Swi5-Sfr1 protein stimulates Rad51-mediated DNA strand exchange reaction through organization of DNA bases in the presynaptic filament

The Swi5-Sfr1 heterodimer protein stimulates the Rad51-promoted DNA strand exchange reaction, a crucial step in homologous recombination. To clarify how this accessory protein acts on the strand exchange reaction, we have analyzed how the structure of the primary reaction intermediate, the Rad51/single-stranded DNA (ssDNA) complex filament formed in the presence of ATP, is affected by Swi5-Sfr1. Using flow linear dichroism spectroscopy, we observe that the nucleobases of the ssDNA are more perpendicularly aligned to the filament axis in the presence of Swi5-Sfr1, whereas the bases are more randomly oriented in the absence of Swi5-Sfr1. When using a modified version of the natural protein where the N-terminal part of Sfr1 is deleted, which has no affinity for DNA but maintained ability to stimulate the strand exchange reaction, we still observe the improved perpendicular DNA base orientation. This indicates that Swi5-Sfr1 exerts its activating effect through interaction with the Rad51 filament mainly and not with the DNA. We propose that the role of a coplanar alignment of nucleobases induced by Swi5-Sfr1 in the presynaptic Rad51/ssDNA complex is to facilitate the critical matching with an invading double-stranded DNA, hence stimulating the strand exchange reaction.     

Rad51 presynaptic filament stabilization function of the mouse Swi5-Sfr1 heterodimeric complex

Homologous recombination (HR) represents a major error-free pathway to eliminate pre-carcinogenic chromosomal lesions. The DNA strand invasion reaction in HR is mediated by a helical filament of the Rad51 recombinase assembled on single-stranded DNA that is derived from the nucleolytic processing of the primary lesion. Recent studies have found that the human and mouse Swi5 and Sfr1 proteins form a complex that influences Rad51-mediated HR in cells. Here, we provide biophysical evidence that the mouse Swi5-Sfr1 complex has a 1:1 stoichiometry. Importantly, the Swi5-Sfr1 complex, but neither Swi5 nor Sfr1 alone, physically interacts with Rad51 and stimulates Rad51-mediated homologous DNA pairing. This stimulatory effect stems from the stabilization of the Rad51-ssDNA presynaptic filament. Moreover, we provide evidence that the RSfp (rodent Sfr1 proline rich) motif in Sfr1 serves as a negative regulatory element. These results thus reveal an evolutionarily conserved function in the Swi5-Sfr1 complex and furnish valuable information as to the regulatory role of the RSfp motif that is specific to the mammalian Sfr1 orthologs.     

Fission yeast Swi5-Sfr1 protein complex, an activator of Rad51 recombinase, forms an extremely elongated dogleg-shaped structure

In eukaryotes, DNA strand exchange is the central reaction of homologous recombination, which is promoted by Rad51 recombinases forming a right-handed nucleoprotein filament on single-stranded DNA, also known as a presynaptic filament. Accessory proteins known as recombination mediators are required for the formation of the active presynaptic filament. One such mediator in the fission yeast Schizosaccharomyces pombe is the Swi5-Sfr1 complex, which has been identified as an activator of Rad51 that assists in presynaptic filament formation and stimulates its strand exchange reaction. Here, we determined the 1:1 binding stoichiometry between the two subunits of the Swi5-Sfr1 complex using analytical ultracentrifugation and electrospray ionization mass spectrometry. Small-angle x-ray scattering experiments revealed that the Swi5-Sfr1 complex displays an extremely elongated dogleg-shaped structure in solution, which is consistent with its exceptionally high frictional ratio (f/f(0)) of 2.0 ± 0.2 obtained by analytical ultracentrifugation. Furthermore, we determined a rough topology of the complex by comparing the small-angle x-ray scattering-based structures of the Swi5-Sfr1 complex and four Swi5-Sfr1-Fab complexes, in which the Fab fragments of monoclonal antibodies were specifically bound to experimentally determined sites of Sfr1. We propose a model for how the Swi5-Sfr1 complex binds to the Rad51 filament, in which the Swi5-Sfr1 complex fits into the groove of the Rad51 filament, leading to an active and stable presynaptic filament.     

