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• W3122607166 abstract "Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Appendix 1 Data availability References Decision letter Author response Article and author information Metrics Abstract Homologous recombination (HR) is essential for maintaining genome stability. Although Rad51 is the key protein that drives HR, multiple auxiliary factors interact with Rad51 to potentiate its activity. Here, we present an interdisciplinary characterization of the interactions between Rad51 and these factors. Through structural analysis, we identified an evolutionarily conserved acidic patch of Rad51. The neutralization of this patch completely abolished recombinational DNA repair due to defects in the recruitment of Rad51 to DNA damage sites. This acidic patch was found to be important for the interaction with Rad55-Rad57 and essential for the interaction with Rad52. Furthermore, biochemical reconstitutions demonstrated that neutralization of this acidic patch also impaired the interaction with Rad54, indicating that a single motif is important for the interaction with multiple auxiliary factors. We propose that this patch is a fundamental motif that facilitates interactions with auxiliary factors and is therefore essential for recombinational DNA repair. eLife digest The DNA molecule contains the chemical instructions necessary for life. Its physical integrity is therefore vital, yet it is also under constant threat from external and internal factors. As a result, organisms have evolved an arsenal of mechanisms to repair damaged DNA. For instance, when the two complementary strands that form the DNA molecule are broken at the same location, the cell triggers a mechanism known as homologous recombination. A protein known as Rad51 orchestrates this process, helped by an array of other proteins that include Rad55-Rad57, Rad52, and Rad54. These physically bind to Rad51 and activate it in different ways. However, exactly how these interactions take place remained unclear. To find out more, Afshar et al. examined models of the structure of Rad51, revealing that three of the protein’s building blocks create a prominent, negatively charged patch that could be important for DNA repair. Yeast cells were then genetically manipulated to produce a modified version of Rad51 in which the three building blocks were neutralised. These organisms were unable to repair their DNA. Further biochemical tests showed that the modified protein could no longer attach well to Rad55-Rad57 or Rad54, and could not stick to Rad52 at all. In fact, without its negatively charged patch, Rad51 could not find the ends of broken DNA strands, a process which is normally aided by Rad55-Rad57 and Rad52. Taken together, these results suggest that the helper proteins all interact with Rad51 in the same place, even though they play different roles. Faulty DNA repair processes have been linked to devastating consequences such as cell death or cancer. Understanding the details of DNA repair in yeast can serve as a template for research in more complex organisms, opening the possibility of applications for human health. Introduction Exogenous factors such as ionizing radiation and genotoxic chemicals can cause DNA damage. However, endogenous processes such as DNA replication and cellular metabolism can also damage DNA (Lambert and Carr, 2013). A particularly severe form of DNA damage is a DNA double-strand break (DSB), in which a single normal chromosome is separated into two pathological chromosomes. Homologous recombination (HR) is a major mechanism responsible for accurately repairing DSBs. HR is also critically important for DNA replication (Ait Saada et al., 2018). Accordingly, defects in HR lead to genome instability, which drives human diseases such as cancer (Prakash et al., 2015). During HR, the DNA ends that are exposed at a DSB are resected to form 3’ overhangs, which are immediately coated by the single-stranded DNA (ssDNA) binding protein RPA (Symington and Gautier, 2011). The RecA-family recombinase Rad51 then displaces RPA to form a helical nucleoprotein filament known as the presynaptic filament (Sun et al., 2020). This filament