Regulation of Hed1 and Rad54 binding during maturation of the meiosis‐specific presynaptic complex
2018; Springer Nature; Volume: 37; Issue: 7 Linguagem: Inglês
10.15252/embj.201798728
ISSN1460-2075
AutoresJ. Brooks Crickard, Kyle Kaniecki, Youngho Kwon, Patrick Sung, Michael Lisby, Eric C. Greene,
Tópico(s)Microtubule and mitosis dynamics
ResumoArticle14 February 2018free access Source DataTransparent process Regulation of Hed1 and Rad54 binding during maturation of the meiosis-specific presynaptic complex J Brooks Crickard Department of Biochemistry & Molecular Biophysics, Columbia University, New York, NY, USA Search for more papers by this author Kyle Kaniecki Department of Genetics and Development, Columbia University, New York, NY, USA Search for more papers by this author YoungHo Kwon Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Patrick Sung Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Michael Lisby Department of Biology, University of Copenhagen, Copenhagen N, Denmark Search for more papers by this author Eric C Greene Corresponding Author [email protected] orcid.org/0000-0002-7387-824X Department of Biochemistry & Molecular Biophysics, Columbia University, New York, NY, USA Search for more papers by this author J Brooks Crickard Department of Biochemistry & Molecular Biophysics, Columbia University, New York, NY, USA Search for more papers by this author Kyle Kaniecki Department of Genetics and Development, Columbia University, New York, NY, USA Search for more papers by this author YoungHo Kwon Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Patrick Sung Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Michael Lisby Department of Biology, University of Copenhagen, Copenhagen N, Denmark Search for more papers by this author Eric C Greene Corresponding Author [email protected] orcid.org/0000-0002-7387-824X Department of Biochemistry & Molecular Biophysics, Columbia University, New York, NY, USA Search for more papers by this author Author Information J Brooks Crickard1, Kyle Kaniecki2, YoungHo Kwon3, Patrick Sung3, Michael Lisby4 and Eric C Greene *,1 1Department of Biochemistry & Molecular Biophysics, Columbia University, New York, NY, USA 2Department of Genetics and Development, Columbia University, New York, NY, USA 3Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT, USA 4Department of Biology, University of Copenhagen, Copenhagen N, Denmark *Corresponding author. Tel: +1 212 342 2944; E-mail: [email protected] EMBO J (2018)37:e98728https://doi.org/10.15252/embj.201798728 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Most eukaryotes have two Rad51/RecA family recombinases, Rad51, which promotes recombination during mitotic double-strand break (DSB) repair, and the meiosis-specific recombinase Dmc1. During meiosis, the strand exchange activity of Rad51 is downregulated through interactions with the meiosis-specific protein Hed1, which helps ensure that strand exchange is driven by Dmc1 instead of Rad51. Hed1 acts by preventing Rad51 from interacting with Rad54, a cofactor required for promoting strand exchange during homologous recombination. However, we have a poor quantitative understanding of the regulatory interplay between these proteins. Here, we use real-time single-molecule imaging to probe how the Hed1- and Rad54-mediated regulatory network contributes to the identity of mitotic and meiotic presynaptic complexes. Based on our findings, we define a model in which kinetic competition between Hed1 and Rad54 helps define the functional identity of the presynaptic complex as cells undergo the transition from mitotic to meiotic repair. Synopsis Hed1 facilitates the switch from Rad51-dependent repair of DNA double strand breaks to the meiosis-specific recombinase Dmc1. Single-molecule imaging data indicate that kinetic competition between Hed1 and the cofactor Rad54 helps define the identity of the presynaptic complex during the transition from mitotic to meiotic repair. Single-molecule microscopy visualizes Hed1 and Rad54 binding to the Rad51 presynaptic complex in real time. Complexes formed between Rad54 and either Rad51 or Dmc1 are highly stable, and apparently irreversible on biological