Role for the mammalian Swi5-Sfr1 complex in DNA strand break repair through homologous recombination

In fission yeast, the Swi5-Sfr1 complex plays an important role in homologous recombination (HR), a pathway crucial for the maintenance of genomic integrity. Here we identify and characterize mammalian Swi5 and Sfr1 homologues. Mouse Swi5 and Sfr1 are nuclear proteins that form a complex in vivo and in vitro. Swi5 interacts in vitro with Rad51, the DNA strand-exchange protein which functions during HR. By generating Swi5(-/-) and Sfr1(-/-) embryonic stem cell lines, we found that both proteins are mutually interdependent for their stability. Importantly, the Swi5-Sfr1 complex plays a role in HR when Rad51 function is perturbed in vivo by expression of a BRC peptide from BRCA2. Swi5(-/-) and Sfr1(-/-) cells are selectively sensitive to agents that cause DNA strand breaks, in particular ionizing radiation, camptothecin, and the Parp inhibitor olaparib. Consistent with a role in HR, sister chromatid exchange induced by Parp inhibition is attenuated in Swi5(-/-) and Sfr1(-/-) cells, and chromosome aberrations are increased. Thus, Swi5-Sfr1 is a newly identified complex required for genomic integrity in mammalian cells with a specific role in the repair of DNA strand breaks.     

Reconstitution of DNA strand exchange mediated by Rhp51 recombinase and two mediators

In the fission yeast Schizosaccharomyces pombe, genetic evidence suggests that two mediators, Rad22 (the S. pombe Rad52 homolog) and the Swi5-Sfr1 complex, participate in a common pathway of Rhp51 (the S. pombe Rad51 homolog)–mediated homologous recombination (HR) and HR repair. Here, we have demonstrated an in vitro reconstitution of the central step of DNA strand exchange during HR. Our system consists entirely of homogeneously purified proteins, including Rhp51, the two mediators, and replication protein A (RPA), which reflects genetic requirements in vivo. Using this system, we present the first robust biochemical evidence that concerted action of the two mediators directs the loading of Rhp51 onto single-stranded DNA (ssDNA) precoated with RPA. Dissection of the reaction reveals that Rad22 overcomes the inhibitory effect of RPA on Rhp51-Swi5-Sfr1–mediated strand exchange. In addition, Rad22 negates the requirement for a strict order of protein addition to the in vitro system. However, despite the presence of Rad22, Swi5-Sfr1 is still essential for strand exchange. Importantly, Rhp51, but neither Rad22 nor the Swi5-Sfr1 mediator, is the factor that displaces RPA from ssDNA. Swi5-Sfr1 stabilizes Rhp51-ssDNA filaments in an ATP-dependent manner, and this stabilization is correlated with activation of Rhp51 for the strand exchange reaction. Rad22 alone cannot activate the Rhp51 presynaptic filament. AMP-PNP, a nonhydrolyzable ATP analog, induces a similar stabilization of Rhp51, but this stabilization is independent of Swi5-Sfr1. However, hydrolysis of ATP is required for processive strand transfer, which results in the formation of a long heteroduplex. Our in vitro reconstitution system has revealed that the two mediators have indispensable, but distinct, roles for mediating Rhp51 loading onto RPA-precoated ssDNA     

Fission yeast Swi5 protein, a novel DNA recombination mediator

The Schizosaccharomyces pombe Swi5 protein forms two distinct protein complexes, Swi5-Sfr1 and Swi5-Swi2, each of which plays an important role in the related but functionally distinct processes of homologous recombination and mating-type switching, respectively. The Swi5-Sfr1 mediator complex has been shown to associate with the two RecA-like recombinases, Rhp51 (spRad51) and Dmc1, and to stimulate in vitro DNA strand exchange reactions mediated by these proteins. Genetic analysis indicates that Swi5-Sfr1 works independently of another mediator complex, Rhp55-Rhp57, during Rhp51-dependent recombinational repair. In addition, mutations affecting the two mediators generate distinct repair spectra of HO endonuclease-induced DNA double strand breaks, suggesting that these recombination mediators differently regulate recombination outcomes in an independent manner.     

