51, and Rad57. This homologous region in the U. maydis Rec2 protein was found to be functionally sensitive to mutation, lending support to the hypothesis that Rec2 has a functional RecA-like domain essential for activity in recombination and repair. Homologous recombination between plasmid and chromosomal DNA sequences is reduced substantially in the rec2 mutant following transformation. The frequency can be restored to a level approaching, but not exceeding, that observed in the wild-type strain if transformation is performed with cells containing multiple copies of REC2.     

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.     

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.     

The differential extension in dsDNA bound to Rad51 filaments may play important roles in homology recognition and strand exchange

RecA and Rad51 proteins play an important role in DNA repair and homologous recombination. For RecA, X-ray structure information and single molecule force experiments have indicated that the differential extension between the complementary strand and its Watson–Crick pairing partners promotes the rapid unbinding of non-homologous dsDNA and drives strand exchange forward for homologous dsDNA. In this work we find that both effects are also present in Rad51 protein. In particular, pulling on the opposite termini (3′ and 5′) of one of the two DNA strands in a dsDNA molecule allows dsDNA to extend along non-homologous Rad51-ssDNA filaments and remain stably bound in the extended state, but pulling on the 3′5′ ends of the complementary strand reduces the strand-exchange rate for homologous filaments. Thus, the results suggest that differential extension is also present in dsDNA bound to Rad51. The differential extension promotes rapid recognition by driving the swift unbinding of dsDNA from non-homologous Rad51-ssDNA filaments, while at the same time, reducing base pair tension due to the transfer of the Watson–Crick pairing of the complementary strand bases from the highly extended outgoing strand to the slightly less extended incoming strand, which drives strand exchange forward.     

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.     

RecA-mediated strand exchange traverses substitutional heterologies more easily than deletions or insertions

RecA protein in bacteria and its eukaryotic homolog Rad51 protein are responsible for initiation of strand exchange between homologous DNA molecules. This process is crucial for homologous recombination, the repair of certain types of DNA damage and for the reinitiation of DNA replication on collapsed replication forks. We show here, using two different types of in vitro assays, that in the absence of ATP hydrolysis RecA-mediated strand exchange traverses small substitutional heterologies between the interacting DNAs, whereas small deletions or insertions block the ongoing strand exchange. We discuss evolutionary implications of RecA selectivity against insertions and deletions and propose a molecular mechanism by which RecA can exert this selectivity.     

Heteroduplex formation by human Rad51 protein: effects of DNA end-structure, hRP-A and hRad52.

Purified human Rad51 protein (hRad51) catalyses ATP-dependent homologous pairing and strand transfer reactions, characteristic of a central role in homologous recombination and double-strand break repair. Using single-stranded circular and partially homologous linear duplex DNA, we found that the length of heteroduplex DNA formed by hRad51 was limited to approximately 1.3 kb, significantly less than that observed with Escherichia coli RecA and Saccharomyces cerevisiae Rad51 protein. Joint molecule formation required the presence of a 3' or 5'-overhang on the duplex DNA substrate and initiated preferentially at the 5'-end of the complementaryx strand. These results are consistent with a preference for strand transfer in the 3'-5' direction relative to the single-stranded DNA. The human single-strand DNA-binding protein, hRP-A, stimulated hRad51-mediated joint molecule formation by removing secondary structures from single-stranded DNA, a role similar to that played by E. coli single-strand DNA-binding protein in RecA-mediated strand exchange reactions. Indeed, E. coli single-strand DNA-binding protein could substitute for hRP-A in hRad51-mediated reactions. Joint molecule formation by hRad51 was stimulated or inhibited by hRad52, dependent upon the reaction conditions. The inhibitory effect could be overcome by the presence of hRP-A or excess heterologous DNA.     

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.     