time scales. Rad54 can remain bound to ssDNA after the dissociation of Rad51 and may thus interact with the presynaptic complex through direct ssDNA contacts. Hed1 dissociates from the presynaptic complex during disassembly of Rad51 filaments, suggesting that Hed1 binding occurs through direct contact with Rad51. Introduction Homologous recombination (HR) is a universally conserved process that is used to repair double-strand breaks (DSBs), protect genomic integrity, and ensure genetic diversity within populations (Neale & Keeney, 2006; San Filippo et al, 2008; Symington et al, 2014). HR proceeds through the resection of broken double-strand DNA (dsDNA), yielding long single-strand DNA (ssDNA) overhangs, which are paired with a homologous dsDNA elsewhere in the genome, that is used as a template for repair of the damaged DNA (San Filippo et al, 2008; Symington et al, 2014). During HR, the 3′ ssDNA overhang is first bound by the heterotrimeric eukaryotic ssDNA-binding protein RPA (replication protein A), which protects the ssDNA from nucleases, removes secondary structure, and serves as a signal for initiating the DNA damage response (Wold, 1997; San Filippo et al, 2008; Chen & Wold, 2014; Symington et al, 2014). RPA is then replaced by Rad51, which forms an extended right-handed helical filament on the 3′ ssDNA overhang (Conway et al, 2004; Chen et al, 2008; Sheridan et al, 2008; Kowalczykowski, 2015; Morrical, 2015). The resulting nucleoprotein filament is referred to as the presynaptic complex (San Filippo et al, 2008; Heyer et al, 2010; Symington et al, 2014). The Rad51 presynaptic complex pairs the bound ssDNA with the complementary strand of a homologous dsDNA, and this strand invasion reaction results in displacement of the non-complementary strand. The resulting D-loop intermediate can be processed through several different pathways, leading to repair of the damaged DNA (Paques & Haber, 1999; San Filippo et al, 2008; Heyer et al, 2010; Mehta & Haber, 2014; Symington et al, 2014). Rad54 is a member of the Swi/Snf family of dsDNA translocases, it is a required cofactor that stimulates the strand invasion activity of Rad51, and it is one of the most highly conserved eukaryotic HR proteins (Heyer et al, 2006; Mazin et al, 2010; Ceballos & Heyer, 2011; Kowalczykowski, 2015). Rad54 deletion imparts sensitivity to DNA-damaging agents (Petukhova et al, 1999; Wesoly et al, 2006), causes defects in strand invasion (Sugawara et al, 2003; Renkawitz et al, 2013), and leads to the accumulation of toxic HR intermediates (Shah et al, 2010). Rad54 is a key accessory factor for regulating Rad51 activity. Association with the presynaptic complex stimulates the ATP hydrolysis activity of Rad54 and greatly increases the efficiency of Rad51-mediated strand invasion (Jiang et al, 1996; Petukhova et al, 1998; Mazin et al, 2000; Van Komen et al, 2000; Raschle et al, 2004). Collectively, it is believed that protein–protein interactions between Rad51 and Rad54 enhance both the homology search and strand invasion during mitotic HR (Kowalczykowski, 2015). In addition, Rad54 has been implicated in stabilization of the Rad51 presynaptic complex (Mazin et al, 2003), DNA branch migration (Bugreev et al, 2006; Rossi & Mazin, 2008), nucleosome remodeling (Alexiadis & Kadonaga, 2002; Alexeev et al, 2003; Jaskelioff et al, 2003), and removal of Rad51 after completion of strand exchange (Symington & Heyer, 2006; Ceballos & Heyer, 2011; Wright & Heyer, 2014). During meiosis, HR-mediated repair of programmed DSBs creates a physical linkage between homologous chromosomes, helping to ensure proper chromosome segregation and allowing for the creation of new allelic combinations (Lao & Hunter, 2010; Brown & Bishop, 2014; Lam & Keeney, 2014; Zickler & Kleckner, 2015). Meiotic recombination requires a number of meiosis-specific proteins and also coincides with the selective inhibition of Rad51 (Neale & Keeney, 2006; Brown & Bishop, 2014). Rad51 inhibition is achieved through two meiosis-specific regulatory proteins, Hed1 and Mek1 (Niu et al, 2009; Brown & Bishop, 2014). Mek1 is a kinase that phosphorylates a number of proteins during meiosis, including Rad54 (Niu