The Swi5-Sfr1 complex stimulates Rhp51/Rad51- and Dmc1-mediated DNA strand exchange in vitro

Nucleoprotein filaments made up of Rad51 or Dmc1 recombinases, the core structures of recombination, engage in ATP-dependent DNA-strand exchange. The ability of recombinases to form filaments is enhanced by recombination factors termed 'mediators'. Here, we show that the Schizosaccharomyces pombe Swi5-Sfr1 complex, a conserved eukaryotic protein complex, at substoichiometric concentrations stimulates strand exchange mediated by Rhp51 (the S. pombe Rad51 homolog) and Dmc1 on long DNA substrates. Reactions mediated by both recombinases are completely dependent on Swi5-Sfr1, replication protein A (RPA) and ATP, although RPA inhibits the reaction when it is incubated with single-stranded DNA (ssDNA) before the recombinase. The Swi5-Sfr1 complex overcomes, at least partly, the inhibitory effect of RPA, representing a novel class of mediator. Notably, the Swi5-Sfr1 complex preferentially stimulates the ssDNA-dependent ATPase activity of Rhp51, and it increases the amounts of Dmc1 bound to ssDNA.     

Multiple Regulation of Rad51-Mediated Homologous Recombination by Fission Yeast Fbh1

Fbh1, an F-box helicase related to bacterial UvrD, has been proposed to modulate homologous recombination in fission yeast. We provide several lines of evidence for such modulation. Fbh1, but not the related helicases Srs2 and Rqh1, suppressed the formation of crossover recombinants from single HO-induced DNA double-strand breaks. Purified Fbh1 in complex with Skp1 (Fbh1-Skp1 complex) inhibited Rad51-driven DNA strand exchange by disrupting Rad51 nucleoprotein filaments in an ATP-dependent manner; this disruption was alleviated by the Swi5-Sfr1 complex, an auxiliary activator of Rad51. In addition, the reconstituted SCF^Fbh1 complex, composed of purified Fbh1-Skp1 and Pcu1-Rbx1, displayed ubiquitin-ligase E3 activity toward Rad51. Furthermore, Fbh1 reduced the protein level of Rad51 in stationary phase in an F-box-dependent, but not in a helicase domain-independent manner. These results suggest that Fbh1 negatively regulates Rad51-mediated homologous recombination via its two putative, unrelated activities, namely DNA unwinding/translocation and ubiquitin ligation. In addition to its anti-recombinase activity, we tentatively suggest that Fbh1 might also have a pro-recombination role in vivo, because the Fbh1-Skp1 complex stimulated Rad51-mediated strand exchange in vitro after strand exchange had been initiated.     

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.     

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

Rad54, a member of the Swi2/Snf2 protein family, works in concert with the RecA-like recombinase Rad51 during the early and late stages of homologous recombination. Rad51 markedly enhances the activities of Rad54, including the induction of topological changes in DNA and the remodeling of chromatin structure. Reciprocally, Rad54 promotes Rad51-mediated DNA strand invasion with either naked or chromatinized DNA. Here, using various Saccharomyces cerevisiae rad51 and rad54 mutant proteins, mechanistic aspects of Rad54/Rad51-mediated chromatin remodeling are defined. Disruption of the Rad51-Rad54 complex leads to a marked attenuation of chromatin remodeling activity. Moreover, we present evidence that assembly of the Rad51 presynaptic filament represents an obligatory step in the enhancement of the chromatin remodeling reaction. Interestingly, we find a specific interaction of the N-terminal tail of histone H3 with Rad54 and show that the H3 tail interaction domain resides within the amino terminus of Rad54. These results suggest that Rad54-mediated chromatin remodeling coincides with DNA homology search by the Rad51 presynaptic filament and that this process is facilitated by an interaction of Rad54 with histone H3.     