can locate a segment of double-stranded DNA (dsDNA) with substantial sequence similarity (i.e.,homology) to the ssDNA (Greene, 2016). Upon identifying a homologous region, the Rad51 filament invades the intact dsDNA, displacing the non-complementary strand and forming base pairs with the complementary strand. Within the context of this displacement loop (D-loop) recombination intermediate, the 3’-end of the invading strand can be extended by utilizing the complementary strand as a template for DNA synthesis, allowing for the recovery of lost genetic information (McVey et al., 2016). The D-loop can also be expanded by Rad51-driven DNA strand exchange, which increases the extent of base pairing between the two DNA molecules. Consequently, D-loops can be processed to form Holliday junctions, which may be resolved as either crossover or non-crossover products, or they may be disassembled prior to Holliday junction formation, resulting exclusively in non-crossover outcomes (Mehta and Haber, 2014). As the entity capable of identifying homology and driving DNA strand exchange, Rad51 is integral to DNA repair by HR (Shinohara et al., 1992; Muris et al., 1993; Sung, 1994). However, Rad51 does not function alone in vivo. Several other proteins that are required for HR have been identified in the fission yeast Schizosaccharomyces pombe including Rad52, Rad54, the Rad51 paralogs Rad55-Rad57, Swi5-Sfr1, and the lesser studied Shu complex (Ostermann et al., 1993; Muris et al., 1996; Khasanov et al., 1999; Tsutsui et al., 2000; Akamatsu et al., 2003; Khasanov et al., 2004; Martín et al., 2006). These factors are mostly conserved in the budding yeast Saccharomyces cerevisiae despite the large evolutionary distance separating the two yeasts, although it should be noted that the S. cerevisiae homolog of Swi5-Sfr1 (Mei5-Sae3) is only involved in meiotic HR (San Filippo et al., 2008; Hoffman et al., 2015; Argunhan et al., 2017a). This suggests that the requirement for a diverse array of auxiliary factors to promote recombinational DNA repair has been conserved throughout evolution, highlighting its importance. However, our understanding of how auxiliary factors promote Rad51 activity remains incomplete, although they seem to perform largely non-overlapping roles (Zelensky et al., 2014). Sfr1 was first identified in S. pombe as an interactor of Rad51 that forms a complex with Swi5 specifically involved in promoting Rad51-dependent DNA repair (Akamatsu et al., 2003). The Swi5-Sfr1 heterodimer stimulates DNA strand exchange by potentiating Rad51’s ATPase activity and stabilizing Rad51 filaments (Haruta et al., 2006; Kurokawa et al., 2008). In addition to being widely conserved among eukaryotes, the mechanisms through which Swi5-Sfr1 promotes HR appear to be highly similar in yeasts and mammals (Tsai et al., 2012; Su et al., 2014; Su et al., 2016; Argunhan et al., 2017a; Lu et al., 2018). The Rad51 paralogs Rad55-Rad57 are another group of evolutionarily conserved auxiliary factors. Rad55 and Rad57 were identified in S. pombe based on sequence homology and genetic screening, respectively (Khasanov et al., 1999; Tsutsui et al., 2000). Relatively little is known about the molecular function of Rad55-Rad57 due to the biochemical intractability of the complex, although much like Swi5-Sfr1, it is thought to be an obligate heterodimer. The biochemical analysis that has been performed with S. cerevisiae proteins suggests that Rad55-Rad57 promotes Rad51 filament formation on RPA-coated ssDNA and protects the Rad51 filament from disruption by the Srs2 anti-recombinase (Sung, 1997; Liu et al., 2011). This is consistent with cytological observations in both S. cerevisiae and S. pombe indicating that the number of DNA damage-induced Rad51 foci, which represent Rad51 filaments at sites of ongoing DNA repair, are reduced in the absence of Rad55/Rad57 (Gasior et al., 1998; Gasior et al., 2001; Akamatsu et al., 2007). Among recombination auxiliary factors, the absence of Rad52 results in the most severe phenotype, with deletion mutants displaying DNA damage sensitivity exceeding the rad51∆ single mutant; this has been attributed to the absolute dependency of Rad51 on Rad52, as well as