Regulation of DNA strand exchange in homologous recombination

Homologous recombination, the exchange of DNA strands between homologous DNA molecules, is involved in repair of many structural diverse DNA lesions. This versatility stems from multiple ways in which homologous DNA strands can be rearranged. At the core of homologous recombination are recombinase proteins such as RecA and RAD51 that mediate homology recognition and DNA strand exchange through formation of a dynamic nucleoprotein filament. Four stages in the life cycle of nucleoprotein filaments are filament nucleation, filament growth, homologous DNA pairing and strand exchange, and filament dissociation. Progression through this cycle requires a sequence of recombinase-DNA and recombinase protein-protein interactions coupled to ATP binding and hydrolysis. The function of recombinases is controlled by accessory proteins that allow coordination of strand exchange with other steps of homologous recombination and that tailor to the needs of specific aberrant DNA structures undergoing recombination. Accessory proteins are also able to reverse filament formation thereby guarding against inappropriate DNA rearrangements. The dynamic instability of the recombinase-DNA interactions allows both positive and negative action of accessory proteins thereby ensuring that genome maintenance by homologous recombination is not only flexible and versatile, but also accurate.     

Rad51 protein involved in repair and recombination in S. cerevisiae is a RecA-like protein.

The RAD51 gene of S. cerevisiae is involved in mitotic recombination and repair of DNA damage and also in meiosis. We show that the rad51 null mutant accumulates meiosis-specific double-strand breaks (DSBs) at a recombination hotspot and reduces the formation of physical recombinants. Rad51 protein shows structural similarity to RecA protein, the bacterial strand exchange protein. Furthermore, we have found that Rad51 protein is similar to RecA in its DNA binding properties and binds directly to Rad52 protein, which also plays a crucial role in recombination. These results suggest that the Rad51 protein, probably together with Rad52 protein, is involved in a step to convert DSBs to the next intermediate in recombination. Rad51 protein is also homologous to a meiosis-specific Dmc1 protein of S. cerevisiae.     

Human Rad51 amino acid residues required for Rad52 binding.

The Rad51 protein, a homologue of the bacterial RecA protein, is an essential factor for both meiotic and mitotic recombination. The N-terminal domain of the human Rad51 protein (HsRad51) directly interacts with DNA. Based on a yeast two-hybrid analysis, it has been reported that the N-terminal region of the Saccharomyces cerevisiae Rad51 protein binds Rad52;S. cerevisiae Rad51 and Rad52 both activate the homologous pairing and strand exchange reactions. Here, we show that the HsRad51 N-terminal region, which corresponds to the Rad52-binding region of ScRad51, does not exhibit strong binding to the human Rad52 protein (HsRad52). To investigate its function, the C-terminal region of HsRad51 was randomly mutagenized. Although this region includes the two segments corresponding to the putative DNA-binding sites of RecA, all seven of the mutants did not decrease, but instead slightly increased, the DNA binding. In contrast, we found that some of these HsRad51 mutations significantly decreased the HsRad52 binding. Therefore, we conclude that these amino acid residues are required for the HsRad51.HsRad52 binding. HsRad52, as well as S. cerevisiae Rad52, promoted homologous pairing between ssDNA and dsDNA, and higher homologous pairing activity was observed in the presence of both HsRad51 and HsRad52 than with either HsRad51 or HsRad52 alone. The HsRad51 F259V mutation, which strongly impaired the HsRad52 binding, decreased the homologous pairing in the presence of both HsRad51 and HsRad52, without affecting the homologous pairing by HsRad51 alone. This result suggests the importance of the HsRad51.HsRad52 interaction in homologous pairing.     

Mutational analysis of Brh2 reveals requirements for compensating mediator functions

Brh2, a member of the BRCA2 family of proteins, governs homologous recombination in the fungus Ustilago maydis through interaction with Rad51. Brh2 serves at an early step in homologous recombination to mediate Rad51 nucleoprotein filament formation and also has the capability to function at a later step in recombination through its inherent DNA annealing activity. Rec2, a Rad51 paralogue, and Rad52 are additional components of the homologous recombination system, but the absence of either is less critical than Brh2 for operational activity. Here we tested a variety of mutant forms of Brh2 for activity in recombinational repair as measured by DNA repair proficiency. We found that a mutant of Brh2 deleted of the non-canonical DNA-binding domain within the N-terminal region is dependent upon the presence of Rad52 for DNA repair activity. We also determined that a motif first identified in human BRCA2 as important in binding DMC1 also contributes to DNA repair proficiency and cooperates with the BRC element in Rad51 binding.     