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.     

Crystal structure of archaeal recombinase RADA: a snapshot of its extended conformation

Homologous recombination of DNA plays crucial roles in repairing severe DNA damage and in generating genetic diversity. The process is facilitated by a superfamily of recombinases: bacterial RecA, archaeal RadA and Rad51, and eukaryal Rad51 and DMC1. These recombinases share a common ATP-dependent filamentous quaternary structure for binding DNA and facilitating strand exchange. We have determined the crystal structure of Methanococcus voltae RadA in complex with the ATP analog AMP-PNP at 2.0 A resolution. The RadA filament is a 106.7 A pitch helix with six subunits per turn. The DNA binding loops L1 and L2 are located in close proximity to the filament axis. The ATP analog is buried between two RadA subunits, a feature similar to that of the active filament of Escherichia coli RecA revealed by electron microscopy. The disposition of the N-terminal domain suggests a role of the Helix-hairpin-Helix motif in binding double-stranded DNA.     

The F-Box DNA helicase Fbh1 prevents Rhp51-dependent recombination without mediator proteins

A key step in homologous recombination is the loading of Rad51 onto single-stranded DNA to form a nucleoprotein filament that promotes homologous DNA pairing and strand exchange. Mediator proteins, such as Rad52 and Rad55-Rad57, are thought to aid filament assembly by overcoming an inhibitory effect of the single-stranded-DNA-binding protein replication protein A. Here we show that mediator proteins are also required to enable fission yeast Rad51 (called Rhp51) to function in the presence of the F-box DNA helicase Fbh1. In particular, we show that the critical function of Rad22 (an orthologue of Rad52) in promoting Rhp51-dependent recombination and DNA repair can be mostly circumvented by deleting fbh1. Similarly, the reduced growth/viability and DNA damage sensitivity of an fbh1(-) mutant are variously suppressed by deletion of any one of the mediators Rad22, Rhp55, and Swi5. From these data we propose that Rhp51 action is controlled through an interplay between Fbh1 and the mediator proteins. Colocalization of Fbh1 with Rhp51 damage-induced foci suggests that this interplay occurs at the sites of nucleoprotein filament assembly. Furthermore, analysis of different fbh1 mutant alleles suggests that both the F-box and helicase activities of Fbh1 contribute to controlling Rhp51.     

The Rad51/RadA N-terminal domain activates nucleoprotein filament ATPase activity

Proteins in the RecA/RadA/Rad51 family form helical filaments on DNA that function in homologous recombination. While these proteins all have the same highly conserved ATP binding core, the RadA/Rad51 proteins have an N-terminal domain that shows no homology with the C-terminal domain found in RecA. Both the Rad51 N-terminal and RecA C-terminal domains have been shown to bind DNA, but no role for these domains has been established. We show that RadA filaments can be trapped in either an inactive or active conformation with respect to the ATPase and that activation involves a large rotation of the subunit aided by the N-terminal domain. The G103E mutation within the yeast Rad51 N-terminal domain inactivates the filament by failing to make proper contacts between the N-terminal domain and the core. These results show that the N-terminal domains play a regulatory role in filament activation and highlight the modular architecture of the recombination proteins.     

