Fig 1.
Screen of potentially phosphorylated residues in Rad54.
(A). Schematic of residues in Rad54 that were identified as phosphorylated in the SuperPhos database. (B). Yeast complementation spot assay to monitor the effect of phosphomimic mutants (Left) and Alanine mutants (Right) of Rad54 at 0.01% MMS. (C). Serial dilution spot assay to monitor the impact of rad54-S816A, S817A, and rad54-S816D, S817D on sensitivity to 0.01% MMS, 20 mM Hydroxyurea (HU), 0.02 µg/ml 4-NQO, and 10 µM CPT. (D). Schematic diagram illustrating the substrate for an ectopic recombination assay. An HO endonuclease site is located on chromosome V, and the repair template is located on chromosome III. (E). Graph representing the survival percentage for RAD54, rad54∆, rad54-S816A, S817A, and rad54-S816D, S817D. The bars represent the mean, and the error bars represent the standard error measurements of at least seven independent experiments.
Fig 2.
Mutations in Rad54 are separation-of-function mutants.
(A). Alphafold3 structural predictions for Rad54 + dsDNA + ATP (Left) and Rad54 + dsDNA + ADP (Right). Colors of the structure reflect the confidence in the prediction. The pLDDT values are listed below the structures. (B). Serial dilution spot assays to test complementation of rad54∆ with pRS415-RAD54, Empty vector, rad54-D525A, D527A, rad54-D525K, D527K, rad54-D525N, D527N, rad54-D525S, D527S, rad54-S816A, S817A, rad54-D525A, D527A, S816A, S817A, rad54-D525K, D527K, S816A, S817A, rad54-D525N, D527N, S816A, S817A, rad54-D525S, D527S, S816A, S817A, rad54-D525A, rad54-D525K, rad54-D525N, rad54-D525S, rad54-S816D, rad54-D525S, S816D, rad54-S816D, S817D, rad54-D525A, D527A, S816D, S817D, rad54-D525K, D527K, S816D, S817D, rad54-D525N, D527N, S816D, S817D, and rad54-D525S, D527S, S816D, S817D for MMS sensitivity. (C). Schematic diagram illustrating the substrate for an ectopic recombination assay. An HO endonuclease site is located on chromosome V, and the repair template is located on chromosome III. (D). Graph illustrating percentage survival for complementation of rad54∆ with pRS415-RAD54, Empty vector, rad54-D525A, D527A, rad54-D525K, D527K, rad54-D525N, D527N, rad54-D525S, D527S, rad54-S816A, S817A, rad54-D525A, D527A, S816A, S817A, rad54-D525K, D527K, S816A, S817A, rad54-D525N, D527N, S816A, S817A, rad54-D525S, D527S, S816A, S817A, rad54-D525A, rad54-D525K, rad54-D525N, rad54-D525S, rad54-S816D, rad54-D525S, S816D, rad54-S816D, S817D, rad54-D525A, D527A, S816D, S817D, rad54-D525K, D527K, S816D, S817D, rad54-D525N, D527N, S816D, S817D, and rad54-D525S, D527S, S816D, S817D in an ectopic recombination assay. The bars represent the mean, and the error bars represent standard error measurements of at least three independent experiments.
Fig 3.
Mutations in RAD54 impact recombination intermediates.
(A). Schematic diagram illustrating the D-loop capture assay to trap the formation of nascent D-loops during recombination. (B). Graph representing D-loop capture efficiency at 4 hours post-break induction for RAD54, rad54∆, rad54-S816A, S817A, rad54-S816D, S817D, and rad54-D525S, D527S, S816D, S817D. The bar represents the mean, and the error bars represent the standard deviation for at least three independent experiments. (C). Schematic diagram illustrating the assay to monitor D-loop extension. (D). Graph representing D-loop extension at 6 hours for RAD54, rad54∆, rad54-S816A, S817A, rad54-S816D, S817D, and rad54-D525S, D527S, S816D, S817D. The bar represents the mean, and the error bars represent the standard deviation for at least three independent experiments. (E). Graph representing D-loop capture efficiency at 4 hours post-break induction for sgs1∆, rad54-S816A, S817A sgs1∆, rad54-S816D, S817D sgs1∆. The bar represents the mean, and the error bars represent the standard deviation for at least three independent experiments. (F). Graph representing D-loop extension at 6 hours for hours post-break induction for sgs1∆, rad54-S816A, S817A sgs1∆, rad54-S816D, S817D sgs1∆. The bar represents the mean, and the error bars represent the standard deviation for at least three independent experiments. (G). The percentage of dsDNA among total extension products for RAD54, rad54∆, rad54-S816A, S817A, rad54-S816D, S817D, sgs1∆, rad54-S816A, S817A sgs1∆, rad54-S816D, S817D sgs1∆. The error bars represent the standard error measurement for at least three independent experiments.