et al, 2009) and histone H3 (Govin et al, 2010; Kniewel et al, 2017), to help suppress sister-directed repair (Liu et al, 2014). Hed1 is a regulatory factor that interferes with Rad54 binding to Rad51 (Busygina et al, 2008, 2012). Rad54 is a required cofactor for Rad51 strand invasion activity in vivo; therefore, Hed1 binding downregulates the activity of Rad51 in meiosis. Hed1 deletion results in delayed appearance of meiotic DSBs and delayed production of crossover recombination products (Lao et al, 2013). Hed1 is also phosphorylated by Mek1, thereby promoting Hed1 stability (Callender et al, 2016). Meiotic recombination also requires the meiosis-specific recombinase Dmc1 (Bishop et al, 1992; Bishop, 1994; Neale & Keeney, 2006; Brown & Bishop, 2014). Saccharomyces cerevisiae Dmc1 is ~45% identical to Rad51, and Dmc1 is thought to act as the active recombinase during meiosis, with Rad51 acting as an accessory factor to mediate Dmc1 filament assembly (Cloud et al, 2012; Brown & Bishop, 2014). Rdh54 is a Rad54 homolog that, while expressed in mitotic cells, is thought to play a primary role during meiosis, potentially acting as a Dmc1-specific accessory factor (Dresser et al, 1997; Klein, 1997; Nimonkar et al, 2012). Interestingly, Rad54 stimulates Dmc1-mediated strand invasion in vitro (Nimonkar et al, 2012; Busygina et al, 2013) and deletion of RAD54 in S. cerevisiae causes delayed progression through meiosis and decreased spore viability (Shinohara et al, 1997), leading to speculation that these phenotypes may be caused by an interaction between Rad54 and Dmc1. However, genetic evidence suggests the role of Rad54 during meiosis is to repair excess DSBs through sister chromatid recombination and is not required for interhomolog recombination (Shinohara et al, 1997, 2003). It remains unclear whether Rad54 can physically discriminate between Rad51 and Dmc1, and it also remains unclear how the regulatory interplay between Hed1 and Rad54 controls the activities of Rad51 as cells transition into meiosis. Here, we utilize ssDNA curtains to observe the protein–protein and protein–DNA interactions that contribute to the maturation of mitotic and meiotic presynaptic complexes. We find that RPA prevents premature association of both Rad54 and Hed1 with ssDNA, helping to ensure a defined progression of assembly events. However, RPA is readily replaced with either Rad51 or Dmc1 in our assays (Gibb et al, 2014b; Qi et al, 2015; Qi & Greene, 2016; Ma et al, 2017c), allowing Rad54 to associate with the presynaptic complexes. The complexes formed between Rad54 and either Rad51 or Dmc1 are remarkably stable. However, once bound, the continued presence of Rad54 becomes independent of either Rad51 or Dmc1 filament stability, suggesting that Rad54 is loaded directly onto the underlying ssDNA. We also demonstrate that Hed1 binds selectively to Rad51 presynaptic complexes, and these binding interactions are essentially irreversible, so long as the Rad51 filament remains intact. In addition, we demonstrate that the Hed1 ssDNA-binding domain is dispensable for its association with the Rad51 presynaptic complex, but deletion of amino acids responsible for Hed1 ssDNA-binding activity prevents Hed1-mediated inhibition of Rad54 binding. Given these findings, we describe a model in which kinetic competition between Hed1 and Rad54 for binding interactions with Rad51 helps define the functional identity of the resulting presynaptic complexes during the transition into meiosis. Results RPA restricts Rad54 association with single-stranded DNA The interaction between Rad51 and Rad54 is essential for promoting efficient recombination (Tan et al, 2003; Heyer et al, 2006; Mazin et al, 2010; Shah et al, 2010). Rad54 binds to the Rad51 presynaptic complex, but quantitative measures of this interaction remain unavailable (Mazin et al, 2003; Raschle et al, 2004). Therefore, we sought to quantitate the association of Rad54 with the presynaptic complex using ssDNA curtains with total internal fluorescent microscopy (TIRFM) (Gibb et al, 2014b; Ma et al, 2017c). This approach allows us to visualize individual ssDNA molecules that can be sequentially coated with GFP- or