The budding yeast Mei5-Sae3 complex interacts with Rad51 and preferentially binds a DNA fork structure

Meiotic homologous recombination in Saccharomyces cerevisiae involves formation of nucleoprotein filaments of Rad51 and Dmc1 that mediate DNA strand exchange between homologous chromosomes. The Mei5-Sae3 protein complex functions as a recombination mediator to promote nucleation of the Dmc1 recombinase onto replication protein A-coated single-stranded DNA. Here, we have expressed and purified the Mei5 protein, Sae3 protein and the Mei5-Sae3 complex for biochemical studies. We show the Mei5-Sae3 complex preferentially binds a fork-like DNA substrate to 3' overhanging DNA, single-stranded DNA or double-stranded DNA. We demonstrate that Mei5 confers DNA binding activity to the Mei5-Sae3 complex. We determined Mei5-Sae3 interacts with the Rad51 recombinase through the N-terminal domain of Mei5. Unlike Rad52, Mei5-Sae3 lacks recombination mediator activity for Rad51. Importantly, we find that the Mei5-Sae3 complex does not harbor single-strand DNA annealing activity. These properties of the Mei5-Sae3 complex distinguishes it from the Rad52 protein, which serves as the mediator of Rad51 and is involved in the single-strand DNA annealing pathway of homologous recombination.     

Rrp1 translocase and ubiquitin ligase activities restrict the genome destabilising effects of Rad51 in fission yeast

Rad51 is the key protein in homologous recombination that plays important roles during DNA replication and repair. Auxiliary factors regulate Rad51 activity to facilitate productive recombination, and prevent inappropriate, untimely or excessive events, which could lead to genome instability. Previous genetic analyses identified a function for Rrp1 (a member of the Rad5/16-like group of SWI2/SNF2 translocases) in modulating Rad51 function, shared with the Rad51 mediator Swi5-Sfr1 and the Srs2 anti-recombinase. Here, we show that Rrp1 overproduction alleviates the toxicity associated with excessive Rad51 levels in a manner dependent on Rrp1 ATPase domain. Purified Rrp1 binds to DNA and has a DNA-dependent ATPase activity. Importantly, Rrp1 directly interacts with Rad51 and removes it from double-stranded DNA, confirming that Rrp1 is a translocase capable of modulating Rad51 function. Rrp1 affects Rad51 binding at centromeres. Additionally, we demonstrate in vivo and in vitro that Rrp1 possesses E3 ubiquitin ligase activity with Rad51 as a substrate, suggesting that Rrp1 regulates Rad51 in a multi-tiered fashion.     

The Rad51-dependent pairing of long DNA substrates is stabilized by replication protein A

Rad51 protein forms nucleoprotein filaments on single-stranded DNA (ssDNA) and then pairs that DNA with the complementary strand of incoming duplex DNA. In apparent contrast with published results, we demonstrate that Rad51 protein promotes an extensive pairing of long homologous DNAs in the absence of replication protein A. This pairing exists only within the Rad51 filament; it was previously undetected because it is lost upon deproteinization. We further demonstrate that RPA has a critical postsynaptic role in DNA strand exchange, stabilizing the DNA pairing initiated by Rad51 protein. Stabilization of the Rad51-generated DNA pairing intermediates can be can occur either by binding the displaced strand with RPA or by degrading the same DNA strand using exonuclease VII. The optimal conditions for Rad51-mediated DNA strand exchange used here minimize the secondary structure in single-stranded DNA, minimizing the established presynaptic role of RPA in facilitating Rad51 filament formation. We verify that RPA has little effect on Rad51 filament formation under these conditions, assigning the dramatic stimulation of strand exchange nevertheless afforded by RPA to its postsynaptic function of removing the displaced DNA strand from Rad51 filaments.     

