The Rad51 paralog complex Rad55-Rad57 acts as a molecular chaperone during homologous recombination.

TL;DR: In this article, the authors used single-molecule imaging to reveal that the Rad51 paralog complex Rad55-Rad57 promotes assembly of Rad51 recombinase filament through transient interactions, providing evidence that it acts like a classical molecular chaperone.

About: This article is published in Molecular Cell.The article was published on 2021-03-04 and is currently open access. It has received 37 citations till now. The article focuses on the topics: RAD51 & Homologous recombination.

Summary

Introduction

• Homologous recombination (HR) is crucial for the repair of stalled or collapsed replication forks, DNA double–strand breaks (DSB), and chromosome segregation during meiosis (Kowalczykowski, 2015; Mehta and Haber, 2014; San Filippo et al., 2008).
• The key DNA transactions that occur during HR are catalyzed by members of the ATP–dependent Rad51/RecA family of DNA recombinases, which form extended helical filaments on the ssDNA associated with processed DNA breaks (Bianco et al., 1998; Kowalczykowski, 2015; Mehta and Haber, 2014; San Filippo et al., 2008).
• There are five human RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3), and cells lacking any of these proteins are sensitive to DNA damaging agents and exhibit chromosomal abnormalities (Bonilla et al., 2020; Prakash et al., 2015).
• Biochemical experiments show that Rad55–Rad57 promotes Rad51 filament assembly and strand exchange activity by overcoming the inhibitory effect of RPA, and not through a direct stimulation of Rad51 strand exchange activity, as reactions without RPA are not stimulated (Gaines et al., 2015; Sung, 1997).

Results

• Rad55–Rad57 does not interact with RPA–ssDNA complexes.
• The authors next examined the interaction of Rad55–Rad57 with pre–assembled Rad51–ssDNA filaments.
• Surprisingly, the authors saw little to no binding of Rad55–Rad57 to pre–assembled Rad51 filaments (Figure 1G–I).
• The authors first tested whether their ATPase deficient mutants complemented the IR defect of rad55D rad57D cells.
• These data demonstrate that residual Rad55–Rad57 complexes bound to mature Rad51 filaments do not physically obstruct Srs2 translocation.

Discussion

• The authors work establishes that the S. cerevisiae Rad51 paralog complex Rad55–Rad57 interacts with Rad51 only during the earliest stages of Rad51 filament assembly, then promptly dissociates during filament maturation through a mechanism linked to ATP hydrolysis by Rad55.
• Antagonistic relationship between Srs2 and Rad55–Rad57 Current knowledge of Rad55–Rad57 posits two independent pro–HR functions for this protein complex.
• Importantly, the Rad52–Rad51 interaction is required for this effect, suggesting that Rad52 stimulation of Rad51 filament re– assembly underlies the Srs2 antagonism.
• The authors data support such a model where the Rad51 filaments are continuously remodeled through the combined action of recombination mediators and anti–recombinases, which could allow for better quality control of the filament and poise it for repair through the most appropriate pathway.
• The authors findings support a model where RPA and Rad51 compete for the same ssDNA substrate, and Rad55–Rad57 tilts the balance in favor of Rad51 by continuously stimulating its reassembly, rather than making the filaments resistant to Srs2 disruption.

Acknowledgments

• The authors thank Rodney Rothstein and members of the Greene and Sung laboratories for discussion and comments on the manuscript.
• The authors thank Simon Boulton, David Rueda, Ondrej Belan and colleagues for sharing data prior to publication and for comments on their manuscript.
• M.L. was supported by the Danish Council for Independent Research, the Villum Foundation, and the Danish National Research Foundation (DNRF115).
• P.S. is the recipient of a CPRIT REI Award (RR180029) and holder of the Robert A. Welch Distinguished Chair in Biochemistry (AQ–0012).

Author Contributions

• U.R. cloned and purified GFP–tagged Rad55–Rad57, designed and conducted all single molecule assays and conducted bulk biochemical assays.
• Y.K. assisted with Rad55–Rad57 expression and purification.
• M.L. conducted all in vivo measurements of Rad55–Rad57 foci.
• U.R. and E.C.G. co–wrote the manuscript with input from all other co–authors.

Figure Legends

• Figure 1. Rad55–Rad57 does not bind RPA–ssDNA or mature Rad51–ssDNA filaments.
• (E) Wide–field view of RPA–mCherry bound ssDNA 1 min before, during, and after injection of 60 nM GFP–tagged Rad55–Rad57 .
• Figure 3. Rad55–Rad57 stimulates Rad51 filament assembly and promotes depletion of RPA.
• Data is represented as mean normalized RPA intensity; shaded area represents 95% CI.

