Characterization of strand exchange activity of yeast Rad51 protein.

The Saccharomyces cerevisiae RAD51 gene product takes part in genetic recombination and repair of DNA double strand breaks. Rad51, like Escherichia coli RecA, catalyzes strand exchange between homologous circular single-stranded DNA (ssDNA) and linear double-stranded DNA (dsDNA) in the presence of ATP and ssDNA-binding protein. The formation of joint molecules between circular ssDNA and linear dsDNA is initiated at either the 5' or the 3' overhanging end of the complementary strand; joint molecules are formed only if the length of the overhanging end is more than 1 nucleotide. Linear dsDNAs with recessed complementary or blunt ends are not utilized. The polarity of strand exchange depends upon which end is used to initiate the formation of joint molecules. Joint molecules formed via the 5' end are processed by branch migration in the 3'-to-5' direction with respect to ssDNA, and joint molecules formed with a 3' end are processed in the opposite direction.     

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 efficiency of strand invasion by Escherichia coli RecA is dependent upon the length and polarity of ssDNA tails

RecA protein is essential for homologous recombination and the repair of DNA double-strand breaks in Escherichia coli. The protein binds DNA to form nucleoprotein filaments that promote joint molecule formation and strand exchange in vitro. RecA polymerises on ssDNA in the 5'-3' direction and catalyses strand exchange and branch migration with a 5'-3' polarity. It has been reported previously, using D-loop assays, in which ssDNA (containing a heterologous block at one end) invades supercoiled duplex DNA that 3'-homologous ends are reactive, whereas 5'-ends are inactive. This polarity bias was thought to be due to the polarity of RecA filament formation, which results in the 3'-ends being coated in RecA, whereas 5'-ends remain naked. Using a range of duplex substrates containing ssDNA tails of various lengths and polarities, we now demonstrate that when no heterologous block is imposed, 5'-ends are just as reactive as 3'-ends. Moreover, using short-tailed substrates, we find that 5'-ends form more stable D-loops than 3'-ends. This bias may be a consequence of the instability of short 3'-joints. With more physiological substrates containing long ssDNA tails, we find that RecA shows no intrinsic preference for 5' or 3'-ends and that both form D-loop complexes with high efficiency.     

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.     

Branch migration of three-strand recombination intermediates by RecG, a possible pathway for securing exchanges initiated by 3''-tailed duplex DNA.

RecG protein is required for normal levels of recombination and DNA repair in Escherichia coli. This 76 kDa polypeptide is a junction-specific DNA helicase that acts post-synaptically to drive branch migration of Holliday junction intermediates made by RecA during the strand exchange stage of recombination. To gain further insight into the role of RecG, we studied its activity on three-strand intermediates formed by RecA between circular single-stranded and linear duplex DNAs. Once RecA is removed, RecG drives branch migration of these intermediates by a junction-targeted activity that depends on hydrolysis of ATP. RuvAB has a similar activity. However, when RecG is added to a RecA strand exchange reaction it severely reduces the accumulation of joint molecule intermediates by driving branch migration of junctions in the reverse direction to that catalysed by RecA strand exchange. In comparison, RuvAB has little effect on the reaction. We discuss how reverse branch migration by RecG, which acts counter of the 5'-->3' polarity of RecA binding and strand exchange, could serve to promote or abort the early stages of recombination, depending on the orientation of the single DNA strand initiating the exchange relative to the adjacent duplex region.     

ATP hydrolysis and the displaced strand are two factors that determine the polarity of RecA-promoted DNA strand exchange.

When the recA protein (RecA) of Escherichia coli promotes strand exchange between single-stranded DNA (ssDNA) circles and linear double-stranded DNAs (dsDNA) with complementary 5' or 3' ends a polarity is observed. This property of RecA depends on ATP hydrolysis and the ssDNA that is displaced in the reaction since no polarity is observed in the presence of the non-hydrolyzable ATP analog, ATP gamma S, or in the presence of single-strand specific exonucleases. Based on these results a model is presented in which both the 5' and 3' complementary ends of the linear dsDNA initiate pairing with the ssDNA circle but only one end remains stably paired. According to this model, the association/dissociation of RecA in the 5' to 3' direction on the displaced strand determines the polarity of strand exchange by favoring or blocking its reinvasion into the newly formed dsDNA. Reinvasion is favored when the displaced strand is coated with RecA whereas it is blocked when it lacks RecA, remains covered by single-stranded DNA binding protein or is removed by a single-strand specific exonuclease. The requirement for ATP hydrolysis is explained if the binding of RecA to the displaced strand occurs via the dissociation and/or transfer of RecA, two functions that depend on ATP hydrolysis. The energy for strand exchange derives from the higher binding constant of RecA for the newly formed dsDNA as compared with that for ssDNA and not from ATP hydrolysis.     