Fig 4.
Impact of RAD54 mutations on allelic recombination between homologous chromosomes.
(A). Schematic diagram illustrating the DNA reporter used to analyze the effect of Rad54 mutation on allelic recombination. (B). Schematic diagram illustrating the potential gene conversion outcomes and HR based repair pathways during allelic recombination. (C). Graph representing the plating efficiency of strains treated with galactose. These were measured by dividing the number of colonies formed after galactose treatment to a no-galactose control. The bar represents the mean of the data, and the error bars the standard deviation of at least 3 independent experiments. (D). Graph representing the fraction of colonies that undergo chromosome loss for All strains tested. The bar represents the mean and the error bars the standard deviation of at least four independent experiments. (E). Graph representing the percentage of solid red, solid white, and sectored colonies for all strains tested. The bar represents the mean of the data, and the error bars represent the standard deviation of at least three independent experiments. (F). Graph representing the fraction colonies that undergo short tract gene conversion for all strains tested. This data was generated by measuring the colonies that grew on YNB (–Ade) + dextrose. The bar represents the mean, and the error bars the standard deviation of at least four independent experiments.
Fig 5.
Rad54 S816D/S816D has reduced affinity for dsDNA.
(A). Representative 0.5% TBE gel for EMSA with Rad54 and Rad54 S816D/S817D with a 90 bp Atto647N labelled dsDNA. (B). Graph representing the quantification of the EMSE data for Rad54, Rad54 S816A/S817A, Rad54 S816D/S817D, and Rad54 D525S/D527S/S816D/S817D. The data are fit by a hill equation. The error bars represent the standard error measurement of at least four independent experiments. (C). Graph representing the Kd measurements for individual EMSA experiments for Rad54, Rad54 S816A/S817A, Rad54 S816D/S817D, and Rad54 D525S/D527S/S816D/S817D. The bar represents the mean, and the error bars represent the standard error measurement of the experiment. (D). Graph representing the amount of ADP produced per minute in an ATP hydrolysis assay for Rad54, Rad54 S816A/S817A, Rad54 S816D/S817D, and Rad54 D525S/D527S/S816D/S817D. The bar represents the mean, and the error bars represent the standard deviation of four independent experiments. (E). Graph representing the amount of ADP produced per min for Rad54 and Rad54 S816D/S817D. The bar represents the mean, and the error bar represents the standard deviation of 3 independent experiments. (F). Schematic diagram illustrating DNA curtains experiments to test the activity of Rad54 on dsDNA. This image is reproduced through a CC-BY 4.0 license from [95]. (G). Widefield microscope image for GFP-Rad54, GFP-Rad54 S816A/S817A, and GFP-Rad54 S816D/S817D, and Rad54 D525S/D527S/S816D/S817D. (H). Mean fluorescent decay rate for Rad54 (Blue), Rad54 S816A/S817A (Green), Rad54 S816D/S817D (Orange), Rad54 D525S/D527S/S816D/S817D (Magenta) and Rad54 with ADP (Cyan). The black line gives the photobleaching rate. The shade represents the standard error measurement of the data. (I). Graph representing the koff for GFP-Rad54 (+ATP), GFP-Rad54 S816A/S817A, and GFP-Rad54 S816D/S817D, Rad54 D525S/ D527S/S816D/S817D and GFP-Rad54 (+ADP) from dsDNA. The bars represent the mean, and the error bars the standard error measurement of the data.
Fig 6.
Rad54 S816D/S817D is poorly processive.