EXPERIMENTAL MODEL AND SUBJECT DETAILS

• Growth temperature, expression and purification information for each different protein are provided below in the METHOD DETAILS.
• Ura+ transformants were selected on synthetic complete plates lacking uracil (SC–Ura) + 2% glucose.
• Additional details are provided below in the METHODS DETAILS.
• Additional details are provided below in the METHODS DETAILS.

METHOD DETAILS Purification of GFP–Rad55–Rad57.

• Cloning was done by the InFusion HD cloning system (Takara Biotech, Cat No. 638920) using the manufacturer’s instructions.
• All steps were carried out at 4˚C, and 2 mM ATP and 5 mM Mg2+ was maintained in all buffers during lysis and purification steps to minimize the aggregation and precipitation of GFP–Rad55–Rad57.
• Lysate was clarified by ultracentrifugation and proteins precipitated with 40% ammonium sulphate.
• Fractions containing GFP–Rad55–Rad57 were pooled and incubated with 1.5–ml anti–FLAG affinity resin (Sigma, Cat No. A2220) for 1 h, with gentle mixing.
• GFP–Rad55–Rad57 containing fractions were pooled and further purified by gel filtration using a 24 ml Superdex 200 column (GE Healthcare, Cat No. 29321906) in buffer K supplemented with 300 mM KCl.

Purification of Rad51, RPA, Srs2, Hed1 and Dmc1.

• Overexpression cells were harvested and stored at –80˚C.
• The extract was then bound to 1 mL of pre–equilibrated Ni–NTA resin for 1 hour with rotation.
• The purified Rad51 was then stored at –80˚C in single use aliquots.
• The protein was then eluted in nickel A buffer + 200 mM imidazole.
• Fractions containing ScDmc1 (eluting at ∼300 mM KCl) were pooled, concentrated in an Amicon Ultra micro– concentrator (Millipore, Cat No. UFC501096), snap–frozen in liquid nitrogen, and stored at −80˚C.

Camptothecin sensitivity assays

• Ura+ transformants were selected on synthetic complete plates lacking uracil (SC–Ura) + 2% glucose.
• Two independent colonies were picked from each transformation and used to inoculate 5 mL SC–Ura + 2% glucose.
• When cultures reached late log phase, 0.25 mL were pelleted from each culture, washed twice with 1 mL of water and the pellets were re–suspended in 10 mL SC–Ura +2% glucose or 10 mL SC–Ura +1% galactose.
• For the cells grown with glucose, 5 µL from 10–fold serial dilutions of each culture were spotted on SC–Ura + 2% glucose plates containing 0, 0.5, 1 or 5 µg/mL camptothecin (CPT).
• Plates were incubated at room temperature (~23˚C) for 2–3 days before imaging.

IR sensitivity assays

• Ten–fold serial dilutions of overnight cultures of wild–type (ML8–9A) and rad55∆ rad57∆ null mutant cells (ML1153–13B) transformed with empty vector (pRS416) or vectors expressing His– GFP–Rad55 and FLAG–Rad57 transgenes as indicated (pX54, pX55 and pX56) were plated on SC–Ura containing either 2% glucose or 2% raffinose supplemented with 0.02% galactose .
• Prior to microscopy, transformed strains were grown overnight in SC–Ura containing 2% raffinose and 0.02% galactose with shaking at 25˚C, diluted to OD600 = 0.2 and grown for another 3 hours in SC– Ura containing 2% raffinose and 0.02% galactose with shaking at 25˚C.
• One fluorescence image was acquired before photobleaching and a time series of 9 images after photobleaching using a wide–field microscope (DeltaVision Elite; Applied Precision) equipped with a 100× objective lens with a numerical aperture of 1.35 (U–PLAN S–APO, NA 1.4; Olympus), a cooled EMCCD camera (Evolve 512; Photometrics), and a solid–state illumination source (Insight; Applied Precision, Inc).
• Chromium was then deposited on the microscope surface using electron beam evaporation.
• Flow cells were assembled and dsDNA curtains were prepared as previously described (Greene et al., 2010).

In vitro photobleaching controls and Hed1–binding assays

• Data was collected for 15 mins under stopped flow conditions, and images acquired either every 10 s, 20 s, or 60 s. Mean normalized GFP intensity and 95% CI was plotted as a function of time for each frame rate.
• For experiments with Hed1, 2 µM Rad51 with 30 nM GFP–Hed1 was co–injected in HR buffer onto mCherry–RPA–ssDNA filaments.
• Images were acquired every 10 s during a 15 min incubation under stopped flow conditions and mean normalized intensity with 95% CI plotted.