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.     

Double-strand break repair: are Rad51/RecA--DNA joints barriers to DNA replication?

The central step of homologous recombination is the DNA strand exchange reaction catalyzed by bacterial RecA or eukaryotic Rad51. Besides Rad51-mediated synthesis-dependent strand annealing (SDSA), DNA ends can promote replication in Escherichia coli (recombination-dependent replication, RDR) and yeast (break-induced replication, BIR). However, what causes a DNA end to be repaired via SDSA or via BIR/RDR? I propose that Rad51/RecA--DNA plectonemic joints act as barriers to DNA replication and that BIR/RDR is only possible when the DNA polymerase that synthesizes DNA from the invading 3' end does not encounter RecA/Rad51--DNA joints in its path.     

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 preference for a 3' homologous end is intrinsic to RecA-promoted strand exchange

The recA protein (RecA) promotes DNA pairing and strand exchange optimally in the presence of single-stranded binding protein (SSB). Under these conditions, 3' homologous ends are essential for stable joint molecule formation between linear single-stranded DNA (ssDNA) and supercoiled DNA (i.e. 3' ends are 50-60 times more reactive than 5' ends). Linear ssDNAs with homology at the 5' end do not participate in pairing. In the absence of SSB, the strand exchange reaction is less efficient; however, linear ssDNAs with 3' end homology are still 5- to 10-fold more reactive than those with 5' end homology. The preference for a 3' homologous end in the absence of SSB suggests that this is an intrinsic property of RecA-promoted strand exchange. The preferential reactivity of 3' homologous ends is likely to be a consequence of the polarity of polymerization of RecA on ssDNA. Specifically, since RecA polymerizes in the 5'----3' direction, 3' ends are more likely to be coated with RecA and, hence, will be more reactive than 5' ends.     

The human Rad51 protein: polarity of strand transfer and stimulation by hRP-A.

The human Rad51 protein is homologous to the RecA protein and catalyses homologous pairing and strand transfer reactions in vitro. Using single-stranded circular and homologous linear duplex DNA, we show that hRad51 forms stable joint molecules by transfer of the 5' end of the complementary strand of the linear duplex to the ssDNA. The polarity of strand transfer is therefore 3' to 5', defined relative to the ssDNA on which hRad51 initiates filament formation. This polarity is opposite to that observed with RecA. Homologous pairing and strand transfer require stoichiometric amounts of hRad51, corresponding to one hRad51 monomer per three nucleotides of ssDNA. Joint molecules are not observed when the protein is present in limiting or excessive amounts. The human ssDNA binding-protein, hRP-A, stimulates hRad51-mediated reactions. Its effect is consistent with a role in the removal of secondary structures from ssDNA, thereby facilitating the formation of continuous Rad51 filaments.     

The P. furiosus mre11/rad50 complex promotes 5' strand resection at a DNA double-strand break

The Mre11/Rad50 complex has been implicated in the early steps of DNA double-strand break (DSB) repair through homologous recombination in several organisms. However, the enzymatic properties of this complex are incompatible with the generation of 3' single-stranded DNA for recombinase loading and strand exchange. In thermophilic archaea, the Mre11 and Rad50 genes cluster in an operon with genes encoding a helicase, HerA, and a 5' to 3' exonuclease, NurA, suggesting a common function. Here we show that purified Mre11 and Rad50 from Pyrococcus furiosus act cooperatively with HerA and NurA to resect the 5' strand at a DNA end under physiological conditions in vitro. The 3' single-stranded DNA generated by these enzymes can be utilized by the archaeal RecA homolog RadA to catalyze strand exchange. This work elucidates how the conserved Mre11/Rad50 complex promotes DNA end resection in archaea and may serve as a model for DSB processing in eukaryotes.     