Rad51-independent functions of Rad52 (Doe et al., 2004). The rad54∆ mutant also shows severe DNA damage sensitivity that is indistinguishable from rad51∆ (Muris et al., 1997), highlighting the absolute requirement for Rad54 in Rad51-dependent DNA repair. By contrast, the rad57Δ and sfr1Δ mutants show only moderate sensitivity to DNA damage, while the rad57Δ sfr1Δ double mutant is as sensitive as the rad51Δ single mutant. Based on this additivity, it was proposed that Rad55-Rad57 and Swi5-Sfr1 comprise independent sub-pathways of HR that function in parallel to promote Rad51-dependent DNA repair (Akamatsu et al., 2003; Akamatsu et al., 2007), although recent evidence has evoked a re-examination of this model (Argunhan et al., 2020). To learn more about the relationship between Rad51 and its auxiliary factors, we sought to identify regions of Rad51 that are important for interactions with auxiliary factors. This led to the identification of an evolutionarily conserved acidic patch comprised of three residues: E205, E206, and D209. Mutation of all three residues to Ala completely ablates Rad51-dependent DNA repair, as does a single charge-reversal mutation, indicating that the negative character of this patch is critical for DNA repair. Mechanistically, these defects in DNA repair stem from abrogation of the interaction with both Rad55-Rad57 and Rad52, leading to impaired recruitment of Rad51 to sites of DNA damage. Remarkably, biochemical reconstitutions indicate that neutralization of the acidic patch also impairs the interaction with Rad54, demonstrating that a single motif of Rad51 is important for its interaction with Rad55-Rad57, Rad52, and Rad54. We propose that this acidic patch of Rad51 comprises a fundamental motif that is essential for interactions with auxiliary factors and therefore recombinational DNA repair. Results E205, E206, and D209 comprise a protruding acidic patch (PAP) on the exterior of the Rad51 presynaptic filament Several motifs important for the enzymatic activity of Rad51 are located in the highly conserved ATPase core domain, which is characterized by a β-sheet consisting of mixed parallel and antiparallel β-strands (Story et al., 1992; Pellegrini et al., 2002; Shin et al., 2003; Conway et al., 2004). These include the Walker A and B motifs, which are important for ATP binding and hydrolysis (Saraste et al., 1990; Story and Steitz, 1992), and two DNA binding sites: Site 1, which is comprised of Loop 1 and Loop 2, and Site 2 (Howard-Flanders et al., 1984; Story et al., 1992). Examination of a homology (i.e., computational) model of S. pombe Rad51 (SpRad51) revealed that the surface of these regions is enriched in positive charge, consistent with roles in the binding of ATP and DNA (Figure 1A, left). By contrast, mostly negatively charged regions were found on the opposite face of SpRad51, including a protruding acidic patch, which we refer to as the PAP hereafter (Figure 1A, right). The PAP is among the most negatively charged regions on the surface of SpRad51 (Figure 1—figure supplement 1A) and is situated on a short α-helix preceding the outermost β-strand of the central β-sheet (Figure 1B and Video 1). Three acidic residues were seen to project out from this α-helix: E205, E206, and D209 (Figure 1C,D and Video 2). Figure 1 with 1 supplement see all Download asset Open asset E205, E206, and D209 form a protruding acidic patch (PAP) on the exterior of the Rad51 presynaptic filament. (A) Homology model of an SpRad51 monomer (residues 42–360). Surface representation colored according to Coulombic surface charge. The molecule on the left is rotated 180° to visualize the PAP on the right, with the region of interest squared. (B) Ribbon depiction of an SpRad51 monomer with relevant motifs highlighted. The molecules are oriented as in (A). (C) Surface representation colored according to Coulombic surface charge. The PAP is enlarged with a semi-transparent surface revealing residues E205, E206, and D209, which have their side-chains shown. (D) Ribbon depiction of SpRad51 with the α-helix containing the PAP enlarged to illustrate the respective positions of each residue (colored in cyan) with their side-chains revealed (O atoms