mCherry-RPA followed by unlabeled Rad51, in a situation that closely mimics physiological assembly of the presynaptic complex (Fig 1A). Figure 1. GFP-Rad54 binds to Rad51-ssDNA filaments Schematic diagram outlining the ssDNA curtain assays used to test for GFP-Rad54 binding to the Rad51-ssDNA filaments. The cartoon illustrates the progression of steps from RPA-coated ssDNA to Rad51-ssDNA filaments, followed by the binding of Rad54, as indicated. Wide-field images of a single ssDNA molecule bound by RPA-mCherry and chased with 30 nM GFP-Rad54. The left panel shows the GFP-Rad54 signal, the middle panel shows the mCherry-RPA signal, and the right panel shows the merged image. Wide-field TIRFM images of Rad51-ssDNA molecules (unlabeled) bound by GFP-Rad54 (shown in green). The images were collected using 0.1, 1.0, 10, or 30 nM GFP-Rad54 as indicated. Examples of single Rad51-ssDNA molecules (unlabeled) bound by 0.1, 0.3, 1.0, 3.0, 10, or 30 nM GFP-Rad54 (top panel). Graph showing the GFP-Rad54 signal intensity, integrated over the entire lengths of the ssDNA substrates, as a function of GFP-Rad54 concentration (bottom panel). The data were fit by non-linear regression, and error bars represent the standard deviation (s.d.) for individual ssDNA molecules at each concentration of GFP-Rad54. The number of Rad51-ssDNA molecules (n) analyzed at each GFP-Rad54 concentration was as follows: 0.1 nM (n = 30), 0.3 nM (n = 30), 1.0 nM (n = 60), 3.0 nM (n = 48), 10 nM (n = 60), and 30.0 nM (n = 60). Source data are available online for this figure. Source Data for Figure 1 [embj201798728-sup-0002-SDataFig1.pdf] Download figure Download PowerPoint To monitor Rad54 binding, we used GFP-tagged Rad54 (Fig EV1A). Purified GFP-Rad54 was active for DNA-dependent ATP hydrolysis activity and also stimulated Rad51-mediated D-loop formation similar to WT Rad54 (Fig EV1B and D). The RPA-ssDNA complex is one of the earliest intermediates during HR (Heyer et al, 2010; Gibb et al, 2014b), and Rad54 harbors a ssDNA-binding domain (Wright & Heyer, 2014). Therefore, to test that Rad54 recruitment did not occur before Rad51 filament formation, we first tested the ability of GFP-Rad54 to bind mCherry-RPA filaments. Rad54 was unable to bind RPA filaments under the concentration regime tested (up to 30 nM GFP-Rad54) (Fig 1B), indicating that RPA prevents the binding of Rad54 to ssDNA in the absence of Rad51, which is consistent with in vivo observations of recombination foci in S. cerevisiae (Lisby et al, 2004). Click here to expand this figure. Figure EV1. Biochemical activities of GFP-Rad54 Schematic diagram of the GFP-Rad54 fusion construct used in this study. Gel image showing that GFP-Rad54 retains in vitro D-loop activity. The ATP hydrolysis-deficient mutant Rad54 K351R (30 nM) is shown as a negative control. Comparison of ATP hydrolysis activities for 30 nM Rad54 and 30 nM GFP-Rad54. Quantification of ATP hydrolysis rates for 30 nM Rad54 or 30 nM GFP-Rad54. Error bars represent s.d. of three independent experiments (N = 3). Download figure Download PowerPoint Rad54 association with the Rad51-ssDNA presynaptic complex Next, we tested GFP-Rad54 binding to Rad51-ssDNA filaments, which were prepared by exchanging the fluorescently tagged RPA for unlabeled Rad51, as previously described (Gibb et al, 2014b; Qi et al, 2015). In striking contrast to results with RPA-ssDNA, GFP-Rad54 bound to the Rad51-ssNDA filaments at concentrations as low as 0.1 nM (Fig 1C). At low concentrations, we could readily discern GFP-Rad54 as individual stationary, fluorescent puncta bound to the Rad51-ssDNA (Fig 1C and D). With increasing protein concentrations, the binding of Rad54 appeared more uniform along the Rad51-ssDNA filaments and appeared to approach saturation around 30 nM GFP-Rad54 (Fig 1C). We quantitated the cumulative fluorescent signal intensity at equilibrium by integrating the GFP signal intensity over the entire lengths of the Rad51-ssDNA filaments at 0.1, 0.3, 1.0, 3.0, 10, and 30 nM GFP-Rad54. The resulting data were fit by non-linear regression, yielding a dissociation constant (Kd) of 6.1 ± 0.6 nM, a Hill coefficient of 1.51 ± 0.3, and mean maximum signal intensity (Bmax) of ~4.57 ± 0.4 × 105 (a.u.) (Fig 1D). It is important to note that we could not go higher than 30 nM GFP-Rad54, due to high background; thus, values determined from the non-linear regression are based on the extrapolated saturation point. Importantly, we do not observe Rad54 translocation on the ssDNA substrates (even though ATP is present in all of the assays), as has been observed for Rad54 bound to dsDNA (Amitani et al, 2006; Ceballos & Heyer, 2011). The absence of translocation activity supports existing models in which the ssDNA-binding domain interacts with the presynaptic ssDNA, whereas the dsDNA-binding motor domain of Rad54 is oriented outward toward solution to allow for interactions with dsDNA during the homology search and strand invasion (Ceballos & Heyer, 2011; Wright & Heyer, 2014). We next estimated the number of Rad54 molecules bound at saturation. We took advantage of the fact that at low concentrations, we were able to observe individual GFP-Rad54 binding events (Fig EV2A). We could determine the number of GFP molecules associated with these binding events by monitoring the number of photo-bleaching steps. From this analysis, we observed that the majority (~60%) of binding events resulted in single-step photo-bleaching, with a smaller number of events showing two, three, or four photo-bleaching steps (Fig EV2B and C). Control experiments confirmed that these events were due to photo-bleaching and not due to protein dissociation (Fig EV2D). These data indicate that under these conditions, Rad54 binds initially as a monomer to the Rad51-ssDNA filaments. We next measured the total GFP signal intensity associated with the single-step photo-bleaching events and found this to be 147 ± 20 intensity units per molecule of GFP-Rad54 (Fig EV2E). Taking the mean maximum GFP-Rad54 signal intensity (Bmax) from the Rad54 titration curve (Fig 1D) and dividing by average signal intensity per molecule yielded a value of ~3,100 molecules of GFP-Rad54 per ssDNA molecule. For a ssDNA ~36,000 bases in length (Qi et al, 2015), this would give a value of ~1 Rad54 molecule per ~12 nucleotides (nt) or ~1 Rad54 molecule for every ~4 Rad51 monomers. It should be noted that these estimates are based on ssDNA that is completely saturated with Rad51, with each monomer occupying three nucleotides. In our experimental system, it is likely there will be some gaps in the Rad51 filaments, and these discontinuities may alter the ratio of Rad54 to Rad51. Regardless, these estimates provide an indication of the strong association between Rad54 and Rad51 within the presynaptic complex. Click here to expand this figure. Figure EV2. Single-step photo-bleaching assays for GFP-Rad54 binding to Rad51-ssDNA Kymograph depicting binding of GFP-Rad54 (0.1 nM; green) to the Rad51-ssDNA filament (unlabeled). The white arrows indicate individual Rad54 binding and bleaching events. Representative traces showing one- and two-step photo-bleaching events for GFP-Rad54 (0.1 nM) bound to Rad51-ssDNA. Quantification of the number of observed photo-bleaching steps determined from low concentration binding of GFP-Rad54 (0.1 nM). Survival probability of individual molecules of GFP-Rad54 (green squares; n = 181) bound to the Rad51-ssDNA filament compared to rate of GFP-Rad54 photo-bleaching (magenta). Error bars for the survival probability data were derived from bootstrapping analysis using a custom Python script. Distribution of signal intensity drops for individual GFP-Rad54 photo-bleaching events. The data were fit by a log-normal distribution, with a mean of 147 ± 20 intensity units. The error represents a 95% confidence interval for the data fit. Download figure Download PowerPoint Rad54 binding to Rad51 and Dmc1 presynaptic complexes Dmc1 is a meiosis-specific recombinase found in most eukaryotes (Bishop et al, 1992; Neale & Keeney, 2006; Brown & Bishop, 2014). Rad54 is required for normal progression through meiosis (Shinohara et al, 1997), and Rad54 interacts with Dmc1 and stimulates Dmc1 strand exchange activity in vitro (Nimonkar et al, 2012; Busygina et al, 2013). However, genetic evidence suggests that Rad54 and Dmc1 function in alternate pathways (Dresser et