Rad52 protein associates with replication protein A (RPA)-single-stranded DNA to accelerate Rad51-mediated displacement of RPA and presynaptic complex formation

The Rad51 nucleoprotein filament mediates DNA strand exchange, a key step of homologous recombination. This activity is stimulated by replication protein A (RPA), but only when RPA is introduced after Rad51 nucleoprotein filament formation. In contrast, RPA inhibits Rad51 nucleoprotein complex formation by prior binding to single-stranded DNA (ssDNA), but Rad52 protein alleviates this inhibition. Here we show that Rad51 filament formation is simultaneous with displacement of RPA from ssDNA. This displacement is initiated by a rate-limiting nucleation of Rad51 protein onto ssDNA complex, followed by rapid elongation of the filament. Rad52 protein accelerates RPA displacement by Rad51 protein. This acceleration probably involves direct interactions with both Rad51 protein and RPA. Detection of a Rad52-RPA-ssDNA co-complex suggests that this co-complex is an intermediate in the displacement process.     

Gly-103 in the N-terminal domain of Saccharomyces cerevisiae Rad51 protein is critical for DNA binding

Rad51 is a homolog of the bacterial RecA protein and is central for recombination in eukaryotes performing homology search and DNA strand exchange. Rad51 and RecA share a core ATPase domain that is structurally similar to the ATPase domains of helicases and the F1 ATPase. Rad51 has an additional N-terminal domain, whereas RecA protein has an additional C-terminal domain. Here we show that glycine 103 in the N-terminal domain of Saccharomyces cerevisiae Rad51 is important for binding to single-stranded and duplex DNA. The Rad51-G103E mutant protein is deficient in DNA strand exchange and ATPase activity due to a primary DNA binding defect. The N-terminal domain of Rad51 is connected to the ATPase core through an extended elbow linker that ensures flexibility of the N-terminal domain. Molecular modeling of the Rad51-G103E mutant protein shows that the negatively charged glutamate residue lies on the surface of the N-terminal domain facing a positively charged patch composed of Arg-260, His-302, and Lys-305 on the ATPase core domain. A possible structural explanation for the DNA binding defect is that a charge interaction between Glu-103 and the positive patch restricts the flexibility of the N-terminal domain. Rad51-G103E was identified in a screen for Rad51 interaction-deficient mutants and was shown to ablate the Rad54 interaction in two-hybrid assays (Krejci, L., Damborsky, J., Thomsen, B., Duno, M., and Bendixen, C. (2001) Mol. Cell. Biol. 21, 966-976). Surprisingly, we found that the physical interaction of Rad51-G103E with Rad54 was not affected. Our data suggest that the two-hybrid interaction defect was an indirect consequence of the DNA binding defect.     

Involvement of Schizosaccharomyces pombe rrp1+ and rrp2+ in the Srs2- and Swi5/Sfr1-dependent pathway in response to DNA damage and replication inhibition

Previously we identified Rrp1 and Rrp2 as two proteins required for the Sfr1/Swi5-dependent branch of homologous recombination (HR) in Schizosaccharomyces pombe. Here we use a yeast two-hybrid approach to demonstrate that Rrp1 and Rrp2 can interact with each other and with Swi5, an HR mediator protein. Rrp1 and Rrp2 form co-localizing methyl methanesulphonate–induced foci in nuclei, further suggesting they function as a complex. To place the Rrp1/2 proteins more accurately within HR sub-pathways, we carried out extensive epistasis analysis between mutants defining Rrp1/2, Rad51 (recombinase), Swi5 and Rad57 (HR-mediators) plus the anti-recombinogenic helicases Srs2 and Rqh1. We confirm that Rrp1 and Rrp2 act together with Srs2 and Swi5 and independently of Rad57 and show that Rqh1 also acts independently of Rrp1/2. Mutants devoid of Srs2 are characterized by elevated recombination frequency with a concomitant increase in the percentage of conversion-type recombinants. Strains devoid of Rrp1 or Rrp2 did not show a change in HR frequency, but the number of conversion-type recombinants was increased, suggesting a possible function for Rrp1/2 with Srs2 in counteracting Rad51 activity. Our data allow us to propose a model placing Rrp1 and Rrp2 functioning together with Swi5 and Srs2 in a synthesis-dependent strand annealing HR repair pathway.