in red). Sequence alignment shows the corresponding region in S. pombe, S. cerevisiae and H. sapiens (Sp, Sc, and Hs, respectively), with arrows indicating PAP residues in S. pombe and acidic residues highlighted in red. (E) Ribbon depiction of three SpRad51 monomers (alternating orange and purple) bound to ssDNA (9-mer poly-dT in gray) with a near-transparent surface for visualization (left). The side-chains of E205, E206, and D209 are revealed in cyan (O atoms in red) and their positions are squared. The surface is made opaque and colored according to Coulombic surface charge to demonstrate that the PAP constitutes dense, negatively charged regions on the exterior of the ssDNA filament (right). Numbers in the legends for (A,C,E) are in units of kcal/(mol•e). Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg A model of the SpRad51 monomer with motifs labeled. A homology model of the SpRad51 monomer is depicted in ribbon form and labeled as follows: N and C, N- and C-termini; L1 and L2, loop 1 and loop 2 of DNA binding site 1; A and B, Walker A and Walker B motifs; Site 2, DNA binding site 2. Residues E205, E206, and D209, which constitute the protruding acidic patch (PAP), are shown in cyan following 180° rotation of the model in the y-axis. Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Visualization of the protruding acidic patch (PAP) in monomer form. A homology model of the SpRad51 monomer is depicted in ribbon form. The side-chains of residues E205, E206, and D209 are shown, intermittently highlighted in cyan (O atoms in red). The surface is made opaque and colored according to Coulombic surface charge to demonstrate that these residues constitute a dense negatively charged patch that we refer to as the PAP. These residues were also examined in the context of a previously published homology model of the SpRad51 presynaptic filament (Ito et al., 2020) and found to form acidic patches constituting dense negatively charged regions on the exterior of the filament (Figure 1E and Video 3). The equivalent α-helix in the human Rad51 (HsRad51) presynaptic filament—the structure of which was determined by cryo-electron microscopy (Xu et al., 2017)—also had a negative surface charge (Figure 1—figure supplement 1B), as did the corresponding α-helix in the S. cerevisiae Rad51 (ScRad51) presynaptic filament (Figure 1—figure supplement 1C)—the structure of which was determined by X-ray crystallography (Conway et al., 2004). While E205 was replaced with a conservative Asp residue in ScRad51 and D209 was conserved in HsRad51, E206 is the only PAP residue that showed conservation in both ScRad51 and HsRad51 (Figure 1D). Thus, we initially focused on E206. Video 3 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Visualization of the protruding acidic patch (PAP) in filament form. A homology model of the SpRad51-ssDNA filament. Ribbon depiction of three monomers (alternating orange and purple) bound to ssDNA (9-mer poly-dT in gray). The side-chains of residues E205, E206, and D209 are shown, intermittently highlighted in cyan. The surface is made opaque and colored according to Coulombic surface charge to demonstrate that these residues constitute dense negatively charged patches on the exterior of the ssDNA filament. Rad51-E206A is specifically defective in the interaction with Rad55-Rad57 HR plays a particularly important role in the repair of ultraviolet light (UV)-induced DNA damage in S. pombe due to the existence of a UV damage endonuclease pathway (McCready et al., 2000). To examine whether E206 is important for DNA repair, it was mutated to Ala and a strain containing this mutation at the native locus was constructed. rad51-E206A showed the same resistance to UV-induced DNA damage as wild type in a clonogenic survival assay (Figure 2A), suggesting that this mutation does not affect the intrinsic ability of Rad51 to repair DNA. Figure 2 with 1 supplement see all Download asset Open asset The rad51-E206A mutant is specifically defective in the interaction with Rad55-Rad57. (A–C) Following acute UV irradiation, a clonogenic assay was employed to test the survival of rad51+, rad51-E206A, and rad51Δ in the wild-type background (A), the rad57Δ background (B), and the sfr1Δ background (C). Statistical significance at the highest dose of UV was assessed by unpaired two-tailed t-test. n.s., not significant (A, p=0.506; B, p=0.242; C, p=0.593). (D) Tenfold serial dilutions of the indicated strains were spotted onto standard media without treatment or containing hydroxyurea (HU). Following growth for the indicated time at 30°C, plates were imaged. (E) Soluble cell extracts treated with a benzonase-like nuclease were prepared from each strain under native conditions (input). Immunoprecipitation (IP) was then performed with mock (human IgG from non-immunized animal) or anti-V5 antibodies. Tubulin serves as a loading control. Data in (A–C) are means of three independent experiments and error bars depict standard deviation. Figure 2—source data 1 Survival (%) following UV irradiation for data in Figure 2A–C. https://cdn.elifesciences.org/articles/64131/elife-64131-fig2-data1-v1.xlsx Download elife-64131-fig2-data1-v1.xlsx In the rad57∆/rad55∆ background, Rad51-mediated HR is reduced but not abolished, and this remaining recombinational DNA repair is dependent on Swi5-Sfr1. There is a similar reduction in recombinational DNA repair in the sfr1∆/swi5∆ background, where the remaining Rad51-mediated HR is dependent on Rad55-Rad57. However, the rad57∆ sfr1∆ double mutant displays a complete loss of Rad51-mediated DNA repair, phenocopying rad51∆. Thus, employing the rad57∆ and sfr1∆ backgrounds allows for the evaluation of DNA repair promoted by Swi5-Sfr1 and Rad55-Rad57, respectively (Akamatsu et al., 2003). The rad51-E206A rad57∆ strain was no more sensitive to UV than rad57∆ (Figure 2B), suggesting that Rad51-E206A is as proficient as wild-type Rad51 in Swi5-Sfr1–dependent DNA repair. By contrast, rad51-E206A was as sensitive as rad51∆ in the absence of Sfr1 (Figure 2C), indicating that the recombinational DNA repair promoted solely by Rad55-Rad57 is ablated by the Rad51-E206A mutation. The generality of these findings was confirmed by performing spot tests with several different genotoxins that induce replication fork stalling and a variety of lesions in DNA (Figure 2—figure supplement 1A–C). The DNA damage sensitivity of sfr1∆ rad51-E206A is comparable in severity to rad51∆, which itself shows similar sensitivity to rad57∆ sfr1∆ and rad54∆ (Muris et al., 1997; Akamatsu et al., 2003). The rad52∆ mutant is even more sensitive to DNA damage, although this added sensitivity stems from Rad51-independent roles of Rad52 (Doe et al., 2004); a mutation that abolishes the interaction between Rad51 and Rad52 would be expected to phenocopy rad51∆, not rad52∆. Thus, it is possible that the DNA damage sensitivity associated with rad51-E206A, which manifests in the sfr1∆ background, reflects a defect in the interaction of Rad51 with Rad52 or Rad54, rather than with Rad55-Rad57. We sought to distinguish between these possibilities genetically. Rqh1 is a RecQ-family helicase (homologous to Sgs1 in S. cerevisiae and the BLM and WRN helicases in humans) that functions in S phase to prevent the accumulation of toxic recombination intermediates that arise during DNA replication (Murray et al., 1997; Stewart et al., 1997). Accordingly, rqh1∆ cells are sensitive to the ribonucleotide reductase inhibitor hydroxyurea (HU). It was previously shown that rad57∆ and sfr1∆ robustly suppress the HU sensitivity of rqh1∆ whereas rad52∆ and rad54∆ do not, presumably because the former mutations reduce HR while the latter mutations eliminate it completely (Hope et al., 2005). If Rad51-E206A is defective in the interaction with Rad55-Rad57 rather than Rad52/Rad54, then the rqh1∆ rad51-E206A strain should phenocopy rqh1∆ rad57∆ rather than rqh1∆ rad52∆/rad54∆. Consistently, rad51-E206A suppressed the HU sensitivity of rqh1∆ to almost the same degree as rad57∆ (Figure 2D). Furthermore, the suppression conferred by sfr1∆, which is dependent on Rad55-Rad57 (Hope et al., 2005), was ablated by rad51-E206A. By contrast, the suppression imparted by rad57∆ was epistatic to rad51-E206A, suggesting that they involve the same mechanism. At 5 mM HU, rad57∆ sfr1∆ could partially suppress rqh1∆ sensitivity, whereas rad51∆ and rad54∆ could