al, 1997; Bishop et al, 1999; Liu et al, 2014). To help further examine the potential role(s) of Rad54 in meiosis, here we sought to determine whether Rad54 could physically discriminate between Rad51 and Dmc1. We first tested the ability of GFP-Rad54 to bind Dmc1 filaments (Fig 2A) and found a concentration-dependent increase in Rad54 binding under equilibrium binding conditions, which was comparable to Rad54 binding on Rad51 filaments (Fig 2B). Analysis of GFP-Rad54 binding to the Dmc1-ssDNA filaments under increasing GFP-Rad54 concentrations by non-linear regression (Fig 2B) yielded a dissociation constant (Kd) of 3.5 ± 0.73 nM, a Hill coefficient of 1.33 ± 0.5, and mean maximum signal intensity (Bmax) of ~2.13 ± 0.5 × 105 (a.u.) (Fig 2C). Interestingly, the Bmax value for GFP-Rad54 binding to Dmc1-ssDNA was ~twofold lower than that observed for GFP-Rad54 binding to Rad51-ssDNA (Fig 2C), corresponding to ~1 Rad54 molecule per ~24 nucleotides (nt) or ~1 Rad54 molecule for every ~8 Dmc1 monomers. We next monitored the GFP-Rad54 binding kinetics to either Rad51-ssDNA or Dmc1-ssDNA filaments in real time (Fig 2D). Quantitation of the resulting data revealed no discernable difference in the rate of GFP-Rad54 association with either the Rad51- or Dmc1-ssDNA filaments (Fig 2D and E). Analysis of the maximum signal intensity values (Bmax) for GFP-Rad54 binding confirmed a ~two- to threefold lower saturation level for GFP-Rad54 to Dmc1-ssDNA filaments relative to the Rad51-ssDNA filaments (Fig 2F). This finding is in agreement with our equilibrium binding analysis, and together, these findings suggest that Rad51-ssDNA filaments support the binding of roughly twice as much GFP-Rad54 compared to Dmc1-ssDNA filaments of equivalent length. Figure 2. Comparison of GFP-Rad54 binding to Dmc1- and Rad51-ssDNA filaments Wide-field TIRFM image of Dmc1-ssDNA (unlabeled) bound by GFP-Rad4 (10 nM; shown in green). Images of single Dmc1-ssDNA molecules (unlabeled) bound by GFP-Rad54 (left panel). Graph showing the GFP-Rad54 signal intensity, integrated over the entire lengths of the ssDNA substrates, as a function of GFP-Rad54 concentration (right panel). The data were fit by non-linear regression, and error bars represent s.d. for individual ssDNA molecules at each concentration of GFP-Rad54. The number of Dmc1-ssDNA molecules analyzed at each GFP-Rad54 concentration was as follows: 0.1 nM (n = 30); 0.3 nM (n = 30); 1.0 nM (n = 30); 3.0 nM (n = 30); 10 nM (n = 30); and 30.0 nM (n = 30). Note that the curve for Rad51 shown here is reproduced from Fig 1D to allow for ready comparison between the Rad51 and Dmc1 data. Maximum signal intensity (Bmax) for the integrated GFP-Rad54 signal collected from Rad51-ssDNA and Dmc1-ssDNA molecules in panel (B). Values were obtained from non-linear regression analysis of the GFP-Rad54 titration data for Rad51-ssDNA and Dmc1-ssDNA, and the error bars represent 95% confidence intervals for the fit data. Kymographs depicting real-time association of GFP-Rad54 (green) with Rad51-ssDNA (top panel) and Dmc1-ssDNA (bottom panel). Quantification of normalized integrated fluorescence intensity for GFP-Rad54 association kinetics on Rad51-ssDNA (green circles; n = 20) and Dmc1-ssDNA (magenta circles; n = 25) filaments. Error bars represent s.d. for individual Rad51- or Dmc1-ssDNA molecules. Quantification of raw integrated fluorescence intensity for GFP-Rad54 association kinetics on Rad51-ssDNA (green circles; n = 20) and Dmc1-ssDNA (magenta circles; n = 25) filaments. Error bars represent s.d. for individual Rad51- or Dmc1-ssDNA molecules. Kymographs depicting the dissociation of GFP-Rad54 (green) with Rad51-ssDNA (top panel) and Dmc1-ssDNA (bottom panel) after free GFP-Rad54 was flushed from the sample chamber. Normalized fluorescence data comparing the rate of GFP-Rad54 signal loss due to protein dissociation from experiments performed with Rad51-ssDNA (green; n = 30), to the rate of GFP-Rad54 photo-bleaching (black; n = 40); error bars represent s.d. between different ssDNA molecules. Photo-bleaching data were collected by continuously illuminating the sample while collecting images at 100-ms intervals, whereas dissociation