Cancer-Associated Mutations in BRC Domains of BRCA2 Affect Homologous Recombination Induced by Rad51

The tumor suppressor BRCA2 protein plays a major role in the regulation of Rad51-catalyzed homologous recombination. BRCA2 interacts with monomeric Rad51 primarily via conserved BRC domains and coordinates the formation of Rad51 filaments at double-stranded DNA (dsDNA) breaks. A number of cancer-associated mutations in BRC4 and BRC2 domains have been reported. To elucidate their effects on homologous recombination, we studied Rad51 filament formation on single-stranded DNA and dsDNA substrates and Rad51-catalyzed strand exchange, in the presence of wild-type and mutated peptides of either BRC4 or BRC2. While the wild-type BRC2 and BRC4 peptides inhibited filament formation and, thus, strand exchange, the mutated forms decreased significantly these inhibitory effects. These results are consistent with a three-dimensional model for the interface between individual BRC repeats and Rad51. We suggest that mutations at sites crucial for the association between Rad51 and BRC domains impair the ability of BRCA2 to recruit Rad51 to dsDNA breaks, hampering recombinational repair.     

Rad51 and Rad54 ATPase activities are both required to modulate Rad51-dsDNA filament dynamics

Rad51 and Rad54 are key proteins that collaborate during homologous recombination. Rad51 forms a presynaptic filament with ATP and ssDNA active in homology search and DNA strand exchange, but the precise role of its ATPase activity is poorly understood. Rad54 is an ATP-dependent dsDNA motor protein that can dissociate Rad51 from dsDNA, the product complex of DNA strand exchange. Kinetic analysis of the budding yeast proteins revealed that the catalytic efficiency of the Rad54 ATPase was stimulated by partial filaments of wild-type and Rad51-K191R mutant protein on dsDNA, unambiguously demonstrating that the Rad54 ATPase activity is stimulated under these conditions. Experiments with Rad51-K191R as well as with wild-type Rad51-dsDNA filaments formed in the presence of ATP, ADP or ATP-γ-S showed that efficient Rad51 turnover from dsDNA requires both the Rad51 ATPase and the Rad54 ATPase activities. The results with Rad51-K191R mutant protein also revealed an unexpected defect in binding to DNA. Once formed, Rad51-K191R-DNA filaments appeared normal upon electron microscopic inspection, but displayed significantly increased stability. These biochemical defects in the Rad51-K191R protein could lead to deficiencies in presynapsis (filament formation) and postsynapsis (filament disassembly) in vivo.     

The direction of RecA protein assembly onto single strand DNA is the same as the direction of strand assimilation during strand exchange

The RecA protein of Escherichia coli optimally promotes DNA strand exchange reactions in the presence of the single strand DNA-binding protein of E. coli (SSB protein). Under these conditions, assembly of RecA protein onto single-stranded DNA (ssDNA) occurs in three steps. First, the ssDNA is rapidly covered by SSB protein. The binding of RecA protein is then initiated by nucleation of a short tract of RecA protein onto the ssDNA. Finally, cooperative polymerization of additional RecA protein accompanied by displacement of SSB protein results in a ssDNA-RecA protein filament (Griffith, J. D., Harris, L. D., and Register, J. C. (1984) Cold Spring Harbor Symp. Quant. Biol. 49, 553-559). We report here that RecA protein assembly onto circular ssDNA yields RecA protein-covered circles in which greater than 85% are completely covered by RecA protein with no remaining SSB protein-covered segments (as detected by electron microscopy). However, when linear ssDNA is used, 90% of the filaments contain a short segment at one end complexed with SSB protein. This suggests that RecA protein assembly is unidirectional. Visualization of the assembly of RecA protein onto either long ssDNA tails (containing either 5' or 3' termini) or ssDNA gaps generated in double strand DNA allowed us to determine that the RecA protein polymerizes in the 5' to 3' direction on ssDNA and preferentially nucleates at ssDNA-double strand DNA junctions containing 5' termini.     

Coordinated Binding of Single-Stranded and Double-Stranded DNA by UvsX Recombinase

Homologous recombination is important for the error-free repair of DNA double-strand breaks and for replication fork restart. Recombinases of the RecA/Rad51 family perform the central catalytic role in this process. UvsX recombinase is the RecA/Rad51 ortholog of bacteriophage T4. UvsX and other recombinases form presynaptic filaments on ssDNA that are activated to search for homology in dsDNA and to perform DNA strand exchange. To effectively initiate recombination, UvsX must find and bind to ssDNA within an excess of dsDNA. Here we examine the binding of UvsX to ssDNA and dsDNA in the presence and absence of nucleotide cofactor, ATP. We also examine how the binding of one DNA substrate is affected by simultaneous binding of the other to determine how UvsX might selectively assemble on ssDNA. We show that the two DNA binding sites of UvsX are regulated by the nucleotide cofactor ATP and are coordinated with each other such that in the presence of ssDNA, dsDNA binding is significantly reduced and correlated with its homology to the ssDNA bound to the enzyme. UvsX has high affinity for dsDNA in the absence of ssDNA, which may allow for sequestration of the enzyme in an inactive form prior to ssDNA generation.     

Human Rad54 protein stimulates DNA strand exchange activity of hRad51 protein in the presence of Ca2+

Rad51 and Rad54 proteins play a key role in homologous recombination in eukaryotes. Recently, we reported that Ca2+ is required in vitro for human Rad51 protein to form an active nucleoprotein filament that is important for the search of homologous DNA and for DNA strand exchange, two critical steps of homologous recombination. Here we find that Ca2+ is also required for hRad54 protein to effectively stimulate DNA strand exchange activity of hRad51 protein. This finding identifies Ca2+ as a universal cofactor of DNA strand exchange promoted by mammalian homologous recombination proteins in vitro. We further investigated the hRad54-dependent stimulation of DNA strand exchange. The mechanism of stimulation appeared to include specific interaction of hRad54 protein with the hRad51 nucleoprotein filament. Our results show that hRad54 protein significantly stimulates homology-independent coaggregation of dsDNA with the filament, which represents an essential step of the search for homologous DNA. The results obtained indicate that hRad54 protein serves as a dsDNA gateway for the hRad51-ssDNA filament, promoting binding and an ATP hydrolysis-dependent translocation of dsDNA during the search for homologous sequences.     

Effect of the Streptococcus pneumoniae MmsA protein on the RecA protein-promoted three-strand exchange reaction. Implications for the mechanism of transformational recombination

Streptococcus pneumoniae is a naturally transformable bacterium that is able to incorporate DNA from its environment into its own chromosome. This process, known as transformational recombination, is dependent in part on the mmsA gene, which encodes a protein having a sequence that is 40% identical to that of the Escherichia coli RecG protein, a junction-specific DNA helicase believed to be involved in the branch migration of recombinational intermediates. We have developed an expression system for the MmsA protein and have purified the MmsA protein to more than 99% homogeneity. The MmsA protein has DNA-dependent ATP hydrolysis and DNA junction-helicase activities that are similar to those of the E. coli RecG protein. The effect of the MmsA protein on the S. pneumoniae RecA protein-promoted three-strand exchange reaction was also investigated. In the standard direction (circular single-stranded (ss) DNA + linear double-stranded (ds) DNA --> linear ssDNA + nicked circular dsDNA), the MmsA protein appears to promote the branch migration of partially exchanged intermediates in a direction opposite of the RecA protein, resulting in a nearly complete inhibition of the overall strand exchange reaction. In the reverse direction (linear ssDNA + nicked circular dsDNA --> circular ssDNA + linear dsDNA), however, the MmsA protein appears to facilitate the conversion of partially exchanged intermediates into fully exchanged products, leading to a pronounced stimulation of the overall reaction. These results are discussed in terms of the molecular mechanism of transformational recombination.     

Mechanisms of distinctive mismatch tolerance between Rad51 and Dmc1 in homologous recombination

Homologous recombination (HR) is a primary DNA double-strand breaks (DSBs) repair mechanism. The recombinases Rad51 and Dmc1 are highly conserved in the RecA family; Rad51 is mainly responsible for DNA repair in somatic cells during mitosis while Dmc1 only works during meiosis in germ cells. This spatiotemporal difference is probably due to their distinctive mismatch tolerance during HR: Rad51 does not permit HR in the presence of mismatches, whereas Dmc1 can tolerate certain mismatches. Here, the cryo-EM structures of Rad51–DNA and Dmc1–DNA complexes revealed that the major conformational differences between these two proteins are located in their Loop2 regions, which contain invading single-stranded DNA (ssDNA) binding residues and double-stranded DNA (dsDNA) complementary strand binding residues, stabilizing ssDNA and dsDNA in presynaptic and postsynaptic complexes, respectively. By combining molecular dynamic simulation and single-molecule FRET assays, we identified that V273 and D274 in the Loop2 region of human RAD51 (hRAD51), corresponding to P274 and G275 of human DMC1 (hDMC1), are the key residues regulating mismatch tolerance during strand exchange in HR. This HR accuracy control mechanism provides mechanistic insights into the specific roles of Rad51 and Dmc1 in DNA double-strand break repair and may shed light on the regulatory mechanism of genetic recombination in mitosis and meiosis.