not. Importantly, the sfr1∆ rad51-E206A strain was still similar to rad57∆ sfr1∆, strongly suggesting that the defect associated with rad51-E206A is related to Rad55-Rad57. Immunoblotting experiments revealed that the level of Rad51-E206A was comparable to wild-type Rad51 (Figure 2—figure supplement 1D), indicating that the DNA repair defect of rad51-E206A is not caused by reduced protein stability. Previous yeast two-hybrid (Y2H) analysis suggested that Rad51 physically interacts with Rad55-Rad57 (Hays et al., 1995; Johnson and Symington, 1995; Tsutsui et al., 2001). Thus, a feasible explanation for the DNA damage sensitivity of rad51-E206A is that the E206A mutation disrupts the physical interaction between Rad51 and Rad55-Rad57. To test this, a sequence encoding 12 copies of the V5 epitope was fused to rad55+ at its native locus, yielding the rad55-12xV5 strain. This strain was as resistant to DNA damage as the untagged wild type (rad55+; Figure 2—figure supplement 1E), indicating that the tag does not interfere with the function of Rad55-Rad57 in DNA repair. In vivo co-immunoprecipitation (co-IP) experiments were performed in both sfr1+ and sfr1∆ backgrounds. While robust signal was observed for wild-type Rad51, substantially less Rad51-E206A was seen to co-IP with Rad55 (Figure 2E), indicating that the E206A mutation impairs Rad51–Rad55-Rad57 complex formation. The presence of Sfr1 did not have an effect on complex formation, irrespective of the E206A mutation. These results suggest that the DNA repair defect associated with rad51-E206A is related to impaired Rad51–Rad55-Rad57 complex formation and that the suppression of this DNA damage sensitivity by Swi5-Sfr1 is not through enhancing physical binding. Rad51-E206A retains normal recombinase activity and can be stimulated by Swi5-Sfr1 Although E206 does not belong to a canonical motif involved in ATP hydrolysis or DNA binding, it remained formally possible that the E206A mutation impaired the enzymatic activity of Rad51. Rad51-E206A was therefore purified to homogeneity from Escherichia coli to investigate its biochemical properties (Figure 3—figure supplement 1). Rad51-E206A shifted both ssDNA and dsDNA to a similar extent as wild-type Rad51 in electrophoretic mobility shift assays (EMSAs; Figure 3A,B), suggesting that the E206A mutation does not affect the ability of Rad51 to bind DNA. In addition to DNA binding, ATP hydrolysis by Rad51 is important for the DNA strand exchange reaction (Ito et al., 2018). Furthermore, Swi5-Sfr1 stimulates the ATPase activity of Rad51 (Haruta et al., 2006). We therefore examined whether Rad51-E206A is proficient for ATP hydrolysis, both with and without Swi5-Sfr1. In the absence of Swi5-Sfr1, the ATP turnover number (kcat) of both Rad51 and Rad51-E206A was ~0.2 min−1 (Figure 3C). The inclusion of Swi5-Sfr1 elicited an approximately twofold increase in ATP hydrolysis by both proteins, demonstrating that Rad51-E206A can hydrolyze ATP like wild type and is proficient for the ATPase stimulation imparted by Swi5-Sfr1. Figure 3 with 1 supplement see all Download asset Open asset Rad51-E206A retains normal DNA binding and recombinase activity. (A,B) The indicated concentrations of Rad51 (WT) or Rad51-E206A (E206A) were incubated with 30 micromolar nucleotide (µM nt) PhiX174 virion DNA (ssDNA; A) or 20 µM nt of linearized PhiX174 RF I DNA (dsDNA; B), protein-DNA complexes were crosslinked with glutaraldehyde and then resolved by agarose gel electrophoresis. (C) The ATPase activity of 5 µM Rad51 (WT) and Rad51-E206A (EA) was measured in the presence of ssDNA (10 µM nt), with or without Swi5-Sfr1 (0.5 µM), and kcat was calculated. (D) Schematic of the strand exchange assay with full-length DNA substrates (PhiX174 virion ssDNA and ApaLI-linearized PhiX174 RF I dsDNA). (E) Strand exchange reactions were conducted according to the scheme outlined above the gel. Rad51 (WT or E206A), 15 µM. RPA, 1 µM. cssDNA, 30 µM nt. ldsDNA, 20 µM nt. (F) Strand exchange reactions were conducted according to the scheme outlined above the gel. Rad51 (WT or E206A), 5 µM. Swi5-Sfr1 (S5S1), 0.5 µM. RPA, 1 µM. cssDNA, 10 µM nt. ldsDNA, 10 µM nt. Data in (C,E,F) are means of three independent