Srs2 antirecombinase assays

• Sample chambers containing pre–assembled Rad51–ssDNA filaments were flushed with HR buffer for several minutes to remove any unbound Rad51 prior to the Srs2 injections.
• 500 pM Srs2 was injected in HR buffer supplemented with 500 pM RPA and observed under stopped flow conditions.
• In experiments with free Rad51 and/or free Rad55–Rad57 in the buffer, the indicated amount of each protein was co–injected with Srs2 and RPA in 150 µL aliquots using a flow rate of 0.5 mL min–1, and then observed under stopped flow conditions.

Single–molecule data collection and analysis

• For all two–color images, the authors used a custom–built shuttering system to avoid signal bleed–through during image acquisition (De Tullio et al., 2018).
• Images from the green (GFP) and the red channels are recorded independently, these recordings are offset by 100 ms such that when one camera records the red channel image, the green laser is shuttered off, and vice versa.
• Images were captured at an acquisition rate of 1 frame per 10 s (unless indicated otherwise) with a 100– millisecond integration time, and the laser was shuttered between each acquired image to minimize photo–bleaching.
• Raw TIFF images were imported as image stacks into ImageJ, and kymographs were generated from the image stacks by defining a 1–pixel wide region of interest (ROI) along the long–axis of the individual dsDNA molecules.
• All data analysis was performed using the resulting kymographs.

Rad51 assembly kinetics

• For calculating assembly kinetics, kymographs were generated by defining a 1–pixel–wide region of interest (ROI) along the length of individual ssDNA–filaments.
• Integrated signal intensity for each time point was obtained from individual kymographs and normalized using the maximum value for that kymograph.
• The mean and 95% CI of normalized intensities across all ssDNA molecules was plotted for each time point, and the distribution was fit to a single exponential decay curve using GraphPad Prism to obtain the rate constants.

Analysis of Srs2 trajectories

• Raw TIFF files were imported as image stacks into Fiji software for analysis, and kymographs were generated by defining a 1–pixel–wide region of interest (ROI) along the length of individual ssDNA molecules.
• Within each kymograph, the start and end points of individual Srs2 traces were analyzed to obtain the x (time), y coordinates in terms of pixel values.
• Apparent Srs2 translocation velocity was calculated from the number of pixels translocated per unit time = (y2 – y1)/(x2 – x1).
• As described previously (De Tullio et al., 2018), each pixel in the y–axis corresponds to ~725 nucleotides (nt) of the Rad51–ssDNA filament, and each pixel in the y–axis corresponds to 10s.
• To obtain the number of Srs2 molecules per Rad51 filament (Figure 6C,F), a single point on the Rad51–ssDNA filament was observed over a 2.5 min time window (this time window was chosen due to substantial ssDNA breakage at longer times).

Statistical information

• All statistical analysis was carried out using Graphpad Prism.
• (B) Cell lysate, resuspended ammonium sulphate precipitate (A.S. ppt), elution from Ni2+ column, elution from FLAG column and fractions from the gel filtration column (0.5 mL fractions) were loaded on a 10% SDS PAGE gel and stained with Coomassie blue.
• Normalized intensity is plotted and the shaded area represents 95% CI.
• Time at which Rad51–K191R and Rad55–Rad57 were co–injected is indicated with an asterisk (*). (C) Kymographs showing the behavior of mCherry–RPA and GFP–Rad55–Rad57.
• (A) Quantifications were done over a 2.5 min time window (as indicated) starting from the first Srs2 molecule translocation on Rad51–ssDNA filaments.

Frequently asked questions

Q1. What are the contributions mentioned in the paper "Rad51 paralog complex rad55–rad57 acts as a molecular chaperone during homologous recombination" ?

In this paper, the Rad51 recombinase filaments are used for the repair of stalled or collapsed replication forks, DNA doublestrand breaks ( DSB ), and chromosome segregation during meiosis. 

Q2. What is the role of Rad51 paralogs in human cancer?

Rad51 paralogs fulfill a conserved, but undefined role in HR, and their mutations are associated with increased cancer risk in humans. 

Q3. What is the role of Rad51 in the human genome?

Their findings support a model in which Rad51 is in flux between free and ssDNA–bound states, the rate of which is dynamically controlled though the opposing actions of Rad55–Rad57 and Srs2. 

Q4. What is the role of the Rad51 paralog complex?

the authors use single–molecule imaging to reveal that the Saccharomyces cerevisiae Rad51 paralog complex Rad55–Rad57 promotes the assembly of Rad51 recombinase filaments through transient interactions, providing evidence that it acts as a classical molecular chaperone.