Biochemical characterization of the human RAD51 protein. I. ATP hydrolysis

The prototypical bacterial RecA protein promotes recombination/repair by catalyzing strand exchange between homologous DNAs. While the mechanism of strand exchange remains enigmatic, ATP-induced cooperativity between RecA protomers is critical for its function. A human RecA homolog, human RAD51 protein (hRAD51), facilitates eukaryotic recombination/repair, although its ability to hydrolyze ATP and/or promote strand exchange appears distinct from the bacterial RecA. We have quantitatively examined the hRAD51 ATPase. The catalytic efficiency (k(cat)/K(m)) of the hRAD51 ATPase was approximately 50-fold lower than the RecA ATPase. Altering the ratio of DNA/hRAD51 and including salts that stimulate DNA strand exchange (ammonium sulfate and spermidine) were found to affect the catalytic efficiency of hRAD51. The average site size of hRAD51 was determined to be approximately 3 nt (bp) for both single-stranded and double-stranded DNA. Importantly, hRAD51 lacks the magnitude of ATP-induced cooperativity that is a hallmark of RecA. Together, these results suggest that hRAD51 may be unable to coordinate ATP hydrolysis between neighboring protomers.     

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.     

Arabidopsis RAD51C gene is important for homologous recombination in meiosis and mitosis

Rad51 is a homolog of the bacterial RecA recombinase, and a key factor in homologous recombination in eukaryotes. Rad51 paralogs have been identified from yeast to vertebrates. Rad51 paralogs are thought to play an important role in the assembly or stabilization of Rad51 that promotes homologous pairing and strand exchange reactions. We previously characterized two RAD51 paralogous genes in Arabidopsis (Arabidopsis thaliana) named AtRAD51C and AtXRCC3, which are homologs of human RAD51C and XRCC3, respectively, and described the interaction of their products in a yeast two-hybrid system. Recent studies showed the involvement of AtXrcc3 in DNA repair and functional role in meiosis. To determine the role of RAD51C in meiotic and mitotic recombination in higher plants, we characterized a T-DNA insertion mutant of AtRAD51C. Although the atrad51C mutant grew normally during vegetative developmental stage, the mutant produced aborted siliques, and their anthers did not contain mature pollen grains. Crossing of the mutant with wild-type plants showed defective male and female gametogeneses as evidenced by lack of seed production. Furthermore, meiosis was severely disturbed in the mutant. The atrad51C mutant also showed increased sensitivity to gamma-irradiation and cisplatin, which are known to induce double-strand DNA breaks. The efficiency of homologous recombination in somatic cells in the mutant was markedly reduced relative to that in wild-type plants.     

Both RadA and RadB are involved in homologous recombination in Pyrococcus furiosus

RecA and Rad51 proteins are essential for homologous recombination in Bacteria and Eukarya, respectively. Homologous proteins, called RadA, have been described for Archaea. Here we present the characterization of two RecA/Rad51 family proteins, RadA and RadB, from Pyrococcus furiosus. The radA and radB genes were not induced by DNA damage resulting from exposure of the cells to gamma and UV irradiation and heat shock, suggesting that they might be constitutively expressed in this hyperthermophile. RadA had DNA-dependent ATPase, D-loop formation, and strand exchange activities. In contrast, RadB had a very weak ATPase activity that is not stimulated by DNA. This protein had a strong binding affinity for DNA, but little strand exchange activity could be detected. A direct interaction between RadA and RadB was detected by an immunoprecipitation assay. Moreover, RadB, but not RadA, coprecipitated with Hjc, a Holliday junction resolvase found in P. furiosus, in the absence of ATP. This interaction was suppressed in the presence of ATP. The Holliday junction cleavage activity of Hjc was inhibited by RadB in the absence, but not in the presence, of ATP. These results suggest that RadB has important roles in homologous recombination in Archaea and may regulate the cleavage reactions of the branch-structured DNA.