experiments and error bars depict standard deviation. Figure 3—source data 1 kcat values in Figure 3C and strand exchange yield (%) in Figure 3E,F. https://cdn.elifesciences.org/articles/64131/elife-64131-fig3-data1-v1.xlsx Download elife-64131-fig3-data1-v1.xlsx Next, an assay with plasmid-sized DNA substrates was employed to examine the strand exchange activity of Rad51-E206A (Figure 3D). Rad51 drives the pairing of circular ssDNA (css) with homologous linear dsDNA (lds) to yield joint molecule intermediates (JMs). Following strand transfer over the length of the dsDNA substrate, JMs are converted into nicked-circular dsDNA molecules (NCs), which are the products in this assay. The different DNA species can be separated by agarose gel electrophoresis and visualized. Under our standard reaction conditions, wild-type Rad51 cannot promote JM or NC formation in the absence of auxiliary factors (Haruta et al., 2006; Kurokawa et al., 2008). However, when reaction conditions were modified by increasing the concentrations of Rad51 and DNA substrates, both Rad51 and Rad51-E206A were seen to drive the efficient pairing of css and lds to produce JMs in the absence of auxiliary factors (Figure 3E). This allowed us to evaluate whether the E206A mutation impaired the intrinsic strand exchange activity of Rad51. Although neither protein was able to drive the robust accumulation of NC, we nevertheless quantified the total yield (JM + NC) at each time point and found them to be comparable for both Rad51 and Rad51-E206A. Our genetic analysis suggested that Rad51-E206A is proficient for the DNA repair promoted by Swi5-Sfr1. To corroborate these findings, strand exchange reactions were supplemented with Swi5-Sfr1. Note that under these standard assay conditions, which differ from those employed above, Rad51 alone cannot promote JM or NC formation, thus allowing us to better examine the effect of Swi5-Sfr1 (Haruta et al., 2006; Kurokawa et al., 2008). The inclusion of Swi5-Sfr1 efficiently stimulated both Rad51 and Rad51-E206A, with a similar accumulation of JMs and NC observed in both cases (Figure 3F). Taken together, these results indicate that Rad51-E206A retains intrinsic recombinase activity and a functional interaction with Swi5-Sfr1. The PAP is essential for Rad51-dependent DNA repair Our results with Rad51-E206A suggested that the PAP is required specifically for the interaction with Rad55-Rad57. We sought to test whether other mutations in the PAP also affect the interaction with Rad55-Rad57. Strains were constructed in which the rad51+ gene at its native locus was replaced with rad51-E205A, rad51-D209A, or rad51-EED (E205A, E206A, D209A). Structural models revealed that the negative surface charge of the PAP is completely neutralized by the EED mutation without affecting its protruding nature (Figure 4—figure supplement 1A,B). The DNA damage sensitivity of these mutant strains was assessed by spot-test. Like rad51-E206A, the rad51-E205A strain did not show any DNA damage sensitivity in the presence of both Rad55-Rad57 and Swi5-Sfr1 (Figure 4A). By contrast, rad51-D209A showed marked sensitivity to DNA damage, although it was still significantly more resistant than rad51∆. Strikingly, the rad51-EED mutant was as sensitive to DNA damage as rad51∆. Since the E205A and E206A mutations alone did not sensitize cells to DNA damage, but combining them with the partially functional D209A completely incapacitated Rad51, we also examined the rad51-EE strain, in which both Glu residues are mutated to Ala. Unlike rad51-E205A and rad51-E206A, rad51-EE showed moderate sensitivity to DNA damage, although this was milder than the sensitivity of rad51-D209A. These results indicate that neutralization of the PAP completely abolishes Rad51-dependent DNA repair, and while all three residues are important, D209 plays a more prominent role than E205 and E206. Figure 4 with 1 supplement see all Download asset Open asset The PAP" @default.
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• W3122607166 title "Author response: A novel motif of Rad51 serves as an interaction hub for recombination auxiliary factors" @default.
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