rate data were collected at 1-min intervals and the laser was shuttered between each image, as previously described (Gibb et al, 2014a). Normalized fluorescence data comparing the rate of GFP-Rad54 signal loss due to protein dissociation from experiments performed with Dmc1-ssDNA (magenta; n = 25), to the rate of GFP-Rad54 photo-bleaching (black; n = 25); error bars represent s.d. between different ssDNA molecules. Source data are available online for this figure. Source Data for Figure 2 [embj201798728-sup-0003-SDataFig2.pdf] Download figure Download PowerPoint Rad54 binds tightly to the Rad51 and Dmc1 presynaptic complexes We next evaluated the stability of GFP-Rad54 bound to both Rad51- and Dmc1-ssDNA filaments. For these measurements, we incubated GFP-Rad54 with either Rad51- or Dmc1-ssDNA filaments under saturating conditions, flushed the sample chambers with buffer to remove excess GFP-Rad54, and then monitored the change in GFP-Rad54 fluorescent signal over a 1-h time period (Fig 2G–I). Under these conditions, the loss of GFP-Rad54 fluorescence signal loss might be attributed to either the dissociation of GFP-Rad54 or GFP photo-bleaching. To discriminate between these two possibilities, we calculated the rate of GFP signal loss with and without laser shuttering, as previously described (Gibb et al, 2014a; Ma et al, 2017b). If the rate is the same in both cases, then signal loss can be attributed to photo-bleaching, and not to GFP-Rad54 dissociation (Gibb et al, 2014a). In contrast, if the rates are different, then the loss of GFP signal can be attributed to GFP-Rad54 dissociation. These experiments revealed that the loss of GFP-Rad54 signal was the same with and without laser shuttering (Fig 2H and I), indicating that the loss of signal intensity could be attributed to photo-bleaching with little or no GFP-Rad54 dissociation taking place during the 1-h observation window. We conclude that GFP-Rad54 binds very tightly to both Rad51- and Dmc1-ssDNA filaments, with little or no dissociation taking place over our experimental time scales. Interestingly, previous fluorescence recovery after photo-bleaching (FRAP) studies of DNA repair foci induced by ionizing radiation have demonstrated that mammalian RAD54 undergoes rapid (t1/2 = 0.5 s) and complete (100% mobile fraction) turnover at sites of DNA repair, whereas RAD51 does not (Essers et al, 2002). These studies have also shown that mammalian RAD52 undergoes complete turnover, exhibiting a recovery time (t1/2) of ~26 s (Essers et al, 2002). The result with mammalian RAD54 is different from our in vitro observations with yeast Rad54, which is surprising given the broad conservation of Rad54 among eukaryotes (Heyer et al, 2010; Mazin et al, 2010). One possible explanation for this difference is that the yeast and mammalian proteins behave differently with respect to interactions with the presynaptic complex. To test this possibility, we performed FRAP experiments of recombination protein foci in vivo. We find that most of the Rad51 (~63%) present within recombination foci remains immobile, while the remaining 37 ± 14% undergoes turnover with a recovery time (t1/2) of 87 (51–284) s (numbers in parentheses represent 95% confidence intervals; Fig EV3A). Consistent with previous results in mammalian cells (Essers et al, 2002), we also find the recombination mediator protein Rad52 undergoes rapid turnover within recombination foci, with 61 ± 18% of the protein present within a mobile fraction exhibiting a recovery time of 6 (4–17) s, but the remaining ~39% of the Rad52 remains immobile (Fig EV3B). Interestingly, 72 ± 28% of Rad54 within the repair foci undergoes turnover with a recovery time of 79 (60–119) s, and the remaining Rad54 remains immobile (Fig EV3C). These findings indicate that a portion of the S. cerevisiae Rad54 present in DNA damage-induced recombination foci does not undergo turnover, while the remaining Rad54 exchanges at a rate that is ~160 times slower than that observed in mammalian cells. Taken together, our in vitro and in vivo data support a model wherein S. cerevisiae Rad54 may be a more stable component of
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