(A). Schematic diagram illustrating DNA curtains experiments to test the activity of Rad54 on dsDNA. This image is reproduced through a CC-BY 4.0 license from [95]. (B). Representative kymographs illustrating the movement of GFP-Rad54, GFP-Rad54 S816A/S817A, GFP-Rad54 S816D/S817D, GFP-Rad54 D525S/D527S, S816D/S817D with and against buffer flow. (C). Bar graph representing the percentage of Rad54 (166/216), Rad54 S816A/S817A (285/446), and Rad54 S816D/S817D (48/271) and GFP-Rad54 D525S/D527S/S816D/S817D that move against the buffer flow. (D). Graph representing the velocity of translocation in kbp/s for Rad54 (N = 216), Rad54 S816A/S817A (N = 446), Rad54 S816D/S817D (N = 271), and Rad54 D525S/D527S/S816D/S817D (N = 81). The molecules that moved against the flow are above the X-axis, and the molecules that moved with the flow are below the X-axis. The bar represents the mean, and the error bars represent the standard deviation of the data. The negative value indicates the direction. (E). Graph representing the distance moved for Rad54 (N = 216), Rad54 S816A/S817A (N = 446), Rad54 S816D/S817D (N = 271), and Rad54 D525S/D527S/S816D/S817D (N = 81). The molecules that moved against the flow are above the X-axis (positive values), and the molecules that moved with the flow are below the X-axis (negative values). The bar represents the mean, and the error bars represent the standard deviation of the data.
Fig 7.
Mutations lead to PSC slippage during translocation.
(A). Cartoon diagram illustrating the use of DNA curtains to perform experiments to monitor the activity of the presynaptic complex (PSC) on dsDNA. This image is reproduce through a CC-BY 4.0 license from [95]. (B). Representative kymographs for PSCs, PSCs with Rad54 S816A/S817A, or PSCs with Rad54 S816D/S817D. Shown are merged images of RPA-mCherry, GFP-Rad54, and Atto647–90-mer ssDNA. (C). Graph representing the percentage of PSC with Rad54 (183/215), PSC with Rad54 S816A/S817A (326/378), and PSC with Rad54 S816DS817D (710/835) that moved against the buffer flow. (D). Measured translocation velocities for the PSC with Rad54 (N = 215), PSC with Rad54 S816A/S817A (N = 378), and PSC with Rad54 S816D/S817D (N = 835). The PSCs that moved against the flow are above the X-axis, and the PSC values that moved with the flow are below the X-axis. The bars represent the mean, and the error bars represent the standard deviation of the data. (E). Measured translocation distances for the PSC with Rad54 (N = 215), PSC with Rad54 S816A/S817A (N = 378), and PSC with Rad54 S816D/S817D (N = 835). The PSCs that moved against the flow are above the X-axis, and the PSC values that moved with the flow are below the X-axis. The bars represent the mean, and the error bars represent the standard deviation of the data. (F). Schematic illustrating the loop extrusion activity of Rad54 extruding loops that can bind RPA-mCherry. (G). Graph representing the number of PSCs with RPA mCherry localized after a five-minute incubation without buffer flow for PSCs with Rad54 and Rad54 S816D/S817D. (H). A dot plot representing the intensity of RPA bound to bound to PSCs with Rad54 (N = 39) and Rad54 S816D/S817D (N = 28). The bar represents the mean of the data, and the error bars represent the standard deviation.
Fig 8.
Loop extrusion mechanism for D-loop formation.
A cartoon diagram illustrating a proposed mechanism for Rad54 mediated D-loop formation, and the consequences of rad54-S816D, S817D failure during the process. We propose that Rad54 promotes homology search by extruding DNA loops and locally pumping duplex DNA to facilitate Rad51-catalyzed strand invasion, leading to the formation of a D-loop intermediate. Following strand invasion, Rad54 removes Rad51 from the ssDNA 3’end, allowing DNA polymerase-mediated extension that stabilizes the D-loop. In the rad54-S816D, S817D mutant, reduced DNA binding affinity and processivity impair loop extrusion, resulting in smaller or less stable pre-D-loop intermediates and reduced efficiency of D-loop formation and extension.